Apoptosis Manual

Roche Applied Science

Apoptosis, Cell Death andCell Proliferation

3rd edition

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Roche Applied Science

Apoptosis, Cell Death, andCell Proliferation

3rd edition

Apoptosis, Cell Death, and Cell Proliferation ManualII

Table of Contents

1. Introduction .......................................................................................................................................................... 21.1 Terminology of cell death ................................................................................................................................... 21.2 Differences between necrosis and apoptosis ............................................................................................ 31.3 Apoptotic Pathways.............................................................................................................................................. 5

2. Apoptosis Assay Methods............................................................................................................................ 7Method/Product selection guide .................................................................................................................... 8

2.1 Methods for studying apoptosis in cell populations............................................................................. 102.1.1 Assays that measure DNA fragmentation ....................................................................................... 10

� Apoptotic DNA Ladder Kit ............................................................................................................... 13� Cell Death Detection ELISAPLUS .................................................................................................... 15

2.1.2 Assays that measure apoptosis-induced proteases (caspases) ............................................. 18� M30 CytoDEATH ................................................................................................................................... 19� M30 CytoDEATH, Fluorescein ......................................................................................................... 19� Caspase 3 Activity Assay .................................................................................................................. 22� Homogeneous Caspases Assay ..................................................................................................... 25� Anti-PARP ............................................................................................................................................... 27

2.1.3 Summary of methods for studying apoptosis in cell populations ........................................... 302.2 Methods for studying apoptosis in individual cells............................................................................... 32

2.2.1 The TUNEL enzymatic labeling assay ............................................................................................... 33� In Situ Cell Death Detection Kit, Fluorescein ............................................................................. 36� In Situ Cell Death Detection Kit, TMR red .................................................................................. 36� In Situ Cell Death Detection Kit, AP .............................................................................................. 39� In Situ Cell Death Detection Kit, POD .......................................................................................... 39

2.2.2 Assays that measure membrane alterations .................................................................................. 43� Annexin-V-FLUOS ............................................................................................................................... 44� Annexin-V-FLUOS Staining Kit ....................................................................................................... 44� Annexin-V-Alexa 568 .......................................................................................................................... 44� Annexin-V-Biotin .................................................................................................................................. 47

2.2.3 Assays that use DNA stains .................................................................................................................. 49� DAPI, Propidium iodide ...................................................................................................................... 50

2.2.4 Summary of methods for studying apoptosis in individual cells ............................................. 522.3. Detection of apoptosis-related proteins.................................................................................................... 54

� Anti-Fas (CD95/Apo-1) ...................................................................................................................... 55� p53 pan ELISA ...................................................................................................................................... 56

3. Cytotoxicity Assay Methods..................................................................................................................... 583.1 Relationship between cytotoxicity, apoptosis and necrosis ............................................................... 583.2 Methods for studying cytotoxicity................................................................................................................ 59

3.2.1 Assays that measure plasma membrane leakage ........................................................................ 59� Cytotoxicity Detection Kit (LDH) .................................................................................................... 61� Cellular DNA Fragmentation ELISA .............................................................................................. 64

3.2.2 Assays that measure metabolic activity ............................................................................................ 68� Cell Proliferation Kit I (MTT) ............................................................................................................ 85� Cell Proliferation Kit II (XTT) ............................................................................................................ 86� Cell Proliferation Reagent WST-1 ................................................................................................... 87

3.2.3 Summary of methods for studying cytotoxicity .............................................................................. 70

A Cell Death — Apoptosis and Necrosis Page

Table of Contents

IIIIntroduction

1. Introduction ........................................................................................................................................................ 741.1 Terminology of cell proliferation and viability........................................................................................... 741.2 Cell Cycle .............................................................................................................................................................. 75

2. Cell proliferation/viability assay methods......................................................................................... 78Method/Product selection guide ................................................................................................................. 80

2.1 Methods for studying cell proliferation and viability in cell populations...................................... 822.1.1 Assays that measure metabolic activity ........................................................................................... 82

� Cell Proliferation Kit I (MTT) ............................................................................................................ 85� Cell Proliferation Kit II (XTT) ............................................................................................................ 86� Cell Proliferation Reagent WST-1 .................................................................................................... 87

2.1.2 Assays that measure DNA synthesis ................................................................................................ 89� 5�-Bromo-2�-deoxy-uridine Labeling and Detection Kit III .................................................. 91� Cell Proliferation ELISA, BrdU (colorimetric) ............................................................................ 94� Cell Proliferation ELISA, BrdU (chemiluminescence) ............................................................ 94

2.1.3 Summary of methods for studying cell proliferation and cell viability in cell populations 982.1.4 Single reagents for the measurement of DNA synthesis .......................................................... 98

2.2 Methods for studying cell proliferation and viability in individual cells ...................................... 1002.2.1 Assays that measure DNA synthesis................................................................................................ 100

� 5�-Bromo-2�-deoxy-uridine Labeling and Detection Kit I .................................................... 101� 5�-Bromo-2�-deoxy-uridine Labeling and Detection Kit II .................................................. 101� In Situ Cell Proliferation Kit, FLUOS ............................................................................................ 102� Anti-BrdU, formalin grade .............................................................................................................. 105� Anti-BrdU-Fluorescein ..................................................................................................................... 105� Anti-BrdU-Peroxidase, Fab fragment ......................................................................................... 105

2.2.2 Summary of methods for studying cell proliferation and viability in individual cells .... 108

B Cell Proliferation and Viability Page

Table of Contents

Apoptosis, Cell Death, and Cell Proliferation ManualIV

1. Technical Tips ................................................................................................................................................. 1121.1 Selected frequently asked questions (FAQs) about cell death assays ....................................... 1121.2 Technical tips on the TUNEL method ..................................................................................................... 113

1.2.1 TUNEL: Improvement and evaluation of the method for in situ apoptotic cell identification ..................................................................................................................................... 113

1.2.2 TUNEL protocol for tissues which tend to give false positives ............................................. 1141.2.3 Tips for avoiding or eliminating potential TUNEL labeling artifacts .................................... 115

1.3 Technical tips on the use of Annexin-V-Biotin for light microscope detection ....................... 1171.4 Technical tips on the use of the Apoptotic DNA Ladder Kit on tissue samples ..................... 1181.5 Technical tips on the Cell Proliferation ELISA Kits ............................................................................. 118

2. Special applications of cell death and cell proliferation methods .................................. 1192.1 TUNEL assays .................................................................................................................................................. 119

2.1.1 Discrimination between dead and viable apoptotic cells using two-color TdT assay and surface labeling as detected by flow cytometry ................................................................. 119

2.1.2 The use of flow cytometry for concomitant detection of apoptosis and cell cycle analysis ............................................................................................................................................ 119

2.1.3 Comparison of two cell death detection methods: In situ nick translation and TUNEL 1202.1.4 Fixation of tissue sections for TUNEL combined with staining for thymic epithelial

cell marker ................................................................................................................................................. 1202.2 Metabolic assays ............................................................................................................................................ 121

2.2.1 Biochemical and cellular basis of cell proliferation assays that use tetrazolium salts . 1212.3 Annexin assays ................................................................................................................................................ 121

2.3.1 The use of annexin for concomitant detection of apoptosis and cellular phenotype ... 1212.4 BrdU assays ...................................................................................................................................................... 122

2.4.1 Detection of bromodeoxyuridine in paraffin-embedded tissue sections using microwave antigen retrieval is dependent on the mode of tissue fixation ........................ 122

3. References ...................................................................................................................................................... 1233.1 Apoptosis-related parameters – Abbreviations and References .................................................. 1233.2 Examples for applications of Roche Applied Science products ................................................... 1283.3 General references ......................................................................................................................................... 154

4. General abbreviations .............................................................................................................................. 158

5. Ordering Guide ............................................................................................................................................. 161

6. Index ................................................................................................................................................................... 164

C Appendix Page

Table of Contents

VIntroduction

Overview of this Guide

How this guide can help you study cell death and cell proliferation?

When and why do cells die? Does the concentration of environmental pollutants exertcytotoxic or cytostatic effects on cells? What factors influence the rate and timing of cellproliferation? Researchers in basic, industrial, and medical research are asking these ques-tions and looking for answers. Understanding the normal regulation of cell death and cellproliferation will be critical e.g., for the development of new and more successful thera-pies for preventing and treating cancer and for the screening of new anti-cancer com-pounds.

Many assays exist to measure cell death and cell proliferation. However, if you have onlyrecently become interested in cell death or cell proliferation, you may find the diversity ofsuch assays bewildering. You may not be able to determine what each assay measures nordecide which assays are best for your purposes. This guide is designed to help you makesuch decisions. It presents a brief overview of cell death and cell proliferation, along withthe major assays currently available to measure each. In addition, it clearly lists theadvantages and the disadvantages of these assays.

For those who want to eliminate radioactivity from their laboratories, this review alsodescribes a number of non-radioactive assays that can serve as alternatives to radioactiveassays. Wherever possible, the review will compare the sensitivity of the radioactive andnon-radioactive assays.

Overview of this Guide

Apoptosis, Cell Death, and Cell Proliferation ManualVI

CELL DEATH by Andrew H. Wyllie

Over the past five or six years there has been a near-exponential increase in publicationson apoptosis. Around 30 new molecules have been discovered whose known functionsare exclusively to do with the initiation or regulation of apoptosis. A further 20 moleculesat least, although already associated with important roles in signalling or DNA replica-tion, transcription or repair, have been recognised as affecting the regulation of apopto-sis. This article is dedicated to young scientists thinking of entering this exploding area ofbiology, and to those more mature ones who happened to be looking elsewhere when theblast reached them, and consequently are in need of a rapid introduction to the presentstate of affairs.

The term apoptosis first appeared in the biomedical literature in 1972, to delineate astructurally-distinctive mode of cell death responsible for cell loss within living tissues1.The cardinal morphological features are cell shrinkage, accompanied by transient butviolent bubbling and blebbing from the surface, and culminating in separation of the cellinto a cluster of membrane-bounded bodies. Organellar structure is usually preservedintact, but the nucleus undergoes a characteristic condensation of chromatin, initiated atsublamellar foci and often extending to generate toroidal or cap-like, densely heterochro-matic regions. Changes in several cell surface molecules also ensure that, in tissues, apop-totic cells are immediately recognised and phagocytosed by their neighbours. The result isthat many cells can be deleted from tissues in a relatively short time with little to show forit in conventional microscopic sections.

This remarkable process is responsible for cell death in development, normal tissue turn-over, atrophy induced by endocrine and other stimuli, negative selection in the immunesystem, and a substantial proportion of T-cell killing. It also accounts for many cell deathsfollowing exposure to cytotoxic compounds, hypoxia or viral infection. It is a major fac-tor in the cell kinetics of tumors, both growing and regressing. Many cancer therapeuticagents exert their effects through initiation of apoptosis, and even the process of carcino-genesis itself seems sometimes to depend upon a selective, critical failure of apoptosisthat permits the survival of cells after mutagenic DNA damage. Apoptosis probably con-tributes to many chronic degenerative processes, including Alzheimer’s disease, Parkin-son’s disease and heart failure. So how does it work?

Molecular genetic studies on the hard-wired developmental cell death programme of thenematode Caenorhabditis elegans led to discovery of a set of proteins, widely representedby homologues in other species, and responsible for turning on or off the final commit-ment to death2. In the nematode these proteins include the products of the ced3 and ced4genes (which initiate cell suicide), ced9 (which prevents it) and a series of some sevengenes involved in recognition and phagocytosis of the doomed cell.

CED3 is the prototype of a family of around a dozen mammalian proteases, calledcaspases because of the obligatory cysteine in their active site and their predilection forcutting adjacent to aspartate residues. Mammalian caspases appear to constitute an auto-catalytic cascade, some members (notably caspase 8 or FLICE) being “apical” and moresusceptible to modification by endogenous regulatory proteins, whilst others (notablycaspase 3 – also called CPP32, Yama and apopain) enact the final, irreversible commit-ment to death. Study of caspase substrates is providing interesting insights into the waysin which cells dismantle their structure and function. Such substrates include – not sur-prisingly – cytoskeletal proteins such as actin and fodrin and the nuclear lamins, but alsoan array of regulatory and chaperone-like proteins whose function is altered by cleavagein subtle and suggestive ways3. A recent example is the nuclease chaperone ICAD, whosecleavage permits nuclear entry by a distinctive apoptosis nuclease responsible for chro-matin cleavage to oligonucleosome fragments4.

VIIIntroduction

Caspases appear to be present in most if not all cells in inactive pro-enzyme form, await-ing activation by cleavage. One of the killing mechanisms of cytotoxic T cells is a protease,granzyme B, that is delivered to the target cell by the T cell granules and triggers theselatent pro-enzymes. There are endogenous triggers also, and the first to be discovered –the C. elegans CED4 protein and its mammalian homologue – is particularly intriguingbecause of its mitochondrial origin5. Thus CED4 could be the signal that initiates apop-tosis under conditions of shut-down of cellular energy metabolism, or when there is acritical level of cell injury affecting mitochondrial respiration. In this way CED4 may actas the link between agents long known to be associated with mitochondrial injury, suchas calcium and reactive oxygen species, and the initiation of apoptosis.

A second mitochondrial protein of enormous significance in apoptosis is BCL2, a mam-malian homologue of the nematode CED9 protein. BCL2 has the tertiary structure of abacterial pore-forming protein, and inserts into the outer membrane of mitochondria. Itabrogates apoptosis, probably through binding CED4 and another protein BAX, withwhich it forms heterodimers and which, like CED4, is also a “killer” protein6. Both BCL2and BAX have several structurally and functionally similar homologues and some of thisfamily at least also tap into other cell membranes such as the outer nuclear membraneand the endoplasmic reticulum.

So are there other sources of death transducers, activating the caspase cascade because ofinjury to or signals arising in other parts of the cell than mitochondria? There are alreadyexamples that show that the answer is yes. Thus, the oncosuppressor protein p53 is acti-vated following some types of DNA damage and can trigger apoptosis. One way – butonly one of several – whereby this happens is through transcriptional activation of BAX7.The second messenger ceramide, a product of membrane-linked acid sphingomyelinaseactivation, may act as a signal for plasma membrane damage8. And a powerful caspase-activating system is mediated by cytokine receptors of the tumor necrosis factor family,notably fas/apo-1/CD95, TNF receptor I, and others. These receptors, on receiving adeath stimulus from binding their ligand, initiate a series of protein-protein interactions,building a complex (the death initiating signalling complex or DISC) which eventuallyrecruits and activates caspase 89.

These mechanisms for coupling cell injury to apoptosis have mostly depended on activa-tion of pre-formed proteins. Apoptosis can also be initiated (and forestalled) by tran-scriptional mechanisms, although rather little is known about most of them. Anoutstanding example is the Drosophila gene reaper, transcriptionally activated aroundtwo hours prior to developmental and injury-induced deaths in this organism. Droso-phila apoptosis can occur without reaper transactivation, but requires very substantiallyenhanced stimuli, suggesting that reaper adjusts a threshold for apoptosis initiation10.Another gene whose transcription can initiate death is the familiar immediate early genec-myc11. Transcriptional activation of c-myc initiates entry into DNA synthesis and isrequired for sustained re-entry in repeated cell cycles, but c-myc activation in the absenceof concurrent cytokine support triggers apoptosis. This can also be interpreted as a“threshold regulatory” effect: – c-myc expression increases the cellular requirement forsurvival factors such as IGF-1.

Impressive confirmation of the significance of these pathways to apoptosis is availablefrom study of transforming viruses. These are hardened survivors in the labyrinth of cellregulation, and have found keys to allow escape from cell death in a variety of ways. Thusthe transforming papovavirus SV40, adenovirus type 12, Human Papilloma Virus type 16and Epstein-Barr Virus all have proteins that inactivate apoptosis through inactivation ofp53 or binding of BAX12. Even lytic viruses possess mechanisms to postpone death, suchas the cowpox crmA serpin protein and the baculovirus p35 protein, which are caspaseinhibitors.

Apoptosis, Cell Death, and Cell Proliferation ManualVIII

So far so good: there are transcriptional and non-transcriptional pathways for activationof apoptosis, and they play through common effector events mediated by caspases andregulated by members of the BCL2 family. Underlying this simple scheme, however, is anextraordinary complexity. Thus, inactivation of fas signalling appears to neuter the abil-ity of both c-myc and p53 to initiate apoptosis13,14. Maybe fas signalling is yet anotherexample of “threshold regulation”. New proteins have been discovered that are recruitedto the DISC but appear to inhibit rather than activate death15, some of them of viral ori-gin. Many of the proteins mentioned above have alternative splice variants that haveopposite effects. And we still have little idea of the relevance of intracellular location or ofcell lineage to the activity of most of the apoptosis proteins. Susceptibility to apoptosiscan be influenced by many other gene products, including oncoproteins such as RAS andABL16, but in some cases a single oncoprotein may either increase or decrease susceptibil-ity depending on the context. Perhaps it is not surprising that a cellular function asimportant and irreversible as death should be subject to a huge range of coarse and finecontrols. The reagents and protocols in this book should help unravel these.

Andrew H. Wyllie FRS,Professor of Experimental Pathology,Sir Alastair Currie CRC Laboratories, University Medical School,Edinburgh, Scotland

References1. Kerr, J. R. F., Wyllie, A. H., Currie, A. R. (1972) Apoptosis: a basic biological phenomenon with wide-ranging

implications in tissue kinetics. Br. J. Cancer 26, 239–257.2. Hengartner, M. O., Horvitz, H. R. (1994) The ins and outs of programmed cell death during C. elegans develop-

ment. Phil. Trans. R. Soc. Lond. B 345, 243–248.3. Thornberry, N.A. (1997) The caspase family of cysteine proteases. Brit. Med. Bull. 53, 478–490.4. Enari, M., Sakahira, H., Yokoyama, H., Okawa, K., Iwamatsu, A., Nagata, S. (1998) A caspase-activated DNAse

that degrades DNA during apoptosis, and its inhibitor ICAD. Nature 391, 43–50.5. Zou, H., Henzel, W. J., Liu, X., Lutschg, A., Wang, X. (1997) Apaf-1, a human protein homologous to C. elegans

CED-4, participates in cytochrome c-dependent activation of caspase 3. Cell 90, 405–413.6. Oltvai, Z. N., Milliman, C. L., Korsmeyer, S. J. (1993) Bcl-2 heterodimerises in vivo with a conserved homo-

logue BAX, that accelerates programmed cell death. Cell 74, 609–619. 7. Miyashita, T., Reed, J. C. (1995) Tumor suppressor p53 is a direct transcriptional activator of the human bax

gene. Cell 80, 293–299.8. Jarvis, D. W., Kolesnick, R. N., Fornari, F. A., Traylor, R. S., Gewirtz, D. A., Grant, S. (1994) Induction of apoptotic

DNA degradation and cell death by activation of the sphingomyelin pathway. Proc. Natl. Acad. Sci. USA 91,73–77.

9. Muzio, M., Chinnaiyan, A. M., Kischkel, F. C., O’Rourke, K., Shevchenko, A., Ni, J., Scaffidi, C., Bretz, J. D.,Zhang, M., Gentz, R., Mann, M., Krammer, P. H., Peter, M. E., Dixit, V. M. (1996) FLICE, a novel FADD-homo-logous ICE/CED-3-like protease, is recruited to the CD 95 (FAS/APO-1) death-inducing signalling complex.Cell 85, 817–827.

10. White, K., Tahaoglu, E., Steller, H. (1996) Cell killing by the Drosophila gene reaper. Science 271, 805–807.11. Evan, G. I., Wyllie, A. H., Gilbert, C. S., Land, H., Brooks, M., Littlewood, T., Waters, C., Hancock, D. (1992)

Induction of apoptosis in fibroblasts by c-myc protein. Cell 69, 119–128.12. Young, L. S., Dawson, C. W., Eliopoulos, A. G. (1997) Viruses and apoptosis. Brit. Med. Bull. 53, 509–521.13. Hueber, A. O., Zornig, M., Lyon, D., Suda, T., Nagata, S., Evan, G. I. (1997) Requirement for the CD95 receptor-

ligand pathway in c-myc-induced apoptosis. Science 278, 1305–1309.14. Krammer, P. H. (1997) The tumor strikes back: new data on expression of the CD-95 (APO-1/Fas) receptor/

ligand system may cause paradigm changes in our view on drug treatment and tumor immunology. Cell Deathand Differentiation 4, 362–364.

15. Irmler, M., Thorne, M., Hanne, M., Schneider, P., Hofmann, B., Steiner, V., Bodmer, J. L., Schroter, M., Burns, K.,Mattmann, C., Rimoldi, D., French, L. E., Tschopp, J. (1997) Inhibition of death receptor signals by cellular FLIP.Nature 388, 190–195.

16. Evan, G. (1997) A question of DNA repair. Nature 387, 450.

ACell Death-

Apoptosis and Necrosis

A

Apoptosis, Cell Death, and Cell Proliferation Manual2

A

1 Introduction

1.1 Terminology of cell death

Cell death can occur by either of two distinct1,2 mechanisms, necrosis or apoptosis. Inaddition, certain chemical compounds and cells are said to be cytotoxic to the cell, that is,to cause its death.

Someone new to the field might ask, what’s the difference between these terms? To clearup any possible confusion, we start with some basic definitions.

Necrosis and apoptosis

The two mechanisms of cell death may briefly be defined:

Necrosis (“accidental” cell death) is the pathological process which occurs when cells areexposed to a serious physical or chemical insult.

Apoptosis (“normal” or “programmed” cell death) is the physiological process by whichunwanted or useless cells are eliminated during development and other normal biologicalprocesses.

Cytotoxicity

Cytotoxicity is the cell-killing property of a chemical compound (such as a food, cos-metic, or pharmaceutical) or a mediator cell (cytotoxic T cell). In contrast to necrosis andapoptosis, the term cytotoxicity does not indicate a specific cellular death mechanism.

For example, cell-mediated cytotoxicity (that is, cell death mediated by either cytotoxicT lymphocytes [CTL] or natural killer [NK] cells) combines some aspects of both necro-sis and apoptosis3,4.

� Figure 1: Illustration of the morphological features of necrosis and apoptosis.

Introduction

Terminology of cell death

AA

3Cell Death – Apoptosis and Necrosis

1.2 Differences between necrosis and apoptosis

There are many observable morphological (Figure 1, Table 1) and biochemical differ-ences (Table 1) between necrosis and apoptosis2.

Necrosis occurs when cells are exposed to extreme variance from physiological conditions(e.g., hypothermia, hypoxia) which may result in damage to the plasma membrane.Under physiological conditions direct damage to the plasma membrane is evoked byagents like complement and lytic viruses.

Necrosis begins with an impairment of the cell’s ability to maintain homeostasis, leadingto an influx of water and extracellular ions. Intracellular organelles, most notably themitochondria, and the entire cell swell and rupture (cell lysis). Due to the ultimate break-down of the plasma membrane, the cytoplasmic contents including lysosomal enzymesare released into the extracellular fluid. Therefore, in vivo, necrotic cell death is oftenassociated with extensive tissue damage resulting in an intense inflammatory response5.

Apoptosis, in contrast, is a mode of cell death that occurs under normal physiologicalconditions and the cell is an active participant in its own demise (“cellular suicide”). It ismost often found during normal cell turnover and tissue homeostasis, embryogenesis,induction and maintenance of immune tolerance, development of the nervous systemand endocrine-dependent tissue atrophy.

Cells undergoing apoptosis show characteristic morphological and biochemical features6.These features include chromatin aggregation, nuclear and cytoplasmic condensation,partition of cytoplasm and nucleus into membrane bound-vesicles (apoptotic bodies)which contain ribosomes, morphologically intact mitochondria and nuclear material. Invivo, these apoptotic bodies are rapidly recognized and phagocytized by either macroph-ages or adjacent epithelial cells7. Due to this efficient mechanism for the removal of apo-ptotic cells in vivo no inflammatory response is elicited. In vitro, the apoptotic bodies aswell as the remaining cell fragments ultimately swell and finally lyse. This terminal phaseof in vitro cell death has been termed “secondary necrosis” (Figure 1).

Introduction

Differences between necrosis and apoptosis

A


A

� Table 1: Differential features and significance of necrosis and apoptosis.

Necrosis Apoptosis

Morphological features

� Loss of membrane integrity � Membrane blebbing, but no loss of integrity

� Aggregation of chromatin at the nuclear membrane

� Begins with swelling of cytoplasm and mitochondria � Begins with shrinking of cytoplasm and condensation of nucleus

� Ends with total cell lysis � Ends with fragmentation of cell into smaller bodies

� No vesicle formation, complete lysis � Formation of membrane bound vesicles (apoptotic bodies)

� Disintegration (swelling) of organelles � Mitochondria become leaky due to pore formation involving proteins of the bcl-2 family.

Biochemical features

� Loss of regulation of ion homeostasis

� No energy requirement (passive process, also occurs at 4°C)

� Tightly regulated process involving activation and enzymatic steps

� Energy (ATP)-dependent (active process, does not occur at 4°C)

� Random digestion of DNA (smear of DNA after agarose gel electrophoresis)

� Non-random mono- and oligonucleosomal length frag-mentation of DNA (Ladder pattern after agarose gel electrophoresis)

� Postlytic DNA fragmentation (= late event of death) � Prelytic DNA fragmentation

� Release of various factors (cytochrome C, AIF) into cytoplasm by mitochondria

� Activation of caspase cascade

� Alterations in membrane asymmetry (i.e., translocation of phosphatidylserine from the cytoplasmic to the extracellular side of the membrane)

Physiological significance

� Affects groups of contiguous cells

� Evoked by non-physiological disturbances (complement attack, lytic viruses, hypothermia, hypoxia, ischemica, metabolic poisons)

� Phagocytosis by macrophages

� Significant inflammatory response

� Affects individual cells

� Induced by physiological stimuli (lack of growthfactors, changes in hormonal environment)

� Phagocytosis by adjacent cells or macrophages

� No inflammatory response

Introduction

Differences between necrosis and apoptosis

AA


1.3 Apoptotic Pathways

Scientists now recognize that most, if not all, physiological cell death occurs by apoptosis,and that alteration of apoptosis may result in a variety of malignant disorders. Conse-quently, in the last few years, interest in apoptosis has increased greatly. Great progresshas been made in the understanding of the basic mechanisms of apoptosis and the geneproducts involved (Figure 2 below, Table 20, see Appendix, page 123).

� Figure 2: Apoptotic pathways. This apoptotic pathways chart represents a compendium of information ondifferent cell lines, from various sources. As the dynamic field of apoptosis changes, the information shown herewill likely change. Table 20 in the Appendix, page 123 contains a list of sources that can be consulted for moreinformation about the items on this chart. Have a look on our website www.roche-applied-science/apoptosisto find this chart under Scientific Information-Apoptotic Pathways and click on the name of a molecule to getinformation about its function.

Key elements of the apoptotic pathway include:

Death receptors

Apoptosis has been found to be induced via the stimulation of several different cell sur-face receptors in association with caspase activation. For example, the CD95 (APO-1, Fas)receptor ligand system is a critical mediator of several physiological and pathophysiologi-cal processes, including homeostasis of the peripheral lymphoid compartment and CTL-mediated target cell killing. Upon cross-linking by ligand or agonist antibody, the Fasreceptor initiates a signal transduction cascade which leads to caspase-dependent pro-grammed cell death.

Membrane alterations

In the early stages of apoptosis, changes occur at the cell surface and plasma membrane.One of these plasma membrane alterations is the translocation of phosphatidylserine(PS) from the inner side of the plasma membrane to the outer layer, by which PSbecomes exposed at the external surface of the cell.

Introduction

Apoptotic Pathways

A


A

Protease cascade

Signals leading to the activation of a family of intracellular cysteine proteases, thecaspases, (Cysteinyl-aspartate-specific proteinases) play a pivotal role in the initiationand execution of apoptosis induced by various stimuli. Different members of caspases inmammalian cells have been identified. Among the best-characterized caspases iscaspase-1 or ICE (Interleukin-1�-Converting Enzyme), which was originally identified asa cysteine protease responsible for the processing of interleukin 1�.

Mitochondrial changes

Mitochondrial physiology is disrupted in cells undergoing either apoptosis or necrosis.During apoptosis mitochondrial permeability is altered and apoptosis specific proteaseactivators are released from mitochondria. Specifically, the discontinuity of the outermitochondrial membrane results in the redistribution of cytochrome C to the cytosol fol-lowed by subsequent depolarization of the inner mitochondrial membrane. CytochromeC (Apaf-2) release further promotes caspase activation by binding to Apaf-1 and there-fore activating Apaf-3 (caspase 9). AIF (apoptosis inducing factor), released in the cyto-plasm, has proteolytic activity and is by itself sufficient to induce apoptosis.

DNA fragmentation

The biochemical hallmark of apoptosis is the fragmentation of the genomic DNA, anirreversible event that commits the cell to die and occurs before changes in plasma mem-brane permeability (prelytic DNA fragmentation). In many systems, this DNA fragmen-tation has been shown to result from activation of an endogenous Ca2+ and Mg2+-dependent nuclear endonuclease. This enzyme selectively cleaves DNA at sites locatedbetween nucleosomal units (linker DNA) generating mono- and oligonucleosomal DNAfragments.

Note: For more information about the elements of the pathways as well as synonyms andabbreviations, please see Table 20 in the Appendix, page 123.

Introduction

Apoptotic Pathways

AA


2 Apoptosis Assay Methods

Originally, to study both forms of cell death, necrosis and apoptosis, cytotoxicity assayswere used. These assays were principally of two types:

� Radioactive and non-radioactive assays that measure increases in plasma membranepermeability, since dying cells become leaky.

� Colorimetric assays that measure reduction in the metabolic activity of mitochondria;mitochondria in dead cells cannot metabolize dyes, while mitochondria in live cellscan.

Note: For a detailed discussion of both types of cytotoxicity assay, see Section A 3, beginningon page 58 of this guide.

However, as more information on apoptosis became available, researchers realized thatboth types of cytotoxicity assays vastly underestimated the extent and timing of apop-tosis. For instance, early phases of apoptosis do not affect membrane permeability, nor dothey alter mitochondrial activity. Although the cytotoxicity assays might be suitable fordetecting the later stages of apoptosis, other assays were needed to detect the early eventsof apoptosis.

In concert with increased understanding of the physiological events that occur duringapoptosis, a number of assay methods have been developed for its detection. For instance,these assays can measure one of the following apoptotic parameters:

� Fragmentation of DNA in populations of cells or in individual cells, in which apop-totic DNA breaks into different length pieces.

� Alterations in membrane asymmetry. Phosphatidylserine translocates from the cyto-plasmic to the extracellular side of the cell membrane.

� Activation of apoptotic caspases. This family of proteases sets off a cascade of eventsthat disable a multitude of cell functions.

� Release of cytochrome C and AIF into cytoplasm by mitochondria.

For practical reasons, we have divided this chapter into two broad categories: assays thatmeasure apoptosis in cell populations (Section A 2.1 of this guide) and assays that mea-sure apoptosis in individual cells (Section A 2.2 of this guide).For discussions of particular assays, turn to the pages indicated in the product selectionguide (Figure 3).

Apoptosis Assay Methods

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Start

Are you studying

Apoptosis Cytotoxicity*

Are you studying

cell populations single cells

Which techniquesdo you perform?

Do you analyze data by

FACS microscopy

ELISA

DNA fragmentation, quantitative�Cell Death Detection 11 774 425 001

ELISAPLUS 11 920 685 001

�Cell Death Detection ELISA 11 544 675 001

Fluorescence microplate Reader

Caspase activity, quantitative� Caspase 3 Activity Assay 12 012 952 001

� Homogeneous Caspases 03 005 372 001Assay, fluorimetric 12 236 869 001

Western blotting

ICE protease activity� Anti-PARP 11 835 238 001

Gel electrophoresis

DNA fragmentation, qualitative� Apoptotic DNA Ladder Kit 11 835 246 001

DNA fragmentation� In Situ Cell Death Detection Kit, 12 156 792 001

TMR red

� In Situ Cell Death Detection Kit, 11 684 795 001Fluorescein

Membrane modification� Annexin-V-Alexa 568 03 703 126 001

� Annexin-V-FLUOS 11 828 681 001

� Annexin-V-FLUOS Staining Kit 11 858 777 00111 988 549 001

Proteolytic activity� M30 CytoDEATH, Fluorescein 12 156 857 001

� Figure 3: Product Selection Guide

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Do your cells proliferate in vitro?

yes

no

Do your cells proliferate in vitro?

yes

no

Are you studying

cytotoxiticsubstances

cell-mediatedcytotoxicity

Are you studying

cell cultures,blood cells,

etc.

tissue sections

Do you use

fluorescencemicroscopy

lightmicroscopy

� Cellular DNA Fragmentation ELISA 11 585 045 001

� Cell Proliferation Reagent WST-1 11 644 807 001

� Cell Proliferation Kit II (XTT) 11 465 015 001

� Cell Proliferation Kit I (MTT) 11 465 007 001

� Cytotoxicity Detection Kit (LDH) 11 644 793 001





� Cytotoxicity Detection Kit (LDH) 11 644 793 001L

DNA fragmentation� In Situ Cell Death Detection Kit, 12 156 792 001

TMR red

� In Situ Cell Death Detection Kit, 11 684 795 001Fluorescein

� In Situ Cell Death Detection Kit, POD 11 684 817 001

� In Situ Cell Death Detection Kit, AP 11 684 809 001

Proteolytic activity� M30 CytoDEATH 12 140 322 001

12 140 349 001

� M30 CytoDEATH, Fluorescein 12 156 857 001

DNA fragmentation� In Situ Cell Death Detection Kit, POD 11 684 817 001

� In Situ Cell Death Detection Kit, AP 11 684 809 001

Membrane modification� Annexin-V-Biotin 10 828 690 001

Proteolytic activity� M30 CytoDEATH 12 140 322 001

12 140 349 001

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2.1 Methods for studying apoptosis in cell populations

A number of methods have now been developed to study apoptosis in cell populations.We focus on two key apoptotic events in the cell:

� Apoptosis and cell mediated cytotoxicity are characterized by cleavage of the genomicDNA into discrete fragments prior to membrane disintegration. Because DNA cleav-age is a hallmark for apoptosis, assays which measure prelytic DNA fragmentation areespecially attractive for the determination of apoptotic cell death. The DNA fragmentsmay be assayed in either of two ways:

� As “ladders” (with the 180 bp multiples as “rungs” of the ladder) derived from pop-ulations of cells, e.g., with the Apoptotic DNA Ladder Kit (described on page 13 ofthis guide).

� By quantification of histone complexed DNA fragments with an ELISA (describedon page 15 of this guide).

� Further, researchers discovered that proteases were involved in the early stages of apo-ptosis. The appearance of these caspases sets off a cascade of events that disable a mul-titude of cell functions. Caspase activation can be analyzed in different ways:

� By an in vitro enzyme assay. Activity of a specific caspase, for instance caspase 3, canbe determined in cellular lysates by capturing of the caspase and measuring pro-teolytic cleavage of a suitable substrate (described on page 22 of this guide).

� By detection of cleavage of an in vivo caspase substrate. For instance caspase 3 isactivated during early stages (as shown in Figure 2). Its substrate PARP (Poly-ADP-Ribose-Polymerase) and the cleaved fragments can be detected with the anti PARPantibody (described on page 27 of this guide).

If you’re just starting out in the field, however, it may be difficult to decide how best toassay apoptosis in your system. Thus, in the following sections, we will describe details ofeach of these apoptosis assays.

2.1.1 Assays that measure DNA fragmentation

The biochemical hallmark of apoptosis is the fragmentation of the genomic DNA, anirreversible event that commits the cell to die. In many systems, this DNA fragmentationhas been shown to result from activation of an endogenous Ca2+ and Mg2+-dependentnuclear endonuclease. This enzyme selectively cleaves DNA at sites located betweennucleosomal units (linker DNA) generating mono- and oligonucleosomal DNA frag-ments (Figure 4). These DNA fragments reveal, upon agarose gel electrophoresis, a dis-tinctive ladder pattern consisting of multiples of an approximately 180 bp subunit8.

Radioactive as well as non-radioactive methods to detect and quantify DNA fragmenta-tion in cell populations have been developed. In general, these methods are based on thedetection and/or quantification of either low molecular weight (LMW) DNA which isincreased in apoptotic cells or high molecular weight (HMW) DNA which is reduced inapoptotic cells (Figure 5). The underlying principle of these methods is that DNA, whichhas undergone extensive double-stranded fragmentation (LMW DNA) may easily be sep-arated from very large, chromosomal length DNA (HMW DNA), e.g., by centrifugationand filtration.


Methods for studying apoptosis in cell populations

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� Figure 4: The biochemistry of DNA fragmentation and the appearance of the “DNA ladder”.

For the quantification of DNA fragmentation, most methods involve a step in which theDNA of the cells has to be labeled: Prior to the addition of the cell death-inducing agentor of the effector cells, the (target) cells are incubated either with the [3H]-thymidine([3H]-dT) isotope or the nucleotide analog 5-bromo-2’-deoxyuridine (BrdU). DuringDNA synthesis (DNA replication) these modified nucleotides are incorporated into thegenomic DNA. Subsequently, those labeled cells are incubated with cell death-inducingagents or effector cells and the labeled DNA is either fragmented or retained in the cellnucleus. Finally each type of DNA (HMW and LMW) is quantitated. Because the labelingof the cellular DNA has to be done prior to the induction of cell death, this labeling is alsocalled “prelabeling”.

The prelabeling of one cell population (e.g., the target cells) allows the behavior of thelabeled cells to be traced specifically when different cell populations are mixed.

Note: Because cell-mediated cytotoxicity (CMT) proceeds, at least in part, by apoptoticmechanisms, the DNA fragmentation assay may also be used as a CMT assay.

In a study of cell-mediated cytotoxicity the target cell population is labeled before theeffector cells (e.g., CTL) are added. Subsequently, due to pore formation in the target cellplasma membrane, the fragmented LMW DNA is released from the cytoplasm of the tar-get cell into the culture supernatant (Table 2). The cytotoxic potential of the effector cellsis measured by quantification of the label released from the damaged target cells.

Because this metabolic prelabeling of the genomic DNA requires DNA synthesis, onlycells proliferating in vitro (e.g., cell lines) may be labeled in this way; cells which do notproliferate in vitro (e.g., primary cell cultures, tumor cells ex vivo) do not replicate theirDNA and therefore, do not incorporate labeled nucleotides (see also Section A 3.2.1.“Cellular DNA Fragmentation ELISA” page 64).



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To detect fragmented DNA in cells which do not replicate in vitro, the DNA has to be iso-lated and analyzed by agarose gel electrophoresis (“DNA ladder assay”, Figure 6, see alsoFigure 4). Roche Applied Science offers a kit, the Apoptotic DNA Ladder Kit, that simpli-fies this assay.

� Figure 5: Compartmentalization of HMW and LMW DNA in normal and apoptotic cells. ( = decreasing, = increasing)

� Table 2: Distribution of HMW and LMW DNA in cells under-going apoptosis and target cells during cell mediated cytotoxicity.

An alternative method which circumvents the isolation and electrophoretic analysis ofDNA is the immunological detection of LMW DNA (histone-complexed DNA frag-ments) by an immunoassay (Cell Death Detection ELISAPLUS, see page 15).

This nonradioactive immunoassay, offered by Roche Applied Science quantifies that hall-mark of apoptosis. The Cell Death Detection ELISAPLUS has been designed for relativequantification of DNA fragmentation in cells which do not proliferate in vitro (since thekit requires no prelabeling of the cells). This kit measures the enrichment of histone-complexed DNA fragments (mono- and oligonucleosomes) in the cytoplasm of apop-totic cells.

Each of the methods to detect and measure apoptosis has its advantages and limitations.Because the cellular mechanisms that result in apoptosis are complex, most publishedmethods cannot by themselves detect apoptosis unambiguously.

To ensure that the mode of cell death in the individual cell system or experiment is apop-totic, one also has to consider other criteria like the cellular morphology. Morphologiccriteria for apoptotic cell death include, for example, chromatin condensation withaggregation along the nuclear envelope and plasma membrane blebbing followed by sep-aration into small, apoptotic bodies. When internucleosomal DNA fragmentation isaccompanied by these morphological features it provides an additional useful criterion todefine cell death as apoptotic.

Apoptosis Cell mediated cytotoxicity

Compartment HMW DNA

LMW DNA

HMW DNA

LMW DNA

Nucleus + + + +

Cytoplasm – + – +

Supernatant – – – +

Note: In the early phases of apopto-sis, no DNA is released into thesupernatant (prelytic DNA frag-mentation). However, in vitro, theapoptotic cells will lyse (“secondarynecrosis“). Therefore, LMW DNAis found in the supernatant late inapoptosis.



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Apoptotic DNA Ladder KitCat. No. 11 835 246 001 20 tests

Type DNA purification kit

Useful for Preparation of apoptotic DNA fragments for display on electrophoretic gels

Samples Whole blood or cells in culture

Method Cell lysis, followed by binding of cellular DNA on glass fiber, removal of impurities, and DNA recovery

Time DNA preparation: < 20 min (after induction of apoptosis)

Significance of kit: This kit offers the easiest way to isolate apoptotic DNA fragments forDNA ladder analysis. The purification method outlined in the kit is much faster thanother DNA purification methods (e.g., phenol/chloroform extraction, DNA precipita-tion). Purified DNA may be mixed directly with gel loading buffer and analyzed on anagarose gel.

Test principle: Apoptotic DNA binds quickly to glass fiber fleece in the presence of achaotropic salt, guanidine hydrochloride (guanidine HCl). After cellular impurities arewashed off the fleece, the DNA is released from the fleece with a low salt buffer. The pro-cedure (see Flow Chart 1) involves:

Sample Preparation

� Incubating an aliquot of apoptotic cells with an equal volume of binding/lysis buffer. After the incubation, the lysed sample is poured into a filter tube containing glass fiber fleece.

� Using centrifugation to separate the DNA in the lysate (which binds to the glass fiber fleece) from unbound lysate components (which flow through the fleece into a collection tube).

� Washing the bound DNA twice.

� Eluting the purified DNA from the filter tube and collecting it by centrifugation.

Treat sample with apoptosis-inducing agent (1–24 h)

Incubate treated sample with binding/lysis buffer (10 min, RT)

Mix isopropanol with sample and pipette mixture into filter tube

Centrifuge tube assembly (8000 rpm) and discard the flow-through (1 min, RT)

Add wash buffer to the filter tube, then centrifuge as before (1 min, RT)

Repeat the wash step, then add a final high speed spin (13,000 rpm) (1 min, then 10 sec, RT)

Insert the filter tube into a 1.5 ml centrifuge tube, and add warm elution buffer to the filter tube

Collect the eluted DNA by centrifugation (1 min, RT)



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DNA Ladder Assay

� Flow Chart 1: Assay procedure, Apoptotic DNA Ladder Kit.

Sample size: 200 – 300 µl whole blood or cell suspension (for instance, 2 x 106 cells). Thekit allows simultaneous processing of multiple samples.

Yield

Specificity: Only nucleic acid will bind to the glass fiber filters under the conditions out-lined in the kit. Salts, proteins, and other cellular components do not bind.

Kit contents

1. Nucleic acid binding/lysis buffer, ready-to-use2. Washing buffer (ethanol to be added before use)3. Elution buffer, ready-to-use4. Glass fiber filter tubes, 700 µl capacity5. Polypropylene collection tubes, 2 ml (for washes)6. Positive control, apoptotic U937 cells, lyophilized

Typical results: see Figure 6

Other applications: For more examples of how the Apoptotic DNA Ladder Kit can beused in the lab, see Appendix, pages 128–129.

� Figure 6: DNA ladder assayed with the Apoptotic DNA Ladder KitM = Size marker– = Control cells without camptothecin+ = Cells treated with camptothecinC = Positive control from the kit

Mix the eluted DNA sample with gel loading buffer

Apply sample to a 1% agarose gel which contains ethidium bromide

Run the gel in TBE (Tris-borate EDTA) buffer at 75 V (1.5 h, RT)

Place the gel on a UV light box to visualize the DNA ladder pattern

Sample Sample volume Yield of purified DNA

Whole blood (human) 200 µl 3–6 µg

Cultured cells (K562) 2 x 106 cells 10 µg



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Cell Death Detection ELISAPLUS

Cat. No. 11 774 425 001 96 tests

Cat. No. 11 920 685 001 10 x 96 tests

Type One-step sandwich ELISA, colorimetric

Useful for Relative quantification of apoptosis without cell labeling; differentiating apoptosis from necrosis

Samples Cell lysates, cell culture supernatants, serum, or plasma

Method Cell lysis, followed by immunochemical determination of histone-com-plexed DNA fragments in a microplate well (Note: For detection of necrosis, histone-complexed DNA fragments are detected directly in the culture superna-tant, without cell lysis)

Time Approx. 3 h (after induction of apoptosis)

Significance of kit: Use this kit for relative quantification of histone-complexed DNAfragments (mono- and oligonucleosomes) out of the cytoplasm of cells after the induc-tion of apoptosis or when released from necrotic cells. Since the assay does not requireprelabeling of cells, it can detect internucleosomal degradation of genomic DNA duringapoptosis even in cells that do not proliferate in vitro (for example, freshly isolated tumorcells). The antibodies used in the assay are not species-specific, so the kit may be used toassay cells from a wide variety of species (see “Other applications” in this article).

Test principle: The assay uses an one-step sandwich immunoassay to detect nucleosomes.The procedure (Figure 7 and Flow Chart 2) involves:

� Incubating cells in a microplate well (for instance, 104 human cells in 200 µl culture) with an agent that induces cell death (for example, campothecin (CAM)). After the incubation, the cells are pelleted by centrifugation and the supernatant is (containing DNA from necrotic cells that leaked through the membrane during incubation) dis-carded.

� Resuspending and incubating cells in lysis buffer. After lysis, intact nuclei are pelleted by centrifugation.

� Transferring an aliquot of the supernatant to a streptavidin-coated well of a microplate.

� Binding nucleosomes in the supernatant with two monoclonal antibodies, anti-histone (biotin-labeled) and anti-DNA (peroxidase-conjugated). Antibody-nucleo-some complexes are bound to the microplate by the streptavidin.

� Washing the immobilized antibody-histone complexes three times to remove cell components that are not immunoreactive.

� Incubating sample with peroxidase substrate (ABTS).

Determining the amount of colored product (and thus, of immobilized antibody-histone complexes) spectrophotometrically.



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� Figure 7: How the Cell Death Detection ELISAPLUS works.Panel A: Sample preparationPanel B: ELISA

� Flow Chart 2: Assay procedure, Cell Death Detection ELISAPLUS.

Sensitivity: In a model system, nucleosomes were detectable in as few as 600 campothe-cin-induced U937 cells (Figure 8). However, the lower limit for detecting dying/dead cellsin a particular sample varies with the kinetics of the apoptotic process, the cytotoxicagent used, and the number of affected cells in the total cell population.

Treat cells with apoptosis-inducing agent in the well of a microplate (1–24 h, 37°C)

Centrifuge microplate (200 x g ) and remove supernatant (10 min, RT)

Incubate treated cells with lysis buffer (30 min, RT)

Repeat microplate centrifugation (200 x g ) (10 min, RT)

Transfer aliquot of supernatant (lysate) to streptavidin-coated microplate

Incubate supernatant with immunoreagent (containing anti-histone and anti-DNA) (2 h, RT)

Wash microplate wells three times with incubation buffer at RT

Add substrate solution to wells and incubate (approx. 15 min, RT)

Measure absorbance at 405 nm

After incubating cells with an apoptosis-inducing agent, pellet the cells by centrifugation.Discard the supernatant, which may contain necrotic DNA that leaked through the membrane during the incubation.

Incubate cells with lysis buffer.

Pellet the intact nuclei by centrifugation. Take an aliquot of the supernatant (cell lysate) and determine the amount of apoptotic nucleosomes present.

�

�



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� Figure 8: Sensitivity of Cell Death DetectionELISAPLUS.Different cell concentrations of U937 cells were incu-bated with camptothecin (CAM) (2 µg/ml) or withoutCAM for 4 h at 37°C. 20 µl of cell culture supernatantand cell lysates were analyzed in the ELISA. Substratereaction time: 10 min. � Lysate with CAM, � Lysatewithout CAM, � Supernatant with CAM, � Supernatantwithout CAM. Result: The ELISA can clearly detect apoptosis-relatednucleosomes in as few as 600 cells.

Specificity: The ELISA is specific for nucleosomes containing single- or double-strandedDNA (Figure 9). It is not species specific.

� Figure 9: Dose-response experiment analyzed bythe Cell Death Detection ELISAPLUS.U937 cells (104 cells/well, in 200 µl) were incubated withdifferent concentrations of camptothecin (CAM) for 4 hat 37°C. Before and after lysis, cells were centrifuged anda 20 µl aliquot of the supernatant was analyzed with theCell Death Detection ELISAPUS. Results were plotted asdose vs. response. Substrate reaction time: 5 min.� Lysate, � Supernatant, � Enrichment factor of thelysate. Result: Amounts of cytoplasmic oligonucleosomes (anindicator of apoptosis) increase as CAM concentrationincreases. Cell culture supernatants removed from thecells after treatment (but before lysis) gave no signal,indicating that there are no necrotic cells during thetreatment.

Can be used to assay:

� Adherent cells

� Cells in suspension culture

� Cell culture supernatant

� Lysates of cells obtained ex vivo

� Serum, or plasma

Kit contents

1. Anti-histone antibody (clone H11-4), biotin-labeled2. Anti-DNA antibody (clone M-CA-33), peroxidase-conjugated3. DNA-histone complex (positive control)4. Incubation buffer, ready-to-use5. Lysis buffer, ready-to-use6. Substrate buffer, ready-to-use7. ABTS substrate tablets8. Microplate modules (12 x 8 wells)9. Adhesive plate cover

Typical results: see Figure 8 and 9

Other applications: For more examples of how the Cell Death Detection ELISAPLUS andthe Cell Death Detection ELISA can be used in the lab, see Appendix, pages 129–131.



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2.1.2 Assays that measure apoptosis-induced proteases (caspases)

Several caspases (see Table 20, in the Appendix, page 123) are thought to mediate veryearly stages of apoptosis10. For instance, one of these, caspase 3 (CPP32) is required forthe induction of apoptosis by certain effectors [especially tumor necrosis factor and thecytotoxic T cell ligand effector, CD95 (also called Fas)] Enari et al. (1996) , Nature 380,723–726.

These proteases cleave numerous substrates at the carboxy site of an aspartate residue. Allare synthesized as pro-enzymes; activation involves cleavage at aspartate residues thatcould themselves be sites for the caspase family. As caspases are probably the most impor-tant effector molecules for triggering the biochemical events which lead to apoptotic celldeath, assays for determination of caspase activation can detect apoptosis earlier thanmany other commonly used methods.

The most elucidatory assay for these caspases involves western blot detection of pro-teolytic cleavage products found in apoptotic cells. An antibody, Anti-PARP, sold byRoche Applied Science, can be used in such an assay. The antibody can detect intact andcleaved forms of Poly-ADP-Ribose Polymerase, a target for some caspases.

For specific and quantitative measurement of caspase activity Western blotting is notsuitable. To quantify caspase activation enzyme activity assays based on detection ofcleaved caspase substrates have been developed recently. However most of the caspasesubstrates are not exclusively cleaved by a specific caspase but only preferentially, whileother members of the caspases family act on these substrates to a lower extent. RocheApplied Science offers a caspase 3 activity assay with highest specificity by the use of animmunosorbent enzyme assay principle.



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M30 CytoDEATH*Cat. No. 12 140 322 001 50 tests

Cat. No. 12 140 349 001 250 tests

M30 CytoDEATH, FluoresceinCat. No. 12 156 857 001 250 tests

Type Monoclonal antibody, clone M30, IgG2b, mouse

Useful for Detection of apoptosis in epithelial cells and tissues (formalin grade)

Samples Adherent cells, tissue samples (routinely fixed and paraffin-embedded tissue sections, cryostat sections)

Method Detect apoptosis by applying the M30-antibody to fixed samples, then using secondary detection systems. Suitable for immunohistochemistry, immuno-cytochemistry, and flow cytometry

Time 2 h for immunofluorescence on cells, 3.5 h for staining of tissues (excluding dewaxing)

Background: During Apoptosis, vital intracellular proteins are cleaved. The proteasesthat mediate this process are called caspases (Cysteinyl-aspartic acid proteases). Caspasesare expressed as zymogenes, which are activated by different apoptosis inducers. Onceactivated, a single caspase activates a cascade of caspases.

Until recently cytokeratins, in particular cytokeratin 18, were not known to be affected byearly events of apoptosis. Recently, it has been shown that the M30 antibody recognizes aspecific caspase cleavage site within cytokeratin 18 that is not detectable in native CK18 ofnormal cells. Consequently, the M30 CytoDEATH antibody is a unique tool for the easyand reliable determination of very early apoptotic events in single cells and tissue sec-tions.

Significance of reagent: Use the M30 CytoDEATH antibody for the determination ofearly apoptotic events in cells and tissue sections by detection of a specific epitope ofcytokeratin 18 that is presented after cleavage by caspases.

Test principle of M30 CytoDEATHfor formalin-embedded tissue involves:

* The M30 CytoDEATH antibody is made under a license agreement form BEKI AB/BEKI Diagnostics AB, Sweden.

for immunofluorescence and flow cytometry on cells for M30 CytoDEATH and M30 CytoDEATH, Fluorescein:

� Dewax formalin-fixed, paraffin-embedded tissue sections.

� Add Streptavidin-POD.

� Retrieve antigen by heating in citric acid buffer.

� Add substrate solution (DAB or AEC).

� Add M30 antibody. Counterstain with Harries hematoxilin.

� Add Anti-Mouse-Biotin. Analyze under a light microscope.

� Fix cells. � Add Anti-Mouse-Ig-Fluorescein (not nec-essary for M30 CytoDEATH, Fluorescein).

� Add M30 CytoDEATH antibody or M30 CytoDEATH, Fluorescein

� Analyze under a fluorescence microscope or in FACS.



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� Flow Chart 3: Assay procedure, M30 CytoDEATH, immunohistochemistry and cytometry.

Staining procedure for fluorescence microscopy and flow cytometry (FACS)

M30 CytoDEATH M30 CytoDEATH, Fluorescein

Wash cells with PBS

Fix cells in ice-cold pure methanol (–20°C) for 30 min

Wash cells twice with PBS/BSA

Incubate with M30 CytoDEATH working solution (for 30 min, at RT)

Incubate with M30 CytoDEATH, Fluorescein working solution (for 30 min, at RT)

Wash cells twice with PBS

Incubate with Anti-Mouse-Ig-Fluorescein (for 30 min, at RT)

Wash cells twice with PBS

Analyze by fluorescence microscope or flow cytometry (dilute cells in PBS, and store the samples in the dark until analysis).

Staining procedure for immunohistochemistry

Incubate paraffin-embedded sections (over night) at 37°C

Dewax formalin-fixed, paraffin-embedded tissue sections:2x xylol, 2x ethanol (96%), 1x ethanol (70%), 1x methanol/H2O2 (3%)

(10 min, RT). Rinse for 10 min in demineralized water

Antigen retrieval:

Prepare 500 ml of 10 mM citric acid buffer. Incubate in a microwave oven at 750 W until boiling. Place slides into the heated citric acid solution. Incubate once more at 750 W. When solution is boiling, turn setting of microwave

oven to “keep warm” (about 100 W). Incubate for 15 min. Cool the slides down (for 5 min, at RT)

Rinse three times in PBS; incubate 2 min in a separate jar of PBS

Block with PBS + 1% BSA (for 10 min, at RT)

Remove blocking solution. Add M30 CytoDEATH working solution (1 h, RT)

Wash slides three times in PBS

Cover with Anti-Mouse-Biotin (for 30 min, at RT)


Cover with Streptavidin-POD (for 30 min, at RT)


Incubate slides in a freshly prepared substrate solution (DAB or AEC at RT) until a clearly visible color develops

Counterstain with hematoxilin, and mount the section

Analyze by a light microscope



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Specificity: The M30 CytoDEATH antibody binds to a caspase-cleaved, formalin-resis-tant epitope of the cytokeratin 18 (CK 18) cytoskeletal protein.

The immunoreactivity of the M30 CytoDEATH antibody confined to the cytoplasma ofapoptotic cells.

Antibody supplied as: Mouse monoclonal antibody (clone M30), lyophilized, stabi-lized. Formalin grade.

Typical results: see Figures 10–12.

Other applications: For more examples of how the M30 CytoDEATH and M30CytoDEATH, Fluorescein can be used in the lab, see Appendix, pages 131–132.

� Figure 10: Detection of apoptosis in HeLa cells, treated with TNF and Actinomycin D, using M30 CytoDEATH. Secondary detection with Anti-Mouse-Fluorescein and propidium iodide.

� Figure 11: Detection of apoptosis in human colon using M30 CytoDEATH (blue filter). Secondarydetection with Anti-Mouse-Biotin, Streptavidin-POD and AEC as substrate, counterstained with hematoxilin.

� Figure 12: FACS analysis of apoptosis in HeLa cells, using M30 CytoDEATH, Fluorescein. White: untreated control cells. Red: Cells treated with TNF and Actinomycin D.



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Caspase 3 Activity AssayCat. No. 12 012 952 001 96 tests

Type Immunosorbent enzyme assay, fluorometric

Useful for Specific, quantitative in vitro determination of caspase 3 activity

Samples Cell lysates, recombinant caspase 3 (CPP32)

Method Cell lysis, followed by capturing of caspase 3 by a specific antibody and fluorometric determination of proteolytic cleavage of the substrate


Significance of kit: This kit allows specific, quantitative detection of caspase 3 activity incellular lysates after induction of apoptosis. Caspase 3 activation play a key role in initia-tion of cellular events during the early apoptotic process. The immunosorbent enzymeassay principle of this kit guarantees high specificity without cross-reactions with otherknown caspases. The fluorochrome generated by proteolytic cleavage of the caspase sub-strate is proportional to the concentration of activated caspase 3 in the lysates.

Test principle: The assay uses a fluorometric immunosorbent enzyme assay (FIENA)principle. The procedure (Figure 13 and Flow Chart 4) involves:

� Figure 13: How the Caspase 3 Activity Assay works.

� Inducing apoptosis in cells by desired method (for instance 2 x 106 cells). After the induction, the cells are washed and pelleted by centrifugation.

� Preparing samples by resuspending and incubating cells in lysis buffer. After lysis and following centrifugation, samples can be removed for direct analysis or storage.

� Coating microplate with anti-caspase 3 solution and blocking of unspecific binding.

� Transferring a sample to the anti-caspase 3-coated well of a microplate and capturing of caspase 3.

� Washing the immobilized antibody-caspase 3 complexes three times to remove cell components that are not immunoreactive.

� Incubating sample with caspase substrate (Ac-DEVD-AFC) that is proteolytically cleaved into free fluorescent AFC.

Measuring generated AFC fluorometrically.



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� Flow Chart 4: Assay procedure, Caspase 3 Activity Assay.

Sensitivity: In a model system, caspase 3 activity was clearly detectable in lysates of 106

cells with 5 % apoptotic cells (Figure 14). However, the lower limit for determination ofcaspase 3 activity in cellular lysates of dying cells in a particular sample varies with thekinetics of the apoptotic process, the apoptotic agent used, and the number of affectedcells within the total cell population.

Specificity: This fluorometric immunosorbent enzyme assay is highly specific for caspase3 by the use of an anti-caspase 3-specific monoclonal capture antibody in combinationwith a specific caspase substrate. Enzyme activity of natural and recombinant humancaspase 3 is detected by this assay. Cross-reactions with other caspases are not known.


� Lysates of adherent cells, of cells in suspension culture, of cells obtained ex vivo orrecombinant caspase 3.

Sample preparation

Treat sample (2 x 106 cells) with apoptosis-inducing agent. Include a negative control without induction (1–24 h)

Wash treated and control cells with ice-cold PBS and centrifuge (300 x g ) (5 min, RT)

Incubate cell pellet in lysis buffer (1 min, on ice)

Centrifuge at maximum speed in a tabletop centrifuge (1 min, RT)

Remove 100 µl sample for direct analysis or storage for 1 week at –20°C

Coating microplate

Incubate microplate with anti-caspase 3 coating solution (either at 37°C for 1h or at 4°C over night)

Block unspecific binding by incubation with blocking buffer (30 min, RT)

Remove blocking solution and wash 3 times with incubation buffer (3 x 1 min, RT)

Assaying protease activity

Add sample (100 µl lysate, positive control) into microplate well (1 h, 37°C)

Remove sample and wash 3 times with incubation buffer (3 x 1 min, RT)

Add freshly prepared substrate solution (1–3 h, 37°C)

Measure fluorometrically (excitation filter 400 (370–425) nm and emission filter 505 (490–530) nm)

Optional: for calibration, set up a calibration curve with different dilutions of AFC as standard



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Kit contents

1. Coating buffer, 10x2. Anti-caspase-3, 20x3. Blocking buffer, ready-to-use4. Incubation buffer, 5x5. DTT, 100x6. Substrate solution Ac-DEVD-AFC, 20x7. AFC8. Positive control, apoptotic U937 cell lysate9. Microplate modules (12 x 8-wells)

10. Adhesive plate cover

Typical results: see Figures 14–15

Other applications: For more examples of how the Caspase 3 Activity Assay can be usedin the lab, see Appendix, pages 132–133.

The caspase 3 activity assay has been used to detect caspase 3 activation in U937 cellsexposed to different concentrations of the apoptosis inducing agent camptothecin(CAM) (Figure 14, dose response curve). In this model system, the induction of apopto-tis in only 5% of U937 cells is sufficient for detection of caspase 3 activation. Caspase 3activity/fluorochrome development is proportional to the percentage of apoptotic cells.

Figures 14 and 15 demonstrate that Caspase 3 activity and Annexin-V binding correlatevery closely in both dose-response and kinetic studies.

� Figure 14: Dose-response experiment analyzed bythe caspase 3 Activity Assay. U937 cells were exposed todifferent concentrations of camptothecin (CAM) for 4 h at37°C. Lysates were analyzed for caspase 3 activity andstandardized values are plotted versus concentration. Addi-tionally, an aliquot of the same cells was analyzed forAnnexin-V binding.

� Figure 15: Kinetic study of caspase 3 activation bycamptothecin exposure in U937 cells. U937 cells wereexposed to 4 µg/ml camptothecin for different time intervalsat 37°C. Lysates were analyzed for caspase 3 activity andfluorescence (minus fluorescence of blank) is plotted ver-sus time. Additionally, an aliquot of the same cells was ana-lyzed for Annexin-V binding in parallel.



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Homogeneous Caspases AssayCat. No. 03 005 372 001 100 assays (96-well plates)

400 assays (384-well plates)

Cat. No. 12 236 869 001 1000 assays (96-well plates)4000 assays (384-well plates)

Type One step assay, fluorimetric

Useful for Specific, quantitative in vitro determination of caspases in microplates

Samples Cell cultures, recombinant caspases

Method Cell lysis, followed by detection of caspases activity (fluorimeric determintion of proteolytic cleavage of the substrate)


Significance of kit: The Homogeneous Caspases Assay is a fluorimetric assay for thequantitative in vitro determination of caspases activity in microplates, which makes itespecially useful for high throughput screening. Apoptotic cells are incubated withDEVD-Rhodamine 110 for 1–24 h. Upon cleavage of the substrate by activated caspases,fluorescence of the released Rhodamine 110 is measured.

Test principle: The kit can be used for the quantification of activated caspases of humanas well as animal origin, or screening for caspase inhibitors. It is one step assay, includingthe cell lysis step.

� Figure 16: How the Homogeneous Caspases Assay work.

� Flow Chart 5: Assay procedure, Homogeneous Caspase Assay.

Dispense double concentrated apoptosis inducing agent into microplate. Include negative control (diluent only). Volume should be 50 µl (96 well plate) or 12.5 µl (384 well plate).

Onto prediluted apoptosis inducing agents, seed cells (4 x 104 per well, volume 50 µl on 96 well plate or 104 cells per well, volume 12.5 µl on 384 well plate) and incubate for desired interval for induction of apoptosis.

Add 100 µl (96-wells) or 25 µl (384-wells) substrate working solution, freshly prepared. Cover the microplate with a lid and incubate more than 1 h at 37 °C.

Measure with an excitation filter 470–500 nm and emission filter 500–560 nm (maxima �ex = 499 nm and �em = 521 nm)



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Specificity: Specifically detects caspases 2, 3 and 7, caspases 6, 8, 9 and 10 to a lesserextent

Can be used to assay: Cell cultures, recombinant caspases

Kit contents

1. Substrate stock solution, 10x2. Positive control, 10x3. Rhodamine 110, standard4. Incubation buffer

Typical results: see Figures 17–18

Other applications: For more examples of how the Homogeneous Caspases Assay can beused in the lab, see Appendix, page 133.

Figure 17: Kinetics of caspase activation in U937 cells by camptothecin (384-well plate). U937 cellswere exposed to 4 µg/ml camptothecin for different time intervals at 37 °C, analyzed for caspase activity with theHomogeneous Caspases Assay and fluorescence plotted versus time.

Figure 18: Dose-response curve of U937 cells exposed to different concentrations of camptothecin(CAM) (384-well plate). U937 cells were exposed to camtothecin for 4 h at 37 °C, analyzed for caspase activitywith the Homogeneous Caspases Assay and standardized values plotted versus concentration.



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Anti-PARPCat. No. 11 835 238 001 100 µl (50 blots)

Type Polyclonal antiserum, from rabbit

Useful for Detection on Western blots of PARP cleaved by caspases during early stages of apoptosis

Samples Crude cell extracts

Method Western blot of apoptotic cell extracts, followed by indirect immunodetec-tion of PARP cleavage fragment

Time Approx. 5.5 h (immunodetection only)

Significance of reagent: Anti-PARP recognizes Poly-ADP-Ribose-Polymerase (PARP), a113 kD protein that binds specifically at DNA strand breaks. PARP is also a substrate forcertain caspases (for example, caspase 3 and 7) activated during early stages of apoptosis.These proteases cleave PARP to fragments of approximately 89 kD and 24 kD. Detectionof the 89 kD PARP fragment with Anti-PARP thus serves as an early marker of apoptosis.

Test principle: The Anti-PARP antibody may be used to detect the 89 kD PARP fragment(and intact PARP) from apoptotic cell extracts on a Western blot. The procedure (FlowChart 6) involves:

� Preparing crude extracts of apoptotic cells (for instance, by sonication and incubation of 105–107 cells in the presence of urea, 2-mercaptoethanol, and SDS).

� Separating proteins in the crude cell extracts on an SDS-polyacrylamide gel.

� Transferring the separated proteins to a membrane by electroblotting.

� Detecting PARP fragments (and intact PARP) on the membrane with the Anti-PARP antibody.

� Visualizing the antibody-protein complexes with an enzyme-conjugated anti-rabbit IgG secondary antibody and a chromogenic or chemiluminescent enzyme substrate (see Table 3).



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A� Flow Chart 6: Assay of caspase activity with Anti-PARP.

Antibody supplied as: Polyclonal antiserum from rabbit, stabilized.

Sensitivity: PARP cleavage fragments from 3 x 105 apoptotic cells could be detected on aWestern blot (Figure 19).

Specificity: On Western blots, Anti-PARP recognizes intact PARP from primates orrodents, as well as the large PARP fragment generated by caspases. Anti-PARP will immu-noprecipitate intact PARP from primates or rodents.


� Crude cell extracts

Typical results: see Figure 19.

The appearance of a large (89 kD) cleavage fragment is indicative of caspase proteolyticactivity.

Other applications: For more examples of how the Anti-PARP can be used in the lab, seeAppendix, pages 133–134.

� Figure 19: Detection of cleaved PARP in cell extracts of apoptotic CEM T cells. CEM T cells were incu-bated with one of three apoptosis-inducing drugs. Cell extracts from 3 x 105 treated or untreated cells were frac-tionated on an 10% polyacrylamide gel in the presence of SDS. After electrophoresis, proteins on the gel weretransferred to a PVDF membrane by electroblotting and the blot was blocked with 5% powdered milk. Theblocked membrane was incubated with a 1:3000 dilution of Anti-PARP. Subsequent incubations with a peroxi-dase-conjugated anti-rabbit secondary antibody and a peroxidase substrate revealed the presence of PARPcleavage products on the blot. Note that the antibody recognizes both uncleaved PARP (113 kD) and the largercleavage fragment (89 kD). Lane 1: Untreated control cellsLane 2: Cells treated with 100 ng/ml doxorubicin for 24 hLane 3: Cells treated with 1 mg/ml methotrexate for 24 h Lane 4: Cells treated with 1 mg/ml cytarabin for 24 h. (Data is courtesy of Dr. Ingrid Herr, German Cancer Research Institute, department of molecular oncology,Heidelberg, Germany)

Prepare crude extracts from apoptotic cells in extraction buffer (approx. 15 min, RT; then 15 min, 65°C)

Separate proteins in crude extracts by SDS-PAGE

Electroblot proteins to nitrocellulose or PVDF membrane (approx. 1 h, RT)

Incubate membrane with blocking buffer (1 h, RT)

Incubate membrane with Anti-PARP (diluted 1:2000) (2 h, RT)

Wash membrane twice with blocking buffer (25 min, RT)

Visualize antibody-antigen complexes with secondary antibody and chromogenic/chemiluminescent detection (approx. 2 h, RT)



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� Table 3: Related products for visualization of Anti-PARP.

Product Cat. No. Pack Size

BM Chromogenic Western Blotting Kit (Mouse/Rabbit)

11 647 644 001 for 2000 cm2 membrane

BM Chemiluminescence Western Blotting Kit (Mouse/Rabbit)

11 520 709 001 for 2000 cm2 membrane

BM Chemiluminescence Blotting Substrate (POD)

11 500 708 00111 500 694 001

for 1000 cm2 membranefor 4000 cm2 membrane

CSPD (chemiluminescent AP substrate), ready-to-use

11 755 633 001 2 x 50 ml

CDP Star (chemiluminescent AP substrate) ready-to-use

11 685 627 00111 759 051 001

1 ml2 x 1 ml

BM Blue POD Substrate, precipitating 11 442 066 001 100 ml

BM Purple AP Substrate, precipitating 11 442 074 001 100 ml



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Apoptosis, Cell Death, and Cell Proliferation Manual Cell Death – Apoptosis and Necrosis30 31





2.1.3 Summary of methods for studying apoptosis in cell populations

� Table 4: Methods for studying apoptosis in cell populations.

Method/Roche Applied Science product

Parameter analyzed

Label Assay principle Advantages Limitations For product informa-tion, see

DNA Fragmentation Assay, radioactive11, 12

DNA fragmentation (LMW and HMW-DNA)

[3H]-TdR or [125I]- UdR, prelabel

� DNA fragments are released from the cytoplasm of apoptotic cells after lysis with non-ionic detergent.

� The LMW DNA is separated from nuclear HMW DNA by centrifugation.

� The radioactivity in the supernatant and in the pellet is determined by LSC.

� Quantitative measurement over a large range (several orders of magnitude)

� Sensitive (103–104 cells/test required)� Suitable for analysis of cell-mediated (cytotoxicity) effects

� Radioactive isotope� Requires prelabeling and extensive washing of the target

cells � Limited to target cells proliferating in vitro� Increased background due to free [3H]-TdR in the

cytoplasm

DNA Fragmentation Assay, non-radioactive13

Cellular DNA Fragmentation ELISA

DNA fragmentation (LMW DNA)

BrdU, prelabel � DNA fragments are released from the cytoplasm of apoptotic cells after lysis with a non-ionic detergent.


� The supernatant is analyzed by ELISA.

� Sensitive (103–104 cells/test required)� Labeled cells do not have to be washed� Optimal for microtiter plate format� Non-radioactive� Suitable for analysis of cell-mediated (cytotoxicity) effects

� Prelabeling of the target cells required� Can only assay target cells proliferating in vitro� Narrow range of quantitative measurement (only one

order of magnitude)

page 64of this guide

JAM Test14 DNA fragmentation (HMW DNA)

[3H]-TdR, prelabel

� Cells are harvested by vacuum aspiration onto glass fiber filters. While LMW-DNA is washed through the filters, the HMW DNA is retained on these filters.

� The radioactivity retained on the filters is measured by LSC.

� Sensitive (103–104 cells/test required) � Only 1 washing step required for the labeled cells� Low spontaneous release: cytotoxic events causing low

cell lysis over prolonged period of time (8–24 h) can be studied

� Optimal for microtiter plate format

� Radioactive isotope� Prelabeling of the target cells required� Limited to target cells proliferating in vitro� In apoptotic cells, DNA is only partially lost: viable and

damaged cells are separated by only a narrow range of assay values

Alkaline Elution Analysis15 DNA fragmentation (LMW and HMW-DNA)

[3H]-TdR, prelabel

� Cells are loaded onto polycarbonate filters.� The filters are incubated with three different buffer

solutions containing SDS, pH 10, SDS + Proteinase K, pH 7, or SDS, pH 12.3.

� The radioactivity in each fraction (LMW DNA) as well as the radioactivity retained on the filter (HMW DNA) is quantified by LSC.

� Differential elution allows the detection of strand breaks in DNA, DNA-interstrand crosslinks and DNA-protein crosslinks

� Radioactive isotope� Prelabeling and washing of the target cells required� Limited to target cells proliferating in vitro� Insensitive (106 cells/test required) � Labor-intensive and time-consuming: only a few tests

may be performed simultaneously

DNA Ladder Assay16

(LMW and HMW DNA by size)

Apoptotic DNA Ladder Kit

DNA fragmentation

none � Cellular DNA is isolated by extraction and quickly purified.

� Purified total DNA (LMW and HMW DNA) is analyzed by agarose gel electrophoresis and visualized by staining with ethidium bromide.

� Hallmark of apoptosis: demonstration of the mono- and oligonucleosomal DNA fragments (180 bp multimers)

� No prelabeling of the cells required: not limited to cells which proliferate in vitro

� Non-radioactive

� No quantitative measurement � Insensitive: More than 106 cells/test required � Labor-intensive and time-consuming:

only a few tests may be performed simultaneously


Nucleosome Quantification ELISA13


DNA fragmentation (LMW DNA in association with histones)

none � Histone complexed DNA-fragments (mono- and oligonucleosomes, LMW DNA) are released from the cytoplasm of apoptotic cells after lysis.


� The supernatant is analyzed by ELISA.

� Sensitive (102–104 cells/test required) � No prelabeling of the cells required: not restricted to cells

which proliferate in vitro� Non-radioactive � Detection of DNA and histones in one immunoassay dem-

onstrates mono- and oligonucleosomal DNA fragments

� Samples have to be analyzed immediately because storage reduces ELISA signal

� Not recommended for tissue homogenates. Increased background could occur due to activation of nucleases during sample preparation.


DNA Ladder Assay, radioactive 17 DNA fragmentation (LMW and HMW by size)

�-[32P]-ATP, postlabel

� Cellular DNA is isolated by extraction and quickly purified.

� Purified total DNA (LMW and HMW DNA) is labeled at the 5’end with �-[32P]-ATP by T4 Polynucleotide Kinase.

� [32P]-labeled DNA is separated by agarose gel electrophoresis and quantitated in the dried gel by a blot analyzer.

� Definitive marker of apoptosis: demonstration of the mono- and oligonucleosomal DNA fragments (180 bp multimers)

� No prelabeling of the cells required: not limited to cells which proliferate in vitro

� Highly sensitive (1000 x more sensitive than ethidium bro-mide): allows earlier detection of DNA fragmentation after induction of apoptosis

� Labor-intensive and time-consuming: only a few tests may be performed simultaneously

� Radioactive assay (32P) � End-labeling of purified DNA required

Protease Activity Assay

Caspase 3 Acitivity Assay

Activation of caspases (Caspase 3)

none � Apoptotic process including activation of the caspase cascade is induced in cells by desired method.

� Cells are lysed and cell extracts are prepared.� Activated caspase 3 is captured out of cellular lysates

by an Anti-caspase 3 antibody� Quantification of fluorochromes cleaved from a caspase

specific substrate.

� Quantitative assay, cleavage of substrate is proportional to concentration of activated caspase 3 in samples

� Detection of very early stages of apoptosis� Highly specific for caspase 3, no cross reactions with other

members of the caspase family

� High cell numbers needed� Fluorescence reader, equipped with special fluorescence

filters needed


Protease Activity Assay

Anti-PARP

Discrete cleavage of DNA repair enzyme (PARP)

none � Cells are treated with an apoptosis-inducing agent, which leads to induction of caspase 3 and the cleavage of Poly-ADP-Ribose-Polymerase (PARP).

� Cell extracts are prepared with SDS, fractionated by SDS-PAGE, and transferred to a PVDF membrane by western blotting.

� Blot is probed with an antibody to PARP, then with a peroxidase-labeled secondary antibody.

� Cleavage products of PARP (about 85 kD) on the mem-brane are revealed after an incubation with a peroxi-dase substrate.

� Flexible, can be used with many different types of cells � No prelabeling of cells required: not limited to cells which

proliferate in vitro� Non-radioactive � Marker for very early stage of apoptosis

� Insensitive (requires 105–106 cells/test)� Labor-intensive and time-consuming:

only a few tests may be performed simultaneously


2404 Apoptosis Kap_A Tab_04_AK1.fm Seite 1 M ontag, 23. August 2004 11:51 11

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2.2 Methods for studying apoptosis in individual cells

A number of methods have now been developed to study apoptosis in individual cells. Inthe following sections, we will describe details of several of these apoptosis assays.

We focus on two key apoptotic events in the cell:

� DNA fragmentation used to study death in cell populations may also be used to studydeath in individual cells. As described in Section A 2.1.1, DNA cleavage is a hallmarkfor apoptosis, and assays which measure prelytic DNA fragmentation are especiallyattractive for the determination of apoptotic cell death.

The methods used to assess DNA strand breaks are based on labeling/staining the cel-lular DNA. The labeled/stained DNA is subsequently analyzed by flow cytometry, fluo-rescence microscopy or light microscopy (Figure 20). In general, two different labelingmethods may be used to identify DNA in apoptotic cells:

� Enzymatic labeling: Cellular DNA is labeled with modified nucleotides (e.g.,biotin-dUTP, DIG-dUTP, fluorescein-dUTP) using exogenous enzymes (e.g., termi-nal transferase, DNA polymerase). This labeling detects extensive DNA strandbreaks. Enzymatic labeling is discussed in detail below (Section A 2.2.1 of thisguide).

� Staining with fluorochromes: Cellular DNA is stained with fluorescent DNA-bind-ing dyes (DNA-fluorochromes) capable of intercalating into DNA. Upon binding toDNA these dyes become highly fluorescent. Apoptotic cells are binding less dye mol-ecules, since they characteristically lose DNA during the staining process (describedin Section A 2.2.3 of this guide).

� In addition, individual cell death may be studied by assays that measure alterations inplasma membranes (alterations in the asymmetry or permeability of individual cellmembranes, which occur as the membrane shrinks and becomes increasingly convo-luted.) For instance, during apoptosis, phosphatidylserine translocates from the cyto-plasmic side of the membrane to the extracellular side and can be detected withAnnexin-V (described in Section A 2.2.2 of this guide).

� Figure 20: Schematic illustration of the two basic principles for detecting DNA fragmentation in single cells.


Methods for studying apoptosis in individual cells

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2.2.1 The TUNEL enzymatic labeling assay

Extensive DNA degradation is a characteristic event which occurs in the late stages ofapoptosis. Cleavage of the DNA may yield double-stranded, LMW DNA fragments(mono- and oligonucleosomes) as well as single strand breaks (“nicks”) in HMW-DNA.Those DNA strand breaks can be detected by enzymatic labeling of the free 3’-OH ter-mini with modified nucleotides (X-dUTP, X = biotin, DIG or fluorescein). Suitable label-ing enzymes include DNA polymerase (nick translation) and terminal deoxynucleotidyltransferase (end labeling) (Figure 21).

� Figure 21: Schematic illustration of two enzymatic DNA labeling methods nick translation and endlabeling.

Nick translation

DNA polymerase I catalyzes the template dependent addition of nucleotides when onestrand of a double-stranded DNA molecule is nicked. Theoretically, this reaction (In SituNick Translation, ISNT) should detect not only apoptotic DNA, but also the randomfragmentation of DNA by multiple endonucleases occurring in cellular necrosis.

End labeling

Terminal deoxynucleotidyl transferase (TdT) is able to label blunt ends of double-stranded DNA breaks independent of a template. The end-labeling method has also beentermed TUNEL (TdT-mediated X-dUTP nick end labeling)18.

The TUNEL method is more sensitive and faster than the ISNT method. Cells undergo-ing apoptosis were preferentially labeled by the TUNEL reaction, whereas necrotic cellswere identified by ISNT. Thus, experiments suggest the TUNEL reaction is more specificfor apoptosis and the combined use of the TUNEL and nick translation techniques maybe helpful to differentiate cellular apoptosis and necrosis19.

Note: For a comparison of results with the TUNEL and ISNT methods, see Figure 22.

To allow exogenous enzymes to enter the cell, the plasma membrane has to be permeabi-lized prior to the enzymatic reaction. To avoid loss of LMW DNA from the permeabilizedcells, the cells have to be fixed with formaldehyde or glutaraldehyde before permeabiliza-tion. This fixation crosslinks LMW DNA to other cellular constituents and precludes itsextraction during the permeabilization step.



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A � Figure 22: Comparison of TUNEL and ISNT methods for detecting apoptosis in CD8+ T cells fromTcR transgenic mice. F5 mice are transgenic for a T cell receptor (TcR) specific for a peptide derived from anucleoprotein of influenza virus ANT/1968. In this experiment, the nucleopeptide protein was injected into F5mice to activate T cells in vivo. After 4 days, mice were sacrificed and lymphoid organs were removed. Cell sus-pensions were prepared and incubated 4 h at 37°C. To allow detection of T cells which were dying after the in vivoimmune response [Pihlgren, M., Thomas, J. and Marvel, J. (1996) Biochemica, No. 3, 12–14], cells were stained forCD8 (with a fluorescent antibody), fixed, permeabilized, and then labeled by either the TUNEL (TdT-mediateddUTP Nick End Labeling) or the ISNT (In Situ Nick Translation) method. Labeled and control cells were analyzedby flow cytometry, with CD8+ cells gated. Spleen cells from a control (not immunized) mouse (red) and from twomice immunized 4 days earlier (green) are shown.

Result: The TUNEL method detected approximately 15% apoptotic cells among CD8+ T cells from the immunizedmice. No positive cells were found in the control mouse. In contrast, the ISNT method was unable to detect anyapoptotic cells, possibly due to the lower sensitivity of the technique.

If free 3’ ends in DNA are labeled with biotin-dUTP or DIG-dUTP, the incorporatednucleotides may be detected in a second incubation step with (strept)avidin or an anti-DIG antibody. The immunocomplex is easily visible if the (strept)avidin or an anti-DIGantibody is conjugated with a reporter molecule (e.g., fluorescein, AP, POD).

In contrast, the use of fluorescein-dUTP to label the DNA strand breaks allows the detec-tion of the incorporated nucleotides directly with a fluorescence microscope or a flowcytometer20. Direct labeling with fluorescein-dUTP offers several other advantages.Direct labeling produces less nonspecific background with sensitivity equal to indirectlabeling (Figure 23) and, thus, is as powerful as the indirect method in detecting apopto-sis. Furthermore, the fluorescence may be converted into a colorimetric signal if an anti-fluorescein antibody conjugated with a reporter enzyme (Table 5) is added to the sample.

Although the enzymatic labeling methods are time-consuming (due to multiple incuba-tion and washing steps), they are very sensitive and specific21.

Caution: One has to keep in mind that these methods are based on the detection of DNAstrand breaks. There are rare situations when apoptosis is induced without DNA degrada-tion. Conversely, extensive DNA degradation, even specific to the internucleosomal linkerDNA, may accompany necrosis. Thus, one should always use another independent assay,along with the TUNEL method, to confirm and characterize apoptosis.

Roche Applied Science offers four kits for the detection of DNA strand breaks in individ-ual cells using the TUNEL method. Each is described on the following pages.

Note: For technical tips on the TUNEL method, see page 113 of the Appendix.



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� Figure 23: Comparison of direct and indirect labeling of DNA strand breaks in apoptotic cells. PBLwere incubated with 1 µM dexamethasone for 24 h at 37°C and then labeled by TUNEL. Recordings were made atthe same photomultiplier settings.(Data were kindly provided by R. Sgonc, University of Innsbruck, Austria).Result: Direct labeling is as sensitive as indirect labeling, but produces less non-specific background.

� Table 5: Four different kits for the enzymatic labeling of DNA and the secondary detection systemsrequired.

Method/RAS prod-uct

Label Indirect (secondary) detection system

Analysis by

In Situ Cell Death Detection Kit, Fluorescein

Fluorescein-dUTP None (direct detection) Flow cytometry Fluorescence micros-copy

In Situ Cell Death Detection Kit, TMR red

TMR-dUTP None (direct detection) Fluorescence micros-copy

In Situ Cell Death Detection Kit, AP

Fluorescein-dUTP Anti-Fluorescein-AP Light microscopy

In Situ Cell Death Detection Kit, POD

Fluorescein-dUTP Anti-Fluorescein-POD Light microscopy



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In Situ Cell Death Detection Kit, FluoresceinCat. No. 11 684 795 001 50 tests

In Situ Cell Death Detection Kit, TMR redCat. No. 12 156 792 001 50 tests

Type Direct TUNEL labeling assay

Useful for Detection of DNA strand breaks in apoptotic cells by flow cytometry or fluorescence microscopy

Samples Cells in suspension, adherent cells, cell smears, frozen or paraffin-embedded tissue sections

Method End-labeling of DNA with fluorescein-dUTP or tetramethylrhodamine-dUTP (TMR-dUTP), followed by direct analysis of fluorescent cells

Time 1–2 h (+ sample preparation, permeabilization, etc.)

Significance of kit: This two In Situ Cell Death Detection Kits, measure and quantitatecell death (apoptosis) by labeling and detection of DNA strand breaks in individual cellsby flow cytometry or fluorescence microscopy. The kits offer a direct TUNEL detectionmethod, for maximum sensitivity and minimal background.

Test principle: The assays use an optimized terminal transferase (TdT) to label free 3’OHends in genomic DNA with fluorescein-dUTP or TMR-dUTP. The procedure involves:

� Figure 24: Schematic showing the principle of the In Situ Cell Death Detection Kits, Fluorescein and TMR red.

For a detailed overview of the steps in the procedure, see Flow Chart 7.

Sensitivity: The enzymatic labeling allows the detection of an apoptotic event thatoccurs, prior to changes in morphology and even before DNA fragments become detect-able in the cytoplasm22. It detects early stage of DNA fragmentation in apoptotic cells.This is especially important if apoptosis is studied in vivo, e.g., in tissue sections, sinceapoptotic cells are rapidly and efficiently removed in vivo.

Specificity: The amount of DNA strand breaks in apoptotic cells is so large that thedegree of cell labeling in these assays is an adequate discriminator between apoptotic andnecrotic cells19.

� Fixing and permeabilizing apoptotic cells.

� Incubating the cells with the TUNEL reaction mixture containing TdT and fluorescein-dUTP or TMR-dUTP. During this incubation step, TdT catalyzes the attachment of flu-orescein-dUTP or TMR-dUTP to free 3’OH ends in the DNA.

� Visualizing the incorporated fluorescein with a flow cytometer and/or a fluorescence microscope (fluorescein/TMR red).



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� Flow Chart 7: Assay procedure, In Situ Cell Death Detection Kits (Fluorescein or TMR red).


� Cells in suspension (permanent cell lines, normal and tumor cells ex vivo)

� Cytospins, cell smears

� Adherent cells cultured on chamber slides

� Frozen tissue sections

� Formalin-fixed, paraffin-embedded tissue sections

Kit contents

1. Enzyme solution (TdT), 5 tubes2. Labeling solution (nucleotide mix), 5 tubes

Typical results: See Figures 25–28.

Technical tips: For more information on the use of the kit for flow cytometric analysis,see page 119 in the Appendix of this guide.

Other applications: For more examples of how the In Situ Cell Death Detection Kits (Flu-orescein or TMR red) can be used in the lab, see Appendix, pages 134–136.

Cell suspensions Adherent cells, cell smears, cytospins

Frozen tissue section Formalin-fixed, paraffin-embedded tissue sections

Fix samples (1 h, RT) Dewax, rehydrate, and treat with protease

Wash samples

Permeabilize samples (2 min, on ice)

Wash samples

Incubate with TUNEL reaction mixture [enzyme solution + labeling solution] (60 min, 37°C)

Wash samples

Analyze by flow cytometry or fluores-

cence microscopy

Analyze by fluorescence microscopy



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A� Figure 25: Detection of apoptotic cells by flow cytometry using the In Situ Cell Death Detection Kit,Fluorescein. HL60 cells were cultured in the absence (A) or presence (B) of 2 µg/ml Camptothecin for 3 h at37°C. Cells were incubated either with TUNEL reaction mixture (�) or label solution as negative control (�) orPBS for autofluorescence (�).

� Figure 26: Detection of apoptotic cells (green)by fluorescence microscopy in a tissue sectionfrom rat. A tissue section from a rat spinal cord wasprepared and assayed with the In Situ Cell Death Detec-tion Kit, Fluorescein. The treated section was viewedunder a fluorescence microscope. (Photomicrographwas kindly provided by R. Gold, University of Würzburg,Germany.)Result: A subpopulation of apoptotic cells, scatteredthroughout the tissue section, are intensely stained(green) by the TUNEL treatment and are easily visibleunder the microscope.

� Figure 27: Cell suspension stained with the InSitu Cell Death Detection Kit, Fluorescein. U937cells induced with 4 µg/ml camptothecin, showing posi-tive staining of apoptotic nuclei.Note: This figure shows a high number (>80%) ofapoptotic cells. To avoid detecting cells that are under-going secondary necrosis, analyze cells earlier in theprocess after induction of apoptosis.

� Figure 28: Rabbit endometrium, stained withthe In Situ Cell Death Detection Kit, TMR red, andviewed under a fluorescence microscope. Apoptoticnuclei stain bright red, limited fluorescence is visible inbackground tissue.



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In Situ Cell Death Detection Kit, APCat. No. 11 684 809 001 50 tests

In Situ Cell Death Detection Kit, PODCat. No. 11 684 817 001 50 tests

Type Indirect TUNEL labeling assay

Useful for Detection of DNA strand breaks in apoptotic cells under a light microscope

Samples Cell smears, adherent cells, cytospins, frozen or fixed tissue sections

Method End-labeling of DNA with fluorescein-dUTP, followed by detection of incorporated fluorescein with an antibody and visualization of the antibody

Time Approx. 3 h (+ sample preparation, permeabilization, etc.)

Significance of kits: These two In Situ Cell Death Detection Kits measure cell death (apo-ptosis) by detecting DNA strand breaks in individual cells by light microscopy. The APand POD kits offer an indirect TUNEL detection method, which is a fast, sensitive, andspecific light microscopic assay.

Test principle: The assays use an optimized terminal transferase (TdT) to label free 3’OHends in genomic DNA with fluorescein-dUTP. The procedure involves:

Sensitivity: The enzymatic labeling allows the detection of an apoptotic event thatoccurs, prior to changes in morphology and even before DNA fragments become detect-able in the cytoplasm22. It detects early stage of DNA fragmentation in apoptotic cells.This is especially important if apoptosis is studied in vivo, e.g., in tissue sections, sinceapoptotic cells are rapidly and efficiently removed in vivo.

Specificity: The amount of DNA strand breaks in apoptotic cells is so large that thedegree of cell labeling in these assays is an adequate discriminator between apoptotic andnecrotic cells19.

� Fixing and permeabilizing apoptotic cells or tissue sections.

� Incubating the cells with the TUNEL reaction mixture containing TdT and fluorescein-dUTP. During this incubation step, TdT catalyzes the attachment of fluorescein-dUTP to free 3’OH ends in the DNA.

� Detecting the incorporated fluorescein with an anti-fluorescein antibody AP conjugate (In Situ Cell Death Detection Kit, AP) or an anti-fluorescein antibody POD conjugate (In Situ Cell Death Detection Kit, POD).

� Visualizing the immunocomplexed AP or POD with a substrate reaction.

� Figure 29: Schematic showing theprinciple of the In Situ Cell Death Detec-tion Kits, AP and POD.



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� Flow Chart 8: Assay procedure In Situ Cell Death Detection Kits (AP or POD).


� Cells smears, adherent cells

� Cytospins

� Tissue sections (frozen or paraffin-embedded).

Kit contents

In Situ Cell Death Detection Kit, AP

1. Enzyme solution (TdT), 5 tubes2. Labeling solution (nucleotide mix), 5 tubes3. Anti-Fluorescein-AP conjugate, ready to use

In Situ Cell Death Detection Kit, POD

1. Enzyme solution (TdT), 5 tubes2. Labeling solution (nucleotide mix), 5 tubes3. Anti-Fluorescein-POD conjugate, ready to use

Note: For added flexibility and convenience, the components of these kits, as well as severalAP and POD precipitating substrates are also available as single reagents (Table 6).

Typical results: see Figures 30 and 31.

Technical tips: For more information on the use of the kits for light microscopic analysis,see pages 113–116 in the Appendix, of this guide.

Remove tissue or organ and use standard methods to process for:

Frozen sectioning Paraffin embedding

Fix sections in formalin (30 min, RT) Dewax, rehydrate sections by standard methods

Optional: Inactivate endogenous POD/AP activity

Wash slides

Fix section (30 min, RT) Add protease and incubate (30 min, 37°C)

Wash slides

Permeabilize sections (2 min, on ice)

Add TUNEL-reaction mixture and incubate (60 min, 37°C)

Wash slides

Optional: Analyze by fluorescence microscopy

Add Anti-Fluorescein-AP or -POD and incubate (30 min, 37°C)

Wash slides

Add substrate and incubate (5–20 min, RT)

Analyze by lightmicroscopy



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� Figure 30: Detection of apoptotic cells by TUNEL and peroxidase staining in rabbit endometrium.A tissue section from rabbit endometrium was prepared and assayed with the In Situ Cell Death Detection Kit,POD. Slide was counterstained with hematoxylin and viewed under a light microscope.Result: A subpopulation of apoptotic cells, scattered throughout the tissue section, are intensely stained (brown)by the TUNEL treatment and subsequent peroxidase immunostaining.

� Figure 31: Detection of apoptotic cells by TUNEL and alkaline phosphatase staining in rat spinalcord. A tissue section from rat spinal cord was prepared and assayed with the In Situ Cell Death Detection Kit,AP. The slide was viewed under a light microscope (Panel A). After viewing, the same slide was stained with pro-pidium iodide and viewed by fluorescence microscopy (Panel B).Result: A few apoptotic cells (red) are clearly visible after TUNEL treatment and subsequent alkaline phos-phatase immunostaining (Panel A). However, the apoptotic cells are not visible in the same slide after stainingwith propidium iodide (Panel B).

A

B



A


A

Other applications: For more examples of how the In Situ Cell Death Detection Kits (APor POD) can be used in the lab, see Appendix, pages 136–138.

For your convenience, we offer a number of additional single reagents to optimize yourTUNEL reaction (Table 6)

� Table 6: Single reagents available for the TUNEL technique.

Product Cat. No. Pack Size

TUNEL Label Mix 11 767 291 001 3 x 550 µl (30 tests)

TUNEL Enzyme 11 767 305 001 2 x 50 µl (20 tests)

TUNEL POD (Anti-Fluorescein, POD conjugate) 11 772 465 001 3.5 ml (70 tests)

TUNEL AP (Anti-Fluorescein, AP conjugate) 11 772 457 001 3.5 ml (70 tests)

TUNEL Dilution Buffer 11 966 006 001 2 x 10 ml

DAB Substrate, metal enhanced, precipitating(Peroxidase (POD) substrate)

11 718 096 001 1 pack

NBT/BCIP Stock Solution (AP substrate) 11 681 451 001 8 ml

Fast Red Tablets (AP substrate) 11 496 549 001 20 tablets



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2.2.2 Assays that measure membrane alterations

In contrast to necrosis, apoptosis occurs without inflammation. In the end stages ofapoptosis, apoptotic bodies are engulfed by macrophages and other phagocytic cells2

in vivo. Thus, apoptotic cells are removed from the population without spilling their con-tents and eliciting an inflammatory response.

The exact mechanism by which the apoptotic cell becomes a target for phagocytes isunclear. However, it has been shown that a number of changes in cell surface (mem-brane) markers occur during apoptosis, any one of which may signal “remove now” tothe phagocytes. These membrane changes include:

� Loss of terminal sialic acid residues from the side chains of cell surface glycoproteins,exposing new sugar residues23,24.

� Emergence of surface glycoproteins that may serve as receptors for macrophage-secreted adhesive molecules such as thrombospondin25.

� Loss of asymmetry in cell membrane phospholipids, altering both the hydrophobicityand charge of the membrane surface26.

In theory, any of these membrane changes could provide an assay for apoptotic cells. Infact, one of them has – the alteration in phospholipid distribution.

In normal cells (Figure 32, left diagram), the distribution of phospholipids is asymmet-ric, with the inner membrane containing anionic phospholipids (such as phosphati-dylserine) and the outer membrane having mostly neutral phospholipids. In apoptoticcells (Figure 32, right diagram) however, the amount of phosphatidylserine (PS) on theouter surface of the membrane increases, exposing PS to the surrounding liquid27.

Annexin-V, a calcium-dependent phospholipid-binding protein, has a high affinity forPS27. Although it will not bind to normal living cells, Annexin-V will bind to the PSexposed on the surface of apoptotic cells (Figure 33, 34). Thus, Annexin-V has provedsuitable for detecting apoptotic cells28, 29. Roche Applied Science supplies a number ofproducts for the detection of PS translocation by Annexin-V.

� Figure 32: Detection of surface morphology changes during apoptosis. During apoptosis, the distribu-tion of neutral phospholipids (black symbols) and anionic phospholipids such as phosphatidylserine (red sym-bols) in the cell membrane changes. Phosphatidylserine is present in the outer membrane of apoptotic cells, butnot of normal cells. An exogenously added molecule specific for phosphatidylserine, such as Annexin-V-FLUOS,will bind to phosphatidylserine on the outer membrane of apoptotic cells, but cannot react with the phosphati-dylserine of normal cells.



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Annexin-V-FLUOSCat. No. 11 828 681 001 250 tests

Annexin-V-FLUOS Staining KitCat. No. 11 858 777 001 50 tests

11 988 549 001 250 tests

Annexin-V-Alexa 568Cat. No. 03 703 126 001 250 tests

Type Direct fluorescence staining for flow cytometric or microscopic analysis

Useful for Detection of apoptotic cells with membrane alterations (phosphatidylserine translocation); differentiation of apoptotic from necrotic cells

Samples Cell lines (adherent or suspensions), freshly isolated cells

Method Simultaneous staining of cell surface phosphatidylserine [with Annexin-V-FLUOS (green dye) or Annexin-V-Alexa 568 (red dye)] and necrotic cells (with propidium iodide)

Time Approx. 15 min (after induction of apoptosis)

Significance of reagent and kit: Annexin-V is a phospholipid-binding protein with a highaffinity for phosphatidylserine (PS). Detection of cell-surface PS with annexin-V thusserves as a marker for apoptotic cells. Analysis may be by flow cytometry or by fluores-cence microscopy.

Test principle: Annexin-V-FLUOS (green dye) and Annexin-V-Alexa 568 (red dye) servesas a fluorescent probe for apoptotic cells. They will not bind normal, intact cells. However,since necrotic cells are leaky enough to give Annexin-V-FLUOS and Annexin-V-Alexa 568access to inner membrane PS, apoptotic cells have to be differentiated from necrotic cells.Thus, the assay involves simultaneous staining with both Annexin-V-FLUOS (green) andthe DNA stain propidium iodide (red) or Annexin-V-Alexa 568 (red) and BOBO-1(green). Exclusion of propidium iodide or BOBO-1, coupled with binding of Annexin-V-FLUOS or Annexin-V-Alexa 568, indicates an apoptotic cell (Table 7). The procedure(Flow Chart 9) involves:

� Washing suspended cells, then pelleting the cells.

� Resuspending cells in a staining solution containing Annexin-V-FLUOS and propidium iodide or Annexin-V-Alexa 568 and BOBO-1.

Note: Cells may also be labeled with other membrane stains, such as a fluorescein-, phycoerythrin- or TRITC-labeled monoclonal antibody simultaneously.

� Analyzing samples in a flow cytometer or under a fluorescence microscope.



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� Table 7: Distinguishing apoptosis from necrosis using Annexin-V, propidium iodide, or BOBO-1.

� Flow Chart 9: Assay procedure, Annexin-V-FLUOS Staining Kit and Annexin-V-Alexa 568.

Specificity: Annexin-V-FLUOS and Annexin-V-Alexa 568 bind apoptotic cells and leakynecrotic cells. Propidium iodide and BOBO-1 are excluded from apoptotic and normalcells, but is taken up by necrotic cells.


� Cell lines (adherent or suspensions)

� Freshly isolated cells

Reagent contents:

� Annexin-V-FLUOS solution, 50 x concentrated

� Annexin-V-Alexa 568, 50 x concentrated

Kit contents

Annexin-V-FLUOS Staining Kit

1. Annexin-V-FLUOS, 50 x concentrated2. Propidium iodide solution, 50 x concentrated3. Binding buffer for flow cytometry, ready to use


Other applications: For more examples of how the Annexin-V-FLUOS, Annexin-V-FLUOS Staining Kit and Annexin-V-Alexa 568 can be used in the lab, see Appendix,pages 138–141.

Normal cells Apoptotic cells Necrotic cells

Annexin-V staining – + +Propidium iodide staining

– – +

BOBO-1 – – +

Treat sample (106 cells) with apoptosis-inducing agent (1–24 h)

Wash treated cells with PBS and centrifuge (200 x g) (5 min, RT)

Incubate cells in binding buffer containing Annexin-V-FLUOS and propidium iodide or Annexin-V-Alexa 568 and BOBO-1. (10–15 min, RT)

Add binding buffer to stained cells Analyze by fluorescence microscopy

Analyze by flow cytometry



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A� Figure 33: Apoptotic and necrotic U937 cells identified in FACS analysis after staining with Annexin-V-FLUOS and propidium iodide (PI). Cells were then stained with the components of the Annexin-V-FLUOSStaining Kit and analyzed.Panels A (upper and lower), single parameter analysis, Annexin-V-FLUOS only; Panels B, single parameter analysis, propidium iodide (PI) only; Panels C, dual parameter analysis, Annexin-V-FLUOS and propidium iodide. FL1, Annexin-V-FLUOS; FL2,propidium iodide.Result: Flow cytometric analysis clearly differentiates normal (living) cells with low Annexin and low PI staining,apoptotic cells with high Annexin and low PI staining, and necrotic cells with high Annexin and high PI staining.

� Figure 34: Discrimination between apoptotic and necrotic U937 cells treated with camptothecin.Early-stage apoptosis detected with Annexin-V-FLUOS (green), and counterstained with propidium iodide (redcells).Results: The apoptotic cells are visible in green and can be differentiated from necrotic cells by the propidiumstaining in figure 34. Necrotic cells take up propidium iodide and stain orange/green, while apoptotic cells staingreen only.

� Figure 35: Discrimination between apoptotic and necrotic U937 cells treated with camptothecin(CAM) and stained with Annexin-V-Alexa 568 (red) and BOBO-1 (green).Results: The apoptotic cells are visible in red and can be differentiated from necrotic cells by the BOBO-1 stain-ing in figure 35. Necrotic cells take up BOBO-1 and stain green/red, while apoptotic cells stain red only.

necrotic apoptoticapoptotic

necrotic

apoptotic

necrotic



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Annexin-V-BiotinCat. No. 11 828 690 001 250 tests

Type Indirect fluorescence staining for flow cytometric, fluorescence or light microscopic analysis

Useful for Detection of apoptotic cells with membrane alterations (phosphatidylserine translocation); differentiation of apoptotic from necrotic cells

Samples Cell lines (adherent and suspensions), freshly isolated cells

Method Simultaneous staining of cell surface phosphatidylserine (with Annexin-V-Biotin) and necrotic cells (with propidium iodide), followed by detection of biotin (with streptavidin/avidin conjugate)

Time Approx. 75 min (after induction of apoptosis)

Significance of reagent: Annexin-V is a phospholipid-binding protein with a high affinityfor phosphatidylserine (PS). During apoptosis, PS translocates to the outer surface ofapoptotic cells. Detection of cell-surface PS with annexin-V thus serves as a marker forapoptotic cells. Labeling of cells with the Biotin-conjugate of Annexin-V allows fixationafter Annexin-V binding for further analysis of additional cellular parameters in com-bination with detection of apoptosis (van Engeland, M., Ramaekers FCS, Schutte, B &Reutelingsperger, CPM (1996): A Novel Assay to Measure Loss of Plasma MembraneAsymmetry During Apoptosis of Adherent Cells in Culture. Cytometry 24: 131–139). Fordistinguishing apoptosis using Annexin-V, see Table 7, page 45.

Test principle: Annexin-V-Biotin serves as a probe for apoptotic cells. It will not bindnormal, intact cells. However, since necrotic cells are leaky enough to give Annexin-V-Biotin access to inner membrane PS, apoptotic cells have to be differentiated fromnecrotic cells. Thus, the assay involves simultaneous staining with both Annexin-V-Biotin, Avidin-Fluorescein and propidium iodide. Exclusion of propidium iodide, cou-pled with binding of Annexin-V-Biotin, indicates an apoptotic cell. Annexin-V-Biotin isvisualized with a streptavidin/avidin conjugate. Analysis may be by flow cytometry, byfluorescence microscopy, or by light microscopy. The procedure (Flow Chart 10)involves:

Specificity: Annexin-V-Biotin binds apoptotic cells and leaky necrotic cells

� Washing suspended cells, then pelleting the cells.

� Resuspending cells in a staining solution containing Annexin-V-Biotin and propidium iodide.Note: Cells may also be labeled with other membrane stains, such as a fluorescein-, phycoerythrin- or TRITC-labeled monoclonal antibody simultaneously.

� Washing labeled cells.

� Incubating cells with a streptavidin (SA)/avidin conjugate (Table 8).

� Analyzing samples in a flow cytometer, under a fluorescence microscope, or under a light microscope (depending on the SA/avidin conjugate).



A


A� Flow Chart 10: Assay procedure, Annexin-V-Biotin.

Typical results: see Figure 36.

Other applications: For more examples of how the Annexin-V-Biotin can be used in thelab, see Appendix, pages 141–142.

� Figure 36: Flow cytometric analysis of apoptoticU937 cells stained with Annexin-V-Biotin, Avidin-FLUOS and propidium iodide. U937 cells (a leukemiccell line) were cultivated for 4 h with 4 µg/mlcampothecin. Cells were stained with Annexin-V-Biotinand propidium iodide (PI), then incubated with Avidin-fluorescein and analyzed. Single parameter histogramsare shown at the top (Annexin-V-Biotin/Avidin-FLUOS)and on the right side (PI) of the diagram. Two parameterhistograms are shown in quadrants 1–4. PI, propidiumiodide; FLUOS, fluorescein.Result: Flow cytometric analysis clearly differentiatesnormal cells (quadrant 3) with low FLUOS and low PIstaining, apoptotic cells (quadrant 4) with high FLUOSand low PI staining, and necrotic cells (quadrant 2) withhigh FLUOS and high PI staining.

� Table 8: Streptavidin (SA) /Avidin conjugates available for the indirect assay of apoptotic cells withAnnexin-V-Biotin.

Note: Additional substrates can be found in Table 6.

Treat sample (106 cells) with apoptosis-inducing agent (1–24 h)

Wash treated cells with PBS and centrifuge (200 x g) (5 min, RT)

Incubate cells in incubation buffer containing Annexin-V-Biotin and propidium iodide (10–15 min, RT)

Centrifuge stained cells (200 x g) and wash once with incubation buffer (approx. 10 min, RT)

Incubate washed cells with streptavidin/avidin conjugate (20 min, 4°C)

Centrifuge stained cells (200 x g) and wash once with incubation buffer (approx. 10 min, RT)

Add incubation buffer to stained cells

Analyze by fluorescence microscopy

Add substrate (5–15 min, RT) and wash

Analyze by flow cytometry Analyze by light microscopy

Can be used to assay: Reagent contents:� Cell lines (adherent/suspensions) � Annexin-V-Biotin solution, 50 x concen-

trated.� Freshly isolated cells

Product Application Cat. No. Pack Size

Avidin-Fluorescein fluorescence microscopy, flow cytometry

11 975 595 001 1 mg

Avidin-Rhodamine fluorescence microscopy, flow cytometry

11 975 609 001 1 mg

SA-Peroxidase light microscopy 11 089 153 001 500 U (1 ml)

SA-Alkaline Phos-phatase

light microscopy 11 089 161 001 1000 U (1 ml)

SA-�-Galactosidase light microscopy 11 112 481 001 500 U



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2.2.3 Assays that use DNA stains

One can differentiate between three methods for studying cell death that use DNA stains:dye exclusion method, profile of DNA content, morphological changes.

Dye exclusion method

Viable (intact plasma membrane) and dead (damaged plasma membrane) cells can bediscriminated by differential staining. Cells with disturbed plasma membrane perme-ability are stained, whereas undamaged (viable) cells are not stained with dyes that do notpenetrate the plasma membrane (“exclusion dyes”). The most frequently used dye forexclusion tests is trypan blue. In addition, the fluorescent dye, propidium iodide (PI)which becomes highly fluorescent after binding to DNA, can be used in the same man-ner. The stained and unstained cells are counted with a standard light microscope (try-pan blue), or flow cytometer (PI) (Table 9).

Profile of DNA content

If cells are permeabilized, the LMW DNA inside the cytoplasm of apoptotic cells leaks outduring the subsequent rinse and staining procedure. The lower DNA content of thesecells means they contain less DNA stained by the fluorochrome. Thus, cells with lowerDNA staining than that of G1 cells (the so-called “sub-G1 peaks”, “A0” cells) have beenconsidered apoptotic. The reduction in staining/DNA content of these cells is measuredby flow cytometry (Figure 37). The major disadvantage of this technique is that apoptoticG2-Phase cells exhibit a reduced DNA content, which could represent the DNA contentof a G1-cell. Therefore it may not be detected as apoptotic. This would result in an under-estimation of the apoptotic population.

� Table 9: Common fluorochromes used to stain the genomic DNA of viable and/or non-viable cells.

DNA-binding dyes (Fluoro-chromes)

Dye enters Dye stains

Viable cells Non viable cells Nucleus (DNA) Cytoplasm (RNA)

Acridine orange Yes Yes Green Red-orange

Hoechst 33342 Yes Yes Blue No

Hoechst 33258 No Yes Blue No

DAPI No Yes Bright blue No

Ethidium bromide

No Yes Orange Slightly red

Propidium iodide

No Yes Red No



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A� Figure 37: Typical flow cytometric profile of the DNA content in normal (A) and apoptotic cells (B),stained with PI. Result: A prominent “sub-G1” peak (earliest peak) appears in apoptotic cells, but not in normal cells.

Morphological changes

On the other hand, the bisbenzimidazole dye, Hoechst 33342 (and also acridine orange),penetrates the plasma membrane and stains DNA in cells; without permeabilization. Incontrast to normal cells, the nuclei of apoptotic cells have highly condensed chromatinthat is uniformly stained by Hoechst 33342. This can take the form of crescents aroundthe periphery of the nucleus, or the entire nucleus can appear to be one or a group of fea-tureless, bright spherical beads. These morphological changes in the nuclei of apoptoticcells may be visualized by fluorescence microscopy. They are also visible in permeabilizedapoptotic cells stained with other DNA binding dyes like DAPI (Figure 38).

Dive et al.30 have reported that during a short exposure to Hoechst 33342, apoptotic cellshave stronger blue fluorescence compared to non-apoptotic cells. Co-staining of the cellswith propidium iodide (PI) allows the discrimination of dead cells from apoptotic cells. If7-amino-actinomycin is used instead of PI, cell surface antigens immunostained with flu-orescein and phycoerythrin may be quantitated simultaneously31.

One drawback of using any vital staining method for measuring apoptosis is the variabil-ity of active dye uptake in different cells and its possible change during certain treat-ments. Therefore, the ability of Hoechst 33342 to discriminate apoptotic cells fromnormal cells by increased uptake of dye has to be tested for each new cell system31.

� Table 10: Fluorescent dyes that stain double-stranded DNA*Only sold in the US

Reagent Cat. No. Pack size

Fluorescence Typical results

Propidium iodide* 11 348 639 001 20 ml red orange See Table 9, Figure 33 and 34

DAPI4’,6-Diamidine-2’-phenylindole dihydrochloride

10 236 276 001 10 mg blue See Table 9 andFigure 38



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� Figure 38: Fluorescent microscopic analysis of apoptotic cells stained with DAPI. DAPI stains thenucleic of all cells (blue).Result: The characteristic condensed nuclei of apoptotic cells are clearly visible here.

� Figure 39: Fluorescent microscopic analysis of mitotic cells stained with ethidium bromide. DNA wasstained with ethidium bromide (orange). Mitotic spindles were stained with anti-tubulin antibody (green).Result: Mitotic cells (with condensed DNA) are brightly stained. Without the double stain, mitotic cells could bemistaken for apoptotic cells, since both have condensed DNA.



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Apoptosis, Cell Death, and Cell Proliferation Manual Cell Death – Apoptosis and Necrosis52 53





2.2.4 Summary of methods for studying apoptosis in individual cells

� Table 11: Methods for studying apoptosis in individual cells.


Parameter analyzed Assay principle Advantages Limitations For product informa-tion, see

Staining of chromosomal DNA after permeabilization (DNA content)22, 32

DAPI, propidium iodide*

DNA fragmentation (HMW DNA, DNA content)

� Apoptotic cells are permeabilized with ethanol or deter-gent. During this procedure the LMW DNA inside the apoptotic cell leaks out and is removed during the sub-sequent washing steps.

� The HWM DNA retained in the cells is stained with a DNA binding dye such as propidium iodide.

� The amount of HMW DNA is quantified by flow cyto-metry (“sub G1” or “A0” peak).

� Quick and cheap� Minimal overlap between the peak representing apoptotic

(“sub G1”) and normal G1 cells � Estimation of position in cell cycle allows cell cycle speci-

ficity of apoptosis to be studied � Applicability to any DNA fluorochrome and instrument

� Degree of extraction of LMW DNA during the washing and staining procedure not always reproducible

� Not specific for apoptosis: Sub G1 peak can also represent mechanically damaged cells, cells with different chromatin structure, or normal cells with lower DNA content in heterogeneous cell populations

� May not detect cells induced to apoptosis in G2� No discrimination of apoptotic cells from dead cells

which have lost their membrane integrity


Staining of chromosomal DNA Chromatin morphology � Apoptotic cells are stained by the addition of DNA fluorochromes which are able to cross the intact plasma membrane, such as acridine orange. Can be done with DAPI on fixed cells.

� The stained DNA allows the altered morphology of the nuclear chromatin to be visualized by fluorescence microscopy.

� Quick and cheap � Discrimination between viable and dead cells when

counterstained with propidium iodide using vital dyes (acridine orange, Hoechst 33342)

� Simultaneous staining of cell surface antigens with standard fluorescein and phycoerythrin conjugates possible if Hoechst 33342 is combined with 7-amino-actinomycin D

� No quantitative measurement� Subjective: no clear cut-off point between normal and

apoptotic cells� Clear morphologically distinct apoptotic nuclei appear

late during apoptosis: May lead to an underestimation of apoptotic cells

Active labeling of cells by nick translation (ISNT)34

DNA strand breaks (nicks) and DNA fragmentation (staggered DNA ends)

� Apoptotic cells are fixed with formaldehyde and subsequently permeabilized.

� DNA strand breaks are labeled with modified nucleotides using exogenous DNA polymerase (nick translation).

� The incorporated nucleotides are visualized with a sec-ondary detection system which has a reporter molecule (e.g. fluorescein, AP, POD).

� Counterstaining with DNA fluorochrome (profile of DNA content) allows cell cycle specificity of apoptosis to be studied (only by flow cytometry)

� Identification of apoptosis at a molecular level (DNA strand breaks)

� Suitable for tissue sections

� Labor-intensive and time-consuming; only a few tests may be performed simultaneously

� Undefined cell loss during fixation procedure (loss of specific cell population?)

� Many cells (2–5 x 106/test) required

Active labeling of cells by end labeling (TUNEL)20, 35

In Situ Cell Death Kit, Fluorescein, TMR, AP, POD

DNA strand breaks (nicks) and DNA fragmentation (staggered DNA ends)

� Apoptotic cells are fixed with formaldehyde and subsequently permeabilized.

� DNA strand breaks are labeled with modified nucleotides using exogenous terminal transferase (end labeling).

� The incorporated nucleotides are visualized directly (e.g. Fluorescein-dUTP) or with a secondary detection system which has a reporter molecule (e.g. fluorescein, AP, POD).

� Advantages like in situ nick translation; in addition:� More sensitive: maximum intensity of labeling (cell

staining) of apoptotic cells is higher compared to ISNT� Fast kinetics of dUTP incorporation in comparison with the

DNA polymerase method: 30 min is sufficient for the labeling reaction

� Higher sensitivity for apoptosis: TUNEL preferentially labels apoptosis in comparison to necrosis

� Direct detection possible by the use of Fluorescein-dUTP (without secondary detection system) for maximum sensitivity and minimal background

� With the direct TUNEL labeling assay less working steps are required than with the indirect assay.

� Labor-intensive and time-consuming; only a few tests may be performed simultaneously

� Many cells (2–5 x 106/test) required � Undefined cell loss during fixation procedure

(loss of specific cell population?)

pages 36-42 of this guide

Detection of translocated membrane component27

Annexin-V-FLUOSAnnexin-V-BiotinAnnexin-V-Staining Kit Annexin-V-Alexa 568

Detection of phosphatidylserine on surface of apoptotic cells

� Apoptotic cells are incubated with an assay protein (e.g. annexin-V) conjugated to a reporter molecule.

� Assay protein binds a membrane component (e.g. phosphatidylserine) found on the outer surface of apoptotic cells only.

� A DNA strain is added to distinguish necrotic cells (permeable) from apoptotic cells (impermeable).

� Apoptotic cells are made visible by assay of reporter molecule in flow cytometer or under a microscope.

� Unique marker for apoptosis related plasma membrane changes

� Allows analysis by flow cytometry fluorescence microscopy, or light microscopy

� Allows simultaneous labeling of other cell surface anti-gens

� Annexin-V-Biotin allows fixation following Annexin-V binding for further analysis of additional cellular parameters

� Not specific for apoptosis: Annexin-V can stain inner membrane of ruptured cells; must distinguish apoptotic from necrotic cells with an additional DNA stain

� Many cells (106/test) required � Cannot be used on tissue sections or any fixed samples

pages 44-48 of this guide

Trypan Blue Exclusion Assay Damage/leakage of plasma membrane

� Cells are incubated with dye.� Dead cells take up dye; living cells do not.� Stained (dead or damaged) cells are determined under

a light microscope.� Dye binds to intracellular proteins of leaky cells.

� The classical standard method to distinguish viable from dead cells by light microscopy

� Quick and cheap� Only a small fraction of total cells from a cell population is

required

� Each individual sample has to be counted: only a few tests may be performed simultaneously

� Subjective evaluation � Not suitable for detection of apoptosis� Stains only necrotic cells or very late apoptotic cells

(secondary necrosis)

Propidium Iodide Exclusion Assay

Propidium iodide solution*

Damage/leakage of plasma membrane

� Cells are incubated with fluorescent dye. � Dead cells take up dye; living cells do not. � Stained (dead or damaged) cells are determined under

a microscope or by flow cytometry.� Dye binds to DNA of leaky cells.

� The standard method to distinguish viable from dead cells by fluorescence microscopy and flow cytometry

� Double labeling procedures possible: simultaneous detection of e.g. surface antigens

� Quick and cheap � Only a small fraction of total cells from a cell population is

required

� Each individual sample has to be counted: only a few tests may be performed simultaneously

� Not specific for apoptosis

page 49 of this guide

*Sold only in the US


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2.3 Detection of apoptosis-related proteins

There are a number of genes that regulate apoptosis. That is, the products of these genesinterfere with apoptotic pathways. Assays to detect the proteins encoded by these genescan complement the assays described in the previous sections.

The study of apoptosis-regulating genes and gene products is still evolving. The pictureso far is complex (as summarized in Section A 1.3 of this guide). For instance, in somecases, the same gene has an effect on both the survival of normal cells and the develop-ment of cancers by preventing apoptosis36. A detailed discussion of the field is beyondthe scope of this guide, but is covered in a number of reviews36, 37. As an introduction tothe field, we discuss the characteristics of a few of these apoptosis-regulating proteins.

The relationship of the ced (caenorhabditis elegans cell death) genes to apoptosis in thenematode Caenorhabditis elegans has been extensively studied38. Of these, the ced-3 andced-4 genes39 encode proteins that must be active to initiate apoptosis. In contrast, theced-9 gene product protects cells from apoptotic cell death, ensuring their survival40. Inother words, apoptosis is more likely when levels of ced-3 and ced-4 protein are high orwhen levels of ced-9 protein are low.

In mammalian systems, the Bcl-2 protooncogene serves much the same function as ced-9, blocking the induction of cell death41. The Bcl-2 oncoprotein also protects against thecytotoxic effects of certain drugs42.

The Bcl-2 protein can dimerize with itself or with the product of the bax gene43. Thepresence of the bax protein seems to counteract the anti-apoptotic activity of Bcl-2. Insummary, apoptosis is more likely when bax protein levels are high or when Bcl-2 proteinlevels are low.

Another mammalian gene product, p53, is a tumor suppressor because it induces apop-tosis in potentially malignant cells44. Absence or mutation of the p53 gene product led tomalignant transformation and immortalization of the cell.

Increases in expression of a growth stimulating gene, the c-myc proto-oncogene, actuallyinduces apoptosis in the absence of other growth factors45, 46. High levels of the Bcl-2protein can counteract the effect of the c-myc protein.

For the analysis of apoptosis-regulating proteins, Roche Applied Science offers an ELISAkit for the detection of p53 in fluids or extracts and antibodies to detect c-myc and Fas(CD95/Apo-1).

Cell surface receptor genes (APO-1/Fas/CD95), other growth-stimulating genes (e.g.,Ras), and other tumor-suppressing genes (e.g., Rb) have also been implicated in the regu-lation of apoptosis2, 37. The Fas (CD95/APO-1) molecule has originally been identified asa cell surface receptor that could mediate apoptotic cell death of transformed cells andcause regression of experimental tumors growing in nude mice. The function of Fas wasassessed by establishment of mouse cell transformants that constitutively expressedhuman Fas. When the transformed cells were treated with the antibody to human Fas, thecells died by apoptosis within 5 hours, which indicated that Fas can transduce an apop-totic signal and that anti-Fas works as an agonist. The subsequent purification of humanAPO-1 antigen and molecular cloning of its cDNA established the identity of APO-1 andFas. Meanwhile, numerous reports have shown a pivotal role of Fas in various physiolog-ical and pathological processes. The Anti-Fas provided by Roche Applied Science is suit-able for the induction of apoptosis as well as for the detection of the Fas receptor.


Detection of apoptosis-related proteins

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Anti-Fas (CD95/Apo-1)Cat. No. 11 922 432 001 100 µg

Type Monoclonal antibody, clone 2R2, IgG3, mouse

Useful for Apoptosis induction in Fas expressing cells

Samples Cell suspensions, adherent cells

Method Direct induction of apoptosis by adding antibody to cell cultures

Time Approx. 3–5 h (induction of apoptosis)

Significance of reagent: The antibody may be used for the induction of apoptosis in cellcultures through Fas by imitating the Fas-ligand. The Fas (CD95/Apo-1) molecule hasbeen identified as a cell surface receptor that could induce apoptotic cell death of trans-formed cells upon activation by its ligand and cause regression of experimental tumors inmice.

Test principle: The antibody may be used for induction of apoptosis:

Antibody supplied as: Mouse monoclonal antibody (clone 2R2, IgG3) in cell culturesupernatant; sterile filtered.

Sensitivity: The antibody is suitable for induction of apoptosis at 0.5 µg/ml in SKW6.4and Jurkat cells. If secondary crosslinking with an anti-mouse IgG is used, the apoptosisinducing concentration could be reduced to 100 ng/ml. In Fas transfectants apoptosis isinduced without crosslinking at 100 ng/ml. It has to be mentioned, that some Jurkat sub-clones do not or only in high doses respond to Anti-Fas induction of apoptosis.

Specificity: The antibody was generated by immunizing mice with transformed murineL-cells bearing recombinant human Fas-receptor. On Western blots, Anti-Fas binds thehuman Fas/Apo-1 (CD95).

Can be used for:

� Induction of apoptosis through the Fas-receptor

� Add antibody (1 µg/ml) into culture medium of Fas-bearing cells

� Incubation for 3–5 hours

� Detection of apoptosis by various assays



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p53 pan ELISACat. No. 11 828 789 001 96 tests

Type One-step sandwich ELISA, colorimetric

Useful for Quantitation of p53 protein, both wild-type and mutant forms

Samples Tissue homogenates, cell lysates, serum, or plasma

Method Lysis/homogenization of sample, followed by immunochemical determination of p53 in a microplate

Time Approx. 2.5 h

Significance of kit: This kit uses antibodies to quantitate the levels of human, mouse, orrat p53 in smear/plasma, in tumor tissue, or in tumor cell lines. Wild type p53 is a proteinthat suppresses malignant transformation by inducing apoptosis. Mutations of the p53gene which increases its stability allow transformation (immortalization) of the cell andare quite commonly found in malignancies. The p53 protein seems also to be involved incell death induced by cytotoxic drugs.

Test principle: The assay uses a one-step sandwich immunoassay (Figure 40) to detectwild-type and mutant p53. The procedure (Flow Chart 11) involves:

� Figure 40: How the p53 pan ELISA works.

� Preparing sample (e.g., detergent lysis of cells, homogenization of tissue), followed by centrifugation.

� Transferring an aliquot of the sample supernatant to streptavidin-coated well of a microplate (precoated with anti-p53-biotin).

� Binding p53 in the supernatant with two antibodies, a biotin-labeled monoclonal anti-p53 (capture antibody), which is pre-bound to the streptavidin-coated plate, and a peroxidase-labeled polyclonal anti-p53 (detection antibody).

� Washing the immobilized antibody-p53 complexes five times to remove sample components that are not immunoreactive.

� Incubating sample with peroxidase substrate (tetramethylbenzidine, TMB).

� Determining the amount of colored product (and thus, of immobilized antibody-p53 complexes) spectrophotometrically.



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� Flow Chart 11: Assay procedure, p53 pan ELISA.

Sensitivity: In 4 independent assays, the lower limit of detection for the assay was deter-mined to be 9 pg/ml.

Specificity: The biotin-labeled capture antibody from mouse recognizes a conserved,pantropic, denaturation stable antigenic determinant of the p53 protein (human, mouse,rat). The peroxidase-labeled detection antibody is highly specific for wild-type andmutant p53 from different species.


� Tissue homogenates

� Cell lysates

� Serum or plasma

Kit contents

1. Anti-human-p53 pan, polyclonal, peroxidase-labeled2. Human p53 standards (six)3. Incubation buffer/Sample diluent, ready-to-use4. Washing buffer, 10 x concentrated5. TMB substrate solution6. TMB stop solution7. Streptavidin-coated microplate, precoated with monoclonal Anti-p53 (biotin-labeled)8. Adhesive plate cover foils

Prepare sample (for example, cell lysate or tissue homogenate)

Centrifuge sample (10,000 x g) (10 min, RT)

Transfer aliquot of supernatant to Anti-p53 precoated microplate

Incubate supernatant with Anti-p53-peroxidase (1:15 diluted) (2 h, RT)

Wash microplate wells five times with washing buffer (5 x 1 min, RT)

Add substrate solution to wells and incubate (approx. 10–20 min, RT)

Measure absorbance at 370 nm Add stop reagent to wells (1 min, RT)

Measure absorbance at 450 nm



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3. Cytotoxicity Assay Methods

3.1 Relationship between cytotoxicity, apoptosis and necrosis

As discussed in Section A 1.1 of this guide, there are two experimentally distinguishablemechanisms of cell death: necrosis, the “accidental” cell death that occurs when cells areexposed to a serious physical or chemical insult, and apoptosis, the “normal” cell deaththat removes unwanted or useless cells.

In contrast to these two cell death processes, cytotoxicity does not define a specific cellu-lar death mechanism. Rather, cytotoxicity is simply the cell-killing property of a chemicalcompound (such as a food, cosmetic, or pharmaceutical) or a mediator cell (such as acytotoxic T cell), independent from the mechanisms of death.

Note: Cytotoxicity may also be used, as it is in this guide, to denote a laboratory method fordetecting dead cells, regardless of the mechanism of their death.

� Figure 41: Schematic illustration of the three basic principles to assess plasma membrane leakage.

Example of cytotoxicity

A common example of cytotoxicity is cell-mediated cytotoxicity. Cells of the immune sys-tem [such as cytotoxic T cells, natural killer (NK) cells, and lymphokine-activated (LAK)cells] can recognize and destroy damaged, infected and mutated target cells. Although therecognition machinery used by these cells is very different, their mechanism of target celldestruction may be very similar.

Two possible cytocidal mechanisms have been proposed for cell-mediated cytotoxicity:(i) the apoptotic mechanism, in which the effector cell triggers an autolytic cascade in thetarget cell and the genomic DNA fragments before cell lysis; and (ii) the lytic mechanism,in which lytic molecules, notably perforin, are secreted by the effector cell into the inter-cellular space and polymerize to form pores in the target cell membrane, leading to celllysis3, 47. These two mechanisms are not mutually exclusive and, quite possibly, are com-plementary.

Cytotoxicity Assay Methods

Relationship between cytotoxicity, apoptosis and necrosis

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3.2 Methods for studying cytotoxicity

Most current assays for measuring cytotoxicity are based on alterations of plasma mem-brane permeability and the consequent release (leakage) of components into the super-natant or the uptake of dyes, normally excluded by viable cells (Figure 41) (see alsoA 2.2.3, on page 49 “Dye exclusion method”). A serious disadvantage of such permeabil-ity assays is that the initial sites of damage of many, if not most cytotoxic agents are intra-cellular. Therefore, cells may be irreversibly damaged and committed to die and theplasma membrane is still intact. Thus, these assays tend to underestimate cellular damagewhen compared to other methods. Despite this fact, some permeability assays have beenwidely accepted for the measurement of cytotoxicity.

Alternatively, dead cells are unable to metabolize various tetrazolium salts (see also Sec-tion A 2.1.1). This allows the use of the colorimetric assays MTT, XTT, or WST-1 to mea-sure cell survival. Apoptosis, however, is an active mode of cell death requiring themetabolism of cells. Thus, like the permeability assays mentioned above, the colorimet-ric assays may underestimate cellular damage and detect cell death only at the later stagesof apoptosis when the metabolic activity of the cells is reduced.

Regardless of this disadvantage, the colorimetric assays are very useful for quantitatingfactor-induced cytotoxicity within a 24 to 96 h period of cell culture. However, these col-orimetric assays are of limited value for measuring cell mediated cytotoxicity, since mosteffector cells become activated upon binding to the target cells. This activation results inan increased formazan production by the effector cell, which tends to mask the decreasedformazan production resulting from target cell death.

Note: Assays for cytotoxicity can be, and frequently are, used to measure cell necrosis.

3.2.1 Assays that measure plasma membrane leakage

Widely used standard methods for measuring plasma membrane leakage are based on theuptake or exclusion of molecules with special light-absorbing or fluorescent properties.Viable (intact plasma membrane) and dead (damaged plasma membrane) cells can bediscriminated by differential staining. Because dyes stain individual cells, each sample hasto be analyzed by flow cytometry or microscopy. This kind of single cell analysis is notsuitable if many different samples have to be measured. In contrast, assays which quanti-tate plasma membrane disintegration in cell populations allow many different samples tobe handled simultaneously in a single MTP.

One group of standard assays performed in a MTP is based on the release of radioactiveisotopes ([51Cr], [3H]-thymidine, [3H]-proline, [35S]- or [75Se]-methionine, 5-[125I]-2-deoxy-uridine) or fluorescent dyes (bis-carboxyethyl-carboxyfluorescein (BCECF) or cal-cein-AM) from prelabeled target cells48, 49, 50. The disadvantages of such assays however,are (i) the use of radioactive isotopes in most of them, (ii) the necessity for prelabeling ofthe target cells, and (iii) the high spontaneous release of most labels from the prelabeledtarget cells.

Another group of assays is based on the measurement of cytoplasmic enzymes released bydamaged cells. The amount of enzyme activity detected in the culture supernatant corre-sponds to the proportion of lysed cells51, 52. Enzyme release assays have been describedfor alkaline and acid phosphatase, for glutamate-oxalacetate transaminase, for glutamatepyruvate transaminase, and for argininosuccinate lyase. However, their use has beenhampered by the low amount of those enzymes present in many cells and by the elaboratekinetic assays required to quantitate most enzyme activities.


Methods for studying cytotoxicity

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In contrast to the above mentioned cytoplasmic enzymes, lactate dehydrogenase (LDH) isa stable cytoplasmic enzyme present in all cells. It is rapidly released into the cell culturesupernatant when the plasma membrane is damaged. With the Cytotoxicity DetectionKit (see page 61), LDH activity can easily be measured in culture supernatants by a singlepoint assay. The use of a spectrophotometric microplate reader (ELISA plate reader)allows the simultaneous measurement of multiple probes and thereby guarantees the easyprocessing of a large number of samples (Figure 42).

� Figure 42: Measurement of LDH activity (�) using the microplate format (see also Flow Chart 12).



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Cytotoxicity Detection Kit (LDH)Cat. No. 11 644 793 001 2000 tests

Type Colorimetric assay, microplate format

Useful for Quantitation of LDH activity released from damaged/dying cells

Samples Cell-free supernatants from cells in culture

Method Preparation of cell-free supernatant, followed by incubation of supernatant with INT to form colored formazan, a product which may be quantitated colorimetrically

Time 0.5–1 h (+ induction of cell death)

Significance of kit: The Cytotoxicity Detection Kit measures cytotoxicity and cell lysis bydetecting LDH activity released from damaged cells. The assay is performed in a 96-wellmicroplate. The kit can be used in many different in vitro cell systems where damage ofthe plasma membrane occurs. Examples are:

� Detection and quantification of cell mediated cytotoxicity.

� Determination of mediator-induced cytolysis.

� Determination of the cytotoxic potential of compounds in environmental and medicalresearch, and in the food, cosmetic, and pharmaceutical industries.

� Determination of cell death in bioreactors.

Test principle: The assay is based on the cleavage of a tetrazolium salt when LDH ispresent in the culture supernatant. The procedure involves:

For a detailed overview of the steps involved in the procedure, see Figures 42 and 43 andFlow Chart 12.

� Flow Chart 12: Assay procedure, Cytotoxicity Detection Kit (LDH).

� Incubating the cells in culture to allow cell death to occur. An increase in the amount of dead or plasma membrane-damaged cells during the assay results in an increase of LDH in the culture supernatant.

� Collecting the cell-free culture supernatant.

� Adding the substrate mixture from the kit to the culture supernatant. Any LDH released into the supernatant during Step 1 will reduce the tetrazolium salt INT to formazan by a coupled enzymatic reaction. Thus, release of LDH into the supernatant directly correlates to the amount of formazan formed in this step.

� Quantitating the formazan dye formed in an ELISA plate reader. The formazan dye formed is water-soluble and shows a broad absorption maximum at about 500 nm.

Incubate cells in a microplate or batch culture for a certain period of time (optional: induction of death by treatment with compounds or test substances)

Centrifuge cells

Remove 100 ml supernatant and transfer it to another microplate

Add 100 µl/well reaction mixture to each supernatant and incubate for 0.5 h

Measure absorbance using an ELISA plate reader



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A� Figure 43: Biochemistry of the Cyto-toxicity Detection Kit (LDH): In the first enzymatic reaction LDHreduces NAD+ to NADH + H+ by oxidation of lactate to pyruvate; in the second enzymatic reaction the catalyst(diaphorase) transfers H/H+ from NADH + H+ to the tetrazolium salt INT.


� Cell-free supernatants obtained from cells cultured in 96-well microplates or batchcultures.

Kit contents

1. Catalyst (Diaphorase/NAD + mixture)2. Dye solution (INT and sodium lactate)

Note: To prepare the reaction mixture, mix catalyst with dye solution prior to use.


Other applications: For more examples of how the Cytotoxicity Detection Kit (LDH) canbe used in the lab, see Appendix, pages 142–143.



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� Figure 44. Determination of the cytolytic activity of allogen-stimulated cytotoxic T lymphocytes(CTLs) using the Cytotoxicity Detection Kit (LDH). A) Spleen cells of C57/BI 6 mice (H-2b) were stimulated invitro with P815 cells (H-2d). Viable CTLs were purified and titrated in the microplate as described in the packageinsert. Target cells (1 x 104 cells/well) were incubated in the presence or absence (effector cell controls) of effec-tor cells for 4 hours. Culture supernatant samples (100 µl/well) for effector controls ( and the effector-targetcell mix (�) were assayed for LDH activity. The middle curve is generated when the background control valuesare subtracted from the effector-target cell values (). B) Calculated percentage of cell-mediated lysis.

� Figure 45: Correlation of cell death (defined by increased plasma membrane permeability) and LDHrelease. Ag8 cells (starting cell concentration: 2 x 105 /ml) were cultured and after 1, 2, 3 and 5 days, aliquotswere removed. The amount of viable (�) and dead (�) cells was determined by trypan blue exclusion. LDH activ-ity in cell free culture supernatant was determined using the Cytotoxicity Detection Kit (�).Result: Increased LDH release clearly correlated with the increase of dead cells.

A B



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Cellular DNA Fragmentation ELISACat. No. 11 585 045 001 500 tests

Type Sandwich ELISA, colorimetric

Useful for Quantitation of BrdU-labeled DNA fragments either released from cells during necrosis or cell-mediated cytotoxicity, or within the cytoplasm of apoptotic cells

Samples Cell-free supernatants from cultured cells or cytoplasmic lysates of cells, prelabeled with BrdU

Method Prelabeling of cells with BrdU, followed by immunodetection of BrdU-labeled DNA fragments in sample

Time 4.5–5.5 h (+ BrdU labeling and induction of cell death)

Significance of kit: The Cellular DNA Fragmentation ELISA measures apoptosis, necro-sis, or cell mediated cytotoxicity by quantitating the fragmentation and/or release ofBrdU-labeled DNA. The kit detects DNA fragments:

� In the cytoplasm of apoptotic cells, thus providing a non-radioactive alternative to the[3H]-thymidine-based DNA fragmentation assay.

� Released into the culture supernatant during cell mediated cytotoxicity, thus providinga non-radioactive alternative to the [3H]-thymidine- and [51Cr]-release assays.

Test principle: The assay is a sandwich enzyme-linked immunosorbent assay (ELISA). Ituses two mouse monoclonal antibodies: one directed against DNA the other againstBrdU (Figure 46). The procedure (Flow Chart 13) involves:

� Prelabeling of cells with BrdU.

� Incubating the labeled cells in the presence of either an apoptosis inducing agent or effector cells (for cell mediated cytotoxicity). At the end of the incubation, cells are centrifuged and either supernatant is analyzed (for cell mediated cytotoxicity or necrosis) or cellular lysate is analyzed for apoptosis. The supernatant, containing LMW-DNA is used for the assay. If desired, both sample types can be prepared and assayed (see Flow Chart 13).

� Adsorbing the Anti-DNA antibody onto the wells of a microplate.

� Adding the supernatant of Step 2 to the microplate. BrdU-labeled DNA fragments in the sample bind to the immobilized Anti-DNA antibody.

� Denaturing the immunocomplexed BrdU-labeled DNA-fragments by microwave irradiation or nuclease treatment. This procedure is necessary for the accessibility of the BrdU antigen.

� Reacting Anti-BrdU antibody peroxidase conjugate (Anti-BrdU-POD) with the BrdU-labeled DNA to form an immunocomplex.

Quantitating the bound Anti-BrdU-POD in the immunocomplex with a peroxidase substrate (TMB).



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� Figure 46: How the Cellular DNA Fragmentation ELISA works.

Sample preparation:

� Flow Chart 13: Assay procedure, Cellular DNA Fragmentation ELISA.

Label cells in tissue culture flask with BrdU (2–18 h, 37°C)

Aliquot BrdU-labeled cells into the microplate

Measurement of apoptosis Measurement of cell mediated cytotoxicity or necrosis

Add cell death-inducing agent Add effector cells

Incubate cells (1–24 h, 37°C)

Add lysis buffer and incubate (30 min, RT)

Centrifuge microplate (10 min, 250 x g)

Remove supernatant and analyze by ELISA

ELISACoat microplate with Anti-DNA antibody (1 h, RT)

Recoat microtiter plate with blocking solution (30 min, RT)

Wash microplate, then add supernatant and incubate (90 min, RT)

Option 1 Option 2

Wash microplate, then denature/fix sample by microwave radiation (5 min)

Wash microplate, then add nuclease and incubate (30 min, 37°C)

Wash MTP

Add Anti-BrdU-POD and incubate (90 min, RT)

Wash microplate, then add substrate and incubate (10–30 min, RT)

Measure absorbance using an ELISA plate reader (2 min, RT)



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A� Flow Chart 14: Simultaneous analysis of apoptosis and necrosis in the same sample with the Cellular DNA Fragmentation ELISA.

Sensitivity

Apoptosis: When HL60/CAM is used as a model system for apoptosis, the ELISA candetect BrdU-labeled DNA fragments in the cytoplasm of 1 x 103 cells/well (Figure 42).

Cell mediated cytotoxicity: When allogeneic-stimulated cytotoxic T cells are used aseffector cells to lyse P815 target cells in a cell mediated cytotoxicity assay, the ELISA candetect BrdU-labeled DNA fragments from 2 x 103 target cells/well.

Note: The ability to detect a minimum number of dying/dead cells in a particular samplestrongly depends on the kinetics of cell death, the cytotoxic agent or the effector cells used toinduce cell death, and the amount of BrdU incorporated into the target cells.

Specificity

� The Anti-DNA antibody binds to single-and double-stranded DNA. It shows no cross-reactivity with BrdU.

� The conjugated antibody (Anti-BrdU-POD, Fab fragments) will bind to BrdU-labeledDNA after the DNA is partially denatured. The antibody specifically recognizes5-bromo-2’-deoxy-uridine. The antibody conjugate shows no cross-reactivity withany endogenous cellular components such as thymidine or uridine.

� The ELISA specifically detects BrdU-labeled DNA fragments in culture supernatantand cytoplasm. The ELISA can detect BrdU-labeled DNA from any species, so theassay is not species-restricted.


� Culture supernatant and cytoplasmic fractions (lysates) of cells whose DNA have beenmetabolically prelabeled with BrdU (e.g., cell lines and other in vitro proliferatingcells). Thus, only cells which proliferate in vitro can be used.

Label cells with BrdU

Add cell death-inducing agent

Split sample into two parts

Centrifuge cells

Remove supernatant totally Transfer 100 ml supernatant into Anti-DNA precoated microplate

Lyse cells Assay supernatant for BrdU-labeled DNA

Centrifuge lysate (to pellet HMW DNA) Result: Measurement of necrosis

Assay supernatant for oligonucleosomes containing BrdU-labeled LMW DNA

Result: Measurement of apoptosis



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Kit contents

1. Anti-DNA antibody (clone M-CA-33)2. Anti-BrdU-POD, Fab fragments (clone BMG 6H8)3. Coating buffer4. Washing buffer5. Incubation buffer6. Substrate solution7. BrdU labeling reagent8. Adhesive cover foils


� Figure 47: Kinetics of camptothecin (CAM) induced cell death in HL60 cells. Cells were prelabeledwith BrdU overnight. Then, cells (1 x 104/well) were incubated either in the presence of 200 ng/ml CAM (�, �) orwithout CAM (�, �) for 1–8 h. Supernatant (100 µl/well) was removed, then cells were lysed and both superna-tant (�, �) and lysate (�, �) were analyzed by Cellular DNA Fragmentation ELISA. Result: Apoptosis clearly occurs after 3–4 h incubation. After 6–8 h, secondary necrosis begins to be seen.

� Figure 48: Kinetics of cytotoxic T lymphocyte (CTL)-mediated cytotoxicity in P815 target cells quan-tified with the Cellular DNA Fragmentation ELISA. 2 x 104 BrdU-labeled target cells/well were incubated withCTLs at different effector-to-target ratios (E/T) for varying times. After incubation for 1 h (�), 2 h (�), 4 h (�), and6 h (�), culture supernatant samples (100 µl/well) were assayed for DNA fragments.

Other applications: For more examples of how the Cellular DNA Fragmentation ELISAcan be used in the lab, see Appendix, page 143.



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3.2.2 Assays that measure metabolic activity

Living (metabolically active) cells reduce tetrazolium salts to colored formazan com-pounds; dead cells do not. Thus, tetrazolium salt-based colorimetric assays detect viablecells exclusively. Because they are sensitive, these assays can readily be performed in amicroplate with relatively few cells.

Since a cytotoxic factor will reduce the rate of tetrazolium salt cleavage by a population ofcells, these metabolic activity assays are frequently used to measure factor-induced cyto-toxicity or cell necrosis53, 54. Applications include:

� Assessment of growth-inhibitory or cytotoxic effects of physiological mediators (Figure 49).

� Analysis of the cytotoxic and cytostatic effects of potential anti-cancer and other drugs(Figure 50).

� Analysis of cytopathic effects of viruses and screening of compounds with potentialanti-viral activity.

� Screening of antibodies for growth-inhibiting potential.

Roche Applied Science offers three microplate-based metabolic activity assays. All may beused to assay factor-induced cytotoxicity or necrosis. They are:

� Cell Proliferation Kit I (MTT), Cat. No. 11 465 007 001, in which metabolically activecells cleave the tetrazolium salt MTT to a water-insoluble formazan that can be solubi-lized and quantitated with an ELISA plate reader (for a more detailed description ofthis kit, see page 85 in this guide).

� Cell Proliferation Kit II (XTT), Cat. No. 11 465 015 001, in which metabolically activecells cleave the modified tetrazolium salt XTT to a water-soluble formazan, which maybe directly quantitated with an ELISA plate reader (for a more detailed description ofthis kit, see page 86 in this guide).

� Cell Proliferation Reagent WST-1, Cat. No. 11 644 807 001, a modified tetrazoliumsalt that can be cleaved by metabolically active cells to a water-soluble formazan, whichmay be directly quantitated with an ELISA plate reader (for a more detailed descrip-tion of this reagent, see page 87 in this guide).

Note: Since proliferating cells are metabolically more active than non-proliferating (resting)cells, these tetrazolium salt-based assays are also frequently used to measure cell activationand proliferation. For a full discussion of this application, see Section B 2.1.1 on page 82 ofthis guide.

For a more complete discussion of the principles behind these metabolic assays, see thetopic, “Biochemical and cellular basis of cell proliferation assays that use tetrazoliumsalts“ (Appendix, page 121) in this guide.



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� Figure 49: Measurement of the cytotoxic effects of human Tumor Necrosis Factor alpha (hTNF-�)on the mouse fibrosarcoma cell line WEHI-164. Cells in culture (106 cells/ml) were preincubated with actino-mycin C (1 µg/ml) for 3 h. Aliquots of these pretreated cells were transferred to a microtiter plate (5 x 104 cells/well) and incubated with actinomycin C and various amounts of hTNF-alpha for 24 h. Cellular response to TNFwas measured with the Roche Applied Science Cell Proliferation Kit II (XTT) and plotted against TNF concentra-tion.Result: Under the assay conditions, 50% of the WEHI-164 cells were killed by a TNF concentration of 35–40 pg/ml.

� Figure 50: Differentiation of cytotoxic and cytostatic effects of actinomycin D. A549 cells were addedto a microtiter plate (104 cells/well) and incubated with (�) or without (�) actinomycin D (10 ng/ml) for 20 h.

Graph A: Some aliquots of actinomycin-treated cells were assayed for cytotoxic effects (changes in metabolicactivity). These cells were assayed with either the Cell Proliferation Kit I (MTT), Cell Proliferation Kit II (XTT), orCell Proliferation Reagent WST-1 (WST). Cells were incubated with each tetrazolium salt for 4 h, then analyzed onan ELISA plate reader. Graph B: Other aliquots of actinomycin-treated cells were assayed for cytostatic effects (suppression of DNAsynthesis). These cells were incubated with either non-radioactive bromodeoxyuridine (BrdU) or tritiated thymi-dine ([3H]-TdR). Incorporation of BrdU into DNA was determined with the Cell Proliferation ELISA, BrdU (colori-metric). Incorporation of [3H]-TdR into DNA was determined by liquid scintillation counting.Result: Although actinomycin D is not significantly cytotoxic (as indicated by graph A) under these conditions, itdoes have a profound cytostatic (proliferation-inhibiting) effect (as indicated by graph B).



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Apoptosis, Cell Death, and Cell Proliferation Cell Death – Apoptosis and Necrosis70 71





3.2.3 Summary of methods for studying cytotoxicity

� Table 12: Methods for studying cytotoxicity.


Label Parameter analyzed

Assay principle Advantages Limitations For product informa-tion, see

[51Cr] Release Assay55 [51Cr] prelabel Damage/leakage of plasma membrane

� Viable cells are incubated with Na2[51Cr]O4, which binds tightly to most intracellular proteins (prelabeling).

� After washing, cells are incubated with cytotoxic agent. During this period, labeled proteins are released into the culture supernatant (SN) due to plasma membrane damage.

� The radioactivity in the SN is determined with a gamma-counter.

� Labeling of proteins by [51Cr] generally independent of the rate of protein synthesis

� Generally not restricted to target cell type: non-proli-ferating or slow turn-over populations can be studied

� Quantitative measurement over a large logarithmic range� Measurement of cell death in mixed cell populations; may

be used to quantitate cell-mediated cytotoxicity

� Radioactive isotope� Requires prelabeling and extensive washing of the target

cells� High spontaneous release: assay limited to cytotoxic

events causing high cell lysis over short period of time (2–5 h)

� For proper intracellular binding [51Cr]6+ has to be converted to [51Cr]3+: cells with low metabolic activity may not label sufficiently

[3H]-Thymidine ([3H]-TdR) Release Assay56

[3H]-TdR prelabel Damage/leakage of plasma membrane and DNA fragmenta-tion

� Cells proliferating in vitro are incubated with [3H] TdR, which is incorporated into the genomic DNA.

� After they are washed, cells are incubated with a cytotoxic agent. During this period, [3H] labeled DNA is released into the culture SN due to plasma membrane damage.

� The radioactivity in the SN and in the pellet is determined with a scintillation �-counter.

� Quantitative measurement over a large logarithmic range� Low spontaneous release: cytotoxic events causing low

cell lysis over a prolonged period of time (8–24 h) can be studied

� Measurement of cell death in mixed cell populations; may be used to quantitate cell-mediated cytotoxicity

� Radioactive isotope � Requires prelabeling and extensive washing of the target

cells� Limited to target cells proliferating in vitro

DNA Release Assay, nonradioactive

Cellular DNA Fragmentation ELISA

BrdU prelabel Damage/leakage of plasma membrane and DNA fragmenta-tion

� Cells proliferating in vitro are incubated with BrdU, which is incorporated into the genomic DNA.

� Cells are incubated with a cytotoxic agent or non-labeled effector cells (for cell-mediated cytotoxicity).

� During this period, BrdU-labeled DNA is released into the cytoplasm of apoptotic cells or into the culture SN due to plasma membrane leakage of damaged cells.

� The BrdU-labeled DNA in the SN or cytoplasm is deter-mined with an enzyme linked immunosorbent assay.

� Sensitive (103–104 cells/test required)� Labeled cells do not have to be washed� Optimal for microplate format� Non-radioactive� Measurement of cell death in mixed cell populations; may

be used to quantitate cell-mediated cytotoxicity� Possible to measure apoptosis and necrosis in parallel

� Prelabeling of the target cells required� Can only assay target cells proliferating in vitro� Narrow range of quantitative measurement

(only one order of magnitude)


LDH Release Assay51

Cytotoxicity Detection Kit, LDH

none Damage/leakage of plasma membrane

� Target cells are incubated with cytotoxic agent. During this period, cytoplasmic LDH is released into the culture SN due to plasma membrane damage.

� The LDH activity in the culture SN is measured by a substrate reaction and quantitated with an ELISA plate reader.

� Constitutively expressed ubiquitous protein: assay gener-ally not restricted by target cell type

� Does not require prelabeling and extensive washing of the target cells

� LDH activity in serum: special assay medium (reduced serum concentrations or BSA instead of serum) required

� Spontaneous release of LDH by target cells and effector cells: assay limited to cytotoxic events causing high cell lysis over short period of time (2–8 h)


Metabolic activity assays

Cell Proliferation Kit I (MTT), Kit II (XTT), Reagent (WST-1)

BrdU Reduced metabolic activity

� Dye solution is added to cells cultured in MTP and cells are incubated. Viable (metabolically active) cells cleave tetrazolium salts to colored formazan compounds; dead cells do not.

� Amount of formazan is quantitated with an ELISA plate reader.

� No cell type restriction� Does not require prelabeling and washing of the cells� Optimal for microplate format

� Increased metabolic activity of effector cells may mask target cell death during cell-mediated cytotoxicity

� No changes in the metabolic activity during the early phases of apoptosis

pages 85–87 of this guide


Seite 72 - Vakat

BCell Proliferation

and Viability

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1. Introduction

Rapid and accurate assessment of viable cell number and cell proliferation is an impor-tant requirement in many experimental situations involving in vitro and in vivo studies.Examples of where determination of cell number is useful include the analysis of growthfactor activity, serum batch testing, drug screening, and the determination of the cyto-static potential of anti-cancer compounds in toxicology testing. In such toxicologicalstudies, in vitro testing techniques are very useful to evaluate the cytotoxic, mutagenic,and carcinogenic effects of chemical compounds on human cells.

1.1 Terminology of cell proliferation and viability

Usually, one of two parameters is used to measure the health of cells: cell viability or cellproliferation. In almost all cases, these parameters are measured by assaying for “vitalfunctions” that are characteristic of healthy cells.

Cell Viability

Cell viability can be defined as the number of healthy cells in a sample. Whether the cellsare actively dividing or are quiescent is not distinguished. Cell viability assays are oftenuseful when non-dividing cells (such as primary cells) are isolated and maintained in cul-ture to determine optimal culture conditions for cell populations.

The most straightforward method for determining viable cell number is a direct countingof the cells in a hemocytometer. Sometimes viable cells are scored based on morphologyalone; however, it is more helpful to stain the cells with a dye such as trypan blue. In thiscase, viability is measured by the ability of cells with uncompromised membrane integ-rity to exclude the dye.

Alternatively, metabolic activity can be assayed as an indication of cell viability. Usuallymetabolic activity is measured in populations of cells by incubating the cells with a tetra-zolium salt (MTT, XTT, WST-1) that is cleaved into a colored formazan product by met-abolic activity.

Cell Proliferation

Cell proliferation is the measurement of the number of cells that are dividing in a culture.One way of measuring this parameter is by performing clonogenic assays. In these assays,a defined number of cells are plated onto the appropriate matrix and the number of colo-nies that are formed after a period of growth are enumerated. Drawbacks to this type oftechnique are that it is tedious and it is not practical for large numbers of samples. Inaddition, if cells divide only a few times and then become quiescent, colonies may be toosmall to be counted and the number of dividing cells may be underestimated. Alterna-tively, growth curves could be established, which is also time-consuming and laborious.

Another way to analyze cell proliferation is the measurement of DNA synthesis as amarker for proliferation. In these assays, labeled DNA precursors (3H-thymidine or bro-modeoxyuridine) are added to cells and their incorporation into DNA is quantified afterincubation. The amount of labeled precursor incorporated into DNA is quantified eitherby measuring the total amount of labeled DNA in a population, or by detecting thelabeled nuclei microscopically. Incorporation of the labeled precursor into DNA isdirectly proportional to the amount of cell division occurring in the culture.

Cell proliferation can also be measured using more indirect parameters. In these tech-niques, molecules that regulate the cell cycle are measured either by their activity (e.g.,CDK kinase assays) or by quantifying their amounts (e.g., Western blots, ELISA, orimmunohistochemistry).

Introduction

Terminology of cell proliferation and viability

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75Cell Proliferation and Viability

1.2 Cell Cycle

In an organism, the rate of cell division is a tightly regulated process that is intimatelyassociated with growth, differentiation and tissue turnover. Generally, cells do notundergo division unless they receive signals that instruct them to enter the active seg-ments of the cell cycle. Resting cells are said to be in the G0 phase (quiescence) of the cellcycle (Figure 51). The signals that induce cells to divide are diverse and trigger a largenumber of signal transduction cascades.

A thorough discussion of the types of signals and the variety of responses they can elicitare beyond the scope of this guide (Table 13). Generally, signals that direct cells to enterthe cell cycle are called growth factors, cytokines, or mitogens.

� Figure 51: Cell cycle: A schematic overview.

� Table 13: Published sources that contain more information about cell proliferation.

Abbreviation Description Reference

RTK Receptor Tyrosine Kinase Marshall, (1995) Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell 80:179–185.

RAS GTP exchange protein White, M. A. et al. (1995) Multiple Ras functions can contribute to mamma-lian cell transformation. Cell 80: 533–541.

RAF MAP kinase kinase kinase Avruch, J. et al. (1994) Raf meets Ras-Completing the framework of a sig-nal transduction pathway. Trends Biochem. Sci. 19: 279–283.

MEK MAP kinase kinase or MAPk/Erk kinase

Marshall, C. J. (1994) MAP kinase kinase kinase, MAP kinase kinase, and MAP kinase. Curr. Opin. Genet. Dev. 4: 82–89.

MAPK Mitogen activated protein kinase or Erk

Marshall, C. J. (1994) MAP kinase kinase kinase, MAP kinase kinase, and MAP kinase. Curr. Opin. Genet. Dev. 4: 82–89.

PKC Protein Kinase C Blobe, G. et al. (1996) Protein Kinase C isoenzymes: regulation and func-tion. Cancer Surveys 27: 213–248.

JAK Just Another Kinase or Janus Kinase

Ihle, J. N. et al. (1994) Signaling by the cytokine receptor superfamily: Jaks and STATs. TIBS 19: 222–227.

STAT Signal Transducers and Activators of Transcription

Ihle, J. N. et al. (1994) Signaling by the cytokine receptor superfamily: Jaks and STATs. TIBS 19: 222–227.

Cyclins Marx, J. (1994) How cells cycle toward cancer. Science 263: 319–321.

CDK Cyclin Dependent Kinase MacLachlan, T. K., Sang, N., and Giordano, A. (1995) Cyclins, cyclin-depen-dent kinases and cdk inhibitors: implications in cell cycle control and can-cer. Crit. Rev. Eukaryot. Gene Expr. 5: 127–156.

CDC2 Cell division cycle mutant MacLachlan, T. K., Sang, N., and Giordano, A. (1995) Cyclins, cyclin-depen-dent kinases and cdk inhibitors: implications in cell cycle control and can-cer. Crit. Rev. Eukaryot. Gene Expr. 5: 127–156.

CAK CDK Activating Kinase Morgan, D. O. (1995) Principles of CDK Regulation. Nature 374: 131–134.

Introduction

Cell Cycle

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Signal Transduction Pathways

Three major types of signal transduction pathways are activated in cells in response togrowth factors or mitogenic stimuli. The response to these stimuli varies from cell type tocell type and the pathways continue to grow more and more complex. These types ofpathways continue to be the focus of a great deal of research and, considering the impor-tance of cell cycle regulation in biology, the pathways will continue to grow in complexityfor some time to come.

� The MAP kinase (MAPK) type of pathways are triggered through a cascade of phos-phorylation events that begins with a growth factor binding to a tyrosine kinase recep-tor at the cell surface. This causes dimerization of the receptor and an intermolecularcross-phosphorylation of the two receptor molecules. The phosphorylated receptorsthen interact with adaptor molecules that trigger downstream events in the cascade.The cascade works through the GTP exchange protein RAS, the protein kinase RAF(MAPKKK), the protein kinase MEK (MAPKK), and MAP kinase (Erk). MAPK thenphosphorylates a variety of substrates that control transcription, the cell cycle, or rear-rangements of the cytoskeleton.

� The protein kinase C (PKC) pathways consist of a family of phospholipid dependentprotein kinases. PKC is regulated by a large variety of metabolic pathways involvingphospholipids and calcium levels within a cell. The main regulator of the pathway isdiacylglycerol (DAG) which appears to recruit PKC to the plasma membrane andcause its activation. The activity of DAG is mimicked by the phorbol-ester tumor pro-moters. Once activated, PKC can phosphorylate a wide variety of cellular substratesthat regulate cell proliferation and differentiation. Responses to PKC appear to varywith the types of PKCs expressed and the types of substrates available within a cell.Some evidence shows that the PKC pathway may interact with and exert effectsthrough the MAPK pathway.

� The JAK/STAT pathway is activated by cytokine interaction with a family of receptorscalled the cytokine receptor superfamily. These receptors do not contain a proteinkinase domain themselves, but they associate with and activate a family of proteinkinases called the JAK (Just Another Kinase or JAnus Kinase) family. JAK family mem-bers are recruited to receptor complexes that are formed as a result of ligand binding.The high concentration of JAK in the complex leads to a cross-phosphorylation ofJAK and thus activation. JAK then phosphorylates members of another protein familycalled STAT (signal transducers and activators of transcription). These proteins thentranslocate to the nucleus and directly modulate transcription.

Control of the Cell Cycle

Once the cell is instructed to divide, it enters the active phase of the cell cycle, which canbe broken down into four segments:

� During G1 (G = gap), the cell prepares to synthesize DNA. In the latter stages of G1,the cell passes through a restriction point (R) and is then committed to complete thecycle.

� During S phase the cell undergoes DNA synthesis and replicates its genome.

� During G2 the cell prepares to undergo division and checks its replication using DNArepair enzymes.

� During M phase, the cell undergoes division by mitosis or meiosis and then re-entersG1 or G0.

Introduction

Cell Cycle

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In most instances, the decision for a cell to undergo division is regulated by the passage ofa cell from G1 to S phase. Progression through the cell cycle is controlled by a group ofkinases called cyclin-dependent kinases (CDKs), (see Figure 51). CDKs are thought tophosphorylate cellular substrates, such as the retinoblastoma gene, that are responsiblefor progression into each of the phases of the cell cycle. CDKs are activated by associatingwith proteins whose levels of expression change during different phases of the cell cycle.These proteins are called cyclins. Once associated with cyclins, CDKs are activated byphosphorylation via CDK-activating kinase (CAKs) or by dephosphorylation via a phos-phatase called CDC25.

D-types cyclins are the primary cyclins that respond to external cellular factors. Their lev-els start off low during G1 and increase towards the G1/S boundary. Cyclin D regulatesCDK4 and CDK6. Cyclin E is expressed transiently during the G1/S transition and is rap-idly degraded once the cell enters S. Cyclin E regulates CDK2 and perhaps CDK3. When Sphase begins, levels of cyclin A increase and activate CDK2. The cyclin A/CDK2 complexis thought to have a direct role in DNA replication. The progression through mitosisis regulated by the presence of cyclin B. Cyclin B associates with CDC2 and forms the pri-mary kinase present during mitosis (MPF = “M-phase/maturation promoting factor”).During anaphase cyclin B is degraded. This degradation of cyclin B appears to regulate thecell’s progression out of mitosis and into G1.

Introduction

Cell Cycle

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2. Cell proliferation/viability assay methods

A variety of methods have been devised that measure the viability or proliferation of cellsin vitro and in vivo. These can be subdivided into four groups:

� Reproductive assays can be used to determine the number of cells in a culture that arecapable of forming colonies in vitro. In these types of experiments, cells are plated atlow densities and the number of colonies is scored after a growth period. These clono-genic assays are the most reliable methods for assessing viable cell number57, 58, 59.These methods, however, are very time-consuming and become impractical whenmany samples have to be analyzed.

� Permeability assays involve staining damaged (leaky) cells with a dye and counting via-ble cells that exclude the dye. Counts can either be performed manually using ahemocytometer and for example trypan blue (Figure 52). This method is quick, inex-pensive, and requires only a small fraction of total cells from a cell population. There-fore, this method is generally used to determine the cell concentration (cell number/ml) in batch cell cultures. This is helpful in ensuring that cell cultures have reached theoptimal level of growth and cell density before routine sub-culture, freezing, or anyexperiment60.

Or counts can be performed mechanically using for example a flow cytometer and pro-pidium iodide. Alternatively, membrane integrity can be assayed by quantifying therelease of substances from cells when membrane integrity is lost, e. g., Lactate dehydroge-nase (LDH) or 51Cr (described in section A 3.2.1 starting on page 59 of this guide).

� Metabolic activity can be measured by adding tetrazolium salts to cells. These salts areconverted by viable cells to colored formazan dyes that are measured spectrophoto-metrically (described in section B 2.1.1 starting on page 82 of this guide).

� Direct proliferation assays use DNA synthesis as an indicator of cell growth. Theseassays are performed using either radioactive or nonradioactive nucleotide analogs.Their incorporation into DNA is then measured (nonradioactive assays are describedin section B 2.1.2 starting on page 89 of this guide).

� Figure 52: Measurement of proliferation by counting the cells with a hemocytometer. The addition oftrypan blue helps to distinguish viable, unstained cells () from non-viable, blue-stained cells (�).

Cell proliferation/viability assay methods

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The first section describes those assays designed to study cell proliferation in whole pop-ulations of cells, followed by a section covering proliferation assays designed to measureproliferation in individual cells (in situ).

For a discussion of the advantages and limitations of all types of cell proliferation assays,read Sections B 2.1.3 and B 2.2.2 of this guide.

For discussions of particular assays, turn to the pages indicated in the following methodselection guide.


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Start

Are you studyingCell Proliferationor Cytotoxicity*

Are you studyingCell Proliferation

in vivo in vitro

Are you studying

cell populations single cells

Do you want to measurepresence of cell

cycle associatedantigens

DNA synthesis

Are you studyingcellpopulations

single cells


microscopy flow cytometry

Do you use

light microscopy fluorescence microscopy

� BrdU Labeling and Detection Kit II, AP 11 299 964 001 � In Situ Cell Proliferation Kit, FLUOS 11 810 740 001

� BrdU Labeling and Detection Kit I, 11 296 736 001Fluorescein

� Figure 53: Product Selection Guide

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� In Situ Cell Proliferation Kit, FLUOS 11 810 740 001

� BrdU Labeling and Detection Kit I, 11 296 736 001Fluorescein

� BrdU Labeling and Detection Kit II, AP 11 299 964 001




� Cell Proliferation ELISA, BrdU 11 669 915 001(chemiluminescent)

� Cell Proliferation ELISA, BrdU 11 647 229 001(colormetric)

� BrdU Labeling and Detection Kit III, 11 444 611 001POD

chemilumi-nescent assay

colorimetricassay

Do you want to use a

othercellular changes

DNA synthesis

cell cycleassociatedantigens

metabolic activityof living cells

Do you want to measure


DNAsynthesis

presence of cellcycle associatedantigens


flowcytometry

microscopy


fluorescencemicroscopy

lightmicroscopy

Do you use

yes

no

yes

no

Do your cells proliferate in vitro ?

Do your cells proliferate in vitro ?

Are you studying

cytotoxiticsubstances

cell-mediatedcytotoxicity





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2.1 Methods for studying cell proliferation and viability in cell populations

A number of methods have been developed to study cell viability and proliferation in cellpopulations. The most convenient modern assays have been developed in a microplateformat (96-well plates). This miniaturization allows many samples to be analyzed rapidlyand simultaneously. The microplate format also reduces the amount of culture mediumand cells required as well as cost of plasticware. Colorimetric assays allow samples to bemeasured directly in the microplate with an ELISA plate reader.

Microplate assays have been developed based on different parameters associated with cellviability and cell proliferation. The most important parameters used are metabolic activ-ity and DNA synthesis for microplate format.

� Cellular damage will inevitably result in loss of the ability of the cell to maintain andprovide energy for metabolic cell function and growth. Metabolic activity assays arebased on this premise. Usually they measure mitochondrial activity. The cells areincubated with a colorometric substrate (MTT, XTT, WST-1) (described on pages 82-87 of this guide.

� As outlined above, during the S phase the cell undergoes DNA synthesis and replicatesits genome. If labeled DNA precursors, in our case BrdU, are added to the cell culture,cells that are about to divide incorporate BrdU into their DNA. The incorporatedBrdU can then be detected by a quantitative cellular enzyme immunoassay using mon-oclonal antibodies against BrdU (described on pages 88-107 of this guide).

In the following sections we will describe details of each of these cell viability and prolif-eration assays.

2.1.1 Assays that measure metabolic activity

One parameter used as the basis for colorimetric assays is the metabolic activity of viablecells. For example, a microtiter plate assay which uses the tetrazolium salt MTT is nowwidely used to quantitate cell proliferation and cytotoxicity53, 65.

Because tetrazolium salts are reduced to a colored formazan only by metabolically activecells, these assays detect viable cells exclusively. For instance, in the MTT assay, MTT isreduced by viable cells to a colored, water-insoluble formazan salt. After it is solubilized,the formazan formed can easily and rapidly be quantitated in a conventional ELISA platereader at 570 nm (maximum absorbance).

[Author’s note: MTT is cleaved to formazan by the “succinate-tetrazolium reductase” system(EC 1.3.99.1) which belongs to the mitochondrial respiratory chain and is active only in via-ble cells. Interestingly however, recent evidence suggests that mitochondrial electron transportmay play a minor role in the cellular reduction of MTT. Since most cellular reduction occursin the cytoplasm and probably involves the pyridine nucleotide cofactors NADH andNADPH, the MTT assay can no longer be considered strictly a mitochondrial assay.]

More recently, modified tetrazolium salts like XTT62, 67, MTT68, and WST-1 (Figure 54)have become available. The major advantage of these compounds is that viable cells con-vert them to a water-soluble formazan. Thus, a metabolic assay with any of these com-pounds requires one less step (solubilization of product) than an assay with MTT. Inaddition, WST-1 is stable enough to be packaged as a ready-to-use solution.


Methods for studying cell proliferation and viability in cell populations

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� Figure 54: Molecular structure of MTT, XTT, WST-1 and their corresponding reaction products.

Since proliferating cells are metabolically more active than non-proliferating (resting)cells, the assays are suitable not only for the determination of cell viability and factor-mediated cytotoxicity (see Section A 3.2.2.) but also for the determination of cell activa-tion and proliferation. However, one has to keep in mind that under non-ideal cell cul-ture conditions (such as the pH and D-glucose concentration in culture medium), theMTT response may vary greatly in viable cells due to the metabolic state of the cells (e.g.,cellular concentration of pyridine nucleotides)65, 69.

These colorimetric assays are very rapid and convenient. Because this technique needs nowashing or harvesting of the cells, the complete assay from the start of the microcultureto data analysis in an ELISA plate reader is performed in the same microplate. In addi-tion, an ELISA plate reader linked with a computer allows rapid and automated data pro-cessing (Figure 55).



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Roche Applied Science offers three microplate-based assays similar to the ones describedin this section. All three assays are suitable for measurement of cell proliferation inresponse to growth factors, cytokines, mitogens and nutrients.

One of these assays uses MTT, which forms an insoluble formazan product; the other twouse tetrazolium salts (XTT and WST-1) that form soluble formazan products. All threeassays are described on the following pages.

Note: For a more detailed discussion of the principles behind these metabolic assays, see thetopic, “Biochemical and cellular basis of cell proliferation assays that use tetrazolium salts”(Appendix, page 121) in this guide.

� Figure 55: Measurement of metabolic activity using the tetrazolium salts MTT, XTT and WST-1.



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Cell Proliferation Kit I (MTT)Cat. No. 11 465 007 001 2500 tests

Type Colorimetric, microplate format

Useful for Quantitation of cell viability and proliferation as well as cytotoxicity

Samples Adherent or suspension cell cultures

Method Incubation of cells with MTT, followed by solubilization and spectrophoto-metric assay of colored product

Time 5–28 h

Significance of kit: The Cell Proliferation Kit I (MTT) measures the metabolic activity ofviable cells. The assay is nonradioactive and can be performed entirely in a microplate. Itis suitable for measuring cell proliferation, cell viability or cytotoxicity.

Test principle: The assay is based on the reduction of the tetrazolium salt MTT by viablecells. The reaction produces a water-insoluble formazan salt which must be solubilized.The procedure involves:


� Adherent and suspension cells cultured in microplate.

Kit contents

1. MTT labeling reagent2. Solubilization solution


Other applications: For more examples of how the Cell Proliferation Kit I (MTT) can beused in the lab, see Appendix, page 144.

Culturing the cells in a 96-well microplate, then incubating them with MTT solution for approx. 4 h. During this incubation period, viable cells convert MTT to a water-insoluble formazan dye.

� Solubilizing the formazan dye in the microplate.

� Quantitating the dye with an ELISA plate reader. The absorbance directly correlates with the cell number.

� Figure 56: Measurement of human Interleukin 6(hIL-6) activity on the mouse hybridoma cell line7TD1. Cells (2 x 103/well) were incubated in the pres-ence of various amounts of hIL-6. After 4 days incuba-tion, cell proliferation was analyzed by Cell ProliferationKit I (MTT).



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Cell Proliferation Kit II (XTT)Cat. No. 11 465 015 001 2500 tests


Useful for Quantitation of cell viability, proliferation, or cytotoxicity


Method Incubation of cells with XTT, followed by spectrophotometric assay of colored product

Time 4 h

Significance of kit: The Cell Proliferation Kit II (XTT) measures the metabolic activity ofviable cells. The assay is nonradioactive and can be performed entirely in a microplate. Itis suitable for measuring cell proliferation, cell viability, or cytotoxicity.

Test principle: The assay is based on the reduction of the tetrazolium salt XTT by viablecells in the presence of an electron coupling reagent. The reaction produces a soluble for-mazan salt. The procedure involves:



Kit contents

1. XTT Labeling reagent2. Electron-coupling reagent

Note: To prepare XTT labeling mixture, mix XTT labeling reagent with electron-couplingreagent prior to use.

Typical results: see Figure 57 and 58.

Other applications: For more examples of how the Cell Proliferation Kit II (XTT) can beused in the lab, see Appendix, pages 144–146.

Culturing the cells in a 96-well microplate, then incubating them with XTT solution for approx. 4 h. During this incubation period, viable cells convert XTT to a water-soluble formazan dye.

� Quantitating the formazan dye in the microplate with an ELISA plate reader. The absorbance directly correlates with the cell number.

� Figure 57: Measurement of human tumor necrosis fac-tor � (hTNF�) activity on the mouse fibrosarcoma cellline WEHI-164. After preincubation of the cells (1 x 106/ml)with actinomycin C (1 µg/ml) for 3 h, cells (5 x 104/well) wereincubated in the presence of actinomycin C and variousamounts of hTNF� for 24 h. The cellular response was ana-lyzed by Cell Proliferation Kit II (XTT).



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Cell Proliferation Reagent WST-1Cat. No. 11 644 807 001 2500 tests


Useful for Quantitation of cell viability, proliferation, or cytotoxicity


Method Incubation of cells with WST-1, followed by spectrophotometric assay of colored product

Time 0.5–4 h

Significance of reagent: The Cell Proliferation Reagent WST-1 is a ready-to-use substratewhich measures the metabolic activity of viable cells. The WST-1 assay is nonradioactiveand can be performed entirely in a microplate. It is suitable for measuring cell prolifera-tion, cell viability or cytotoxicity.

Test principle: The assay is based on the reduction of WST-1 by viable cells. The reactionproduces a soluble formazan salt. The procedure involves:

For a detailed comparison of the WST-1 assay procedure with the MTT and XTT assays,see Flow Chart 15 and Figure 58.




Culturing the cells in a 96-well microplate, then incubating them with WST-1 for approx. 0.5–4 h. During this incubation period, viable cells convert WST-1 to a water-soluble formazan dye.

� Quantitating the formazan dye in the microplate with an ELISA plate reader. The absorbance directly correlates with the cell number.

� Figure 58: Comparison of the sensitivity of various tet-razolium salts. P815 cells were preincubated at various con-centrations for 20 h before MTT (�), XTT (�) or CellProliferation Reagent WST-1 (�) was added. After 4 h sub-strate reaction, the absorbance was determined at therespective wavelength with an ELISA plate reader.



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Other applications: For more examples of how the Cell Proliferation WST-1 can be usedin the lab, see Appendix, pages 146–147.

Flow Chart 15: Assay procedures for Cell Proliferation Kit I (MTT), Cell Proliferation Kit II (XTT), and Cell Proli-feration Reagent WST-1.

Culture cells in a MTP for a certain period of time (37°C)

Kit I (MTT) Kit II (XTT) WST-1

Prepare XTT labeling mixture

Add MTT labeling reagent Add XTT labeling mixture Add Cell Proliferation Reagent WST-1

Incubate cells (0.5–4 h, 37°C)

Add solubilized solution and incubate (1–24 h, 37°C)


� Figure 59: Combined use of the Cell ProliferationReagent WST-1 and the Cell Proliferation ELISA, BrdU(colorimetric) for the simultaneous measurement ofcell viability and cell proliferation. A549 cells (1 x 104/wellin 100 µl) were incubated in the presence of various amountsof actinomycin D for 20 h. After labeling the cells with BrdUfor 2 h, additionally Cell Proliferation Reagent WST-1 (�) wasadded and cells were reincubated for another 2 h. Thereafter,the formazan formed was quantitated at 450 nm with anELISA plate reader. Subsequently, BrdU incorporation wasdetermined using the Cell Proliferation ELISA, BrdU (colori-metric) (�).Result: Actinomycin D inhibits DNA synthesis (�), but itdoes not inhibit the metabolic activity of the cell (�). Thus,actinomycin D is cytostatic (inhibition of DNA synthesis) butnot cytotoxic (no inhibition of metabolic activity).



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2.1.2 Assays that measure DNA synthesis

During cell proliferation the DNA has to be replicated before the cell is devided into twodaughter cells.

This close association between DNA synthesis and cell doubling (Figure 60) makes themeasurement of DNA synthesis very attractive for assessing cell proliferation. If labeledDNA precursors are added to the cell culture, cells that are about to divide incorporate thelabeled nucleotide into their DNA. Traditionally, those assays involve the use of radiola-beled nucleosides, particularly tritiated thymidine ([3H]-TdR). The amount of [3H]-TdRincorporated into the cellular DNA is quantitated by liquid scintillation counting(LSC)63, 70.

� Figure 60: Cell proliferation, a close association between DNA synthesis and cell doubling.

Experiments have shown that the thymidine analogue 5-bromo-2’-deoxy-uridine (BrdU)is incorporated into cellular DNA like thymidine (Figure 61). The incorporated BrdUcould be detected by a quantitative cellular enzyme immunoassay using monoclonal anti-bodies directed against BrdU64. The use of BrdU for such proliferation assays circum-vents the disadvantages associated with the radioactive compound [3H]-TdR.

The first report of this technique involved the extraction and partial purification of DNAfrom BrdU-labeled proliferating cells, followed by an enzyme immunoassay in a separateassay71. Because this method was relatively laborious, the entire BrdU-based procedurewas adapted to a 96 well microplate72. This adaptation required no harvesting of the cells;the complete assay from the start of the microculture to data analysis by an ELISA platereader was performed in the same microplate (Figure 62).

� Figure 61: Molecular structure of thymidine and BrdU.



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� Figure 62: Measurement of DNA synthesis using modified nucleotides [3H]-TdR and BrdU.

Roche Applied Science offers three kits that use the convenient BrdU-based assay and themicroplate format. The BrdU Labeling and Detection Kit III is a first generation assay.The colorimetric and chemiluminescence Cell Proliferation ELISAs, are second genera-tion assays that offer fewer steps, a faster assay, and greater sensitivity than the first gener-ation assay (Table 14). These three kits are described on the following pages.



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5�-Bromo-2�-deoxy-uridine Labeling and Detection Kit IIICat. No. 11 444 611 001 1000 tests

Type 1st generation ELISA with colorimetric detection

Useful for Quantitation of DNA synthesis during cell activation and proliferation

Sample Adherent or suspension cell cultures

Method Incubation of cells with BrdU, followed by partial digestion of DNA and immunodetection of incorporated BrdU label

Time 2.5–6 h (+ cell labeling)

Significance of kit: The BrdU Labeling and Detection Kit III measures cell proliferationby quantitating BrdU incorporated into the newly synthesized DNA of replicating cells. Itoffers a nonradioactive alternative to the [3H]-thymidine-based cell proliferation assay.

Test principle: The assay is a cellular immunoassay which uses a mouse monoclonal anti-body directed against BrdU (Figure 63 and Flow Chart 16).

Note: This kit belongs to the first generation of kits used to measure DNA synthesis. The sameassay procedure has been optimized and improved in the second generation Cell ProliferationELISA, BrdU (colorimetric) kit (see Table 14).

� Figure 63: How the BrdU Labeling and Detection Kit III works.



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� Flow Chart 16: Assay procedure, BrdU Labeling and Detection Kit III.

Sensitivity: The BrdU Labeling and Detection Kit III is almost as sensitive as the [3H]-thymidine-based cell proliferation assay. The ability to detect a minimum number of pro-liferating cells in a certain sample strongly depends on the amount of BrdU incorporatedinto the cells and thus on the labeling period. In most cases, detection requires a labelingperiod of 2 to 4 h.

Specificity: The antibody conjugate (Anti-BrdU-POD, Fab fragments) will bind to BrdU-labeled DNA after the DNA is denatured. The antibody is conjugated to peroxidase andspecifically recognizes 5-bromo-2’-deoxyuridine; it shows no cross-reactivity with anyendogenous cellular components such as thymidine or uridine.

Label cells with BrdU (2–18 h, 37°C)

Suspension cells Adherent cells

Remove labeling medium using a cannula Remove labeling medium by inverting the micro-plate

Air dry cells (180 min, 60°C)

Add fixative and incubate (30 min, –20°C)

Wash microplate 3 times (15 min, RT)

Add Nuclease and incubate (30 min, 37°C)


Add Anti BrdU-POD and incubate (30 min, 37°C)






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� Adherent cells as well as cells cultured in suspension in 96-well microplates (e.g., celllines, activated peripheral blood lymphocytes and other in vitro proliferating cells).

Kit contents

1. BrdU labeling reagent (1000 x), sterile 5. Nucleases2. Anti-BrdU-POD Fab fragments 6. Substrate buffer3. Incubation buffer (ready-to-use) 7. ABTS substrate tablets4. Washing buffer (10 x) 8. Substrate enhancer

Typical results:

Result: Figures 64 and 65 illustrate the equivalent sensitivity of the BrdU and [3H]-thymidine methods in measur-ing proliferation, as shown in hEGF and mIL-6 stimulation assays.

Other applications: For examples of how the BrdU Labeling and Detection Kit III can beused in the lab, see Appendix, pages 149–150.

� Table 14: Improvements of the assay procedure using the Cell Proliferation ELISA, BrdU (colori-metric) and Cell Proliferation ELISA, BrdU (chemiluminescence) described on the following pages.

� Figure 64: Proliferation of AKR-2B cells (mouse fibroblast cell line) in response to recombinant human epidermal growth factor (hEGF) using the BrdU Labeling and Detection Kit III (�), or the [3H]-thymi-dine method (�), respectively.

� Figure 65: Proliferation of 7TD1 cells (mouse-mouse hybridoma) in response to recombinant mouse interleukin-6 (mlL-6) using the BrdU Labeling and Detection Kit III (�), or the [3H]-thymidine method (�), respectively.

Parameter BrdU Labeling and Detection Kit III

Cell Proliferation ELISA BrdU (colorimetric) Cell Proliferation ELISA, BrdU (chemilumines-cence)

Incubation steps

3 2

Washing steps 3–4 1

Working solutions

6 (4 included in the kit) 4 (all included in the kit)

Assay time 2.5–6 h 1.5–2.5 h

Incubation temperatures

–20°C: FixationRT: Substrate reaction 37°C: Nuclease treatment 60°C: Air drying

For Cell Proliferation ELISA, BrdU (colorimetric) each step at RT

Measuring range

Absorbance: 0.1–2.5 U (factor 25)

Same as BrdU Kit III For Cell Proliferation ELISA, BrdU (chemiluminescence): rlu/s: 103–106 (factor 1000)

Sensitivity Almost as sensitive as[3H]-TdR

As sensitive as [3H]-TdR



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Cell Proliferation ELISA, BrdU (colorimetric)Cat. No. 11 647 229 001 1000 tests

Cell Proliferation ELISA, BrdU (chemiluminescence)Cat. No. 11 669 915 001 1000 tests

Type 2nd generation ELISAs with colorimetric or chemiluminescent detection

Useful for Quantitation of DNA synthesis during cell activation and proliferation


Method Incubation of cells with BrdU, followed by immunodetection of incorpo-rated BrdU label

Time 1.5–2.5 h (+ cell labeling)

Note: These two kits belong to the second, improved generation of kits for measuring DNAsynthesis (see Tables 14 and 15)

Significance of the kits: The two Cell Proliferation ELISAs measure cell proliferation byquantitating BrdU incorporated into the newly synthesized DNA of replicating cells.They offer a nonradioactive alternative to the [3H]-thymidine-based cell proliferationassay with comparable sensitivity.

Test principle: The assay is a cellular immunoassay which uses a mouse monoclonal anti-body directed against BrdU. The procedure (Figures 66 and 67, Flow Chart 17) involves:.

Culturing the cells in a 96-well microtiterplate and pulse-labeling them with BrdU. Only proliferating cells incorporate BrdU into their DNA.

� Fixing the cells with FixDenat solution. This FixDenat solution also denatures the genomic DNA, exposing the incorporated BrdU to immunodetection.

� Locating the BrdU label in the DNA with a peroxidase-conjugated anti-BrdU antibody (anti-BrdU-POD).

� Quantitating the bound anti-BrdU-POD with a peroxidase substrate. TMB is used as a substrate in the Cell Proliferation, BrdU (colorimetric). Luminol/4-iodophenol is used as a substrate in the Cell Proliferation, BrdU (chemiluminescence).

� Figure 66: Principle of the Cell Proliferation ELISA,BrdU (colorimetric).

� Figure 67: Principle of the Cell Proliferation ELISA, BrdU(chemiluminescent).



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� Flow Chart 17: Assay procedures for Cell Proliferation ELISA, BrdU (colorimetric) and Cell ProliferationELISA, BrdU (chemiluminescence).

Sensitivity: The Cell Proliferation ELISA BrdU (colorimetric) and Cell ProliferationELISA, BrdU (chemiluminescence) are as sensitive as the [3H]-thymidine-based cell pro-liferation assay.

Note: The ability to detect a minimum number of proliferating cells in a certain sampledepends on the amount of BrdU incorporated into the cells and thus on the labeling period.In most cases, detection requires a labeling period of 2 to 24 h.

The use of a chemiluminescence substrate allows the measurement of cell proliferationover a broad range. This range is directly comparable to the measuring range of the [3H]-thymidine-based cell proliferation assay.

Specificity: The anti-BrdU antibody peroxidase-conjugate (anti-BrdU-POD, Fab frag-ments) will bind to BrdU-labeled DNA after the DNA is denatured. The antibody specifi-cally recognizes 5-bromo-2’-deoxyuridine; it shows no cross-reactivity with anyendogenous cellular components such as thymidine or uridine.


� Adherent cells as well as cells in suspension cultured in 96-well microplates (e.g., celllines, activated peripheral blood lymphocytes and other in vitro proliferating cells).

Kit contentsCell Proliferation ELISA, BrdU (colorimetric):

1. BrdU labeling reagent (1000 x), sterile2. Anti-BrdU-POD Fab fragments3. Antibody dilution solution (ready-to-use)4. Washing buffer (10 x)5. FixDenat (ready-to-use)6. TMB substrate solution (ready-to-use)

Label cells with BrdU (2–18 h, 37°C)

Suspension cells Adherent cells

Remove labeling medium using a needle Remove labeling medium by inverting the microplate

Air dry cells (10 min, hairdryer)

Add FixDenat solution and incubate (30 min, RT)

Tap microplate to remove FixDenat then add Anti-BrdU-POD and incubate (30–90 min, RT)



Cell Proliferation ELISA, BrdU (colorimetric)

Cell Proliferation ELISA, BrdU (chemiluminescence)


Measure light emission using a luminometer (2 min, RT)



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Cell Proliferation ELISA, BrdU (chemiluminescence):

1. BrdU labeling reagent (1000 x), sterile2. Anti-BrdU-POD Fab fragments3. Antibody dilution solution (ready-to-use)4. Washing buffer (10 x)5. FixDenat (ready-to-use)6. Substrate component A (luminol/4-iodophenol)7. Substrate component B (peroxide)

Note: The FixDenat solution (included in the kits) is also available as a separate reagent(Cat. No. 11 758 764 001, 4 x 100 ml [enough for 2000 tests]). This ready-to-use solution sim-plifies detection of BrdU-labeled DNA in ELISA applications, since it simultaneously fixescells and denatures DNA to expose BrdU epitopes.

Typical results: see Figures 68 – 71.

� Figure 68: Comparison of the sensitivity of theCell Proliferation ELISA, BrdU (colorimetric) andthe radioactive thymidine incorporation assay formeasuring proliferation in various concentrationsof cells. Various concentrations of L929 cells werecultured in the wells of a microtiter plate. Duplicatecultures of each cell concentration were labeled for 4h with either bromodeoxyuridine (BrdU) or tritiatedthymidine ([3H]-TdR). The cells were assayed for cellproliferation with either the Cell Proliferation ELISA,BrdU (BrdU labeling, �) or a standard filtration/liquidscintillation counting protocol ([3H]-TdR labeling, ).Result: The Cell Proliferation ELISA, BrdU (colori-metric) measures proliferation with a sensitivity com-parable to the radioactive thymidine assay at all cellconcentrations.

� Figure 69: Comparison of the Cell Prolifera-tion ELISA, BrdU (colorimetric) and the radioac-tive thymidine incorporation assay for measuringstimulation of various concentrations of mitogen.Human peripheral blood lymphocytes were cultured inthe presence of varying concentrations of phytohe-magglutinin (PHA) in the wells of a microtiter plate.Duplicate cultures from each PHA concentration werelabeled for 4 h with either bromodeoxyuridine (BrdU)or tritiated thymidine ([3H]-TdR). The cells wereassayed for cell proliferation with either the Cell Prolif-eration ELISA, BrdU (BrdU labeling, �) or a standardfiltration/liquid scintillation counting protocol ([3H]-TdR labeling, ).Result: The Cell Proliferation ELISA, BrdU (colori-metric) is able to detect mitogen-stimulation with asensitivity comparable to the radioactive thymidineassay.

� Figure 70: Reduced PBL proliferation of an immuno-suppressed patient in response to various mitogens.Cells (1 x 105/well) from a healthy volunteer (�) or an immu-nosuppressed individual ( �) were incubated in the presenceof various mitogens for 56 h. Cells were labeled with BrdU for16 h, then cell proliferation was analyzed by Cell ProliferationELISA, BrdU (colorimetric). The error bars indicate the maxi-mum and minimum values of triplicate microcultures (datafrom T. Brüning, [1994] Klin. Lab. 40, 917–927, Figure 3). Mito-gens used were: PHA (phytohemagglutinin), OKT3 (anti-CD3monoclonal antibody), Con A (concanavalin A), PWM(pokeweed mitogen), and SAC (Staphylococcus aureasCowan I). Result: The BrdU ELISA clearly detected the difference inresponse between the healthy and immunosuppressed sub-jects.



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� Figure 71: Measurement of the proliferation of antigen-activated PBL. Cells (1 x 105/well) were incu-bated in the presence of various viral antigens on culture medium alone for 5 days. After labeling with BrdU ( �)or [3H]-TdR (�) for 16 h, cell proliferation was analyzed by Cell Proliferation ELISA BrdU (chemiluminescence)(�) or LSC (�). Antigens used were: INV-KA (influenza control antigen), INV-A (influenza virus A), INV-B, (nflu-enza virus B), RUV (Rubella virus), and HSV (herpes simplex virus type I).Result: The Cell Proliferation ELISA, BrdU (chemiluminescence) detected antigen stimulation with a sensitivitycomparable to the radioactive thymidine assay.

Other applications: For more example of how the Cell Proliferation ELISAs can be usedin the laboratory, see Appendix, pages 150–151.



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Apoptosis, Cell Death, and Cell Proliferation Manual Cell Proliferation and Viability98 99





2.1.3 Summary of methods for studying cell proliferation and cell viability in cell populations

DNA Synthesis

� Table 15: Summary of methods to study DNA synthesis in cell populations.

Metabolic activity

� Table 16: Summary of methods to study metabolic activity in cell populations.

2.1.4 Single reagents for the measurement of DNA synthesis


Label Assay Principle Advantages Limitations For product informa-tion, see

[3H]-TdR Proliferation Assay [3H]-TdR � [3H]-TdR is added to cells cultured in MTP and the cells are incubated (usually for 2–24 h). During this labeling period, [3H]-TdR is incorporated into the DNA of proliferating cells.

� Cells are harvested by vacuum aspiration onto glass fiber filters. While free [3H]-TdR is washed through the filters, the [3H]-TdR incorporated in the DNA is retained.

� The radioactivity retained on the filters is measured by liquid scintillation counting (LSC).

� Sensitive (103–104 cells/test required)� Linear measurement of cell proliferation over a broad,

logarithmic range� Low background

� Radioactive isotope handling and storage problems� Long half life � Radioactive waste disposal costs

BrdU incorporation assay

BrdU Labeling and Detection Kit III

BrdU � BrdU is added to cells cultured in MTP and the cells are incubated (usu-ally for 2–24 h). During this labeling period BrdU is incorporated into the DNA of proliferating cells.

� After the culture supernatant is removed, the cells are fixed and then incubated with an anti-BrdU antibody conjugated with peroxidase (anti-BrdU-POD).

� This antibody binds to BrdU which has been incorporated into the DNA.� Bound anti-BrdU-POD is detected by a substrate reaction and quantified

by an ELISA plate reader.

� No transfer of the cells; the entire assay is performed in a single MTP

� Non-radioactive

� Assay is not linear over a broad logarithmic range of cell proliferation (limitation of the ELISA plate reader)

� 3 washing and incubation steps� Longer assay time

page 91-93of this guide


Cell Proliferation ELISA, BrdU (colorimetric)

BrdU See above (BrdU incorporation assay) � No transfer of the cells; the entire assay is performed in a single MTP

� 1 washing and 2 incubation steps only� Short assay time � Robust system: low standard deviation� Sensitive (103–104 cells/test required)

� Assay is not linear over a broad logarithmic range of cell proliferation (limitation of the ELISA plate reader)



Cell Proliferation ELISA, BrdU (chemiluminescence)

BrdU See above (BrdU incorporation assay) � [see also Cell Proliferation ELISA, BrdU (colorimetric)]� Linear measurement of cell proliferation over a broad,

logarithmic range

� For chemiluminescence measurement special MTP (Black with clear, flat bottom) required

page 94-97 of this guide


Label Assay Principle Advantages Limitations For product informa-tion, see

MTT Assay61

Cell Proliferation Kit I (MTT)

Non-isotopic � MTT solution is added to cells cultured in MTP and the cells are incubated (usually for 4 h). During this period, MTT is converted into a colored, water-insoluble formazan salt by the metabolic activity of viable cells.

� The insoluble formazan is solubilized.� The amount of formazan is quantified by an ELISA plate reader at

550–600 nm.


� MTT is metabolized by all cells; the assay can be used with all cell types

� Inexpensive

� Assay is not linear over a broad logarithmic range of cell proliferation due to the ELISA plate reader

� Insoluble reaction product; resolubilization of the reac-tion product required

� Connot take multiple time points in a single assay� Cells with low metabolic activity (e.g., lymphocytes) must

be used in high numbers


XTT Assay62

Cell Proliferation Kit II (XTT)

Non-isotopic � XTT solution is added to cells cultured in MTP and the cells are incubated (usually for 2–4 h). During this period, XTT is converted into a colored, soluble formazan salt by the metabolic activity of viable cells.

� The amount of formazan is quantified by an ELISA plate reader at 450–500 nm.


� Soluble reaction product� Can take multiple time points in a single assay


� XTT working solution has to be prepared shortly before use

� XTT is not metabolized by all cell types


WST-1 Assay

Cell Proliferation Reagent (WST-1)

Non-isotopic � WST-1 solution is added to cells cultured in MTP and the cells are incu-bated (usually for 0.5–2 h). During this period, WST-1 is converted into a colored, soluble formazan salt by the metabolic activity of viable cells.

� The amount of formazan is quantified by an ELISA plate reader at 420–480 nm.


� Soluble reaction product� Repeated measurement of the assay� Ready-to-use solution


� WST-1 is not metabolized by all cell types


Product Cat. No. Pack Size Product Cat. No. Pack Size

FixDenat 11 758 764 001 4 x 100 ml (2000 tests) Anti-Bromodeoxyuridine formalin grade 11 170 376 001 50 µg (250 tests*)

Anti-Bromodeoxyuridine-Peroxidase, Fab fragments, formalin grade

11 585 860 001 15 U** Anti-Bromodeoxyuridine-Fluorescein formalin grade

11 202 693 001 50 µg (100 tests**)

� Table 17: Single reagents available �for detection of DNA fragmentation.

* Flow cytometry** 0.2 U/ml (ELISA), 1.5 U/ml

(Immunochemistry)

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2.2 Methods for studying cell proliferation and viability in individual cells

As mentioned in Section B 1, the viability as well as proliferation of individual cells can beassessed by standard microscopic methods. For instance, cells may be treated with a vitalstain or exclusion dye and counted directly in a hemocytometer. The same cell parame-ters may be determined by flow cytometry if the cells are differentially stained with fluo-rescent dyes that bind DNA (DNA fluorochromes), see also section A 2.2.3 on page 49 ofthis guide.

In the following section we will describe details of the following proliferation assays:

� Assays that measure DNA synthesis: As outlined above, if labeled DNA precursors areadded to the cell culture, cells that are about to divide incorporate this precursor intotheir DNA (described on the following pages of this guide)

2.2.1 Assays that measure DNA synthesis

Studies of cell proliferation in vivo as well as on individual cells in vitro frequently employ[3H]-TdR to label the DNA of replicating cells and autoradiography to reveal the radioac-tive label. As a nonradioactive alternative, bromodeoxyuridine (BrdU) can be used tolabel proliferating cells in vivo and in vitro. Incorporated BrdU can be detected byimmunohistochemistry, immunocytochemistry or flow cytometry.

Immunochemical techniques allow both the visualization of dividing cells and the detec-tion of tissue morphology by counterstaining (e.g., with hematoxylin and/or eosin).Thus, it is possible to visualize cells which have incorporated BrdU into DNA in its natu-ral environment and to localize cell position in the tissue73, 74.

As only those cells which are actually in the S-phase (DNA-synthesis) of the cell cycle willbe labeled, the so-called “labeling index” can be determined if the labeled nucleotide([3H]-TdR or BrdU) is present for only short periods of time (e.g., 15–60 minutes). The“labeling index” (proportion of S-phase cells in an asynchronously growing population)is calculated by dividing the number of labeled cells by the total number of cells in theentire population.

While short labeling periods (pulse labeling) are suitable to quantify the percentage of S-phase cells within a cellular population, longer labeling periods (e.g., for a whole cell cycletransition) can be used to determine a replicating population.

Roche Applied Science offers several kits and reagents for measuring proliferating cells byBrdU incorporation. These products are described on the following pages.


Methods for studying cell proliferation and viability in individual cells

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5�-Bromo-2�-deoxy-uridine Labeling and Detection Kit ICat. No. 11 296 736 001 100 tests

5�-Bromo-2�-deoxy-uridine Labeling and Detection Kit IICat. No. 11 299 964 001 100 tests

Type 1st generation immunostaining assays for fluorescence (Kit I) or light (Kit II) microscopy

Useful for Detection of BrdU-labeled DNA in proliferating individual cells

Samples Cultured or freshly isolated cells, tissue explants or sections

Method Incubation of cells with BrdU, or injection into an animal, followed by nuclease digestion of DNA of cells or tissue sections and indirect immunodetection (with anti-BrdU and a secondary antibody) of incorpo-rated BrdU label

Time approx. 2–3 h (+ BrdU labeling)

Significance of kits: The BrdU Labeling and Detection Kits I and II offer an indirectimmunostaining method for visualizing proliferating cells under a fluorescence micro-scope (Kit I) or under a light microscope (Kit II). The kits detect BrdU-labeled DNA withan anti-BrdU antibody, then make the antibody-labeled DNA visible with either a fluo-rescein-labeled (Kit I) (Figure 72) or an alkaline phosphatase-labeled anti-mouse sec-ondary antibody (Kit II) (Figure 73).

Note: These kits belong to the first generation of kits used to measure DNA synthesis. Thesame assay procedure has been optimized and improved in the second generation of kits,namely the In Situ Cell Proliferation Kit, FLUOS (for flow cytometry and fluorescencemicroscopy) and the In Situ Cell Proliferation Kit, AP (for light microscopy). For a detaileddescription of these second generation kits, see the following pages.

Other applications: For examples of how the BrdU Labeling and Detection Kits I and IIcan be used in the laboratory, see Appendix, pages 147–149.

� Figure 72: Principle of the BrdU Labeling andDetection Kit I (Fluorescein).

� Figure 73: Principle of the BrdU Labeling andDetection Kit II (AP).



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In Situ Cell Proliferation Kit, FLUOSCat. No. 11 810 740 001 100 tests

Type Direct immunofluorescence staining for flow cytometry or fluorescence microscopy



Method Incubation of cells with BrdU, or injection of BrdU into an animal followed by denaturation of DNA of cells or tissue sections and direct immunodetec-tion of incorporated BrdU label

Time approx. 2 h (+ 0.5–4 h BrdU labeling)

Significance of kit: Bromodeoxyuridine (BrdU) is only incorporated into the DNA ofproliferating cells. Short periods (15–60 min) of incubation in vitro with BrdU will tagonly cells actually going through the S phase of the cell cycle. Alternatively, BrdU can beinjected into an animal to label growing cells in vivo. The In Situ Cell Proliferation Kit,FLUOS can detect proliferating cells in culture or in tissues which have been tagged by invitro or in vivo BrdU labeling. Analysis can be done by flow cytometry or by fluorescencemicroscopy.

Test principle: The BrdU solution and fluorescein-conjugated anti-BrdU antibody sup-plied in the kit allow BrdU labeling and detection of proliferating cells. The procedure(Figure 74 and Flow Chart 18) involves:

� Figure 74: Principle of the In Situ Cell Proliferation Kit, FLUOS.

A: Incubating growing animal tissue or cells in vitro with BrdU— or —B: Injecting BrdU into whole animals for in vivo labeling, then sacrificing the animal and preparing tissue sections.

Note: Only proliferating cells incorporate BrdU into their DNA.

� Fixing BrdU-labeled tissue or cells.

� Denaturing cellular DNA with acid.

� Detecting incorporated BrdU with fluorescein-labeled anti-BrdU monoclonal antibody.

� Analyzing the antibody-labeled samples with a flow cytometer or a fluorescence microscope.



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Specificity: The antibody conjugate (anti-BrdU-fluorescein, F(ab’)2 fragments) will bindto BrdU-labeled DNA after the DNA is denatured and partially degraded with acid. Theantibody specifically recognizes 5-bromo-2’-deoxyuridine; it shows no cross-reactivitywith any endogenous cellular components such as thymidine or uridine.


� Cell lines (in adherent or suspension cell culture)

� Freshly isolated cells

� Tissue explants labeled with BrdU in vitro

� Frozen or paraffin-embedded tissue sections from animals labeled with BrdU in vivo.

Kit contents

1. BrdU labeling reagent (1000 x), sterile2. Anti-BrdU-fluorescein, monoclonal, F(ab’)2 fragments3. Antibody incubation buffer

Typical results: see Figures 75–77.

Other applications: For examples of how the In Situ Cell Proliferation Kit, FLUOS can beused in the lab, see Appendix, pages 151–152.

� Figure 75: Flow cytometric measurement oftotal DNA and incorporated BrdU with the In SituCell Proliferation Kit, FLUOS. Exponentially growingU937 cells were incubated with BrdU for 30 min.Incorporated BrdU was measured flow cytometricallywith the fluorescein-conjugated anti-BrdU antibody(<BrdU>fluos) from the In Situ Cell Proliferation Kit,FLUOS. Total DNA was counterstained with 1 µg/mlpropidium iodide (PI). The phase of the cell cyclerepresented by each population of cells is indicatedon the flow cytometric histogram. FL1-H, fluoresceinintensity (relative BrdU content); FL3-H, propidiumiodide intensity (relative DNA content).Result: BrdU labeling is confined exclusively to the S-phase (DNA synthesis) of the cell cycle.



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� Figure 76: In vivo labeling and analysis of dorsal, hyperproliferative epidermis tissue from mousewith the In Situ Cell Proliferation Kit, FLUOS. Undiluted BrdU labeling solution from the kit was injected intra-peritoneally into a mouse (1 ml BrdU solution/100 g body weight). After 2 h of in vivo BrdU labeling, the mousewas sacrificed and 5 µm thick, paraffin-embedded tissue sections were prepared. Sections were deparaffinizedand rehydrated according to standard methods, then digested with trypsin (15 min). DNA was partially denaturedwith HCl (20 min) and detected with anti-BrdU-fluorescein. Each section was analyzed by differential interferencemicroscopy (upper photo) and epifluorescence microscopy (lower photo). Magnification, 530 x. (Data kindly pro-vided by S. Kaiser and M. Blessing, I. Med. Klinik der Universität Mainz, Germany.)Result: Proliferating cells (green spots) are clearly visible throughout the tissue under epifluorescence microscopy.

� Figure 77: In vitro labeling and analysis of proliferating HeLa cells with the In Situ Cell ProliferationKit, FLUOS. HeLa cells in culture were labeled with BrdU and the BrdU-labeled DNA detected with anti-BrdU-fluorescein, according to the package insert of the In Situ Cell Proliferation Kit, FLUOS. The labeled cell prepara-tion was analyzed under a light microscope (upper photo) and a fluorescence microscope (lower photo).Result: Proliferating cells (bright green nuclei) within the HeLa preparation are clearly visible under the fluores-cence microscope.



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Anti-BrdU, formalin gradeCat. No. 11 170 376 001 50 µg

Anti-BrdU-FluoresceinCat. No. 11 202 693 001 50 µg

Anti-BrdU-Peroxidase, Fab fragmentCat. No. 11 585 860 001 15 units

Type Monoclonal antibodies, from mouse



Method Incubation of samples with BrdU, followed by denaturation of DNA, detec-tion of BrdU label with anti-BrdU antibody, and (if necessary) visualization of anti-BrdU antibody with secondary antibody

Time Variable (depending on sample and antibody used)

Significance of antibodies: Bromodeoxyuridine (BrdU) is only incorporated into the DNAof proliferating cells. Short periods (15–60 min) of incubation in vitro with BrdU will tagonly cells going through the S phase of the cell cycle. Alternatively, BrdU can be injectedinto an animal to label growing cells in vivo. Conjugated or unconjugated anti-BrdU anti-body may be used to detect proliferating cells or tissues which have been tagged by invitro or in vivo BrdU labeling. Depending on the sample and the antibody used, analysiscan be by flow cytometry, fluorescence microscopy, or light microscopy.

Test principle: The anti-BrdU antibodies may be used to detect BrdU-labeled DNA inproliferating cells. The procedure involves (Flow Chart 19):

A: Incubating growing animal tissue or cells in vitro with BrdU— or —B: Injecting BrdU into whole animals for in vivo labeling, then sacrificing the animal and preparing tissue sections.Note: Only proliferating cells (cells in S-phase) incorporate BrdU into their DNA.

� Fixing BrdU-labeled tissue or cells.

� Denaturing cellular DNA.

� Detecting incorporated BrdU with conjugated or unconjugated anti-BrdU monoclonal antibody.

� (Option) A: Localizing unconjugated anti-BrdU antibody with a secondary antibody detection system— or —(Option) B: Localizing enzyme-conjugated anti-BrdU antibody with an enzyme substrate.

� Analyzing the antibody-labeled samples with a flow cytometer, a fluorescence microscope, or a light microscope.



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� Flow Chart 18: Assay procedure, in vivo labeling of proliferating cells with BrdU.

� Flow Chart 19: Immunostaining procedure, Anti-BrdU antibody and conjugates.

Specificity: Conjugated or unconjugated anti-BrdU antibody will bind to BrdU-labeledDNA after the DNA is denatured and partially degraded (e.g., with DNase, acid or micro-waves). The antibody specifically recognizes 5-bromo-2’-deoxyuridine; it shows no cross-reactivity with any endogenous cellular components such as thymidine or uridine.


� Cell lines (in adherent or suspension cell culture)

� Freshly isolated cells, or tissue explants labeled with BrdU in vitro

� Frozen or paraffin-embedded tissue sections from animals labeled with BrdU in vivo.

Inject the animal with BrdU labeling reagent intraperitoneally

Sacrifice the animal (approx. 1–4 h later) and remove tissue samples or organ

Process tissue samples or organ for:

Frozen sectioning Paraffin embedding

Freeze tissue samples/organ immediately after removal

Fix tissue samples/organ in formalin immediately after removal

Store sample frozen until required for sectioning

Use standard dehydration and paraffin embed-ding procedures to process fixed sample

Cut sections of frozen sample in a cryostat Cut sections of embedded sample on a microtome

Transfer sections onto a glass slide and fix Transfer sections onto a glass slide and use stand-ard procedures to dewax and rehydrate sections

Proceed with the immunostaining procedure

Fixed, BrdU-labeled sample

Denature sample DNA e.g., with HCl (10–20 min, RT) or microwaves (15 min, 100 °C)

Incubate sample with Anti-BrdU-Fluorescein

Incubate sample with Anti-BrdU

Incubate sample with Anti-BrdU-POD

Analyze sample by flow cytometry or fluores-cence microscopy.

Incubate sample with anti-mouse-fluores-cein or anti-mouse-

enzyme (+ enzyme substrate)

Add peroxidase substrate and incubate until color forms

Analyze sample by flow cytometry, fluores-

cence or light micros-copy

Analyze sample by light microscopy



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Typical results: The anti-BrdU antibody has been used to determine the cell cycle posi-tion of apoptotic cells76.

Briefly, the experimental procedure was as follows: Cultured mouse thymocytes weretreated with 0.5 µM ionomycin (2 h or 12 h) to induce apoptosis. After treatment, thecells were harvested, fixed in paraformaldehyde and ethanol (two-step fixation), and ana-lyzed for apoptosis and cell cycle position by flow cytometry. As a measure of apoptoticcells, fragmented DNA content was quantitated with the In Situ Cell Death Detection Kit,Fluorescein (TUNEL method, according to the kit package insert). Either of two flowcytometric techniques was used to determine the cell cycle position of the cells: 1) Rela-tive DNA content was determined by treating the cells with 5 µg/ml propidium iodideand 200 µg/ml ribonuclease (30 min, room temperature). 2) Cells going through S-phasewere identified by labeling with BrdU (10 µM BrdU, 30 min), detection of BrdU-labeledcells with anti-BrdU monoclonal antibody (30 min, 37°C), and visualization of thosecells with R-phycoerythrin-conjugated goat anti-mouse antibody (30 min, 37°C). Forresults, see Figure 78.

Other applications: For examples of how the Anti-BrdU conjugates and the antibodiesmay be used in the lab, see Appendix, pages 152 and 153.



Figure 78: Concomitant flow cytomet-ric analysis of apoptosis and cellcycle position with the anti-BrdUantibody, propidium iodide, and the InSitu Cell Death Detection Kit, Fluores-cein. Cultured mouse thymocytes weretreated with ionomycin (2 h or 12 h) toinduce apoptosis. After treatment, thecells were harvested, fixed, and analyzedfor apoptosis and cell cycle position byflow cytometry. Histograms A, C, and Eshow data obtained from cells after 2 htreatment with ionomycin. Histograms B,D, and F show data obtained from cellsafter 12 h treatment with ionomycin. His-tograms A and B show fluorescein inten-sity (green fluorescence) alone, ameasure of DNA fragmentation. Histo-grams C and D show a two-parameteranalysis of fluorescein intensity (greenfluorescence, DNA fragmentation) andpropidium iodide intensity (red fluores-cence, DNA content). Histograms E and Fshow a two-parameter analysis of fluo-rescein intensity (green fluorescence,DNA fragmentation) and phycoerythrinintensity (orange fluorescence, BrdUcontent). The percentage of positive cellsis indicated in each panel. [Data fromHanon, E., Vanderplasschen, A. and Pas-toret, P.-P. (1996) Biochemica No. 2, 25–27.]Result: The ionomycin-treated cells con-tained about 13% apoptotic cells (histo-gram A) after 2 h and about 29%apoptotic cells (histogram B) after 12 hexposure. Concomitant analysis of apop-tosis and total DNA content (histogramsC and D) showed that apoptotic cellscontained about as much DNA as cells inG0/G1 or early S-phase. Concomitantanalysis of apoptosis and BrdU contentafter 12 h ionomycin treatment (histo-gram F) showed that 6% of the apoptoticcells went through S phase (that is, werepositive for BrdU) while 21% of apoptoticcells remained in G0/G1 (that is, werenegative for BrdU).

�

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Apoptosis, Cell Death, and Cell Proliferation Cell Death – Apoptosis and Necrosis108 109





2.2.2 Summary of methods for studying cell proliferation and viability in individual cells

DNA Synthesis

� Table 18: Summary of methods to study DNA synthesis in individual cells.


Assay principle Advantages Limitations For product informa-tion, see

Autoradiography � The samples are incubated with [3H]-TdR for a certain period of time. If [3H]-TdR is present for 1 h or less, only those cells which are in the S-phase (DNA synthesis) of the cell cycle will be labeled.

� The samples are fixed and immersed in emulsion. � The radiolabel is visualized as black grains on the film.

� Quantitative detection of S phase cells: Determination of growing fraction in population

� Long exposure time (days) required � Radioactive isotope, handling and storage problems

Immunocytochemistry (fluorescence microscopy)

In Situ Cell Proliferation Kit, FLUOS

BrdU Labeling and Detection Kit I

� The samples are incubated with BrdU for a certain period of time. If BrdU is present for 1 h or less, only those cells which are in the S-phase (DNA synthesis) of the cell cycle will be labeled.

� The samples are fixed and the DNA is denatured. � Incorporated BrdU is bound by a fluorescein-conjugated monoclonal

antibody against BrdU.� Bound Anti-BrdU-Fluorescein is detected by fluorescence microscopy or

flow cytometry.

� Quantitative detection of S-phase cells: Determination of growing fraction in population

� Results within a few hours� Can counterstain the tissue simultaneously to reveal tissue

morphology

� Stained samples cannot be stored for long periods of time

� Histological tissue organization cannot be observed simultaneously

pages 101- 104 of this guide

Immunocyto/histochemistry (light microscopy)

BrdU Labeling and Detection Kit II

� The samples are incubated with BrdU for a certain period of time. If BrdU is present for 1 h or less, only those cells which are in the S-phase (DNA synthesis) of the cell cycle will be labeled.

� The samples are fixed and the DNA is denatured. � Incorporated BrdU is bound by an alkaline phosphate (AP)-conjugated

monoclonal antibody against BrdU.� Bound anti-BrdU AP is detected by a substrate reaction and visualized by

light microscopy.

� Quantitative detection of S-phase cells: Determination of growing fraction in population

� Results within a few hours� Can counterstain the tissue simultaneously to reveal tissue

morphology


2404 Apoptosis Kap_B Tab_18_AK1.fm Seite 1 M ontag, 23. August 2004 12:28 12

Seite 110 - Vakat

Appendix

CCApoptosis, Cell Death, and Cell Proliferation Manual112

1. Technical tips

1.1 Selected frequently asked questions (FAQs) about cell death assays

The questions below were chosen from those received by our Technical Services represen-tatives. Wherever possible, the answers will direct you to pages and sections of this guidewhich can provide more information.

� Can I determine the number of apoptotic cells using the Cell Death Detection ELISAPLUS?

A: No. The ELISA data is interpreted as a change in the level of death in an apoptoticpopulation compared to an uninduced control population. It does not provide data onindividual cells.

� What is the best way to get rid of non-specific (false-positive) background in theTUNEL (In Situ Cell Death Detection) kits?

A: The best approach to reducing background depends on the results you obtain with thecontrols:

� If cells incubated with fluorescein-dUTP but without terminal transferase are falsepositive, try washing the cells more thoroughly, reducing the concentration of fluo-rescein-dUTP, or using an alternative permeabilization procedure.

� If false positives are produced only in reactions which include both fluorescein-dUTP and terminal transferase, the best means of reducing false positives is areduction in enzyme concentration or a change in permeabilization procedure.

Note: For further tips on obtaining the best results with the TUNEL method, see page 113of this Appendix.

� What types of sample can be assayed with the TUNEL method?

A: Tissue sections, adherent cell cultures, cytospins and cell smears have all been usedwith this assay (page 33, Section A 2.2.1). Note, however, that the sample materialmust be preserved with a cross-linking fixative (such as paraformaldehyde).

� Why isn’t substrate included in the TUNEL kits (In Situ Cell Death Detection Kits, AP or POD)?

A: These kits will work with a variety of common alkaline phosphatase or peroxidasesubstrates. Since many laboratories already have these substrates, and know how thesesubstrates work in “their” system we decided to leave them out. In addition this givesthe researcher the flexibility for secondary staining.

� How long and at what temperature can I store my samples before analyzing them withthe various kits that you offer?

A: Table 19 gives some general guidelines for sample storage. Note however that somesamples may be more or less stable than others.

� Is a special wash/stop buffer required for the TUNEL kits?

A: Our procedure does not require an equilibration buffer. Our wash buffer is PBS, acommonly used solution.

Technical tips

Selected frequently asked questions (FAQs) about cell death assays

CC113Appendix

� Table 19: Storage of samples for apoptosis assay

1.2 Technical tips on the TUNEL method

1.2.1 TUNEL: Improvement and evaluation of the method for in situapoptotic cell identification

[from Adrien Negoescu, Philippe Lorimier, Francoise Labat-Moleur, Laurent Azoti, Catherine Robert, ChristianeGuillermat, Christian Brambilla, and Elisabeth Brambilla; of Groupe de recherche sur le cancer du poumon, Insti-tut Albert Bonniot, Faculte de Medecine, Domaine de la Merci, 38706 Grenoble cedex, France, and Laboratoire dePathologie cellulaire, CHRU, BP 217X, 38043 Grenoble cedex 09, France]

Note: This is a summary of an article that appeared in the Biochemica No. 2 (1997). For fur-ther experimental detail and background, see the full Biochemica article.

Summary: TUNEL or terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling, is a preferred method for rapid identification and quantification of the apop-totic cell fraction in cultured cell preparations. However, the accessibility of DNA breaksto enzymatic reactions is reduced by the nuclear protein environment (Kerrigan et al.,1987) and impaired by cell fixation (Gold et al., 1994) and postfixation (Gorczyca et al.,1994). Thus, several sample pretreatments have been devised to improve TUNEL sensi-tivity (Desjardins and MacManus, 1995; Kerrigan et al., 1987; Lucassen et al., 1995). Anoptimized TUNEL protocol for cultured cells has been developed.

Kit Storage of samples before assay

Cell Death Detection ELISAPLUS Purified cytoplasmic samples can be stored at –20°C for 2 weeks (with some reduction of signal)

Apoptotic DNA Ladder Kit Purified DNA can be stored at –20°C for 1 year

Annexin-V-FLUOS Staining Kit; Annexin V-Biotin

Cells must be used live, directly after induction of apoptosis

Cellular DNA Fragmentation ELISA Purified cytoplasmic samples can be stored at –20°C for at least 2 weeks

Technical tips

Technical tips on the TUNEL method


1.2.2 TUNEL protocol for tissues which tend to give false positives

The protocol given below has been found to eliminate the TUNEL labeling “false posi-tives” seen with certain paraffin-embedded tissue sections (for example, of rabbitendometrium). The key step is pretreatment of the slide with microwave irradiationrather than proteinase K.

Sample: Paraffin-embedded tissue sections (e.g., of rabbit endometrium)

Reagents: In Situ Cell Death Detection Kit, POD, Cat. No. 11 684 817 001DAB Substrate, Cat. No. 11 718 096 001

Dewax paraformaldehyde- or formalin-fixed tissue sections according to standard procedures.

� Place the slide(s) in a plastic jar containing 200 ml 0.1 M citrate buffer, pH 6.0, put the jar in a microwave oven, and apply 750 W (high) microwave irradiation for 1 min. For rapid cooling, immediately add 80 ml redist. water (20°–25°C) to the jar, then transfer the slide(s) into PBS (20°–25°C).

Caution: DO NOT perform a proteinase K treatment!

� Immerse the slide(s) for 30 min at room temperature (RT) in a blocking solution containing 0.1 M Tris-HCl, 3% BSA, and 20% normal bovine serum, pH 7.5.

� Rinse the slide(s) twice with PBS at RT. Let excess fluid drain off.

� Apply 50 µl of TUNEL reaction mixture to the section and incubate for 60 min at 37°C in a humidified atmosphere.

� Rinse slide(s) three times in PBS (5 min for each wash).

Note: At this stage, you can evaluate the section under a fluorescence microscope.

� Block endogenous peroxidase activity by incubating slides for 10 min at RT with 0.3% H2O2 in methanol.

� Repeat steps 3 and 4 to block nonspecific binding of the anti-fluorescein-antibody to the tissue.

� Add 50 µl Converter-POD, pre-diluted 1:2 in blocking solution (from Step 3), and incubate for 30 min at 37°C in a humidified atmosphere.

� Rinse slide(s) three times in PBS at RT for 5 min each.

� Add 50 µl DAB substrate solution and incubate for 1–3 min at RT.

� Wash slide(s) extensively in tap water and counterstain if needed.

Technical tips


CC115Appendix

1.2.3 Tips for avoiding or eliminating potential TUNEL labeling artifacts

To avoid this artifact Which may be caused by Try the following

Nonspecific TUNEL labeling

� DNA strand breaks induced by UV irradia-tion during tissue embedding (UV used to polymerize tissue embedding material such as methacrylate)

� Use a different embedding material, which does not require UV irradiation

� Use an alternate polymerization method

� Acid tissue fixatives (e.g., mathacarn, Carnoy’s fixative) cause DNA strand breaks

� Use buffered 4% paraformaldehyde as fixative

� Endogenous nuclease activity which occurs soon after tissue preparation (e.g., in smooth muscle tissue slices)

� Fix tissue immediately after organ harvest

� Perfuse fixative through liver vein in intact animal

� TdT concentration too high during TUNEL labeling

� Reduce concentration of TdT by diluting it 1:2 or 1:3 with TUNEL Dilution Buffer (Cat.No. 1966 006) containing 30 mM Tris (pH 7.2) containing 140 mM sodium caco-dylate and 1 mM CoCl2

� Endogenous alkaline phosphatase activity during converter reaction

� Block endogenous AP activity by adding 1 mM levamisole to the AP substrate solution

� Endogenous peroxidase activity during converter reaction

� Before permeabilizing cells, block endoge-nous POD activity by immersing the slides in a solution of 0.3% H202 in methanol

� Nonspecific binding of anti-fluorescein anti-body conjugate during converter reaction

� Block nonspecific sites with normal anti-sheep serum

� Block nonspecific sites with PBS containing 3% BSA (20 min)

� Use 1:2 dilution of converter solution in PBS

High background � Formalin fixation, which causes yellow stain-ing of cells containing melanin precursors

� Use methanol fixation

� Note: This fixation may lead to a reduction in TUNEL labeling sensitivity

� TUNEL labeling mix too concentrated (e.g.,for carcinomas)

� Reduce concentration of labeling mix by diluting it 1:2 with TUNEL Dilution Buffer (Cat. No. 1966 006) containing 30 mM Tris (pH 7.2) containing 140 mM sodium caco-dylate and 1 mM CoCl2

� Endogenous alkaline phosphatase activity during converter reaction

� Block endogenous AP activity by adding 1 mM levamisole to the AP substrate solution

� Endogenous peroxidase activity during converter reaction

� Before permeabilizing cells, block endoge-nous POD activity by immersing the slides in a solution of 0.3% H202 in methanol

� Nonspecific binding of anti-fluorescein antibody conjugate during converter reaction

� Block nonspecific sites with normal anti-sheep serum

� Block nonspecific sites with PBS containing 3% BSA (20 min)

� Use 1:2 dilution of converter solution in PBS

Technical tips



Low TUNEL labeling (low sensitivity)

� Ethanol and methanol fixation � Use buffered 4% paraformaldehyde as fixative

� Extensive crosslinking during prolonged fixation reactions

� Reduce fixation time

� Use buffered 2% paraformaldehyde as fixative

� Insufficient permeabilization of cells, so TUNEL reagents cannot reach nuclei

� Pretreat with proteinase K (concentration and time must be optimized empirically) Note: To avoid possible nuclease contamina-tion, use only Proteinase K from Roche Applied Science, Cat. No. 03 115 836 001

� Pretreat with 0.01 M sodium citrate for 30 min at 70°C

� Increase TUNEL incubation time

� Restricted access of TUNEL reagents to nuclei, caused by paraffin-embedding

� After dewaxing tissue sections, treat with proteinase K (concentration, time, and tem-perature must be optimized empirically) Note: To avoid possible nuclease contamina-tion, use only Proteinase K from Roche Applied Science, Cat. No 03 115 836 001

� Immerse dewaxed tissue sections in 200 ml 0.01 M citrate buffer (pH 6.0) and treat with microwave irradiation (370 W, 5 min) Note: Conditions must be experimentally optimized for each tissue

No signal on positive control

� Inadequate DNase treatment (DNase concentration too low)

� For cryosections, apply 1 µg/ml DNase

� For paraffin-embedded tissue sections, apply 0.5 mg/ml DNase

� For many other samples, apply 1 U/ml DNase in a solution of 10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 5 mM MnCl2, 0.1 mM CaCl2, 25 mM KCl; incubate 30 min at 37°C

� As an alternative DNase buffer, use a solution of 10 mM Tris-HCl (pH 7.5), 1 mM MgCl2, 1 mg/ml BSA

Diminished TUNEL staining during DNA counterstaining

� Quenching of fluorescein signal by propidium iodide (PI)

� Use 0.5 µg/ml PI as DNA stain

� Substitute TO-PRO-3 (from Molecular Probes) in place of PI

To avoid this artifact Which may be caused by Try the following

Technical tips


CC117Appendix

1.3 Technical tips on the use of Annexin-V-Biotin for light microscope detection

The following protocol provides a method for the detection of Annexin-V-Biotin-binding to cell culture cells with light microscopy. The percentage of necrotic cells isdetermined by trypan blue staining.

Preparation of solutions

� Annexin-V-Biotin working solution: Dilute 20 µl. Annexin-V-Biotin labeling reagentin 1000 µl incubation buffer (sufficient for 10 samples).

� HEPES buffer: Prepare according to the instructions in the Annexin-V-Biotin packinsert.

All steps can be performed at room temperature

Incubate 1 x 106 cells in 100 µl Annexin-V-Biotin working solution for 10–15 min.

� Wash 2 times with HEPES buffer. For suspension cells: Continue with step 3.For adherent cells: Continue with step 4.

� Resuspend suspension cells in 1 ml HEPES buffer. Transfer 2 x 105 cells to a slide using cytospin device.

� Air dry cells. Fix with methanol/ethanol 1:1 for 90 sec.

� Air dry cells. Add 100 µl Streptavidin-POD (Cat. No. 11 089 153 001) working solution, incubate for 1 h.

� Rinse with HEPES buffer

� Add DAB substrate solution (Cat. No. 11 718 096 001) working solution, incubate for 10–15 min.

� Rinse with HEPES buffer

� Analyze samples under a light microscope.

Technical tips

Technical tips on the use of Annexin-V-Biotin for light microscope detection


1.4 Technical tips on the use of the Apoptotic DNA Ladder Kit on tissue samples

The package insert for our Apoptotic DNA-Ladder Kit, Cat. No. 11 835 246 001, des-cribes the purification of nucleic acids from whole blood and cultured cells. By followingthe modified procedure decribed here it is also possible to use tissue samples.

Preliminary Information

� Weight of sample: The tissue sample should weigh between 25 and 50 mg.

� Additional required solutions:

– Lysis buffer: Prior to extraction of DNA, prepare a lysis buffer. 200 µl of this bufferare sufficient for one tissue sample. The lysis buffer consists of 4 M urea, 100 mMTris, 20 mM NaCl and 200 mM EDTA, pH 7.4 (25°C).

– Proteinase K solution: 20 mg/ml in 50 mM Tris-HCl (pH 8.0) and 1 mM CaCl2.

Protocol for isolation of DNA from tissue samples

Note: Be aware, that apoptosis is a single cell event, and therefore in most tissues you will notfind a sufficient number of apoptotic cells to produce a DNA ladder.

1.5 Technical tips on the Cell Proliferation ELISA Kits

How to interrupt the proliferation assay

The detection of BrdU-labeled DNA with the Cell Proliferation ELISAs does not takemore than 1.5–3 hours. Nevertheless, the labeling period which may vary between 2 and24 hours can get the scientist in time trouble. Our assay can be interrupted after the label-ing process: After the removal of the culture medium, the protocol proceeds with the dry-ing of the labeled cells using e.g., a hair-dryer. The dry cells stay safe and sound up to oneweek when stored at 4°C in the microtiter plate before they are fixed and denaturedaccording to the provided protocol.

A tip for measuring lymphocyte proliferation

To study the proliferation of lymphocytes, the cells are stimulated e.g., with growth fac-tors, cytokines or mitogens. The increase in cell numbers can (in special cases) lead tocluster formation of the lymphocytes: Cells from the same progenitor stick together andform aggregates in the culture plate. This effect may disturb the antibody recognition ofthe ELISA system and thereby result in an underestimation of response. To avoid signalvariation: Carefully resuspend the cells after the BrdU-labeling period and before centrif-ugation for removing the culture medium. This will enable the equal accessibility of eachcell for the antibody recognizing the BrdU-label.

Add 200 µl lysis buffer and 40 µl proteinase K solution to 25–50 mg tissue, mix.

� Incubate for 1 h at 55°C.

� Add 200 µl binding buffer, mix.

� Incubate for 10 min at 72°C.

� Proceed with the addition of 100 µl isopropanol as described in the pack insert (3rd. step of section 5).

Technical tips

Technical tips on the use of the Apoptotic DNA Ladder Kit and the Cell Proliferation ELISA Kits

CC119Appendix

2. Special applications of cell death and cell proliferation methods

This section of the Appendix contains condensed versions of articles that appeared in theRoche Applied Science Biochemica newsletter. For further experimental detail and back-ground, see the full Biochemica articles.

2.1 TUNEL assays

2.1.1 Discrimination between dead and viable apoptotic cells using two-color TdT assay and surface labeling as detected by flow cytometry

[from Earl A. Timm, Jr. and Carleton C. Stewart, Laboratory of Flow Cytometry, Roswell Park Cancer Institute,Buffalo, N.Y., USA]

Note: This article appeared in Biochemica No. 1 (1996), 44–47.

Summary: The TUNEL method uses terminal dideoxynucleotidyl transferase (TdT) toincorporate hapten-tagged nucleotides into the 3’-strand breaks that occur in DNA dur-ing apoptosis (Gorczyca et al., 1993; Chapman et al., 1995). If these nucleotides are cou-pled to a fluorescent molecule, or if the hapten can be detected by a fluorescent secondaryreagent, the apoptotic cells can be analyzed by flow cytometry.

Flow cytometry permits not only the detection of apoptotic populations, but also thesimultaneous detection and immunophenotyping of necrotic populations. The drawbackto using the current TdT method, however, is that the ethanol permeabilization of thecells is incompatible with immunophenotyping because it denatures cellular epitopes(Darzynkiewicz et al., 1992; Li et al., 1995).

A protocol has been developed that both preserves the surface markers and detects apop-totic cells. In addition, it is possible to discriminate between dead apoptotic cells and via-ble apoptotic cells with a second hapten-tagged nucleotide that labels dead cells. Themethod also can distinguish dead apoptotic cells from cells that have died by other mech-anisms (e.g., necrosis).

2.1.2 The use of flow cytometry for concomitant detection of apoptosis and cell cycle analysis

[from E. Hanon, A. Vanderplasschen and P.-P. Pastoret, Department of Immunology/Vaccinology, Faculty of Veterinary Medecine, University of Liège, Liège, Belgium]


Summary: Two distinct modes of cell death, apoptosis and necrosis, can be distinguishedon the basis of differences in morphological, biochemical, and molecular changes occur-ing in the dying cells (Duvall and Wyllie, 1986).

Cells undergoing apoptosis display a characteristic pattern of structural changes in thenucleus and cytoplasm, including rapid blebbing of the plasma membrane and nucleardisintegration (Duvall and Wyllie, 1986). Extensive damage to chromatin and cleavage ofDNA into oligonucleosomal-length fragments both occur during apoptosis (Duvall andWyllie, 1986).

Several flow cytometric methods for identifying cells undergoing DNA fragmentationhave been described recently. These include DNA content analysis and in situ labeling ofDNA fragments with tracer-dUTP. The former is based on the accumulation of ethanol-

Special applications of cell death and cell proliferation methods

TUNEL assays


fixed apoptotic cells in the sub-G0/G1 peak of DNA content histogram as a result of lossof DNA fragments out of the cells and because of a reduced DNA “stainability” (Telfordet al.,1991, 1992). The latter uses exogenous terminal deoxynucleotidyl transferase (TdT)to label in situ the DNA strand breaks with a tracer-dUTP (Gorczyca et al., 1993; Sgonc etal., 1994).

Recent observations have revealed a profound regulatory interrelationship between apo-ptosis and the cell cycle (Gorczyca et al.). The investigation of this relationship ideallyrequires techniques that permit concomitant apoptosis detection and cell cycle analysis ata single-cell level.

Two flow cytometric techniques are usually used to investigate the cell cycle: DNA quan-tification to identify the cell cycle position (Vindelov et al., 1990) and detection of bro-modeoxyuridine (BrdU) incorporation to reveal cells going through the S phase(Gratzner, 1982). In this investigation, the development of flow cytometric techniquesthat permit concomitant detection of apoptosis and cellular DNA content or BrdU con-tent analysis by adapting the apoptosis detection protocol of the Roche Diagnoctics InSitu Cell Death Detection Kit, Fluorescein is reported.

2.1.3 Comparison of two cell death detection methods: In situ nick translation and TUNEL

[from Maria Pihlgren, Joelle Thomas, and Jaqueline Marvel, Immunologie cellulaire, Lyon Cedex, France]


Summary: Apoptosis is a form of regulated cell death characterized by specific morpho-logical changes. These include cell shrinkage, membrane blebbing, chromatin condensa-tion, and cell fragmentation into small apoptotic bodies. At the molecular level, theactivation of an endogenous endonuclease results in the fragmentation of cellular DNAinto oligosomal length fragments (Martin et al. 1994). These can be readily detected byDNA gel electrophoresis. However, gel electrophoresis does not allow the detection ofapoptosis in individual cells.

In contrast, techniques that use enzymatic labeling of DNA strand breaks can provideinformation regarding apoptosis at a single-cell level. The TdT-mediated dUTP Nick EndLabeling (TUNEL) technique uses terminal deoxnucleotidyl transferase (TdT) andallows the labeling of double-stranded DNA breaks (free 3’-OH DNA ends), while the InSitu Nick Translation (ISNT) method employs DNA Polymerase I and detects single-stranded DNA breaks. Another advantage of these techniques is that they can be used incombination with cell surface staining or cell cycle analysis. The abilities of the TUNELand ISNT techniques to detect apoptosis in two types of cells: the IL-3-dependent cell lineBAF-3 and freshly isolated CD8+ lymphocytes from mouse spleen are compared.

2.1.4 Fixation of tissue sections for TUNEL combined with staining for thymic epithelial cell marker

[from Olav Schreurs, Trond S. Halstensen, Zlatko Dembic, Bjarne Bogen, Karl Schenck, Department of OralBiology, University of Oslo, Oslo, Norway]


Summary: In the thymus, positive and negative selection of thymocytes are importantforces that shape the repertoire of mature T lymphocytes in the immune system. In stud-ies on negative selection, it is of great interest to determine whether apoptotic cells residein thymic cortex or medulla (Surh et al. 1994; Kisielow et al. 1995). Terminal dUTP nickend labeling (TUNEL) is a technique, that is well suited to demonstrate apoptosis in situ,and the method may be combined with labeling of other markers. In order to distinguishbetween thymic cortex and medulla, differential expression of cytokeratin, MHC class II


TUNEL assays

CC121Appendix

molecules and epithelial cell markers have been used (Surh et al. 1994; Wack et al. 1996;Douek et al. 1996). In the course of an investigation on deletion of tumor-specific TCR-transgenic T-cells in the thymus (Lauritzsen et al.), TUNEL has been combined withcommercially available monoclonal antibodies, that are monospecific for thymic epithe-lial cells, to unambiguously localize T-cell deletion. During the course of these studies, weestablished fixating conditions, that gave us superior results.

2.2 Metabolic assays, Annexin assays

2.2.1 Biochemical and cellular basis of cell proliferation assays that use tetrazolium salts

[from Michael V. Berridge, An S. Tan, Kathy D. McCoy, and Rui Wang, Malaghan Institute of Medical Research,Wellington School of Medicine, Wellington South, New Zealand]


Summary: Tetrazolium salts (such as MTT, XTT, and WST-1) are used extensively in cellproliferation and cytotoxicity assays, enzyme assays, histochemical procedures, and bac-teriological screening. In each, these tetrazolium salts are metabolically reduced to highlycolored end products called formazans. Yet, the nature of their cellular bioreduction ispoorly understood despite their long-time use (Stoward and Pearse, 1991).

In our laboratory, we demonstrated that most cellular reduction of MTT was dependenton the reduced pyridine nucleotides NADH and NADPH, not on succinate as had beenpreviously believed (Berridge et al., 1993, 1994; Berridge and Tan, 1993). Cellular reduc-tion of MTT was associated with enzymes of the endoplasmic reticulum and was morerelated to NADH production through glycolysis than to respiration.

Recently, assays have been introduced based on tetrazolium salts (such as XTT and WST-1) that are reduced to soluble formazans. These assays depend on intermediate electronacceptors such as phenazine methosulfate (PMS).

The question arises: Is the cellular reduction of these new salts similar to that of MTT? Inthis article, the answer to that question is attempted.

In summary, it could be shown that, unlike MTT, XTT and WST-1 are efficiently reducedby NADH and NADPH in the absence of cells or enzymes, and their reduction involvessuperoxide. Cellular reduction of WST-1 occurs at the cell surface and also involvessuperoxide.

2.3 Annexin-V assays

2.3.1 The use of annexin for concomitant detection of apoptosis and cellular phenotype

[from S. Hoornaert, E. Hanon, J. Lyaku, and P.-P. Pastoret, Department of Immunology/Vaccinology, Faculty ofVeternary Medicine, University of Liège, Liège, Belgium]


Summary: Two distinct modes of cell death, apoptosis and necrosis, can be distinguishedon the basis of differences in morphological and biochemical characteristics. Under theelctron microscope, cells undergoing apoptosis display cell shrinkage, apoptotic bodyformation, and chromatin condensation. Biochemically, the apoptotic process is chara-terized by fragmentation of DNA into oligonucleosomal fragments. Furthermore, during


Metabolic and Annexin-V assays


the early stages of apoptosis, changes also occur at the cell surface membrane (Andree etal. 1990; Creutz, 1992; Fadok et al. 1992). One of these plasma membrane alterations isthe translocation of phosphatidylserine (PS) from the inner part to the outer layer of theplasma (Vermes et al., 1995), thus exposing PS at the external surface of apoptotic cells,where it can be specifically recognized by macrophage (Fadok et al. 1992).

Annexin-V, a Ca2+-dependent phospholipid-binding protein, possesses high affinity forPS (Vermes et al., 1995) and can thus be used for detecting early apoptotic cells (Koop-man et al. 1992, Verhoven et al. 1995, Vermes et al. 1995, Homburg et al. 1995). Sinceannexin-V can also detect necrotic cells as a result of the loss of membrane integrity, apo-ptotic cells have to be differentiated from these necrotic cells by the use of propidiumiodide (PI). Indeed, PI selectively labels necrotic, but not apoptotic cells.

Several studies have revealed a correlation between apoptosis and cell phenotype (Car-bonari et al. 1995, Lewis, et al. 1994). The investigation of this relationship ideallyrequires techniques that permit the concomitant detection of apoptosis and cell pheno-type analysis at a single cell level. In this report, the development of a procedure whichpermits concomitant detection of apoptosis and cell phenotype characterization by flowcytometry is described.

2.4 BrdU assays

2.4. Detection of bromodeoxyuridine in paraffin-embedded tissue sections using microwave antigen retrieval is dependent on the mode of tissue fixation

[from Wesley M. Garrett and H.D. Guthrie, Germplasm and Gamete Physiology, Agricultural Research Service,Beltsville, United States]


Summary: A simple routine microwave antigen retrieval procedure allows the sensitivedetection of incorporated BrdU in pulse labeled cells. Of the two fixatives tested, Carnoy’soffers superior nuclear morphology, but with a sacrifice of immunostaining intensity. Forinvestigations where animals are sacrificed within several hours after pulse labeling, Car-noy’s fixative may prove adequate for a general fixative, but it is not known what effect ithas on cellular antigens of interest. For our purposes, 10% neutral buffered formalin wasfound to be a superior fixative, because of its ability to cross-link nuclear proteins andassociated chromatin, resulting in more intense immunostaining for BrdU. In addition,we have found that formalin fixation coupled with microwave antigen retrieval is com-pletely compatible with immunostaining of other antigens of interest.


BrdU assays

CC123Appendix

3. References

3.1 Apoptosis-related parameters – Abbreviations and References

Parameter Full length name Reference Roche Applied Science product

AIF Apoptosis inducing factor

�Susin S. A. et al. (1996) J. Exp. Med. 184, 1331.

Apaf Apoptotic protease activating factor

�Zou H. et al. (1997) Cell 90, 405.�Li P. et al. (1997) Cell 91, 479.

APO-2 (L) Apoptosis receptor/ligand

�Masters S. A. et al. (1996) Curr. Biol. 6, 750.�Pit R. M. et al. (1996) J. Biol. Chem. 271, 12687.

APO-3 (L) Apoptosis receptor/ligand

�Masters S. A. et al. (1996) Curr. Biology 6, 1669.�Chinnaiyan A. M. et al. (1996) Science 274, 990.

Apopain �Schlegel J. et al. (1996) J. Biol. Chem. 271, 1841.

Bad �Yang E. et al. (1995) Cell 80, 285.

Bak �Sattler M. et al. (1997) Science 275, 983.�Orth R. & Dixit V. M. (1997) J. Biol. Chem. 272, 8841.

Bax �Bargou R. C. et al. (1995) Eur. J. Immunol. 25, 770.�Zhan Q. M. et al. (1994) Oncogene 9, 3743.�Yang E. et al. (1995) Cell 80, 285.

Bcl-2 �Craig W. C. (1995) Cancer Biology 6, 35.�Yang E. et al. (1995) Cell 80, 285.

Bcl-xL �Yang E. et al. (1995) Cell 80, 285.

Bcl-xS �Williams G. T. & Smith C. A. (1993) Cell 74, 777.�Yang E. et al. (1995) Cell 80, 285.

bik �Orth R. & Dixit V. M. (1997) J. Biol. Chem. 272, 8841.

Ca2+ �McConkey D. J. et al. (1995) J. Immunology 155, 5133.�Kataoka A. et al. (1995) FEBS Letters 364, 264.�Sokolova I. A. et al. (1995) Biochimica et Biophysica

Acta – Mol. Cell Res. 1266, 135.

CAD Caspase activated DNase

�Enari, M. et al. (1998) Nature 391, 43.

Calpain �Kikuchi H. & Imajohohmi S. (1995) Cell Death and Differentiation 2, 195.

�Slukvin I. I. & Jerrelis T. R. (1995) Immunopharmacology31, 43.

Calpain inhibitor I, Cat. No. 11 086 090 001Calpain inhibitor II,Cat. No. 11 086 103 001

Caspase Cysteine protease cleaving an aspartic acid residue

�Cohen G. M. (1997) Biochem. J. 326, 1.�Alnemri E. S. et al. (1996) Cell 87, 171.�Nicholson D. W. & Thornberry N. A. (1997) TIBS 22, 299.

ced-3 Caenorhabditis ele-gans cell death gene

�Yuan J. et al. (1993) Cell 75, 641.�Miura M. et al. (1993) Cell 75, 653.

ced-9 Caenorhabditis ele-gans cell death gene

�Henegartner M. O. & Horovitz H. R. (1994) Cell 76, 665.

Ceramide �Wiegmann K. et al. (1994) Cell 78, 1005.

c-Jun �Grand R. J. A. et al. (1995) Exp. Cell Res. 218, 439.

c-Myc �Wang Y. et al. (1993) Cell Growth Differ. 4, 467.�Schwartz L. M. & Osborne B. A. (1993) Immunol.

Today 14, 582.

CPP32 �Darmon A. J. et al. (1995) Nature 377, 446. Anti-PARP, Cat. No. 11 835 238 001

crm A Cytokine response modifier A

�Zhou Q. et al. (1997) J. Biol. Chem. 272, 7797.�Ogasawara J. et al. (1993) Nature 364, 806.

Cytochrome C �Liu X. et al. (1996) Cell 86, 147.�Krippner A. et al. (1996) J. Biol. Chem. 271, 21629.�Yang J. et al. (1997) Science 275, 1129.�Li P. et al. (1997) Cell 91, 479.

References

Apoptosis-related parameters – Abbreviations and References


D4-GDP-DI DI = dissociation inhibitor

�Danley D. E. et al. (1996) J. Immunology 157, 500.

Daxx Death-domain-associ-ated protein xx

�Yang X. L. et al. (1997) Cell 89.

DcR1 Decoy receptor 1 �Pan G. et al. (1997) Science 277, 815.�Sheridan J. P. et al. (1997) Science 277.

DD Death Domain �Muzio M. et al. (1996) Cell, 85, 817.

DED Death Effector Domain �Chinnaiyan A. M. et al. (1996) J. Biol. Chem. 271, 4961.

DISC Death Inducing Signal Complex

�Muzio M. et al. (1996) Cell, 85, 817.

DNA-Fragmentation

�Wyllie A. H. et al. (1980) Int. Rev. of Cytol. 68, 251.�Burgoyne L. A. et al. (1974) Biochem. J. 143, 67.�Stach R. W. et al. (1979) J. Neurochem. 33, 257.

Apoptotic DNA Ladder Kit, Cat. No. 11 835 246 001Cell Death Detection ELISAPLUS,Cat. No. 11 744 425 001Cell Death Detection ELISA, Cat. No. 11 544 675 001Cellular DNA Fragmentation ELISA, Cat. No. 11 585 045 001In Situ Cell Death Detection Kit, Fluorescein, Cat. No. 11 684 795 001In Situ Cell Death Detection Kit, TMR, Cat. No. 12 156 792 001In Situ Cell Death Detection Kit, AP, Cat. No. 11 684 809 001In Situ Cell Death Detection Kit, POD, Cat. No. 11 684 817 001

DNA-PKCS DNA-dependent pro-tein kinase catalytic subunit

�Casiolarosen L. et al. (1996) J. Exp. Med. 183, 1957.

DNA-repair �De Murcia G. & De Murcia J. (1994) TIBS 19, 172. Anti-PARP, Cat. No. 11 835 238 001

DR3 Death Receptor �Chinnaiyan A. M. et al. (1996) Science 274, 990.

DR4 Death Receptor �Pan G. H. et al. (1997) Science 276, 111.

DR5 Death Receptor �Walczak H. et al. (1997) EMBO J. 16, 5386.�Sheridan J. P. et al. (1997) Science 277.

Endonuclease �Walker P. R. & Sikorska (1994) Biochem. and Cell Biology 72, 615.

�Dini L. et al. (1996) Exp. Cell Res. 223, 340.

Nuclease S7, Cat. No. 10 107 921 001Nuclease P1, Cat. No. 10 236 225 001Nuclease S1, Cat. No. 10 818 348 001DNase I, RNase free, Cat. No. 10 776 785 001DNase I, grade I, Cat. No. 10 104 132 001DNase I, grade II, Cat. No. 10 104 159 001

FADD/MORT-1

FADD = Fas-associated death domain

�Chinnaiyan A. M. et al. (1995) Cell 81, 505.�Chinnaiyan A. M. et al. (1996) J. Biol. Chem. 271, 4961.�Vincenz C. & Dixit V. M. (1997) J. Biol. Chem. 272, 6578.

FAK Focal adhesion kinase

�Crouch D. H. et al. (1996) Oncogene 12, 2689.�Hungerford J. E. et al. (1996) J. Cell Biol. 135, 1383.

Fas Synonyms: Fas = CD 95 = Apo1

�Trauth et al. (1989) Science 245, 301. Anti-Fas, Cat. No. 11 922 432 001

Fas-ligand CD 95/fas (receptor)

Synonyms: Fas = CD 95 = Apo1

�Nagata S. & Goldstein P. (1995) Science 267, 1449.�Lynch D. H. et al. (1995) Immunol. Today 16, 569.�Tanaka M. et al. (1998) Nature Medicine 4, 1, 31.

FLICE/MACH FADD like ICE �Muzio M. et al. (1996) Cell 85, 817.�Boldin M. P. et al. (1996) Cell 85, 803.�Fernandes-Alnemri T. et al. (1996) Proc. Natl. Acad. Sci.

USA 93, 7464.�Scaffidi C. et al. (1997) J. Biol. Chem. 272, 43, 26953.


References


CC125Appendix

FLIP FLICE-inhibitory proteins

�Thome M. et al. (1997) Nature 386, 517.� Irmler M. et al (1997) Nature 388, 190.

Fodrin �Martin S. J. et al. (1995) J. Biol. Chemistry 270, 6425.

fos �Smeyne R. J. et al. (1995) Nature 363, 166 and Erratum Nature 365, 279.

�Colotta F. et al. (1992) J. Biol. Chem. 267, 18278.

G-Actin �Boone D. L. & Tsang B. K. (1997) Biology and Reproduction 57, 813.

Gas-2 �Brancolini C. et al. (1997) Cell Death and Diff. 4, 247.

Gelsolin �Kothakota S. et al. (1997) Science 278, 294.

Glucocorticoid/Glucocorticoid-Receptor

�Schwartzman R. A. & Cidlowski J. A. (1994) Int. Arch. of Allergy and Immunology 105, 347.

�Perrinwolff M. et al. (1995) Biochem. Pharmacology 50,103.

�Kiefer J. et al. (1995) J. Immunology 155, 4525.

Granzyme A, B

� Irmler M. et al. (1995) J. Exp. Med. 181, 1917.�Peitsch M. C. & Tschopp J. (1994) Proteolytic Enzymes

244, 80.�Nakajima H. et al. (1995) J. Exp. Med. 181, 1037.�Smyth M. J. & Trapani J. A. (1995) Immunology Today

16, 202.�Darmon A. J. et al. (1995) Nature 377, 446.�Quan L. T. et al. (1996) Proc. Nat. Acad. Sci. 93, 1972.

hnRNPs C1/C2

Heteronuclear Ribonucleoproteins

�Waterhaus N. et al. (1996) J. Biol. Chem. 271, 29335.

ICAD Inhibitor of CAD �Enari M. et al. (1998) Nature 391, 43.

ICE Interleukin-1�/converting enzyme

�Whyte M. & Evan G. (1995) Nature 376, 17.�Atkinson E. A. & Bleackley R. C. (1995) Critical Reviews

in Immunology 15, 359.�Kumar S. & Harvey N. L. (1995) FEBS Letters 375, 169.

Interleukin-1�, human, Cat. No. 11 457 756 001Interleukin-1�, mouse, Cat. No. 11 444 590 001

JNK Jun N-terminal kinase

�Hibi M. et al. (1993) Genes Dev. 7 (11), 2135.

Lamin A, B �Weaver V. M. et al. (1996) J. of Cell Science 109, 45.

MAP Mitogen activated pro-tein kinase

�Meyer C. F. et al. (1996) J. Biol. Chem. 271, 8971.

MCL-1 �Williams G. T. & Smith C. A. (1993) Cell 74, 777.

Mdm-2 �Chen J. D. et al. (1996) Mol. and Cellular Biol. 16, 2445.�Yu K. et al. (1997) Cell Growth & Diff. 8.

MEKK-1 MAP Kinase Kinase 1

�Cardone M. H. et al. (1997) Cell 90.�Meyer C. F. et al. (1996) J. Biol. Chem. 271, 8971.

MORT-1 (see FADD)

�Boldin M. P. (1995) J. Biol. Chem. 270, 7795.�Chinnaiyan A. M. et al. (1995) Cell 81, 505.�Chinnaiyan A. M. et al. (1996) J. Biol. Chem. 271, 4961.

NEDD �Gu Y. et al. (1995) J. Biol. Chemistry 270, 18715.

NF-�B Nuclear factor kappaB

�Wiegmann K. et al. (1994) Cell 78, 1005.

NuMa Nuclear matrix protein �Guethhallonet C. et al. (1997) Exp. Cell Res. 233.�Weaver V. M. et al. (1996) J. Cell science 109, 45.�Hsu H. L. & Yeh N. H. (1996) J. Cell science 109, 277.

p53 �Yonish-Rouach E. et al. (1993) Mol. Cell Biol. 13, 1415.�Zambetti G. P. (1993) FASEB J. 7, 855.�Lowe S. W. et al. (1993) Cell 74, 957.

p53 ELISA, Cat. No. 11 828 789 001

PAK-2 p21 activated kinase �Rudel T. & Bokoch G. M. (1997) Science 276.

PARP Poly-ADP-ribose-polymerase

�Lippke J. A. et al. (1996) J. Biol. Chem. 271, 1825.�De Murcia G. & De Murcia J. (1994) TIBS 19, 172.

Perforin �Nakajima H. et al. (1995) J. Exp. Med. 181, 1037.�Schroter M. et al. (1995) Europ. J. Immunol. 25, 3509.�Lowin B. et al. (1996) Int. Immunology 8, 57.


References



� Table 20: Published sources that contain more information about the components of the apoptosis pathways (Figure 2, page 5).

Phosphatidyl-serine

�Vermes I. et al. (1995) J. Immunol. Methods 184, 39. Annexin-V-Alexa 568, Cat. No. 11 985 485 001,(new Cat. No. 03 703 126 001)Annexin-V-FLUOS, Cat. No. 11 828 681001Annexin-V-Biotin, Cat. No. 11 828 690 001

PITSLRE �Beyaert R. et al. (1997) J. Biol. Cem. 272, 11694.

PKC � Protein kinase C �Emoto Y. et al. (1995) EMBO J. 14, 6148.�Ghayur T. et al. (1996) J. Exp. Med. 184, 2399.

pRb Retinoblastoma protein

�Hansen R. et al. (1995) Oncogene 11, 2535.�Haaskogan D. A. et al. (1995) EMBO J. 14, 461.�Picksley S. M. (1994) Curr. Opinion in Cell Biology 6, 853.

Presenilin �Loetscher H. et al. (1997) J. Biol. Chem. 272.

prICE �Smyth M. J. et al. (1996) Biochem. Journal 316, 25.

RAIDD RIP associated ICH-1/CED-3 homologous protein with a death domain

�Duan & Dixit (1997) Nature 385, 86.

Ras �Krueger G. R. F. et al. (1995) Pathologe 16, 120.�Wang H. G. et al. (1995) J. Cell Biol. 129, 1103.�Fernandez A. et al. (1995) Oncogene 10, 769.

RIP Receptor interacting protein

�Stanger B. Z. et al. (1995) Cell 81, 513.�Hsu H. et al. (1996) Immunity 4, 387.�Grimm S. et al. (1996) Proc. Natl. Acad. Sci. 93, 10923.

Sphingo-myelinase

�Heller R. A. & Kronke M. (1994) J. Cell Biol. 126, 5.�Kolesnik R. & Golde D. W. (1994) Cell 77, 325.

SREBPs Sterol-regulatory element binding proteins

�Wang X. D. et al. (1996) EMBO J. 15, 1012.

TNF-� Tumor necrosis factor

�Leist M. et al. (1994) J. Immunol. 153, 1778. TNF-�, human, Cat. Nos. 11 371 843 001,11 088 939 001

TNF-�receptor

�Nagata S. (1997) Cell, 88, 355.�Tartaglia L. A. et al. (1993) Cell 74, 845.

TNF-�, mouse, Cat. No. 11 271 156 001TNF-� ELISA, human, Cat. No. 11 425 943 001

TRADD TNFR1-associated death domain

�Hsu H. et al. (1995) Cell 81, 495.

TRAF2 TNF receptor associated factor

�Liu Z.-G. et al. (1996) Cell 87, 565.

TRAIL -R1, -R2, -R3

TNF-related apoptosis inducing ligand

�Wiley S. R. et al. (1995) Immunity 3, 673.�Walczak H. et al. (1997) EMBO Journal 16, 5386.�Deglli-Esposti M. A. et al. (1997) J. Exp. Med. 186, 1165.�Sheridan J. P. et al. (1997) Science 277, 818.

Trans-glutaminase

�Zhang L.-X. et al. (1995) J. Biol. Chemistry 270, 6022.�Melino G. et al. (1994) Mol. and Cell Biology 14, 6584.

U1-70 kDa snRNP

U1 small nuclear ribonucleoprotein protein

�Rosena & Casciolarosen L. (1997) J. Biol. Chem. 64, 50.

YAMA Synonyms:CPP32, Apopain

�Tewari M. et al (1995) Cell 81, 801.


References


CC127Appendix

Synonyms

Proteases Synonyms

Caspase-1 ICE

Caspase-2 ICH-1

Caspase-3 CPP32, Yama, Apopain

Caspase-4 ICErel-II, TX, ICH-2

Caspase-5 ICErel-III, TY

Caspase-6 Mch2

Caspase-7 Mch3, ICE-LAP3, CMH-1

Caspase-8 FLICE, MACH, Mch5

Caspase-9 ICE-LAP6, Mch6

Caspase-10 Mch4 / FLICE 2

Caspase-11 ICH-3

Caspase-12

Caspase-13 ERICE

Caspase-14 MICE

Granzyme B CTL proteinase-1, Fragmentin-2, RNKP-1

Receptor Synonyms

CD95 APO-1, Fas

DcR1 TRID, LIT and TRAIL-R3

DcR2 TRAIL-R4

DcR3

DR-3 APO-3, TRAMP, WSL-1, LARD

DR-4 TRAIL-R1

DR-5 TRAIL-R2, TRICK2, KILLER

DR-6

DR-1 TNF-R1

DR-2 CD95

RANK

Ligands

CD95L Fas ligand, APO-1L

TRAIL APO-2L

TWEAK APO-3L

RANK L TRANCE

Apaf Synonyms

Apaf-1 (no alternative, homologue to ced-4)

Apaf-2 Cytochrome C

Apaf-3 Caspase 9 (homologue to ced-3)

References



3.2 Examples for applications of Roche Applied Science products

Cell Death

Apoptotic DNA-Ladder Kit, Cat.-No. 11 835 246 001

Jennifer Wessells, Shoshana Yakar, and Peter F. Johnson Critical Prosurvival Roles for C/EBPß and Insulin-Like Growth Factor I in Macrophage Tumor CellsMol. Cell. Biol., Apr 2004; 24: 3238 - 3250.

Renu A. Kowluru, Anjaneyulu Kowluru, Subrata Chakrabarti, and Zia Khan Potential Contributory Role of H-Ras, a Small G-Protein, in the Development of Retinopathy in Diabetic RatsDiabetes, Mar 2004; 53: 775 - 783.

Li Wen, Jian Peng, Zhenjun Li, and F. Susan Wong The Effect of Innate Immunity on Autoimmune Diabetes and the Expression of Toll-Like Receptors on Pancreatic IsletsJ. Immunol., Mar 2004; 172: 3173 - 3180.

Athanassios Argiris, Chun-Xia Wang, Steve G. Whalen, and Michael P. DiGiovannaSynergistic Interactions between Tamoxifen and Trastuzumab (Herceptin)Clin. Cancer Res., Feb 2004; 10: 1409 - 1420.

Renu A. Kowluru and Saiyeda Noor Abbas Diabetes-Induced Mitochondrial Dysfunction in the RetinaInvest. Ophthalmol. Vis. Sci., Dec 2003; 44: 5327 - 5334.

Laura Suomalainen, Jukka K. Hakala, Virve Pentikäinen, Marjut Otala, Krista Erkkilä, Markku O. Pentikäinen, and Leo Dunkel Sphingosine-1-Phosphate in Inhibition of Male Germ Cell Apo-ptosis in the Human TestisJ. Clin. Endocrinol. Metab., Nov 2003; 88: 5572 - 5579.

Katare Gopalrao Rajesh, Shiro Sasaguri, Ryoko Suzuki, and Hironori MaedaAntioxidant MCI-186 inhibits mitochondrial permeability transi-tion pore and upregulates Bcl-2 expressionAm J Physiol Heart Circ Physiol, Nov 2003; 285: 2171 - 2178.

R Horváth, H Lochmüller, C Scharfe, B H Do, P J Oefner, J Müller-Höcker, B G Schoser, D Pongratz, D P Auer, and M Jaksch A tRNAAla mutation causing mitochondrial myopathy clinically resembling myotonic dystrophyJ. Med. Genet., Oct 2003; 40: 752 - 757.

K. Erkkilä, L. Suomalainen, M. Wikström, M. Parvinen, and L. Dunkel Chemical Anoxia Delays Germ Cell Apoptosis in the Human TestisBiol. Reprod., Aug 2003; 69: 617 - 626.

Irina A. Vasilevskaya, Tatiana V. Rakitina, and Peter J. O’DwyerGeldanamycin and its 17-Allylamino-17-Demethoxy Analogue Antagonize the Action of Cisplatin in Human Colon Adenocar-cinoma Cells: Differential Caspase Activation as a Basis for InteractionCancer Res., Jun 2003; 63: 3241 - 3246.

Maria Elena Dell'Aquila, Maria Albrizio, Filippo Maritato, Paolo Minoia, and Katrin Hinrichs Meiotic Competence of Equine Oocytes and Pronucleus Forma-tion after Intracytoplasmic Sperm Injection (ICSI) as Related to Granulosa Cell ApoptosisBiol. Reprod., Jun 2003; 68: 2065 - 2072.

Kan Koizumi, Vassiliki Poulaki, Sven Doehmen, Gerhard Welsandt, Sven Radetzky, Alexandra Lappas, Norbert Kociok, Bernd Kirchhof, and Antonia M. Joussen Contribution of TNF- to Leukocyte Adhesion, Vascular Leakage, and Apoptotic Cell Death in Endotoxin-Induced Uveitis In VivoInvest. Ophthalmol. Vis. Sci., May 2003; 44: 2184 - 2191.

Wendy E. Smith, Anne V. Kane, Sausan T. Campbell, David W. K. Acheson, Brent H. Cochran, and Cheleste M. Thorpe Shiga Toxin 1 Triggers a Ribotoxic Stress Response Leading to p38 and JNK Activation and Induction of Apoptosis in Intestinal Epithelial CellsInfect. Immun., Mar 2003; 71: 1497 - 1504.

Hanping Feng, Yi Zeng, Michael W. Graner, Anna Likhacheva, and Emmanuel Katsanis Exogenous stress proteins enhance the immunogenicity of apoptotic tumor cells and stimulate antitumor immunityBlood, Jan 2003; 101: 245 - 252.

Lawrence Y. Lee, Yuko J. Miyamoto, Bradley W. McIntyre, Mag-nus Höök, Kirk W. McCrea, Damien McDevitt, and Eric L. Brown The Staphylococcus aureus Map protein is an immunomodula-tor that interferes with T cell–mediated responsesJ. Clin. Invest., Nov 2002; 110: 1461 - 1471.

Fabienne Vernejoul, Patrick Faure, Naoual Benali, Denis Calise, Gérard Tiraby, Lucien Pradayrol, Christiane Susini, and Louis Buscail Antitumor Effect of in Vivo Somatostatin Receptor Subtype 2 Gene Transfer in Primary and Metastatic Pancreatic Cancer ModelsCancer Res., Nov 2002; 62: 6124 - 6131.

William C. Gordon, Douglas M. Casey, Walter J. Lukiw, and Nicolas G. Bazan DNA Damage and Repair in Light-Induced Photoreceptor DegenerationInvest. Ophthalmol. Vis. Sci., Nov 2002; 43: 3511 - 3521.

Julien Davaille, Liying Li, Ariane Mallat, and Sophie Lotersztajn Sphingosine 1-Phosphate Triggers Both Apoptotic and Survival Signals for Human Hepatic MyofibroblastsJ. Biol. Chem., Sep 2002; 277: 37323 - 37330.

Jenny M. Kim, Junjie Fang, Susan Rheingold, Richard Aplenc, Robert Wasserman, and Stephan A. Grupp Cytoplasmic µ Heavy Chain Confers Sensitivity to Dexametha-sone-induced Apoptosis in Early B-lineage Acute Lymphoblas-tic LeukemiaCancer Res., Aug 2002; 62: 4212 - 4216.

HENRIKKA AITO, KRISTIINA T. AALTO, and KARI O. RAIVIO Biphasic ATP Depletion Caused by Transient Oxidative Exposure Is Associated with Apoptotic Cell Death in Rat Embryonal Corti-cal NeuronsPediatr. Res., Jun 2002; 52: 40 - 45.

M. Otala, K. Erkkilä, T. Tuuri, J. Sjöberg, L. Suomalainen, A-M. Suikkari, V. Pentikäinen, and L. Dunkel Cell death and its suppression in human ovarian tissue cultureMol. Hum. Reprod., Mar 2002; 8: 228 - 236.

K. Erkkilä, H. Aito, K. Aalto, V. Pentikäinen, and L. Dunkel Lactate inhibits germ cell apoptosis in the human testisMol. Hum. Reprod., Feb 2002; 8: 109 - 117.

References

Examples for applications of Roche Applied Science products

CC129Appendix

Virve Pentikäinen, Laura Suomalainen, Krista Erkkilä, Eeva Martelin, Martti Parvinen, Markku O. Pentikäinen, and Leo Dunkel Nuclear Factor-B Activation in Human Testicular ApoptosisAm. J. Pathol., Jan 2002; 160: 205 - 218.

Maria Laura Garcia-Bermejo, Federico Coluccio Leskow, Teruhiko Fujii, Qiming Wang, Peter M. Blumberg, Motoi Ohba, Toshio Kuroki, Kee-Chung Han, Jeewoo Lee, Victor E. Marquez, and Marcelo G. Kazanietz Diacylglycerol (DAG)-lactones, a New Class of Protein Kinase C (PKC) Agonists, Induce Apoptosis in LNCaP Prostate Cancer Cells by Selective Activation of PKCJ. Biol. Chem., Jan 2002; 277: 645 - 655.

Robert X.-D. Song, Gil Mor, Fred Naftolin, Robert A. McPherson, Joon Song, Zhenguo Zhang, Wei Yue, JiPing Wang, and Richard J. Santen Effect of Long-Term Estrogen Deprivation on Apoptotic Responses of Breast Cancer Cells to 17-EstradiolJ Natl Cancer Inst, Nov 2001; 93: 1714 - 1723.

Cell Death Detection ELISA, Cat.-No. 11 544 675 001

Jeffrey A. Rudolph, Julia L. Poccia, and Mitchell B. Cohen Cyclic AMP Activation of the Extracellular Signal-regulated Kinases 1 and 2: IMPLICATIONS FOR INTESTINAL CELL SURVIVAL THROUGH THE TRANSIENT INHIBITION OF APOPTOSISJ. Biol. Chem., Apr 2004; 279: 14828 - 14834.

Riichiro Abe, Tadamichi Shimizu, Sho-ichi Yamagishi, Akihiko Shibaki, Shinjiro Amano, Yosuke Inagaki, Hirokazu Watanabe, Hiroshi Sugawara, Hideki Nakamura, Masayoshi Takeuchi, Tsutomu Imaizumi, and Hiroshi Shimizu Overexpression of Pigment Epithelium-Derived Factor Decreases Angiogenesis and Inhibits the Growth of Human Malignant Melanoma Cells in VivoAm. J. Pathol., Apr 2004; 164: 1225 - 1232.

Ho-Seong Kim, Angela R. Ingermann, Junko Tsubaki, Stephen M. Twigg, Gillian E. Walker, and Youngman Oh Insulin-Like Growth Factor-Binding Protein 3 Induces Caspase-Dependent Apoptosis through a Death Receptor-Mediated Pathway in MCF-7 Human Breast Cancer CellsCancer Res., Mar 2004; 64: 2229 - 2237.

W K Leung, A H C Bai, V Y W Chan, J Yu, M W Y Chan, K-F To, J-R Wu, K-K Chan, Y-G Fu, F K L Chan, and J J Y Sung Effect of peroxisome proliferator activated receptor ligands on growth and gene expression profiles of gastric cancer cellsGut, Mar 2004; 53: 331 - 338.

Samuel K. Kulp, Ya-Ting Yang, Chin-Chun Hung, Kuen-Feng Chen, Ju-Ping Lai, Ping-Hui Tseng, Joseph W. Fowble, Patrick J. Ward, and Ching-Shih Chen 3-Phosphoinositide-Dependent Protein Kinase-1/Akt Signaling Represents a Major Cyclooxygenase-2-Independent Target for Celecoxib in Prostate Cancer CellsCancer Res., Feb 2004; 64: 1444 - 1451.

Nathalie Méthot, JingQi Huang, Nathalie Coulombe, John P. Vaillancourt, Dita Rasper, John Tam, Yongxin Han, John Colucci, Robert Zamboni, Steven Xanthoudakis, Sylvie Toulmond, Donald W. Nicholson, and Sophie Roy Differential Efficacy of Caspase Inhibitors on Apoptosis Markers during Sepsis in Rats and Implication for Fractional Inhibition Requirements for TherapeuticsJ. Exp. Med., Jan 2004; 199: 199 - 207.

Steve Gendron, Julie Couture, and Fawzi Aoudjit Integrin 21 Inhibits Fas-mediated Apoptosis in T Lymphocytes by Protein Phosphatase 2A-dependent Activation of the MAPK/ERK PathwayJ. Biol. Chem., Dec 2003; 278: 48633 - 48643.

PART XIV. CELL DEATH AND NEURODEGENERATIVE DIS-EASES:A CORSARO, S THELLUNG, V VILLA, D ROSSI PRINCIPE, D PALUDI, S ARENA, E MILLO, D SCHETTINI, G DAMONTE, A ACETO, G SCHETTINI, and T FLORIO Prion Protein Fragment 106-126 Induces a p38 MAP Kinase—Dependent Apoptosis in SH-SY5Y Neuroblastoma Cells Inde-pendently from the Amyloid Fibril FormationAnn. N.Y. Acad. Sci., Dec 2003; 1010: 610 - 622.

Nicoletta Ferrari, Monica Morini, Ulrich Pfeffer, Simona Ming-helli, Douglas M. Noonan, and Adriana Albini Inhibition of Kaposi’s Sarcoma in Vivo by FenretinideClin. Cancer Res., Dec 2003; 9: 6020 - 6029.

PART XIII. INFECTION-INDUCED APOPTOSIS:PACO PINO, IOANNIS VOULDOUKIS, NATHALIE DUGAS, GERALDINE HASSANI-LOPPION, BERNARD DUGAS, and DOMINIQUE MAZIER Redox-Dependent Apoptosis in Human Endothelial Cells after Adhesion of Plasmodium falciparum-Infected ErythrocytesAnn. N.Y. Acad. Sci., Dec 2003; 1010: 582 - 586.

Shruti Nagar, Leslie E. Smith, and William F. Morgan Mechanisms of cell death associated with death-inducing fac-tors from genomically unstable cell linesMutagenesis, Nov 2003; 18: 549 - 560.

Barinder P. S. Kang, Arunas Urbonas, Andrew Baddoo, Stuart Baskin, Ashwani Malhotra, and Leonard G. Meggs IGF-1 inhibits the mitochondrial apoptosis program in mesangial cells exposed to high glucoseAm J Physiol Renal Physiol, Nov 2003; 285: 1013 - 1024.

Jiyun Yoo, Mayshan Ghiassi, Ludmila Jirmanova, Arthur G. Balliet, Barbara Hoffman, Albert J. Fornace, Jr., Dan A. Liebermann, Erwin P. Böttinger, and Anita B. Roberts Transforming Growth Factor--induced Apoptosis Is Mediatedby Smad-dependent Expression of GADD45b through p38 ActivationJ. Biol. Chem., Oct 2003; 278: 43001 - 43007.

Tsungda Hsu, Suzanne M. Hingley-Wilson, Bing Chen, Mei Chen, Annie Z. Dai, Paul M. Morin, Carolyn B. Marks, Jeevan Padiyar, Celia Goulding, Mari Gingery, David Eisenberg, Robert G. Russell, Steven C. Derrick, Frank M. Collins, Sheldon L. Morris, C. Harold King, and William R. Jacobs, Jr. The primary mechanism of attenuation of bacillus Calmette–Guérin is a loss of secreted lytic function required for invasion of lung interstitial tissuePNAS, Oct 2003; 100: 12420 - 12425.

Nilesh M. Dagia and Douglas J. Goetz A proteasome inhibitor reduces concurrent, sequential, and long-term IL-1- and TNF--induced ECAM expression and adhesionAm J Physiol Cell Physiol, Oct 2003; 285: 813 - 822.

Alex C. Chin, Nathalie Vergnolle, Wallace K. MacNaughton, John L. Wallace, Morley D. Hollenberg, and Andre G. Buret Proteinase-activated receptor 1 activation induces epithelial apoptosis and increases intestinal permeabilityPNAS, Sep 2003; 100: 11104 - 11109.

References



M. Lonergan, D. Aponso, K. W. Marvin, R. J. A. Helliwell, T. A. Sato, M. D. Mitchell, T. Chaiwaropongsa, R. Romero, and J. A. Keelan Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL), TRAIL Receptors, and the Soluble Receptor Osteopro-tegerin in Human Gestational Membranes and Amniotic Fluid during Pregnancy and Labor at Term and PretermJ. Clin. Endocrinol. Metab., Aug 2003; 88: 3835 - 3844.

Hideharu Tomita, Michael Nazmy, Katsuya Kajimoto, Ghassan Yehia, Carlos A. Molina, and Junichi Sadoshima Inducible cAMP Early Repressor (ICER) Is a Negative-Feedback Regulator of Cardiac Hypertrophy and an Important Mediator of Cardiac Myocyte Apoptosis in Response to ß-Adrenergic Receptor StimulationCirc. Res., Jul 2003; 93: 12 - 22.

GLYCOBIOLOGY AND EXTRACELLULAR MATRICES:Liliana Schaefer, Karl-Friedrich Beck, Igor Raslik, Sebastian Walpen, Daniel Mihalik, Miroslava Micegova, Katarina Macak-ova, Elke Schönherr, Daniela G. Seidler, Georg Varga, Roland M. Schaefer, Hans Kresse, and Josef Pfeilschifter Biglycan, a Nitric Oxide-regulated Gene, Affects Adhesion, Growth, and Survival of Mesangial CellsJ. Biol. Chem., Jul 2003; 278: 26227 - 26237.

Arno Hueber, Michael Weller, Gerhard Welsandt, Norbert Kociok, Bernd Kirchhof, and Peter Esser Characterization of Daunorubicin-Induced Apoptosis in Retinal Pigment Epithelial Cells: Modulation by CD95LInvest. Ophthalmol. Vis. Sci., Jul 2003; 44: 2851 - 2857.

Ryuya Shimoda, William E. Achanzar, Wei Qu, Takeaki Naga-mine, Hitoshi Takagi, Masatomo Mori, and Michael P. Waalkes Metallothionein Is a Potential Negative Regulator of ApoptosisToxicol. Sci., Jun 2003; 73: 294 - 300.

Rui-Dong Duan, Yajun Cheng, Gert Hansen, Erik Hertervig, Jian-Jun Liu, Ingvar Syk, Hans Sjöström, and Åke Nilsson Purification, localization, and expression of human intestinal alkaline sphingomyelinaseJ. Lipid Res., Jun 2003; 44: 1241 - 1250.

Lara Longobardi, Monica Torello, Caroline Buckway, Lynda O’Rear, William A. Horton, Vivian Hwa, Charles T. Roberts, Jr., Francesco Chiarelli, Ron G. Rosenfeld, and Anna Spagnoli A Novel Insulin-Like Growth Factor (IGF)-Independent Role for IGF Binding Protein-3 in Mesenchymal Chondroprogenitor Cell ApoptosisEndocrinology, May 2003; 144: 1695 - 1702.

Li Hua Wang, Xiao Yi Yang, Xiaohu Zhang, Kelly Mihalic, Weihua Xiao, and William L. Farrar The cis Decoy against the Estrogen Response Element Sup-presses Breast Cancer Cells via Target Disrupting c-fos not Mitogen-activated Protein Kinase ActivityCancer Res., May 2003; 63: 2046 - 2051.

Dong Xiao, Sanjay K. Srivastava, Karen L. Lew, Yan Zeng, Pam-ela Hershberger, Candace S. Johnson, Donald L. Trump, and Shivendra V. Singh Allyl isothiocyanate, a constituent of cruciferous vegetables, inhibits proliferation of human prostate cancer cells by causing G2/M arrest and inducing apoptosisCarcinogenesis, May 2003; 24: 891 - 897.

Cell Death Detection ELISAPLUS, Cat.-No. 11 774 425 001

Raffaella Spagnuolo, Monica Corada, Fabrizio Orsenigo, Lucia Zanetta, Ulrich Deuschle, Peter Sandy, Claudio Schneider, Christopher J. Drake, Ferruccio Breviario, and Elisabetta Dejana Gas1 is induced by VE-cadherin and vascular endothelial growth factor and inhibits endothelial cell apoptosisBlood, Apr 2004; 103: 3005 - 3012.

Dharminder Chauhan, Guilan Li, Klaus Podar, Teru Hideshima, Reshma Shringarpure, Laurence Catley, Constantine Mitsiades, Nikhil Munshi, Yu Tzu Tai, Nanjoo Suh, Gordon W. Gribble, Tadashi Honda, Robert Schlossman, Paul Richardson, Michael B. Sporn, and Kenneth C. Anderson The bortezomib/proteasome inhibitor PS-341 and triterpenoid CDDO-Im induce synergistic anti–multiple myeloma (MM) activity and overcome bortezomib resistanceBlood, Apr 2004; 103: 3158 - 3166.

Chadrick E. Denlinger, Michael D. Keller, Marty W. Mayo, R. Michael Broad, and David R. Jones Combined proteasome and histone deacetylase inhibition in non–small cell lung cancerJ. Thorac. Cardiovasc. Surg., Apr 2004; 127: 1078 - 1086.

Harry A. Rogoff, Mary T. Pickering, Fiona M. Frame, Michelle E. Debatis, Yolanda Sanchez, Stephen Jones, and Timothy F. Kowalik Apoptosis Associated with Deregulated E2F Activity Is Depen-dent on E2F1 and Atm/Nbs1/Chk2Mol. Cell. Biol., Apr 2004; 24: 2968 - 2977.

Anna Csiszar, Zoltan Ungvari, Akos Koller, John G. Edwards, and Gabor Kaley Proinflammatory phenotype of coronary arteries promotes endothelial apoptosis in agingPhysiol Genomics, Mar 2004; 17: 21 - 30.

Renu A. Kowluru, Anjaneyulu Kowluru, Subrata Chakrabarti, and Zia Khan Potential Contributory Role of H-Ras, a Small G-Protein, in the Development of Retinopathy in Diabetic RatsDiabetes, Mar 2004; 53: 775 - 783.

Ioannis P. Trougakos, Alan So, Burkhard Jansen, Martin E. Gleave, and Efstathios S. Gonos Silencing Expression of the Clusterin/Apolipoprotein J Gene in Human Cancer Cells Using Small Interfering RNA Induces Spontaneous Apoptosis, Reduced Growth Ability, and Cell Sensitization to Genotoxic and Oxidative StressCancer Res., Mar 2004; 64: 1834 - 1842.

Stefan Hammerschmidt, Hartmut Kuhn, Thomas Grasenack, Christian Gessner, and Hubert Wirtz Apoptosis and Necrosis Induced by Cyclic Mechanical Stretch-ing in Alveolar Type II CellsAm. J. Respir. Cell Mol. Biol., Mar 2004; 30: 396 - 402.

Sujoy Bhattacharya, Ramesh M. Ray, and Leonard R. Johnson Prevention of TNF--induced apoptosis in polyamine-depleted IEC-6 cells is mediated through the activation of ERK1/2Am J Physiol Gastrointest Liver Physiol, Mar 2004; 286:479 - 490.

References


CC131Appendix

Masahiko Watabe, Keiichi Hishikawa, Atsushi Takayanagi, Nobuyoshi Shimizu, and Toshio Nakaki Caffeic Acid Phenethyl Ester Induces Apoptosis by Inhibition of NFB and Activation of Fas in Human Breast Cancer MCF-7 CellsJ. Biol. Chem., Feb 2004; 279: 6017 - 6026.

Geoffrey A. Walford, Rose-Laure Moussignac, Anne W. Scribner, Joseph Loscalzo, and Jane A. Leopold Hypoxia Potentiates Nitric Oxide-mediated Apoptosis in Endot-helial Cells via Peroxynitrite-induced Activation of Mitochon-dria-dependent and -independent PathwaysJ. Biol. Chem., Feb 2004; 279: 4425 - 4432.

Sylvie Toulmond, Keith Tang, Yves Bureau, Helen Ashdown, Sarah Degen, Ruth O'Donnell, John Tam, Yongxin Han, John Colucci, André Giroux, Yanxia Zhu, Mathieu Boucher, Bill Pikounis, Steven Xanthoudakis, Sophie Roy, Michael Rigby, Robert Zamboni, George S Robertson, Gordon Y K Ng, Donald W Nicholson, and Jean-Pierre FlückigerNeuroprotective effects of M826, a reversible caspase-3 inhibi-tor, in the rat malonate model of Huntington's diseaseBr. J. Pharmacol., Feb 2004; 141: 689 - 697.

Qiang Liu and Baolu Zhao Nicotine attenuates ß-amyloid peptide-induced neurotoxicity, free radical and calcium accumulation in hippocampal neuronal culturesBr. J. Pharmacol., Feb 2004; 141: 746 - 754.

Michishige Harada, Ken-ichiro Seino, Hiroshi Wakao, Sakura Sakata, Yuko Ishizuka, Toshihiro Ito, Satoshi Kojo, Toshinori Nakayama, and Masaru Taniguchi Down-regulation of the invariant V14 antigen receptor in NKT cells upon activationInt. Immunol., Feb 2004; 16: 241 - 247.

Masahito Shimizu, Masumi Suzui, Atsuko Deguchi, Jin T. E. Lim, and I. Bernard Weinstein Effects of Acyclic Retinoid on Growth, Cell Cycle Control, Epi-dermal Growth Factor Receptor Signaling, and Gene Expression in Human Squamous Cell Carcinoma CellsClin. Cancer Res., Feb 2004; 10: 1130 - 1140.

Chia-Ron Yang, Shie-Liang Hsieh, Che-Ming Teng, Feng-Ming Ho, Wen-Ling Su, and Wan-Wan Lin Soluble Decoy Receptor 3 Induces Angiogenesis by Neutraliza-tion of TL1A, a Cytokine Belonging to Tumor Necrosis Factor Superfamily and Exhibiting Angiostatic ActionCancer Res., Feb 2004; 64: 1122 - 1129.

Pak-Shan Leung, William J. Aronson, Tung H. Ngo, Lawrence A. Golding, and R. James Barnard Exercise alters the IGF axis in vivo and increases p53 protein in prostate tumor cells in vitroJ Appl Physiol, Feb 2004; 96: 450 - 454.

Ulrich Laufs, Nikos Werner, Andreas Link, Matthias Endres, Sven Wassmann, Kristina Jürgens, Eckart Miche, Michael Böhm, and Georg Nickenig Physical Training Increases Endothelial Progenitor Cells, Inhibits Neointima Formation, and Enhances AngiogenesisCirculation, Jan 2004; 109: 220 - 226.

Catherine Kern, Jean-François Cornuel, Christian Billard, Ruoping Tang, Danielle Rouillard, Virginie Stenou, Thierry Defrance, Florence Ajchenbaum-Cymbalista, Pierre-Yves Simonin, Sophie Feldblum, and Jean-Pierre Kolb Involvement of BAFF and APRIL in the resistance to apoptosis of B-CLL through an autocrine pathwayBlood, Jan 2004; 103: 679 - 688.

Kimberly A. Carlson, Gary Leisman, Jenae Limoges, Garrett D. Pohlman, Masahide Horiba, James Buescher, Howard E. Gen-delman, and Tsuneya Ikezu Molecular Characterization of a Putative Antiretroviral Tran-scriptional Factor, OTK18J. Immunol., Jan 2004; 172: 381 - 391.

Salvatore Cortellino, David Turner, Valeria Masciullo, Filippo Schepis, Domenico Albino, Rene Daniel, Anna Marie Skalka, Neal J. Meropol, Christophe Alberti, Lionel Larue, and Alfonso Bellacosa From The Cover: The base excision repair enzyme MED1 medi-ates DNA damage response to antitumor drugs and is associ-ated with mismatch repair system integrityPNAS, Dec 2003; 100: 15071 - 15076.

George W. Small, Sivagurunathan Somasundaram, Dominic T. Moore, Yue Y. Shi, and Robert Z. Orlowski Repression of Mitogen-Activated Protein Kinase (MAPK) Phos-phatase-1 by Anthracyclines Contributes to Their Antiapoptotic Activation of p44/42-MAPKJ. Pharmacol. Exp. Ther., Dec 2003; 307: 861 - 869.

Stephanie Brändlein, Tina Pohle, Nele Ruoff, Ewa Wozniak, Hans-Konrad Müller-Hermelink, and H. Peter Vollmers Natural IgM Antibodies and Immunosurveillance Mechanisms against Epithelial Cancer Cells in HumansCancer Res., Nov 2003; 63: 7995 - 8005.

Maria V. Papadopoulou and William D. Bloomer NLCQ-1 (NSC 709257): Exploiting Hypoxia with a Weak DNA-Intercalating Bioreductive DrugClin. Cancer Res., Nov 2003; 9: 5714 - 5720.

Erding Hu, Edward Dul, Chiu-Mei Sung, Zunxuan Chen, Robert Kirkpatrick, Gui-Feng Zhang, Kyung Johanson, Ronggang Liu, Amparo Lago, Glenn Hofmann, Ricardo Macarron, Maite De Los Frailes, Paloma Perez, John Krawiec, James Winkler, and Michael Jaye Identification of Novel Isoform-Selective Inhibitors within Class I Histone DeacetylasesJ. Pharmacol. Exp. Ther., Nov 2003; 307: 720 - 728.

M30 CytoDEATH, Cat.-No. 12 140 322 001

M30 CytoDEATH, Fluorescein, Cat.-No. 12 156 857 001

Leonieke Terpstra, Josée Prud'homme, Alice Arabian, Shu Takeda, Gérard Karsenty, Shoukat Dedhar, and René St-Arnaud Reduced chondrocyte proliferation and chondrodysplasia in mice lacking the integrin-linked kinase in chondrocytesJ. Cell Biol., Jul 2003; 162: 139 - 148.

Ingrid Herr, Esat Ucur, Kerstin Herzer, Stella Okouoyo, Rüdiger Ridder, Peter H. Krammer, Magnus von Knebel Doeberitz, and Klaus-Michael Debatin Glucocorticoid Cotreatment Induces Apoptosis Resistance toward Cancer Therapy in CarcinomasCancer Res., Jun 2003; 63: 3112 - 3120.

Kay-Uwe Wagner, Andrea Krempler, Yongyue Qi, KyungRan Park, MaLinda D. Henry, Aleata A. Triplett, Gregory Riedlinger, Edmund B. Rucker III, and Lothar Hennighausen Tsg101 Is Essential for Cell Growth, Proliferation, and Cell Sur-vival of Embryonic and Adult TissuesMol. Cell. Biol., Jan 2003; 23: 150 - 162.

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Ai Q. Truong-Tran, Richard E. Ruffin, Paul S. Foster, Aulikki M. Koskinen, Peter Coyle, Jeffrey C. Philcox, Allan M Rofe, and Peter D. Zalewski Altered Zinc Homeostasis and Caspase-3 Activity in Murine Allergic Airway InflammationAm. J. Respir. Cell Mol. Biol., Sep 2002; 27: 286 - 296.

Kristin R. Coulter, Andrea Doseff, Patricia Sweeney, Yijie Wang, Clay B. Marsh, Mark D. Wewers, and Daren L. Knoell Opposing Effect by Cytokines on Fas-Mediated Apoptosis in A549 Lung Epithelial CellsAm. J. Respir. Cell Mol. Biol., Jan 2002; 26: 58 - 66.

Michael Kasper, Cora Roehlecke, Martin Witt, Heinz Fehren-bach, Andreas Hofer, Toshio Miyata, Cora Weigert, Richard H. W. Funk, and Erwin D. Schleicher Induction of Apoptosis by Glyoxal in Human Embryonic Lung Epithelial Cell Line L132Am. J. Respir. Cell Mol. Biol., Oct 2000; 23: 485 - 491.

Stacy M. Stabler, Lisa L. Ostrowski, Susan M. Janicki, and Mervyn J. Monteiro A Myristoylated Calcium-binding Protein that Preferentially Interacts with the Alzheimer's Disease Presenilin 2 ProteinJ. Cell Biol., Jun 1999; 145: 1277 - 1292.

Christian Bojarski, Jörg Weiske, Torsten Schöneberg, Werner Schröder, Joachim Mankertz, Jörg-Dieter Schulzke, Peter Florian, Michael Fromm, Rudolf Tauber, and Otmar Huber The specific fates of tight junction proteins in apoptotic epithelial cellsJ. Cell Sci., Mar 2004; 10.1242/jcs.01071.

Matthias Bruewer, Andreas Luegering, Torsten Kucharzik, Charles A. Parkos, James L. Madara, Ann M. Hopkins, and Asma Nusrat Proinflammatory Cytokines Disrupt Epithelial Barrier Function by Apoptosis-Independent Mechanisms J. Immunol., Dec 2003; 171: 6164 - 6172.

Carolyn L. Cannon, Michael P. Kowalski, Kimberly S. Stopak, and Gerald B. Pier Pseudomonas aeruginosa–Induced Apoptosis Is Defective in Respiratory Epithelial Cells Expressing Mutant Cystic Fibrosis Transmembrane Conductance RegulatorAm. J. Respir. Cell Mol. Biol., Aug 2003; 29: 188 - 197.

Alison J. Faragher and Andrew M. Fry Nek2A kinase stimulates centrosome disjunction and is required for formation of bipolar mitotic spindlesMol. Biol. Cell, Jul 2003; 14: 2876 - 2889.

Julie Guillermet, Nathalie Saint-Laurent, Philippe Rochaix, Oliv-ier Cuvillier, Thierry Levade, Andrew V. Schally, Lucien Praday-rol, Louis Buscail, Christiane Susini, and Corinne Bousquet Somatostatin receptor subtype 2 sensitizes human pancreatic cancer cells to death ligand-induced apoptosisPNAS, Jan 2003; 100: 155 - 160.

Guo-Zhong Tao, Lusijah S. Rott, Anson W. Lowe, and M. Bishr Omary Hyposmotic Stress Induces Cell Growth Arrest via Proteasome Activation and Cyclin/Cyclin-dependent Kinase DegradationJ. Biol. Chem., May 2002; 277: 19295 - 19303.

Naoko Yagishita, Yoko Yamamoto, Tatsuya Yoshizawa, Keisuke Sekine, Yoshikatsu Uematsu, Hisashi Murayama, Yumiko Nagai, Wojciech Krezel, Pierre Chambon, Toshio Matsumoto, and Shigeaki Kato Aberrant Growth Plate Development in VDR/RXR Double Null Mutant MiceEndocrinology, Dec 2001; 142: 5332 - 5341.

Rainer Riesenberg, Alexander Buchner, Heike Pohla, and Horst Lindhofer Lysis of Prostate Carcinoma Cells by Trifunctional Bispecific Antibodies (EpCAM x CD3)J. Histochem. Cytochem., Jul 2001; 49: 911 - 918.

Peter Birner, Monika Schindl, Andreas Obermair, Gerhard Breitenecker, and Georg Oberhuber Expression of Hypoxia-inducible Factor 1 in Epithelial Ovarian Tumors: Its Impact on Prognosis and on Response to Chemo-therapyClin. Cancer Res., Jun 2001; 7: 1661 - 1668.

Declan F. McCole, Lars Eckmann, Fabrice Laurent, and Martin F. Kagnoff Intestinal Epithelial Cell Apoptosis following Cryptosporidium parvum InfectionInfect. Immun., Mar 2000; 68: 1710 - 1713.

Caspase 3 Activity Assay, Cat.-No. 12 012 952 001

Yossi Gottfried, Asaf Rotem, Rona Lotan, Hermann Steller, and Sarit Larisch The mitochondrial ARTS protein promotes apoptosis through targeting XIAPEMBO J., Mar 2004; 10.1038/sj.emboj.7600155.

Kyeong Cheon Jung, Weon Seo Park, Hae Jung Kim, Eun Young Choi, Myeong-Cherl Kook, Han-Woong Lee, and Youngmee Bae TCR-Independent and Caspase-Independent Apoptosis of Murine Thymocytes by CD24 Cross-LinkingJ. Immunol., Jan 2004; 172: 795 - 802.

Debabrata Mandal, Veronique Baudin-Creuza, Asima Bhatta-charyya, Shresh Pathak, Jean Delaunay, Manikuntala Kundu, and Joyoti Basu Caspase 3-mediated Proteolysis of the N-terminal Cytoplasmic Domain of the Human Erythroid Anion Exchanger 1 (Band 3)J. Biol. Chem., Dec 2003; 278: 52551 - 52558.

Niels W. C. J. van de Donk, Diana Schotte, Marloes M. J. Kamphuis, Ariënne M. W. van Marion, Berris van Kessel, Andries C. Bloem, and Henk M. Lokhorst Protein Geranylgeranylation Is Critical for the Regulation of Survival and Proliferation of Lymphoma Tumor CellsClin. Cancer Res., Nov 2003; 9: 5735 - 5748.

Toshiki Itoh, Cristin O'Shea, and Stuart Linn Impaired Regulation of Tumor Suppressor p53 Caused by Muta-tions in the Xeroderma Pigmentosum DDB2 Gene: Mutual Reg-ulatory Interactions between p48DDB2 and p53Mol. Cell. Biol., Nov 2003; 23: 7540 - 7553.

P. Kelk, A. Johansson, R. Claesson, L. Hänström, and S. Kalfas Caspase 1 Involvement in Human Monocyte Lysis Induced by Actinobacillus actinomycetemcomitans LeukotoxinInfect. Immun., Aug 2003; 71: 4448 - 4455.

Lin-Tao Jia, Li-Hong Zhang, Cui-Juan Yu, Jing Zhao, Yan-Ming Xu, Jun-Hao Gui, Ming Jin, Zong-Ling Ji, Wei-Hong Wen, Cheng-Ji Wang, Si-Yi Chen, and An-Gang Yang Specific Tumoricidal Activity of a Secreted Proapoptotic Protein Consisting of HER2 Antibody and Constitutively Active Caspase-3Cancer Res., Jun 2003; 63: 3257 - 3262.

Tommer Ravid, Adili Tsaba, Peter Gee, Reuven Rasooly, Edward A. Medina, and Tzipora Goldkorn Ceramide accumulation precedes caspase-3 activation during apoptosis of A549 human lung adenocarcinoma cellsAm J Physiol Lung Cell Mol Physiol, Jun 2003; 284: 1082 - 1092.

References


CC133Appendix

Mizue Nakajoh, Takeyasu Fukushima, Tomoko Suzuki, Mutsuo Yamaya, Katsutoshi Nakayama, Kiyohisa Sekizawa, and Hidetada Sasaki Retinoic Acid Inhibits Elastase-Induced Injury in Human Lung Epithelial Cell LinesAm. J. Respir. Cell Mol. Biol., Mar 2003; 28: 296 - 304.

Thomas Mund, Andreas Gewies, Nicole Schoenfeld, Manuel K.A. Bauer, and Stefan Grimm Spike, a novel BH3-only protein, regulates apoptosis at the endoplasmic reticulumFASEB J, Feb 2003; 10.1096/fj.02-0657fje.

Andreas Gewies and Stefan Grimm UBP41 Is a Proapoptotic Ubiquitin-specific ProteaseCancer Res., Feb 2003; 63: 682 - 688.

Christelle Forcet, Xin Ye, Laure Granger, Véronique Corset, Hwain Shin, Dale E. Bredesen, and Patrick Mehlen The dependence receptor DCC (deleted in colorectal cancer) defines an alternative mechanism for caspase activationPNAS, Mar 2001; 98: 3416 - 3421.

Christelle Forcet, Xin Ye, Laure Granger, Véronique Corset, Hwain Shin, Dale E. Bredesen, and Patrick Mehlen The dependence receptor DCC (deleted in colorectal cancer) defines an alternative mechanism for caspase activationPNAS, Mar 2001; 98: 3416 - 3421.

Michael Gekle, Gerald Schwerdt, Ruth Freudinger, Sigrid Mildenberger, Doris Wilflingseder, Verena Pollack, Martina Dander, and Herbert Schramek Ochratoxin A Induces JNK Activation and Apoptosis in MDCK-C7 Cells at Nanomolar ConcentrationsJ. Pharmacol. Exp. Ther., Jun 2000; 293: 837 - 844.

Quy N. Diep, Hope D. Intengan, and Ernesto L. Schiffrin Endothelin-1 Attenuates 3 Fatty Acid–Induced Apoptosis by Inhibition of Caspase 3Hypertension, Jan 2000; 35: 287 - 291.

Homogeneous Caspases Assay, fluorimetric, Cat.-No. 03 005 372 001

Leng Wen, Li Zhuang, Xia Luo, and Ping Wei TL1A-induced NF-B Activation and c-IAP2 Production Prevent DR3-mediated Apoptosis in TF-1 CellsJ. Biol. Chem., Oct 2003; 278: 39251 - 39258.

Anke Niedernberg, Andree Blaukat, Torsten Schöneberg, and Evi Kostenis Regulated and constitutive activation of specific signalling pathways by the human S1P5 receptorBr. J. Pharmacol., Feb 2003; 138: 481 - 493.

Lucas C. Armstrong, Benny Björkblom, Kurt D. Hankenson, Anthony W. Siadak, Charlotte E. Stiles, and Paul Bornstein Thrombospondin 2 Inhibits Microvascular Endothelial Cell Pro-liferation by a Caspase-independent MechanismMol. Biol. Cell, Jun 2002; 13: 1893 - 1905.

Douglas D. Bannerman, Joan C. Tupper, Ryan D. Erwert, Robert K. Winn, and John M. Harlan Divergence of Bacterial Lipopolysaccharide Pro-apoptotic Signaling Downstream of IRAK-1J. Biol. Chem., Mar 2002; 277: 8048 - 8053.

Anti-Poly (ADP-Ribose) Polymerase, Cat.-No. 11 835 238 001

Christian Bojarski, Jörg Weiske, Torsten Schöneberg, Werner Schröder, Joachim Mankertz, Jörg-Dieter Schulzke, Peter Florian, Michael Fromm, Rudolf Tauber, and Otmar Huber The specific fates of tight junction proteins in apoptotic epithelial cellsJ. Cell Sci., Mar 2004; 10.1242/jcs.01071.

Ju-Hyung Woo, Young-Ho Kim, Yun-Jung Choi, Dae-Gon Kim, Kyung-Seop Lee, Jae Hoon Bae, Do Sik Min, Jong-Soo Chang, Yong-Jin Jeong, Young Han Lee, Jong-Wook Park, and Taeg Kyu Kwon Molecular mechanisms of curcumin-induced cytotoxicity: induction of apoptosis through generation of reactive oxygen species, down-regulation of Bcl-XL and IAP, the release of cytochrome c and inhibition of AktCarcinogenesis, Jul 2003; 24: 1199 - 1208.

Michael D. Gober, Cynthia C. Smith, Kaori Ueda, Jeffrey A. Toretsky, and Laure Aurelian Forced Expression of the H11 Heat Shock Protein Can Be Regu-lated by DNA Methylation and Trigger Apoptosis in Human CellsJ. Biol. Chem., Sep 2003; 278: 37600 - 37609.

Kyung-Hee Chun, Jerome W. Kosmeder, II, Shihua Sun, John M. Pezzuto, Reuben Lotan, Waun Ki Hong, and Ho-Young Lee Effects of Deguelin on the Phosphatidylinositol 3-Kinase/Akt Pathway and Apoptosis in Premalignant Human Bronchial Epi-thelial CellsJ Natl Cancer Inst, Feb 2003; 95: 291 - 302.

Olivier Sordet, Cédric Rébé, Stéphanie Plenchette, Yaël Zermati, Olivier Hermine, William Vainchenker, Carmen Garrido, Eric Solary, and Laurence Dubrez-Daloz Specific involvement of caspases in the differentiation of mono-cytes into macrophagesBlood, Dec 2002; 100: 4446 - 4453.

Victor T. Solovyan, Zinayida A. Bezvenyuk, Antero Salminen, Caroline A. Austin, and Michael J. Courtney The Role of Topoisomerase II in the Excision of DNA Loop Domains during ApoptosisJ. Biol. Chem., Jun 2002; 277: 21458 - 21467.

George Zachos, Margy Koffa, Chris M. Preston, J. Barklie Clements, and Joe Conner Herpes Simplex Virus Type 1 Blocks the Apoptotic Host Cell Defense Mechanisms That Target Bcl-2 and Manipulates Acti-vation of p38 Mitogen-Activated Protein Kinase To Improve Viral ReplicationJ. Virol., Mar 2001; 75: 2710 - 2728.

Gareth J. Inman, Ulrich K. Binné, Gillian A. Parker, Paul J. Farrell, and Martin J. Allday Activators of the Epstein-Barr Virus Lytic Program Concomi-tantly Induce Apoptosis, but Lytic Gene Expression Protects from Cell DeathJ. Virol., Mar 2001; 75: 2400 - 2410.

Ricky W. Johnstone, Mark Gerber, Theresa Landewe, Anne Tollefson, William S. Wold, and Ali Shilatifard Functional Analysis of the Leukemia Protein ELL: Evidence for a Role in the Regulation of Cell Growth and SurvivalMol. Cell. Biol., Mar 2001; 21: 1672 - 1681.

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Michael Schmidt, Norbert Lügering, Andreas Lügering, Hans-Gerd Pauels, Klaus Schulze-Osthoff, Wolfram Domschke, and Torsten Kucharzik Role of the CD95/CD95 Ligand System in Glucocorticoid-Induced Monocyte ApoptosisJ. Immunol., Jan 2001; 166: 1344 - 1351.

Gareth J. Inman and Martin J. Allday Apoptosis Induced by TGF-ß1 in Burkitt’s Lymphoma Cells Is Caspase 8 Dependent But Is Death Receptor IndependentJ. Immunol., Sep 2000; 165: 2500 - 2510.

Gareth J. Inman and Martin J. Allday Resistance to TGF-1 correlates with a reduction of TGF- type II receptor expression in Burkitt’s lymphoma and Epstein–Barr virus-transformed B lymphoblastoid cell linesJ. Gen. Virol., Jun 2000; 81: 1567 - 1578.

M. Maddalena Di Somma, Francesca Somma, Maria Saveria Gilardini Montani, Rosamaria Mangiacasale, Enrico Cundari, and Enza Piccolella TCR Engagement Regulates Differential Responsiveness of Human Memory T Cells to Fas (CD95)-Mediated ApoptosisJ. Immunol., Apr 1999; 162: 3851 - 3858.

In Situ Cell Death Detection Kit, Fluorescein, Cat.-No. 11 684 795 001

Chi-Huang Chen, Shang-Sen Lee, Da-Chang Chen, Hsin-Hsuan Chien, I-Ching Chen, Yung-Ning Chu, Jah-Yao Liu, Wei-Hwa Chen, and Gwo-Jang Wu Apoptosis and Kinematics of Ejaculated Spermatozoa in Patients With VaricoceleJ Androl, May 2004; 25: 348 - 353.

Gerhard Schratt, Ulrike Philippar, Dirk Hockemeyer, Heinz Schwarz, Siegfried Alberti, and Alfred Nordheim SRF regulates Bcl-2 expression and promotes cell survival dur-ing murine embryonic developmentEMBO J., Apr 2004; 10.1038/sj.emboj.7600188.

Pascal Froment, Christophe Staub, Stéphanie Hembert, Claudine Pisselet, Michèle Magistrini, Bernadette Delaleu, Danielle Seurin, Jon E. Levine, Larry Johnson, Michel Binoux, and Philippe Monget Reproductive Abnormalities in Human Insulin-Like Growth Fac-tor-Binding Protein-1 Transgenic Male MiceEndocrinology, Apr 2004; 145: 2080 - 2091.

Nasrin Mesaeli and Clark Phillipson Impaired p53 Expression, Function, and Nuclear Localization in Calreticulin-deficient CellsMol. Biol. Cell, Apr 2004; 15: 1862 - 1870.

Deepak Raina, Surender Kharbanda, and Donald Kufe The MUC1 oncoprotein activates the anti-apoptotic P13K/Akt and Bcl-xL pathways in rat 3Y1 fibroblastsJ. Biol. Chem., Mar 2004; 10.1074/jbc.M310538200.

Stefania Croci, Lorena Landuzzi, Annalisa Astolfi, Giordano Nicoletti, Angelo Rosolen, Francesca Sartori, Matilde Y. Follo, Noelynn Oliver, Carla De Giovanni, Patrizia Nanni, and Pier-Luigi Lollini Inhibition of Connective Tissue Growth Factor (CTGF/CCN2) Expression Decreases the Survival and Myogenic Differentia-tion of Human Rhabdomyosarcoma CellsCancer Res., Mar 2004; 64: 1730 - 1736.

Christian Kannemeier, Nadia Al-Fakhri, Klaus T. Preissner, and Sandip M. Kanse Factor VII activating protease (FSAP) inhibits growth factor-mediated cell proliferation and migration of vascular smooth muscle cells. FASEB J, Feb 2004; 10.1096/fj.03-0898fje.

Antonio Parrado, Macarena Robledo, M. Rosa Moya-Quiles, Luis A. Marín, Christine Chomienne, Rose Ann Padua, and M. Rocío Álvarez-López The promyelocytic leukemia zinc finger protein down-regulates apoptosis and expression of the proapoptotic BID protein in lymphocytesPNAS, Feb 2004; 101: 1898 - 1903.

Hervé Le Stunff, Rodolphe ;Auger, Jean Kanellopoulos, and Marie-Noëlle Raymond The Pro-451 to Leu polymorphism within the C-terminal tail of P2X7 receptor impairs cell death but not phospholipase D acti-vation in murine thymocytesJ. Biol. Chem., Feb 2004; 10.1074/jbc.M313064200.

Erica D. Smith, Yanfei Xu, Brett N. Tomson, Cindy G. Leung, Yuko Fujiwara, Stuart H. Orkin, and John D. Crispino More than blood, a Novel Gene Required for Mammalian Postimplantation DevelopmentMol. Cell. Biol., Feb 2004; 24: 1168 - 1173.

Maryam Diarra-Mehrpour, Samuel Arrabal, Abdelali Jalil, Xavier Pinson, Catherine Gaudin, Geneviève Piétu, Amandine Pitaval, Hugues Ripoche, Marc Eloit, Dominique Dormont, and Salem Chouaib Prion Protein Prevents Human Breast Carcinoma Cell Line from Tumor Necrosis Factor -Induced Cell DeathCancer Res., Jan 2004; 64: 719 - 727.

Takashi Maeda, Caroline M. Alexander, and Andreas Friedl Induction of Syndecan-1 Expression in Stromal Fibroblasts Pro-motes Proliferation of Human Breast Cancer CellsCancer Res., Jan 2004; 64: 612 - 621.

Rodrigo Cuervo and Luis Covarrubias Death is the major fate of medial edge epithelial cells and the cause of basal lamina degradation during palatogenesisDevelopment, Jan 2004; 131: 15 - 24.

T. T. T. Nguyen, E. Tran, T. H. Nguyen, P. T. Do, T. H. Huynh, and H. Huynh The role of activated MEK-ERK pathway in quercetin-induced growth inhibition and apoptosis in A549 lung cancer cellsCarcinogenesis, Dec 2003; 10.1093/carcin/bgh052.

Benjamin D. Yu, Michelle Becker-Hapak, Eric L. Snyder, Marc Vooijs, Catherine Denicourt, and Steven F. Dowdy Distinct and nonoverlapping roles for pRB and cyclin D:cyclin-dependent kinases 4/6 activity in melanocyte survivalPNAS, Dec 2003; 100: 14881 - 14886.

Bernhard Stockmeyer, Thomas Beyer, Winfried Neuhuber, Roland Repp, Joachim R. Kalden, Thomas Valerius, and Martin Herrmann Polymorphonuclear Granulocytes Induce Antibody-Dependent Apoptosis in Human Breast Cancer CellsJ. Immunol., Nov 2003; 171: 5124 - 5129.

Kevin H. Mayo, Ruud P. M. Dings, Carolee Flader, Irina Nes-melova, Balasz Hargittai, Daisy W. J. van der Schaft, Loes I. van Eijk, Dinesha Walek, Judy Haseman, Thomas R. Hoye, and Arjan W. Griffioen Design of a Partial Peptide Mimetic of Anginex with Antiangio-genic and Anticancer ActivityJ. Biol. Chem., Nov 2003; 278: 45746 - 45752.

References


CC135Appendix

John W. Calvert, Changman Zhou, Anil Nanda, and John H. ZhangEffect of hyperbaric oxygen on apoptosis in neonatal hypoxia-ischemia rat modelJ Appl Physiol, Nov 2003; 95: 2072 - 2080.

Marjorie A. Robbins, Lola Maksumova, Emma Pocock, and Janet K. Chantler Nuclear Factor-B Translocation Mediates Double-Stranded Ribonucleic Acid-Induced NIT-1 ß-Cell Apoptosis and Up-Reg-ulates Caspase-12 and Tumor Necrosis Factor Receptor-Asso-ciated Ligand (TRAIL)Endocrinology, Oct 2003; 144: 4616 - 4625.

XINGZHI XU and DAVID F. STERN1 NFBD1/MDC1 regulates ionizing radiation-induced focus for-mation by DNA checkpoint signaling and repair factorsFASEB J, Oct 2003; 17: 1842 - 1848.

Xianmin Meng, John F. Klement, Dominic A. Leperi, David E. Birk, Takako Sasaki, Rupert Timpl, Jouni Uitto, and Leena Pulkkinen Targeted Inactivation of Murine Laminin 2-Chain Gene Recapit-ulates Human Junctional Epidermolysis BullosaJ. Invest. Dermatol., Oct 2003; 121: 720 - 731.

Andrew Zloza, Yvonne B. Sullivan, Elizabeth Connick, Alan L. Landay, and Lena Al-Harthi CD8+ T cells that express CD4 on their surface (CD4dimCD8bright T cells) recognize an antigen-specific target, are detected in vivo, and can be productively infected by T-tropic HIVBlood, Sep 2003; 102: 2156 - 2164.

Annette Lasham, Stephanie Moloney, Tracy Hale, Craig Homer, You Fang Zhang, J. Greg Murison, Antony W. Braithwaite, and James Watson The Y-box-binding Protein, YB1, Is a Potential Negative Regula-tor of the p53 Tumor SuppressorJ. Biol. Chem., Sep 2003; 278: 35516 - 35523.

Jinn-Yang Chen, Chin-Wen Chi, Hui-Ling Chen, Chiung-Pei Wan, Wu-Chang Yang, and An-Hang Yang TNF- renders human peritoneal mesothelial cells sensitive to anti-Fas antibody-induced apoptosisNephrol. Dial. Transplant., Sep 2003; 18: 1741 - 1747.

Man-Seong Park, Adolfo García-Sastre, Jerome F. Cros, Christo-pher F. Basler, and Peter Palese Newcastle Disease Virus V Protein Is a Determinant of Host Range RestrictionJ. Virol., Sep 2003; 77: 9522 - 9532.

In Situ Cell Death Detection Kit, TMR red, Cat.-No. 12 156 792 001

Gerry Melino, Francesca Bernassola, Marco Ranalli, Karen Yee, Wei Xing Zong, Marco Corazzari, Richard A. Knight, Doug R. Green, Craig Thompson, and Karen H. Vousden p73 Induces Apoptosis via PUMA Transactivation and Bax Mitochondrial TranslocationJ. Biol. Chem., Feb 2004; 279: 8076 - 8083.

Wulin Aerbajinai, Y. Terry Lee, Urszula Wojda, Valarie A. Barr, and Jeffery L. Miller Cloning and Characterization of a Gene Expressed during Ter-minal Differentiation That Encodes a Novel Inhibitor of GrowthJ. Biol. Chem., Jan 2004; 279: 1916 - 1921.

Shane W. Rau, Dena B. Dubal, Martina Böttner, Lynnette M. Gerhold, and Phyllis M. Wise Estradiol Attenuates Programmed Cell Death after Stroke-Like InjuryJ. Neurosci., Dec 2003; 23: 11420 - 11426.

Gregory Kennedy, Jun Komano, and Bill Sugden Epstein-Barr virus provides a survival factor to Burkitt's lympho-masPNAS, Nov 2003; 100: 14269 - 14274.

Jiu Jiang, Lisa L. Lau, and Hao Shen Selective Depletion of Nonspecific T Cells During the Early Stage of Immune Responses to Infection J. Immunol., Oct 2003; 171: 4352 - 4358.

Jiu Jiang, Farvardin Anaraki, Kenneth J. Blank, and Donna M. Murasko Cutting Edge: T Cells from Aged Mice Are Resistant to Deple-tion Early During Virus Infection

J. Immunol., Oct 2003; 171: 3353 - 3357.

May Nour, Alexander B. Quiambao, Ward M. Peterson, Muayyad R. Al-Ubaidi, and Muna I. Naash P2Y2 Receptor Agonist INS37217 Enhances Functional Recov-ery after Detachment Caused by Subretinal Injection in Normal and rds MiceInvest. Ophthalmol. Vis. Sci., Oct 2003; 44: 4505 - 4514.

Timur O. Yarovinsky, Linda S. Powers, Noah S. Butler, Mary A. Bradford, Martha M. Monick, and Gary W. Hunninghake Adenoviral Infection Decreases Mortality from Lipopolysaccha-ride-Induced Liver Failure Via Induction of TNF- Tolerance J. Immunol., Sep 2003; 171: 2453 - 2460.

Elisabeth Larsen, Christine Gran, Barbro Elisabet Sæther, Erling Seeberg, and Arne Klungland Proliferation Failure and Gamma Radiation Sensitivity of Fen1 Null Mutant Mice at the Blastocyst StageMol. Cell. Biol., Aug 2003; 23: 5346 - 5353.

Yanhong Hao, Liangxue Lai, Jiude Mao, Gi-Sun Im, Aaron Bonk, and Randall S. Prather Apoptosis and In Vitro Development of Preimplantation Porcine Embryos Derived In Vitro or by Nuclear TransferBiol. Reprod., Aug 2003; 69: 501 - 507.

Nicole Maas-Szabowski, Anja Stärker, and Norbert E. Fusenig Epidermal tissue regeneration and stromal interaction in HaCaT cells is initiated by TGF-J. Cell Sci., Jul 2003; 116: 2937 - 2948.

D. Magnus Eklund and Johan Edqvist Localization of Nonspecific Lipid Transfer Proteins Correlate with Programmed Cell Death Responses during Endosperm Degradation in Euphorbia lagascae SeedlingsPlant Physiology, Jul 2003; 132: 1249 - 1259.

E. A. Eugenin, T. G. D'Aversa, L. Lopez, T. M. Calderon, and J. W. Berman MCP-1 (CCL2) protects human neurons and astrocytes from NMDA or HIV-tat-induced apoptosisJ. NeuRochem., Jun 2003; 85: 1299 - 1311.

Adi Inbal, Daniel Levanon, and Adi Salzberg Multiple roles for u-turn/ventral veinless in the development of Drosophila PNSDevelopment, Jun 2003; 130: 2467 - 2478.

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Wolfgang Fierlbeck, Ailian Liu, Raluca Coyle, and Barbara J. Ballermann Endothelial Cell Apoptosis during Glomerular Capillary Lumen Formation In VivoJ. Am. Soc. Nephrol., May 2003; 14: 1349 - 1354.

Charvi A. Patel, Muhammad Mukhtar, and Roger J. Pomerantz Human Immunodeficiency Virus Type 1 Vpr Induces Apoptosis in Human Neuronal CellsJ. Virol., Oct 2000; 74: 9717 - 9726.

In Situ Cell Death Detection Kit, AP, Cat.-No. 11 684 809 001

Xiao-Nan Li, Suhag Parikh, Qin Shu, Hye-Lim Jung, Chi-Wan Chow, Laszlo Perlaky, Hon-Chiu Eastwood Leung, Jack Su, Susan Blaney, and Ching C. Lau Phenylbutyrate and Phenylacetate Induce Differentiation and Inhibit Proliferation of Human Medulloblastoma CellsClin. Cancer Res., Feb 2004; 10: 1150 - 1159.

Estíbaliz L. Fernández, Anne-Lee Gustafson, Maria Andersson, Bjorn Hellman, and Lennart Dencker Cadmium-Induced Changes in Apoptotic Gene Expression Lev-els and DNA Damage in Mouse Embryos Are Blocked by ZincToxicol. Sci., Nov 2003; 76: 162 - 170.

Wolfgang Zink, Christoph Seif, Jürgen R. E. Bohl, Nicola Hacke, Peter M. Braun, Barbara Sinner, Eike Martin, Rainer H. A. Fink, and Bernhard M. Graf The Acute Myotoxic Effects of Bupivacaine and Ropivacaine After Continuous Peripheral Nerve BlockadesAnesth. Analg., Oct 2003; 97: 1173 - 1179.

Christophe Van de Wiele, Christophe Lahorte, Hubert Vermeersch, D. Loose, Kris Mervillie, Neil D. Steinmetz, Jean-Luc Vanderheyden, Claude A. Cuvelier, Guido Slegers, and Rudi A. Dierck Quantitative Tumor Apoptosis Imaging Using Technetium-99m–HYNIC Annexin V Single Photon Emission Computed Tomogra-phyJ. Clin. Oncol., Sep 2003; 21: 3483 - 3487.

Robert A. Ritzel and Peter C. Butler Replication Increases ß-Cell Vulnerability to Human Islet Amy-loid Polypeptide-Induced ApoptosisDiabetes, Jul 2003; 52: 1701 - 1708.

Francesca Mascia, Valentina Mariani, Giampiero Girolomoni, and Saveria Pastore Blockade of the EGF Receptor Induces a Deranged Chemokine Expression in Keratinocytes Leading to Enhanced Skin Inflam-mationAm. J. Pathol., Jul 2003; 163: 303 - 312.

Xian-Yong Ma, Hong Wang, Bo Ding, Haihong Zhong, Sankar Ghosh, and Peter Lengyel The Interferon-inducible p202a Protein Modulates NF-B Activity by Inhibiting the Binding to DNA of p50/p65 Heterodimers and p65 Homodimers While Enhancing the Binding of p50 HomodimersJ. Biol. Chem., Jun 2003; 278: 23008 - 23019.

Michael Abdo, Susan Hisheh, and Arun Dharmarajan Role of Tumor Necrosis Factor-Alpha and the Modulating Effect of the Caspases in Rat Corpus Luteum ApoptosisBiol. Reprod., Apr 2003; 68: 1241 - 1248.

Kathrin Maedler, José Oberholzer, Pascal Bucher, Giatgen A. Spinas, and Marc Y. Donath Monounsaturated Fatty Acids Prevent the Deleterious Effects of Palmitate and High Glucose on Human Pancreatic ß-Cell Turn-over and FunctionDiabetes, Mar 2003; 52: 726 - 733.

C. Kitamura, Y. Ogawa, T. Nishihara, T. Morotomi, and M. Terashita Transient Co-localization of c-Jun N-terminal Kinase and c-Jun with Heat Shock Protein 70 in Pulp Cells during ApoptosisJ. Dent. Res., Feb 2003; 82: 91 - 95.

Birgitte Viuff, Kirsten Tjørnehøj, Lars E. Larsen, Christine M. Røntved, Åse Uttenthal, Leif Rønsholt, and Soren Alexandersen Replication and Clearance of Respiratory Syncytial Virus: Apop-tosis Is an Important Pathway of Virus Clearance after Experi-mental Infection with Bovine Respiratory Syncytial VirusAm. J. Pathol., Dec 2002; 161: 2195 - 2207.

Koji Shinozaki, Toshihiko Miyagi, Michio Yoshida, Takaki Miyata, Masaharu Ogawa, Shinichi Aizawa, and Yoko Suda Absence of Cajal-Retzius cells and subplate neurons associated with defects of tangential cell migration from ganglionic emi-nence in Emx1/2 double mutant cerebral cortexDevelopment, Jul 2002; 129: 3479 - 3492.

Lori M. Roberts, Jenny A. Visser, and Holly A. Ingraham Involvement of a matrix metalloproteinase in MIS-induced cell death during urogenital developmentDevelopment, Mar 2002; 129: 1487 - 1496.

Hitoshi Akiba, Jeanne Kehren, Marie-Thérèse Ducluzeau, Maya Krasteva, Françoise Horand, Dominique Kaiserlian, Fumio Kaneko, and Jean-François Nicolas Skin Inflammation During Contact Hypersensitivity Is Mediated by Early Recruitment of CD8+ T Cytotoxic 1 Cells Inducing Keratinocyte ApoptosisJ. Immunol., Mar 2002; 168: 3079 - 3087.

Lisa Wise-Faberowski, Mohan K. Raizada, and Colin Sumners Oxygen and Glucose Deprivation-Induced Neuronal Apoptosis is Attenuated by Halothane and IsofluraneAnesth. Analg., Nov 2001; 93: 1281 - 1287.

Toshimasa Yamauchi, Junji Kamon, Hironori Waki, Koji Murakami, Kiyoto Motojima, Kajuro Komeda, Tomohiro Ide, Naoto Kubota, Yasuo Terauchi, Kazuyuki Tobe, Hiroshi Miki, Atsuko Tsuchida, Yasuo Akanuma, Ryozo Nagai, Satoshi Kimura, and Takashi Kadowaki The Mechanisms by Which Both Heterozygous Peroxisome Proliferator-activated Receptor (PPAR) Deficiency and PPAR Agonist Improve Insulin ResistanceJ. Biol. Chem., Oct 2001; 276: 41245 - 41254.

Annemiek Beverdam, Antje Brouwer, Mark Reijnen, Jeroen Kor-ving, and Frits Meijlink Severe nasal clefting and abnormal embryonic apoptosis in Alx3/Alx4 double mutant miceDevelopment, Oct 2001; 128: 3975 - 3986.

Dana G. Mordue, Fernando Monroy, Marie La Regina, Charles A. Dinarello, and L. David Sibley Acute Toxoplasmosis Leads to Lethal Overproduction of Th1 CytokinesJ. Immunol., Oct 2001; 167: 4574 - 4584.

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CC137Appendix

Daniela Dyntar, Monika Eppenberger-Eberhardt, Kathrin Maedler, Martin Pruschy, Hans M. Eppenberger, Giatgen A. Spinas, and Marc Y. Donath Glucose and Palmitic Acid Induce Degeneration of Myofibrils and Modulate Apoptosis in Rat Adult CardiomyocytesDiabetes, Sep 2001; 50: 2105 - 2113.

Kathrin Maedler, Giatgen A. Spinas, Roger Lehmann, Pavel Sergeev, Markus Weber, Adriano Fontana, Nurit Kaiser, and Marc Y. Donath Glucose Induces ß-Cell Apoptosis Via Upregulation of the Fas Receptor in Human IsletsDiabetes, Aug 2001; 50: 1683 - 1690.

Annett Jungmann, Hermann Nieper, and Hermann Müller Apoptosis is induced by infectious bursal disease virus replica-tion in productively infected cells as well as in antigen-negativecells in their vicinityJ. Gen. Virol., May 2001; 82: 1107 - 1115.

J. ALLEN D. COOPER, Jr., A. LYNN RIDGEWAY, JOHN PEARSON, and RACHEL R. CULBRETH Attenuation of Interleukin 8-induced Nasal Inflammation by an Inhibitor PeptideAm. J. Respir. Crit. Care Med., Apr 2001; 163: 1198 - 1205.

Batya Cohen, Dalit Barkan, Yinon Levy, Iris Goldberg, Eduard Fridman, Juri Kopolovic, and Menachem Rubinstein Leptin Induces Angiopoietin-2 Expression in Adipose TissuesJ. Biol. Chem., Mar 2001; 276: 7697 - 7700.

Françoise Trousse, Pilar Esteve, and Paola Bovolenta BMP4 Mediates Apoptotic Cell Death in the Developing Chick EyeJ. Neurosci., Feb 2001; 21: 1292 - 1301.

Antony W. Wood and Glen J. Van Der Kraak Apoptosis and Ovarian Function: Novel Perspectives from the TeleostsBiol. Reprod., Jan 2001; 64: 264 - 271.

In Situ Cell Death Detection Kit, POD, Cat.-No. 11 684 817 001

Ken-ichi Kimura, Akitoshi Kodama, Yosihiro Hayasaka, and Takumi Ohta Activation of the cAMP/PKA signaling pathway is required for post-ecdysial cell death in wing epidermal cells of Drosophila melanogasterDevelopment, Apr 2004; 131: 1597 - 1606.

Ivan Orlandi, Maurizio Bettiga, Lilia Alberghina, and Marina Vai Transcriptional Profiling of ubp10 Null Mutant Reveals Altered Subtelomeric Gene Expression and Insurgence of Oxidative Stress ResponseJ. Biol. Chem., Feb 2004; 279: 6414 - 6425.

Satomi Kuramochi-Miyagawa, Tohru Kimura, Takashi W. Ijiri, Taku Isobe, Noriko Asada, Yukiko Fujita, Masahito Ikawa, Naomi Iwai, Masaru Okabe, Wei Deng, Haifan Lin, Yoichi Matsuda, and Toru Nakano Mili, a mammalian member of piwi family gene, is essential for spermatogenesisDevelopment, Feb 2004; 131: 839 - 849.

Claudia Orelio, Kirsty N Harvey, Colin Miles, Robert A Oosten-dorp, Karin van der Horn, and Elaine Dzierzak The role of apoptosis in the development of AGM hematopoie-tic stem cells revealed by Bcl-2 overexpressionBlood, Feb 2004; 10.1182/blood-2003-06-1827.

Shuji Takiguchi, Norihiro Sugino, Kikue Esato, Ayako Karube-Harada, Aki Sakata, Yasuhiko Nakamura, Hitoshi Ishikawa, and Hiroshi Kato Differential Regulation of Apoptosis in the Corpus Luteum of Pregnancy and Newly Formed Corpus Luteum after Parturition in RatsBiol. Reprod., Feb 2004; 70: 313 - 318.

Hidenori Ozaki, Kazuaki Nakamura, Jun-ichi Funahashi, Keiko Ikeda, Gen Yamada, Hisashi Tokano, Hiro-oki Okamura, Ken Kitamura, Shigeaki Muto, Hayato Kotaki, Katsuko Sudo, Reiko Horai, Yoichiro Iwakura, and Kiyoshi Kawakami Six1 controls patterning of the mouse otic vesicleDevelopment, Feb 2004; 131: 551 - 562.

OV Anichtchik, J Kaslin, N Peitsaro, M Scheinin, and P Panula Neurochemical and behavioural changes in zebrafish Danio rerio after systemic administration of 6-hydroxydopamine and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.J Neurochem, Jan 2004; 88(2): 443-53.

Luciano Dalla Libera, Barbara Ravara, Maurizio Volterrani, Valerio Gobbo, Mila Della Barbera, Annalisa Angelini, Daniela Danieli Betto, Elena Germinario, and Giorgio Vescovo Beneficial effects of GH/IGF-1 on skeletal muscle atrophy and function in experimental heart failureAm J Physiol Cell Physiol, Jan 2004; 286: 138 - 144.

Juan F. Medina, Sergio Recalde, Jesús Prieto, Jon Lecanda, Elena Sáez, Colin D. Funk, Paola Vecino, Marian A. van Roon, Roelof Ottenhoff, Piter J. Bosma, Conny T. Bakker, and Ronald P. J. Oude Elferink Anion exchanger 2 is essential for spermiogenesis in micePNAS, Dec 2003; 100: 15847 - 15852.

Arturo Chavez-Reyes, John M. Parant, Lisa L. Amelse, Roberto Montes de Oca Luna, Stanley J. Korsmeyer, and Guillermina Lozano Switching Mechanisms of Cell Death in mdm2- and mdm4-null Mice by Deletion of p53 Downstream TargetsCancer Res., Dec 2003; 63: 8664 - 8669.

HE Paczoska-Eliasiewicz, A Gertler, M Proszkowiec, J Proud-man, A Hrabia, A Sechman, M Mika, T Jacek, S Cassy, N Ravner, and J Rzasa Attenuation by leptin of the effects of fasting on ovarian func-tion in hens (Gallus domesticus)Reproduction, Dec 2003; 126: 739 - 751.

Robert D. Knight, Sreelaja Nair, Sarah S. Nelson, Ali Afshar, Yashar Javidan, Robert Geisler, Gerd-Joerg Rauch, and Thomas F. Schilling lockjaw encodes a zebrafish tfap2a required for early neural crest developmentDevelopment, Dec 2003; 130: 5755 - 5768.

Ilka B. Schiffer, Susanne Gebhard, Carolin K. Heimerdinger, Annette Heling, Jochem Hast, Ursula Wollscheid, Barbara Seliger, Berno Tanner, Sandra Gilbert, Thomas Beckers, Silke Baasner, Walburgis Brenner, Christian Spangenberg, Dirk Prawitt, Tatjana Trost, Wolfgang G. Schreiber, Bernhard Zabel, Manfred Thelen, Hans-Anton Lehr, Franz Oesch, and Jan G. Hengstler Switching Off HER-2/neu in a Tetracycline-Controlled Mouse Tumor Model Leads to Apoptosis and Tumor-Size-DependentRemissionCancer Res., Nov 2003; 63: 7221 - 7231.

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Cristiane G. Lin, Shr-Jeng Leu, Ningyu Chen, Christopher M. Tebeau, Shao-Xia Lin, Cho-Yau Yeung, and Lester F. Lau CCN3 (NOV) Is a Novel Angiogenic Regulator of the CCN Pro-tein FamilyJ. Biol. Chem., Jun 2003; 278: 24200 - 24208.

Hiroyuki Koizumi, Noritaka Yamaguchi, Mitsuharu Hattori, Tomo-o Ishikawa, Junken Aoki, Makoto M. Taketo, Keizo Inoue, and Hiroyuki Arai Targeted Disruption of Intracellular Type I Platelet Activating Factor-acetylhydrolase Catalytic Subunits Causes Severe Impairment in SpermatogenesisJ. Biol. Chem., Mar 2003; 278: 12489 - 12494.

Torsten Wuestefeld, Christian Klein, Konrad L. Streetz, Ulrich Betz, Jörg Lauber, Jan Buer, Michael P. Manns, Werner Müller, and Christian Trautwein Interleukin-6/Glycoprotein 130-dependent Pathways Are Pro-tective during Liver RegenerationJ. Biol. Chem., Mar 2003; 278: 11281 - 11288.

Geert Hamer, Hermien L. Roepers-Gajadien, Annemarie van Duyn-Goedhart, Iris S. Gademan, Henk B. Kal, Paul P.W. van Buul, Terry Ashley, and Dirk G. de Rooij Function of DNA-Protein Kinase Catalytic Subunit During the Early Meiotic Prophase Without Ku70 and Ku86Biol. Reprod., Mar 2003; 68: 717 - 721.

Ulrike Wille, Eric N. Villegas, Linden Craig, Robert Peach, and Christopher A. Hunter Contribution of Interleukin-12 (IL-12) and the CD28/B7 and CD40/CD40 Ligand Pathways to the Development of a Patho-logical T-Cell Response in IL-10-Deficient MiceInfect. Immun., Dec 2002; 70: 6940 - 6947.

Hairong Peng, Vonda Wright, Arvydas Usas, Brian Gearhart, Hsain-Chung Shen, James Cummins, and Johnny Huard Synergistic enhancement of bone formation and healing by stem cell–expressed VEGF and bone morphogenetic protein-4J. Clin. Invest., Sep 2002; 110: 751 - 759.

Heung Tae Kim, Byung Chul Kim, Isaac Yi Kim, Mizuko Mamura, Do Hwan Seong, Ja-June Jang, and Seong-Jin Kim Raloxifene, a Mixed Estrogen Agonist/Antagonist, Induces Apo-ptosis through Cleavage of BAD in TSU-PR1 Human Cancer CellsJ. Biol. Chem., Aug 2002; 277: 32510 - 32515.

Byung-Chul Kim, Heung-Tae Kim, Mizuko Mamura, Indu S. Ambudkar, Kyeong-Sook Choi, and Seong-Jin Kim Tumor Necrosis Factor Induces Apoptosis in Hepatoma Cells by Increasing Ca2+ Release from the Endoplasmic Reticulum and Suppressing Bcl-2 ExpressionJ. Biol. Chem., Aug 2002; 277: 31381 - 31389.

Li Zeng, Hervé Kempf, L. Charles Murtaugh, Mie Elissa Sato, and Andrew B. Lassar Shh establishes an Nkx3.2/Sox9 autoregulatory loop that is maintained by BMP signals to induce somitic chondrogenesisGenes & Dev., Aug 2002; 16: 1990 - 2005.

Ken-ichi Sunayama, Hiroyuki Konno, Toshio Nakamura, Hide-humi Kashiwabara, Tsuyoshi Shoji, Toshihiro Tsuneyoshi, and Satoshi Nakamura The role of cyclooxygenase-2 (COX-2) in two different morpho-logical stages of intestinal polyps in APC474 knockout miceCarcinogenesis, Aug 2002; 23: 1351 - 1359.

Mary L. Alpaugh, James S. Tomlinson, Yin Ye, and Sanford H. Barsky Relationship of Sialyl-Lewisx/a Underexpression and E-Cad-herin Overexpression in the Lymphovascular Embolus of Inflam-matory Breast CarcinomaAm. J. Pathol., Aug 2002; 161: 619 - 628.

Alana M. Thackray and Raymond Bujdoso PrPc Expression Influences the Establishment of Herpes Sim-plex Virus Type 1 LatencyJ. Virol., Mar 2002; 76: 2498 - 2509.

Annexin-V-FLUOS, Cat.-No. 11 828 681 001

Gero Kramer, Hamdiye Erdal, Helena J. M. M. Mertens, Marius Nap, Julian Mauermann, Georg Steiner, Michael Marberger, Kenneth Bivén, Maria C. Shoshan, and Stig Linder Differentiation between Cell Death Modes Using Measure-ments of Different Soluble Forms of Extracellular Cytokeratin 18Cancer Res., Mar 2004; 64: 1751 - 1756.

Rung-chi Li, Tereza Cindrova-Davies, Jeremy N. Skepper, and Lynda A. Sellers Prostacyclin Induces Apoptosis of Vascular Smooth Muscle Cells by a cAMP-Mediated Inhibition of Extracellular Signal-Regulated Kinase Activity and Can Counteract the Mitogenic Activity of Endothelin-1 or Basic Fibroblast Growth FactorCirc. Res., Feb 2004; 10.1161/01.RES.0000121568.40692.97.

Gong Yang, Kathy Qi Cai, Jennifer A. Thompson-Lanza, Robert C. Bast, Jr., and Jinsong Liu Inhibition of Breast and Ovarian Tumor Growth through Multiple Signaling Pathways by Using Retrovirus-mediated Small Inter-fering RNA against Her-2/neu Gene ExpressionJ. Biol. Chem., Feb 2004; 279: 4339 - 4345.

Olena Yermolaieva, Rong Xu, Carrie Schinstock, Nathan Brot, Herbert Weissbach, Stefan H. Heinemann, and Toshinori Hoshi Methionine sulfoxide reductase A protects neuronal cells against brief hypoxia/reoxygenationPNAS, Feb 2004; 101: 1159 - 1164.

Shanhong Ling, Aozhi Dai, Rodney J. Dilley, Margaret Jones, Evan Simpson, Paul A. Komesaroff, and Krishnankutty Sudhir Endogenous Estrogen Deficiency Reduces Proliferation and Enhances Apoptosis-Related Death in Vascular Smooth Muscle Cells: Insights From the Aromatase-Knockout MouseCirculation, Feb 2004; 109: 537 - 543.

Mark A. Guthridge, Emma F. Barry, Fernando A. Felquer, Bar-bara J. McClure, Frank C. Stomski, Hayley Ramshaw, and Angel F. Lopez The phosphoserine-585–dependent pathway of the GM-CSF/IL-3/IL-5 receptors mediates hematopoietic cell survival through activation of NF-B and induction of bcl-2Blood, Feb 2004; 103: 820 - 827.

Petranel T. Ferrao, Michelle J. Frost, Shoo-Peng Siah, and Leonie K. Ashman Overexpression of P-glycoprotein in K562 cells does not confer resistance to the growth inhibitory effects of imatinib (STI571) in vitroBlood, Dec 2003; 102: 4499 - 4503.

Yuan Lin, Angela C. Bright, Terri A. Rothermel, and Biao He Induction of Apoptosis by Paramyxovirus Simian Virus 5 Lack-ing a Small Hydrophobic GeneJ. Virol., Mar 2003; 77: 3371 - 3383.

E. López, S. Figueroa, M. J. Oset-Gasque, and M. P. González Apoptosis and necrosis: two distinct events induced by cad-mium in cortical neurons in cultureBr. J. Pharmacol., Mar 2003; 138: 901 - 911.

Hui Xu, Dengping Yin, Bashoo Naziruddin, Libing Chen, Aileen Stark, Yuanyuan Wei, Ying Lei, JiKun Shen, John S. Logan, Guerard W. Byrne, and Anita S.-F. Chong The In Vitro and In Vivo Effects of Anti-Galactose Antibodies on Endothelial Cell Activation and Xenograft Rejection J. Immunol., Feb 2003; 170: 1531 - 1539.

References


CC139Appendix

Pattabhiraman Shankaranarayanan and Santosh Nigam IL-4 Induces Apoptosis in A549 Lung Adenocarcinoma Cells: Evidence for the Pivotal Role of 15-Hydroxyeicosatetraenoic Acid Binding to Activated Peroxisome Proliferator-Activated Receptor Transcription FactorJ. Immunol., Jan 2003; 170: 887 - 894.

Katja Issbrücker, Hugo H. Marti, Stefan Hippenstiel, Georg Springmann, Robert Voswinckel, Andreas Gaumann, Georg Breier, Hannes C. A. Drexler, Norbert Suttorp, and Matthias Clauss p38 MAP Kinase - a molecular switch between VEGF-induced angiogenesis and vascular hyperpermeabilityFASEB J, Dec 2002; 10.1096/fj.02-0329fje.

Margaret Park, Amanda Helip-Wooley, and Jess Thoene Lysosomal Cystine Storage Augments Apoptosis in Cultured Human Fibroblasts and Renal Tubular Epithelial CellsJ. Am. Soc. Nephrol., Dec 2002; 13: 2878 - 2887.

Theocharis Panaretakis, Katja Pokrovskaja, Maria C. Shoshan, and Dan Grandér Activation of Bak, Bax, and BH3-only Proteins in the Apoptotic Response to DoxorubicinJ. Biol. Chem., Nov 2002; 277: 44317 - 44326.

Michelle J. Frost, Petranel T. Ferrao, Timothy P. Hughes, and Leonie K. Ashman Juxtamembrane Mutant V560GKit Is More Sensitive to Imatinib (STI571) Compared with Wild-Type c-Kit Whereas the Kinase Domain Mutant D816VKit Is ResistantMol. Cancer Ther., Oct 2002; 1: 1115 - 1124.

Satoshi Murasawa, Joan Llevadot, Marcy Silver, Jeffrey M. Isner, Douglas W. Losordo, and Takayuki Asahara Constitutive Human Telomerase Reverse Transcriptase Expres-sion Enhances Regenerative Properties of Endothelial Progeni-tor CellsCirculation, Aug 2002; 106: 1133 - 1139.

Ilse Decordier, Lubina Dillen, Enrico Cundari, and Micheline Kirsch-Volders Elimination of micronucleated cells by apoptosis after treatment with inhibitors of microtubulesMutagenesis, Jul 2002; 17: 337 - 344.

Juan J. Muñoz, Luis M. Alonso-C., Rosa Sacedón, Tessa Cromp-ton, Angeles Vicente, Eva Jiménez, Alberto Varas, and Agustín G. Zapata Expression and Function of the Eph A Receptors and Their Ligands Ephrins A in the Rat ThymusJ. Immunol., Jul 2002; 169: 177 - 184.

Chin-Ju Tsi, Yee Chao, Ching-Wen Chen, and Wan Wan Lin Aurintricarboxylic Acid Protects against Cell Death Caused by Lipopolysaccharide in Macrophages by Decreasing Inducible Nitric-Oxide Synthase Induction via IB Kinase, Extracellular Sig-nal-Regulated Kinase, and p38 Mitogen-Activated Protein Kinase InhibitionMol. Pharmacol., Jul 2002; 62: 90 - 101.

PART VIII. NERVOUS SYSTEM AND ARTHRITIS:DAVID S. JESSOP, LOUISE J. RICHARDS, and MICHAEL S. HARBUZ Opioid Peptides Endomorphin-1 and Endomorphin-2 in the Immune System in Humans and in a Rodent Model of Inflam-mationAnn. N.Y. Acad. Sci., Jun 2002; 966: 456 - 463.

Lingge Lu, Kristina Holmqvist, Michael Cross, and Michael Welsh Role of the Src Homology 2 Domain-containing Protein Shb in Murine Brain Endothelial Cell Proliferation and DifferentiationCell Growth Differ., Mar 2002; 13: 141 - 148.

Veronica A. Tovar Sepulveda, Xiaoli Shen, and Miriam Falzon Intracrine PTHrP Protects against Serum Starvation-Induced Apoptosis and Regulates the Cell Cycle in MCF-7 Breast Cancer CellsEndocrinology, Feb 2002; 143: 596 - 606.

Lee Carpenter, Damien Cordery, and Trevor J. Biden Inhibition of Protein Kinase C Protects Rat INS-1 Cells Against Interleukin-1ß and Streptozotocin-Induced Apoptosis Diabetes, Feb 2002; 51: 317 - 324.

Yoshiki Yanagawa, Norifumi Iijima, Kazuya Iwabuchi, and Kazunori Onoé Activation of extracellular signal-related kinase by TNF- con-trols the maturation and function of murine dendritic cellsJ. Leukoc. Biol., Jan 2002; 71: 125 - 132.

Thomas D. Batiuk, Carol Schnizlein-Bick, Zoya Plotkin, and Pierre C. Dagher Guanine nucleosides and Jurkat cell death: roles of ATP deple-tion and accumulation of deoxyribonucleotidesAm J Physiol Cell Physiol, Dec 2001; 281: 1776 - 1784.

Annexin-V-FLUOS Staining Kit, Cat.-No. 11 858 777 001

Caroline Paris, Philippe M. Loiseau, Christian Bories, and Jaque-line Bréard Miltefosine Induces Apoptosis-Like Death in Leishmania dono-vani PromastigotesAntimicrob. Agents Chemother., Mar 2004; 48: 852 - 859.

Yayoi Kaneko, Kenji Kitazato, and Yuji Basaki Integrin-linked kinase regulates vascular morphogenesis induced by vascular endothelial growth factorJ. Cell Sci., Jan 2004; 117: 407 - 415.

Silke Appel, Andreas M. Boehmler, Frank Grünebach, Martin R. Müller, Anette Rupf, Markus M. Weck, Ulrike Hartmann, Volker L. Reichardt, Lothar Kanz, Tim H. Brümmendorf, and Peter Bros-sart Imatinib mesylate affects the development and function of den-dritic cells generated from CD34+ peripheral blood progenitor cellsBlood, Jan 2004; 103: 538 - 544.

Jianwu Pei and Thomas A. Ficht Brucella abortus Rough Mutants Are Cytopathic for Macroph-ages in CultureInfect. Immun., Jan 2004; 72: 440 - 450.

Rena Balzan, Karen Sapienza, Dolores R. Galea, Neville Vas-sallo, Hank Frey, and William H. Bannister Aspirin commits yeast cells to apoptosis depending on carbon sourceMicrobiology, Jan 2004; 150: 109 - 115.

Bing Z. Carter, Steven M. Kornblau, Twee Tsao, Rui-Yu Wang, Wendy D. Schober, Michele Milella, Hsi-Guang Sung, John C. Reed, and Michael Andreeff Caspase-independent cell death in AML: caspase inhibition in vitro with pan-caspase inhibitors or in vivo by XIAP or Survivin does not affect cell survival or prognosisBlood, Dec 2003; 102: 4179 - 4186.

Nicola A. Johnson, Shiladitya Sengupta, Samir A. Saidi, Khasha-yar Lessan, Stephen D. Charnock-Jones, Laurie Scott, Richard Stephens, Tom C. Freeman, Brian D. M. Tom, Michael Harris, Gareth Denyer, Mallik Sundaram, Ram Sasisekharan, Stephen K. Smith, and Cristin G. Print Endothelial cells preparing to die by apoptosis initiate a pro-gram of transcriptome and glycome regulationFASEB J, Nov 2003; 10.1096/fj.03-0097fje.

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Alessio Nencioni, Kirsten Lauber, Frank Grünebach, Luk Van Parijs, Claudio Denzlinger, Sebastian Wesselborg, and Peter Brossart Cyclopentenone Prostaglandins Induce Lymphocyte Apoptosis by Activating the Mitochondrial Apoptosis Pathway Indepen-dent of External Death Receptor Signaling J. Immunol., Nov 2003; 171: 5148 - 5156.

Francesco D'Alo', Lisa M. Johansen, Erik A. Nelson, Hanna S. Radomska, Erica K. Evans, Pu Zhang, Claus Nerlov, and Daniel G. Tenen The amino terminal and E2F interaction domains are critical for C/EBP-�mediated induction of granulopoietic development of hematopoietic cellsBlood, Nov 2003; 102: 3163 - 3171.

Martin Sattler, Yuri B. Pride, Patrick Ma, Jessica L. Gramlich, Stephanie C. Chu, Laura A. Quinnan, Sheri Shirazian, Congxin Liang, Klaus Podar, James G. Christensen, and Ravi Salgia A Novel Small Molecule Met Inhibitor Induces Apoptosis in Cells Transformed by the Oncogenic TPR-MET Tyrosine KinaseCancer Res., Sep 2003; 63: 5462 - 5469.

Toshiaki Hayashi, Teru Hideshima, Masaharu Akiyama, Noopur Raje, Paul Richardson, Dharminder Chauhan, and Kenneth C. Anderson Ex vivo induction of multiple myeloma–specific cytotoxic T lymphocytesBlood, Aug 2003; 102: 1435 - 1442.

Guido Janeke, Wilfried Siefken, Stefanie Carstensen, Gunja Springmann, Oliver Bleck, Hans Steinhart, Peter Höger, Klaus-Peter Wittern, Horst Wenck, Franz Stäb, Gerhard Sauermann, Volker Schreiner, and Thomas Doering Role of Taurine Accumulation in Keratinocyte HydrationJ. Invest. Dermatol., Aug 2003; 121: 354 - 361.

Howard Clark, Nades Palaniyar, Peter Strong, Jess Edmondson, Samuel Hawgood, and Kenneth B. M. Reid Surfactant Protein D Reduces Alveolar Macrophage Apoptosis In VivoJ. Immunol., Sep 2002; 169: 2892 - 2899.

Yoichiro Kakugawa, Tadashi Wada, Kazunori Yamaguchi, Hide-aki Yamanami, Kiyoaki Ouchi, Ikuro Sato, and Taeko Miyagi Up-regulation of plasma membrane-associated ganglioside sialidase (Neu3) in human colon cancer and its involvement in apoptosis suppressionPNAS, Aug 2002; 99: 10718 - 10723.

Toshiaki Hayashi, Teru Hideshima, Masaharu Akiyama, Paul Richardson, Robert L. Schlossman, Dharminder Chauhan, Nikhil C. Munshi, Samuel Waxman, and Kenneth C. Anderson Arsenic Trioxide Inhibits Growth of Human Multiple Myeloma Cells in the Bone Marrow MicroenvironmentMol. Cancer Ther., Aug 2002; 1: 851 - 860.

Volker Brinkmann, Michael D. Davis, Christopher E. Heise, Rainer Albert, Sylvain Cottens, Robert Hof, Christian Bruns, Eva Prieschl, Thomas Baumruker, Peter Hiestand, Carolyn A. Foster, Markus Zollinger, and Kevin R. Lynch The Immune Modulator FTY720 Targets Sphingosine 1-Phos-phate ReceptorsJ. Biol. Chem., Jun 2002; 277: 21453 - 21457.

Srikanth Yellayi, Afia Naaz, Melissa A. Szewczykowski, Tomomi Sato, Jeffrey A. Woods, Jongsoo Chang, Mariangela Segre, Clint D. Allred, William G. Helferich, and Paul S. Cooke The phytoestrogen genistein induces thymic and immune changes: A human health concern?PNAS, May 2002; 99: 7616 - 7621.

Fabrice Bureau, Alain Vanderplasschen, Fabrice Jaspar, Frédéric Minner, Paul-Pierre Pastoret, Marie-Paule Merville, Vincent Bours, and Pierre Lekeux Constitutive nuclear factor-�B activity preserves homeostasis of quiescent mature lymphocytes and granulocytes by controlling the expression of distinct Bcl-2 family proteinsBlood, May 2002; 99: 3683 - 3691.

S. Radulovic, P. W. Price, M. S. Beier, J. Gaywee, J. A. Macaluso, and A. Azad Rickettsia-Macrophage Interactions: Host Cell Responses to Rickettsia akari and Rickettsia typhi Infect. Immun., May 2002; 70: 2576 - 2582.

Han-Ming Shen, Jun Dai, Sin-Eng Chia, Alvin Lim, and Choon-Nam Ong Detection of apoptotic alterations in sperm in subfertile patients and their correlations with sperm qualityHum. Reprod., May 2002; 17: 1266 - 1273.

Claudio Hetz, María Rosa Bono, Luis Felipe Barros, and Rosalba Lagos Microcin E492, a channel-forming bacteriocin from Klebsiella pneumoniae, induces apoptosis in some human cell linesPNAS, Mar 2002; 99: 2696 - 2701.

Min Huang, Piotr Kozlowski, Matthew Collins, Yanhong Wang, Timothy A. Haystead, and Lee M. Graves Caspase-Dependent Cleavage of Carbamoyl Phosphate Synthetase II during ApoptosisMol. Pharmacol., Mar 2002; 61: 569 - 577.

Kaoruko Tada Iida, Hiroaki Suzuki, Hirohito Sone, Hitoshi Shimano, Hideo Toyoshima, Shigeru Yatoh, Tomoichiro Asano, Yukichi Okuda, and Nobuhiro Yamada Insulin Inhibits Apoptosis of Macrophage Cell Line, THP-1 Cells, via Phosphatidylinositol-3-Kinase–Dependent PathwayArterioscler. Thromb. Vasc. Biol., Mar 2002; 22: 380 - 386.

Robert A. Mitchell, Hong Liao, Jason Chesney, Gunter Fingerle-Rowson, John Baugh, John David, and Richard Bucala Macrophage migration inhibitory factor (MIF) sustains mac-rophage proinflammatory function by inhibiting p53: Regulatory role in the innate immune responsePNAS, Jan 2002; 99: 345 - 350.

Taro Matsumoto, Ingela Turesson, Majlis Book, Pär Gerwins, and Lena Claesson-Welsh p38 MAP kinase negatively regulates endothelial cell survival, proliferation, and differentiation in FGF-2–stimulated angiogen-esisJ. Cell Biol., Jan 2002; 156: 149 - 160.

Annexin-V-Alexa 568, Cat.-No. 11 985 485 001(new Cat.-No. 03 703 126 001)

Rauf Latif, Nicole Kerlero de Rosbo, Tany Amarant, Rino Rap-puoli, Gregor Sappler, and Avraham Ben-NunReversal of the CD4+/CD8+ T-Cell Ratio in Lymph Node Cells upon In Vitro Mitogenic Stimulation by Highly Purified, Water-Soluble S3-S4 Dimer of Pertussis ToxinInfect. Immun., May 2001; 69: 3073 - 3081.

Felipe Diaz-Griffero, Steven A. Hoschander, and Jürgen Bro-jatschBystander Killing during Avian Leukosis Virus Subgroup B Infection Requires TVBS3 SignalingJ. Virol., Dec 2003; 77: 12552 - 12561.

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CC141Appendix

Hong-Lin Su, Ching-Len Liao, and Yi-Ling LinJapanese Encephalitis Virus Infection Initiates Endoplasmic Reticulum Stress and an Unfolded Protein ResponseJ. Virol., May 2002; 76: 4162 - 4171.

Amir Abbas Samani, Eric Chevet, Lucia Fallavollita, Jacques Galipeau, and Pnina BrodtLoss of Tumorigenicity and Metastatic Potential in Carcinoma Cells Expressing the Extracellular Domain of the Type 1 Insulin-Like Growth Factor ReceptorCancer Res., May 2004; 64: 3380 - 3385.

Yu-Hauh Wu, Sheue-Fang Shih, and Jung-Yaw LinRicin Triggers Apoptotic Morphological Changes through Caspase-3 Cleavage of BAT3J. Biol. Chem., Apr 2004; 279: 19264 - 19275.

Simona Caporali, Manami Imai, Lucia Altucci, Massimo Cancemi, Silvana Caristi, Luigi Cicatiello, Filomena Matarese, Roberta Penta, Dipak K. Sarkar, Francesco Bresciani, and Alessandro WeiszDistinct Signaling Pathways Mediate Stimulation of Cell Cycle Progression and Prevention of Apoptotic Cell Death by Estrogen in Rat Pituitary Tumor PR1 CellsMol. Biol. Cell, Dec 2003; 14: 5051 - 5059.

Alessio Cardinale, Ilaria Filesi, Sonia Mattei, and Silvia BioccaEvidence for proteasome dysfunction in cytotoxicity mediated by anti-Ras intracellular antibodiesEur. J. Biochem., Aug 2003; 270: 3389 - 3397.

Kamel Izeradjene, Jean-Pierre Revillard, and Laurent GenestierInhibition of thymidine synthesis by folate analogues induces a Fas–Fas ligand-independent deletion of superantigen-reactive peripheral T cellsInt. Immunol., Jan 2001; 13: 85 - 93.

Jaleh Doostzadeh-Cizeron, Shenmin Yin, and David W. Goo-drichApoptosis Induced by the Nuclear Death Domain Protein p84N5 Is Associated with Caspase-6 and NF-�B ActivationJ. Biol. Chem., Aug 2000; 275: 25336 - 25341.

Gautam Bandyopadhyay, Tanusree Biswas, Keshab C Roy, Swapan Mandal, Chhabinath Mandal, Bikas C Pal, Samir Bhattacharya, Srabanti Rakshit, Dilip K Bhattacharya, Utpal Chaudhuri, Aditya Konar, and Santu BandyopadhyayChlorogenic acid inhibits Bcr-Abl tyrosine kinase and triggers p38 mitogen-activated protein kinase-dependent apoptosis in chronic myelogenous leukemic cellsBlood, Jun 2004; 10.1182/blood-2003-11-4065.

Hyungshin Yim, Ying Hua Jin, Byoung Duck Park, Hye Jin Choi, and Seung Ki LeeCaspase-3-mediated Cleavage of Cdc6 Induces Nuclear Local-ization of p49-truncated Cdc6 and ApoptosisMol. Biol. Cell, Oct 2003; 14: 4250 - 4259.

Dominique Garcin, Joseph Curran, Masae Itoh, and Daniel KolakofskyLonger and Shorter Forms of Sendai Virus C Proteins Play Dif-ferent Roles in Modulating the Cellular Antiviral ResponseJ. Virol., Aug 2001; 75: 6800 - 6807.

Yaron Shav-Tal, Michal Cohen, Smadar Lapter, Billy Dye, James G. Patton, Joel Vandekerckhove, and Dov ZiporiNuclear Relocalization of the Pre-mRNA Splicing Factor PSF during Apoptosis Involves Hyperphosphorylation, Masking of Antigenic Epitopes, and Changes in Protein InteractionsMol. Biol. Cell, Aug 2001; 12: 2328 - 2340.

Jaleh Doostzadeh-Cizeron, Nicholas H. A. Terry, and David W. GoodrichThe Nuclear Death Domain Protein p84N5 Activates a G2/M Cell Cycle Checkpoint Prior to the Onset of ApoptosisJ. Biol. Chem., Jan 2001; 276: 1127 - 1132.

Helena Helmby, Gun Jönsson, and Marita Troye-BlombergCellular Changes and Apoptosis in the Spleens and Peripheral Blood of Mice Infected with Blood-Stage Plasmodium chabaudi chabaudi ASInfect. Immun., Mar 2000; 68: 1485 - 1490.

Frank Thévenod, Jenny M. Friedmann, Alice D. Katsen, and Ingeborg A. HauserUp-regulation of Multidrug Resistance P-glycoprotein via Nuclear Factor-�B Activation Protects Kidney Proximal Tubule Cells from Cadmium- and Reactive Oxygen Species-induced ApoptosisJ. Biol. Chem., Jan 2000; 275: 1887 - 1896.

Annexin-V-Biotin, Cat.-No. 11 828 690 001

Mounira Djerbi, Khairul-Bariah Abdul-Majid, Manuchehr Abedi-Valugerdi, Tomas Olsson, Robert A. Harris, and Alf Grandien Expression of the Long Form of Human FLIP by Retroviral Gene Transfer of Hemopoietic Stem Cells Exacerbates Experimental Autoimmune Encephalomyelitis J. Immunol., Feb 2003; 170: 2064 - 2073.

Tom Taghon, Magda De Smedt, Frank Stolz, Maggy Cnockaert, Jean Plum, and Georges Leclercq Enforced Expression of GATA-3 Severely Reduces Human Thy-mic CellularityJ. Immunol., Oct 2001; 167: 4468 - 4475.

Elad Katz, Maureen R. Deehan, Sandra Seatter, Caroline Lord, Roger D. Sturrock, and Margaret M. Harnett B Cell Receptor-Stimulated Mitochondrial Phospholipase A2 Activation and Resultant Disruption of Mitochondrial Mem-brane Potential Correlate with the Induction of Apoptosis in WEHI-231 B CellsJ. Immunol., Jan 2001; 166: 137 - 147.

Masako Nishizawa, Masakazu Kamata, Ryoichi Katsumata, and Yoko Aida A Carboxy-Terminally Truncated Form of the Human Immunode-ficiency Virus Type 1 Vpr Protein Induces Apoptosis via G1 Cell Cycle ArrestJ. Virol., Jul 2000; 74: 6058 - 6067.

Francesco Puppo, Paola Contini, Massimo Ghio, Sabrina Brenci, Marco Scudeletti, Gilberto Filaci, Soldano Ferrone, and Francesco Indiveri Soluble human MHC class I molecules induce soluble Fas ligand secretion and trigger apoptosis in activated CD8+ Fas (CD95)+ T lymphocytesInt. Immunol., Feb 2000; 12: 195 - 203.

Josefa Blanco-Rodríguez and Carmen Martínez-García Apoptosis Is Physiologically Restricted to a Specialized Cyto-plasmic Compartment in Rat SpermatidsBiol. Reprod., Dec 1999; 61: 1541 - 1547.

Bruno Verhasselt, Evelien Naessens, Chris Verhofstede, Magda De Smedt, Sigrid Schollen, Tessa Kerre, Dominique Vanhecke, and Jean Plum Human Immunodeficiency Virus nef Gene Expression Affects Generation and Function of Human T Cells, But Not Dendritic CellsBlood, Oct 1999; 94: 2809 - 2818.

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Cytotoxicity Detection Kit (LDH), Cat.-No. 11 644 793 001

Markus Meissner, Monika Stein, Carmen Urbich, Kerstin Reis-inger, Guntram Suske, Bart Staels, Roland Kaufmann, and Jens Gille PPAR Activators Inhibit Vascular Endothelial Growth Factor Receptor-2 Expression by Repressing Sp1-Dependent DNA Binding and TransactivationCirc. Res., Feb 2004; 94: 324 - 332.

Sabine Morand, Valérie Buchillier, Fabienne Maurer, Christophe Bonny, Yvan Arsenijevic, Francis L. Munier, and Daniel F. Schor-deret Induction of Apoptosis in Human Corneal and HeLa Cells by Mutated BIGH3Invest. Ophthalmol. Vis. Sci., Jul 2003; 44: 2973 - 2979.

Alberto Chiarugi, Elena Meli, Maura Calvani, Roberta Picca, Roberto Baronti, Emidio Camaioni, Gabriele Costantino, Maura Marinozzi, Domenico E. Pellegrini-Giampietro, Roberto Pellic-ciari, and Flavio Moroni Novel Isoquinolinone-Derived Inhibitors of Poly(ADP-ribose) Polymerase-1: Pharmacological Characterization and Neuro-protective Effects in an in Vitro Model of Cerebral IschemiaJ. Pharmacol. Exp. Ther., Jun 2003; 305: 943 - 949.

Nicholas Mitsiades, Constantine S. Mitsiades, Paul G. Richard-son, Ciaran McMullan, Vassiliki Poulaki, Galinos Fanourakis, Robert Schlossman, Dharminder Chauhan, Nikhil C. Munshi, Teru Hideshima, Victoria M. Richon, Paul A. Marks, and Kenneth C. Anderson Molecular sequelae of histone deacetylase inhibition in human malignant B cellsBlood, May 2003; 101: 4055 - 4062.

K Wosik, J Antel, T Kuhlmann, W Bruck, B Massie, and J Nal-bantoglu Oligodendrocyte injury in multiple sclerosis: a role for p53.J Neurochem, May 2003; 85(3): 635-44.

Alexandra Aicher, Winfried Brenner, Maaz Zuhayra, Cornel Badorff, Schirin Massoudi, Birgit Assmus, Thomas Eckey, Eberhard Henze, Andreas M. Zeiher, and Stefanie Dimmeler Assessment of the Tissue Distribution of Transplanted Human Endothelial Progenitor Cells by Radioactive LabelingCirculation, Apr 2003; 107: 2134 - 2139.

Raquel Blanco, Luis Carrasco, and Iván Ventoso Cell Killing by HIV-1 ProteaseJ. Biol. Chem., Jan 2003; 278: 1086 - 1093.

Nicholas Mitsiades, Constantine S. Mitsiades, Vassiliki Poulaki, Dharminder Chauhan, Galinos Fanourakis, Xuesong Gu, Charles Bailey, Marie Joseph, Towia A. Libermann, Steven P. Treon, Nikhil C. Munshi, Paul G. Richardson, Teru Hideshima, and Kenneth C. Anderson Molecular sequelae of proteasome inhibition in human multiple myeloma cellsPNAS, Oct 2002; 99: 14374 - 14379.

Alberto Chiarugi, Giovanni Mario Pitari, Rosa Costa, Margherita Ferrante, Loredana Villari, Matilde Amico-Roxas, Théophile Godfraind, Alfredo Bianchi, and Salvatore Salomone Effect of prolonged incubation with copper on endothelium-dependent relaxation in rat isolated aortaBr. J. Pharmacol., Aug 2002; 136: 1185 - 1193.

Eduardo Correa-Meyer, Liuska Pesce, Carmen Guerrero, and Jacob I. Sznajder Mechanotransduction in the Lung: Cyclic stretch activates ERK1/2 via G proteins and EGFR in alveolar epithelial cellsAm J Physiol Lung Cell Mol Physiol, May 2002; 282: 883 - 891.

RUI F.M. SILVA, CECÍLIA M.P. RODRIGUES, and DORA BRITES Rat Cultured Neuronal and Glial Cells Respond Differently to Toxicity of Unconjugated BilirubinPediatr. Res., Apr 2002; 51: 535 - 541.

Jonathan G. Crowston, Lydia H. Chang, Peter H. Constable, Julie T. Daniels, Arne N. Akbar, and Peng T. Khaw Apoptosis Gene Expression and Death Receptor Signaling in Mitomycin-C–Treated Human Tenon Capsule FibroblastsInvest. Ophthalmol. Vis. Sci., Mar 2002; 43: 692 - 699.

M Nakajima, M Miura, T Aosaki, and T Shirasawa Deficiency of presenilin-1 increases calcium-dependent vulner-ability of neurons to oxidative stress in vitro.J Neurochem, Aug 2001; 78(4): 807-14.

Monika Raisova, Amir M. Hossini, Jürgen Eberle, Christian Riebeling, Thomas Wieder, Isrid Sturm, Peter T. Daniel, Constantin E. Orfanos, and Christoph C. Geilen The Bax/Bcl-2 Ratio Determines the Susceptibility of Human Melanoma Cells to CD95/Fas-Mediated ApoptosisJ. Invest. Dermatol., Aug 2001; 117: 333 - 340.

Stig O.P. Jacobsson and Christopher J. Fowler Characterization of palmitoylethanolamide transport in mouse Neuro-2a neuroblastoma and rat RBL-2H3 basophilic leu-kaemia cells: comparison with anandamideBr. J. Pharmacol., Apr 2001; 132: 1743 - 1754.

HANS-PETER DEIGNER, RALF CLAUS, GABRIEL A. BONATERRA, CHRISTOF GEHRKE, NILOFAR BIBAK, MARKUS BLAESS, MICHAEL CANTZ, JÜRGEN METZ, and RALF KINSCHERF Ceramide induces aSMase expression: implications for oxLDL-induced apoptosisFASEB J, Mar 2001; 15: 807 - 814.

Akihiko Hirata, Masahiko Igarashi, Hiroshi Yamaguchi, Akira Suwabe, Makoto Daimon, Takeo Kato, and Makoto Tominaga Nifedipine suppresses neointimal thickening by its inhibitory effect on vascular smooth muscle cell growth via a MEK-ERK pathway coupling with Pyk2Br. J. Pharmacol., Dec 2000; 131: 1521 - 1530.

Nicolas Ancellin, Chantal Colmont, Joseph Su, Qin Li, Nanette Mittereder, Sung-Suk Chae, Steingrimur Stefansson, Gene Liau, and Timothy Hla Extracellular Export of Sphingosine Kinase-1 Enzyme. SPHIN-GOSINE 1-PHOSPHATE GENERATION AND THE INDUCTION OF ANGIOGENIC VASCULAR MATURATIONJ. Biol. Chem., Feb 2002; 277: 6667 - 6675.

Cristina Cebrián, Cristina Aresté, Antoni Nicolás, Pere Olivé, Ana Carceller, Jaume Piulats, and Anna Meseguer Kidney Androgen-regulated Protein Interacts with Cyclophilin B and Reduces Cyclosporine A-mediated Toxicity in Proximal Tubule CellsJ. Biol. Chem., Jul 2001; 276: 29410 - 29419.

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CC143Appendix

Silke Reinartz, Siegmund Köhler, Harald Schlebusch, Karl Krista, Patrick Giffels, Kirsten Renke, Jens Huober, Volker Möbus, Rolf Kreienberg, Andreas duBois, Paul Sabbatini, and Uwe Wagner Vaccination of Patients with Advanced Ovarian Carcinoma with the Anti-Idiotype ACA125: Immunological Response and Sur-vival (Phase Ib/II)Clin. Cancer Res., Mar 2004; 10: 1580 - 1587.

G. Zhang, P. H. H. Lopez, C. Y. Li, N. R. Mehta, J. W. Griffin, R. L. Schnaar, and K. A. Sheikh Anti-ganglioside antibody-mediated neuronal cytotoxicity and its protection by intravenous immunoglobulin: implications for immune neuropathiesBrain, Feb 2004; 10.1093/brain/awh127.

Rebecca R. Foster, Rachel Hole, Karen Anderson, Simon C. Satchell, Richard J. Coward, Peter W. Mathieson, David A. Gillatt, Moin A. Saleem, David O. Bates, and Steven J. Harper Functional evidence that vascular endothelial growth factor may act as an autocrine factor on human podocytesAm J Physiol Renal Physiol, Jun 2003; 284: 1263 - 1273.

E C Clark, S D Patel, P R Chadwick, G Warhurst, A Curry, and G L Carlson Glutamine deprivation facilitates tumour necrosis factor induced bacterial translocation in Caco-2 cells by depletion of enterocyte fuel substrateGut, Feb 2003; 52: 224 - 230.

Andreas C. Tomac, Alan D. Agulnick, Norman Haughey, Chen-Fu Chang, Yajun Zhang, Cristina Bäckman, Marisela Morales, Mark P. Mattson, Yun Wang, Heiner Westphal, and Barry J. Hoffer Effects of cerebral ischemia in mice deficient in PersephinPNAS, Jul 2002; 99: 9521 - 9526.

Jill C. Todt, Bin Hu, Antonello Punturieri, Joanne Sonstein, Timo-thy Polak, and Jeffrey L. Curtis Activation of Protein Kinase C II by the Stereo-specific Phos-phatidylserine Receptor Is Required for Phagocytosis of Apop-totic Thymocytes by Resident Murine Tissue MacrophagesJ. Biol. Chem., Sep 2002; 277: 35906 - 35914.

Cellular DNA Fragmentation ELISA, Cat.- No. 11 585 045 001

Makio Mogi and Akifumi Togari Activation of Caspases Is Required for Osteoblastic Differentia-tionJ. Biol. Chem., Nov 2003; 278: 47477 - 47482.

Luis Miguel Blanco-Colio, Begoña Muñoz-García, Jose Luis Martín-Ventura, Corina Lorz, Cristina Díaz, Gonzalo Hernández, and Jesús Egido 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Inhibitors Decrease Fas Ligand Expression and Cytotoxicity in Activated Human T LymphocytesCirculation, Sep 2003; 108: 1506 - 1513.

Yong-Taek Jun, Hee-Jung Kim, Min-Jin Song, Ji-Hyang Lim, Dong-Gun Lee, Kyung-Ja Han, Su-Mi Choi, Jin-Hong Yoo, Wan-Shik Shin, and Jung-Hyun Choi In Vitro Effects of Ciprofloxacin and Roxithromycin on Apoptosis of Jurkat T LymphocytesAntimicrob. Agents Chemother., Mar 2003; 47: 1161 - 1164.

Satoshi Hirakawa, Young-Kwon Hong, Natasha Harvey, Vivien Schacht, Kant Matsuda, Towia Libermann, and Michael Detmar Identification of Vascular Lineage-Specific Genes by Transcrip-tional Profiling of Isolated Blood Vascular and Lymphatic Endot-helial CellsAm. J. Pathol., Feb 2003; 162: 575 - 586.

Patrizia Russo, Dario Arzani, Sonya Trombino, and Carla Falugi c-myc Down-Regulation Induces Apoptosis in Human Cancer Cell Lines Exposed to RPR-115135 (C31H29NO4), a Non-Pepti-domimetic Farnesyltransferase InhibitorJ. Pharmacol. Exp. Ther., Jan 2003; 304: 37 - 47.

Antonia M. Joussen, Vassiliki Poulaki, Nicholas Mitsiades, Wen-yi Cai, Izumi Suzuma, John Pak, Shyr-Te Ju, Susan L. Rook, Peter Esser, Constantin Mitsiades, Bernd Kirchhof, Anthony P. Adamis, and Lloyd Paul Aiello Suppression of Fas-FasL-induced endothelial cell apoptosis prevents diabetic blood-retinal barrier breakdown in a model of streptozotocin-induced diabetesFASEB J, Nov 2002; 10.1096/fj.02-0157fje.

M Toyoda, H Takagi, N Horiguchi, S Kakizaki, K Sato, H Takayama, and M Mori A ligand for peroxisome proliferator activated receptor inhibits cell growth and induces apoptosis in human liver cancer cellsGut, Apr 2002; 50: 563 - 567.

Nicoletta Di Simone, Roberta Castellani, Dario Caliandro, and Alessandro Caruso Monoclonal Anti-Annexin V Antibody Inhibits Trophoblast Gonadotropin Secretion and Induces Syncytiotrophoblast Apo-ptosisBiol. Reprod., Dec 2001; 65: 1766 - 1770.

Jingxuan Pan, Guangpu Xu, and Sai-Ching Jim Yeung Cytochrome c Release Is Upstream to Activation of Caspase-9, Caspase-8, and Caspase-3 in the Enhanced Apoptosis of Ana-plastic Thyroid Cancer Cells Induced by Manumycin and Pacli-taxelJ. Clin. Endocrinol. Metab., Oct 2001; 86: 4731 - 4740.

Ching-Len Liao, Yi-Ling Lin, Bi-Ching Wu, Chang-Huei Tsao, Mei-Chuan Wang, Chiu-I Liu, Yue-Ling Huang, Jui-Hui Chen, Jia-Pey Wang, and Li-Kuang Chen Salicylates Inhibit Flavivirus Replication Independently of Block-ing Nuclear Factor Kappa B ActivationJ. Virol., Sep 2001; 75: 7828 - 7839.

Kei Yamamoto, Ryuichi Morishita, Shin-ichiro Hayashi, Hidet-sugu Matsushita, Hidenori Nakagami, Atsushi Moriguchi, Kunio Matsumoto, Toshikazu Nakamura, Yasufumi Kaneda, and Toshio Ogihara Contribution of Bcl-2, but Not Bcl-xL and Bax, to Antiapoptotic Actions of Hepatocyte Growth Factor in Hypoxia-Conditioned Human Endothelial CellsHypertension, May 2001; 37: 1341 - 1348.

Q. Chang and B. L. TeppermanThe role of protein kinase C isozymes in TNF--induced cytotox-icity to a rat intestinal epithelial cell lineAm J Physiol Gastrointest Liver Physiol, Apr 2001; 280: 572 - 583.

XiaoYing Li, Michela Marani, Roberta Mannucci, Berma Kinsey, Francesca Andriani, Ildo Nicoletti, Larry Denner, and Marco Marcelli Overexpression of BCL-XL Underlies the Molecular Basis for Resistance to Staurosporine-induced Apoptosis in PC-3 CellsCancer Res., Feb 2001; 61: 1699 - 1706.

Hidetsugu Matsushita, Ryuichi Morishita, Toshie Nata, Motokuni Aoki, Hironori Nakagami, Yoshiaki Taniyama, Kei Yamamoto, Jitsuo Higaki, Kaneda Yasufumi, and Toshio Ogihara Hypoxia-Induced Endothelial Apoptosis Through Nuclear Fac-tor-B (NF-B)–Mediated bcl-2 Suppression : In Vivo Evidence of the Importance of NF-B in Endothelial Cell RegulationCirc. Res., May 2000; 86: 974 - 981.

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Cell Proliferation

Cell Proliferation Kit I (MTT), Cat.-No. 11 465 007 001

Alexey V. Gordadze, Chisaroka W. Onunwor, RongSheng Peng, David Poston, Elisabeth Kremmer, and Paul D. Ling EBNA2 Amino Acids 3 to 30 Are Required for Induction of LMP-1 and Immortalization MaintenanceJ. Virol., Apr 2004; 78: 3919 - 3929.

K. Mimori, Y. Tanaka, K. Yoshinaga, T. Masuda, K. Yamashita, M. Okamoto, H. Inoue, and M. Mori Clinical significance of the overexpression of the candidate oncogene CYP24 in esophageal cancerAnn. Onc., Feb 2004; 15: 236 - 241.

Liang-Nian Song, Meghan Coghlan, and Edward P. GelmannAntiandrogen Effects of Mifepristone on Coactivator and Core-pressor Interactions with the Androgen ReceptorMol. Endocrinol., Jan 2004; 18: 70 - 85.

Christopher S. Stipp, Tatiana V. Kolesnikova, and Martin E. HemlerEWI-2 regulates 3ß1 integrin–dependent cell functions on laminin-5J. Cell Biol., Dec 2003; 163: 1167 - 1177.

Minji Jo, Keena S. Thomas, Lihua Wu, and Steven L. Gonias Soluble Urokinase-type Plasminogen Activator Receptor Inhib-its Cancer Cell Growth and Invasion by Direct Urokinase-inde-pendent Effects on Cell SignalingJ. Biol. Chem., Nov 2003; 278: 46692 - 46698.

Oliver Planz, Stephan Pleschka, Katja Oesterle, Friederike Berberich-Siebelt, Christina Ehrhardt, Lothar Stitz, and Stephan Ludwig Borna Disease Virus Nucleoprotein Interacts with the Cdc2-Cyclin B1 ComplexJ. Virol., Oct 2003; 77: 11186 - 11192.

Dean H. Hamer, Sven Bocklandt, Louise McHugh, Tae-Wook Chun, Peter M. Blumberg, Dina M. Sigano, and Victor E. Marquez Rational Design of Drugs That Induce Human Immunodefi-ciency Virus ReplicationJ. Virol., Oct 2003; 77: 10227 - 10236.

Santiago Canals, Maria José Casarejos, Sonsoles de Bernardo, Eulalia Rodríguez-Martín, and Maria Angeles Mena Nitric Oxide Triggers the Toxicity due to Glutathione Depletion in Midbrain Cultures through 12-LipoxygenaseJ. Biol. Chem., Jun 2003; 278: 21542 - 21549.

Yin Li, Anthony J. Raffo, Lisa Drew, Yuehua Mao, Andy Tran, Daniel P. Petrylak, and Robert L. Fine Fas-Mediated Apoptosis Is Dependent on Wild-Type p53 Status in Human Cancer Cells Expressing a Temperature-Sensitive p53 Mutant Alanine-143Cancer Res., Apr 2003; 63: 1527 - 1533.

Ulrike Krummrei, Etienne-Emile Baulieu, and Béatrice Chambraud The FKBP-associated protein FAP48 is an antiproliferative molecule and a player in T cell activation that increases IL2 synthesisPNAS, Mar 2003; 100: 2444 - 2449.

Karsten Spiekermann, Ralf J. Dirschinger, Ruth Schwab, Ksenia Bagrintseva, Florian Faber, Christian Buske, Susanne Schnittger, Louise M. Kelly, D. Gary Gilliland, and Wolfgang Hiddemann The protein tyrosine kinase inhibitor SU5614 inhibits FLT3 and induces growth arrest and apoptosis in AML-derived cell lines expressing a constitutively activated FLT3Blood, Feb 2003; 101: 1494 - 1504.

Jennifer J. Schlezinger, Brenda A. Jensen, Koren K. Mann, Heui-Young Ryu, and David H. Sherr Peroxisome Proliferator-Activated Receptor -Mediated NF-B Activation and Apoptosis in Pre-B CellsJ. Immunol., Dec 2002; 169: 6831 - 6841.

Louise McHugh, Stella Hu, B. K. Lee, Kenneth Santora, Paul E. Kennedy, Edward A. Berger, Ira Pastan, and Dean H. Hamer Increased Affinity and Stability of an Anti-HIV-1 Envelope Immunotoxin by Structure-based MutagenesisJ. Biol. Chem., Sep 2002; 277: 34383 - 34390.

Guoxiong Xu, Marie-Josée Guimond, Chandan Chakraborty, and Peeyush K. Lala Control of Proliferation, Migration, and Invasiveness of Human Extravillous Trophoblast by Decorin, a Decidual ProductBiol. Reprod., Aug 2002; 67: 681 - 689.

Seckho Ha, Sulra Lee, Myungil Chung, and Yunjaie Choi Mouse ING1 Homologue, a Protein Interacting with A1, Enhances Cell Death and Is Inhibited by A1 in Mammary Epi-thelial CellsCancer Res., Mar 2002; 62: 1275 - 1278.

Zhong Ma, Donna J. Webb, Minji Jo, and Steven L. Gonias Endogenously produced urokinase-type plasminogen activator is a major determinant of the basal level of activated ERK/MAP kinase and prevents apoptosis in MDA-MB-231 breast cancer cellsJ. Cell Sci., Sep 2001; 114: 3387 - 3396.

Alexey V. Gordadze, RongSheng Peng, Jie Tan, GuoZhen Liu, Richard Sutton, Bettina Kempkes, George W. Bornkamm, and Paul D. Ling Notch1IC Partially Replaces EBNA2 Function in B Cells Immor-talized by Epstein-Barr VirusJ. Virol., Jul 2001; 75: 5899 - 5912.

Wojciech Rzeski, Lechoslaw Turski, and Chrysanthy Ikonomidou From the Cover: Glutamate antagonists limit tumor growthPNAS, May 2001; 98: 6372 - 6377.

Marc Leng, Daniel Locker, Marie-Josèphe Giraud-Panis, Annie Schwartz, Francesco P. Intini, Giovanni Natile, Claudio Pisano, Angelina Boccarelli, Domenico Giordano, and Mauro Coluccia Replacement of an NH3 by an Iminoether in Transplatin Makes an Antitumor Drug from an Inactive CompoundMol. Pharmacol., Dec 2000; 58: 1525 - 1535.

Chaoyong Zhu, Magnus Johansson, and Anna Karlsson Incorporation of Nucleoside Analogs into Nuclear or Mitochon-drial DNA Is Determined by the Intracellular Phosphorylation SiteJ. Biol. Chem., Aug 2000; 275: 26727 - 26731.

Toshihiro Nanki and Peter E. Lipsky Cutting Edge: Stromal Cell-Derived Factor-1 Is a Costimulator for CD4+ T Cell ActivationJ. Immunol., May 2000; 164: 5010 - 5014.

Cell Proliferation Kit II (XTT), Cat.-No. 11 465 015 001

Evguenia S. Svarovskaia, Rebekah Barr, Xuechun Zhang, Godwin C. G. Pais, Christophe Marchand, Yves Pommier, Terrence R. Burke, Jr., and Vinay K. Pathak Azido-Containing Diketo Acid Derivatives Inhibit Human Immu-nodeficiency Virus Type 1 Integrase In Vivo and Influence the Frequency of Deletions at Two-Long-Terminal-Repeat-Circle JunctionsJ. Virol., Apr 2004; 78: 3210 - 3222.

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Y Tsunemitsu, S Kagawa, N Tokunaga, S Otani, T Umeoka, J A Roth, B Fang, N Tanaka, and T Fujiwara Molecular therapy for peritoneal dissemination of xenotrans-planted human MKN-45 gastric cancer cells with adenovirus mediated Bax gene transferGut, Apr 2004; 53: 554 - 560.

Harutaka Katano, Lesley Pesnicak, and Jeffrey I. Cohen Simvastatin induces apoptosis of Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines and delays development of EBV lymphomasPNAS, Mar 2004; 10.1073/pnas.0305149101.

Federica Piccioni, Benjamin R. Roman, Kenneth H. Fischbeck, and J. Paul Taylor A screen for drugs that protect against the cytotoxicity of poly-glutamine-expanded androgen receptorHum. Mol. Genet., Feb 2004; 13: 437 - 446.

Shuhong Wu, Hongbo Zhu, Jian Gu, Lidong Zhang, Fuminori Teraishi, John J. Davis, Dietmar A. Jacob, and Bingliang Fang Induction of Apoptosis and Down-Regulation of Bcl-XL in Cancer Cells by a Novel Small Molecule, 2[ [3-(2,3-Dichlo-rophenoxy)propyl]amino]ethanolCancer Res., Feb 2004; 64: 1110 - 1113.

Takeshi Kawashima, Shunsuke Kagawa, Naoya Kobayashi, Yoshiko Shirakiya, Tatsuo Umeoka, Fuminori Teraishi, Masaki Taki, Satoru Kyo, Noriaki Tanaka, and Toshiyoshi Fujiwara Telomerase-Specific Replication-Selective Virotherapy for Human CancerClin. Cancer Res., Jan 2004; 10: 285 - 292.

Ada Girnita, Leonard Girnita, Fabrizio del Prete, Armando Bartolazzi, Olle Larsson, and Magnus Axelson Cyclolignans as Inhibitors of the Insulin-Like Growth Factor-1 Receptor and Malignant Cell GrowthCancer Res., Jan 2004; 64: 236 - 242.

Stefania Fontana, Daniele Moratto, Surinder Mangal, Maria De Francesco, William Vermi, Simona Ferrari, Fabio Facchetti, Necil Kutukculer, Claudia Fiorini, Marzia Duse, Pranab K. Das, Luigi D. Notarangelo, Alessandro Plebani, and Raffaele Badolato Functional defects of dendritic cells in patients with CD40 defi-ciencyBlood, Dec 2003; 102: 4099 - 4106.

Weibo Zhou, P. Jeanette Simpson, Jill M. McFadden, Craig A. Townsend, Susan M. Medghalchi, Aravinda Vadlamudi, Michael L. Pinn, Gabriele V. Ronnett, and Francis P. Kuhajda Fatty Acid Synthase Inhibition Triggers Apoptosis during S Phase in Human Cancer CellsCancer Res., Nov 2003; 63: 7330 - 7337.

Ming-Yu Cao, Yoon Lee, Ning-Ping Feng, Keyong Xiong, Hong-nan Jin, Ming Wang, Aikaterini Vassilakos, Stéphane Viau, Jim A. Wright, and Aiping H. Young Adenovirus-Mediated Ribonucleotide Reductase R1 Gene Therapy of Human Colon AdenocarcinomaClin. Cancer Res., Oct 2003; 9: 4553 - 4561.

Akira Asai, Yuko Oshima, Yoshihiro Yamamoto, Taka-aki Uochi, Hideaki Kusaka, Shiro Akinaga, Yoshinori Yamashita, Krisztina Pongracz, Ronald Pruzan, Ellen Wunder, Mieczyslaw Piatyszek, Shihong Li, Allison C. Chin, Calvin B. Harley, and Sergei Gryaznov A Novel Telomerase Template Antagonist (GRN163) as a Poten-tial Anticancer AgentCancer Res., Jul 2003; 63: 3931 - 3939.

Leonard Girnita, Ada Girnita, and Olle Larsson Mdm2-dependent ubiquitination and degradation of the insu-lin-like growth factor 1 receptorPNAS, Jul 2003; 100: 8247 - 8252.

Masaru Nawa, Tomohiko Takasaki, Ken-Ichiro Yamada, Ichiro Kurane, and Toshitaka Akatsuka Interference in Japanese encephalitis virus infection of Vero cells by a cationic amphiphilic drug, chlorpromazineJ. Gen. Virol., Jul 2003; 84: 1737 - 1741.

Shivanand P. Lad, Daniel A. Peterson, Ralph A. Bradshaw, and Kenneth E. Neet Individual and Combined Effects of TrkA and p75NTR Nerve Growth Factor Receptors: A ROLE FOR THE HIGH AFFINITY RECEPTOR SITEJ. Biol. Chem., Jun 2003; 278: 24808 - 24817.

Silke Meiners, Dirk Heyken, Andrea Weller, Antje Ludwig, Karl Stangl, Peter-M. Kloetzel, and Elke Krüger Inhibition of Proteasome Activity Induces Concerted Expression of Proteasome Genes and de Novo Formation of Mammalian ProteasomesJ. Biol. Chem., Jun 2003; 278: 21517 - 21525.

Congxiao Zhang, Judit Baffi, Scott W. Cousins, and Karl G. Csaky Oxidant-induced cell death in retinal pigment epithelium cells mediated through the release of apoptosis-inducing factorJ. Cell Sci., May 2003; 116: 1915 - 1923.

Chengchen Lufei, Jing Ma, Guochang Huang, Tong Zhang, Veronica Novotny-Diermayr, Chin Thing Ong, and Xinmin Cao GRIM-19, a death-regulatory gene product, suppresses Stat3 activity via functional interactionEMBO J., Mar 2003; 22: 1325 - 1335.

J. Blake Marriott, Ian A. Clarke, Anna Czajka, Keith Dredge, Kay Childs, Hon-Wah Man, Peter Schafer, Sowmya Govinda, George W. Muller, David I. Stirling, and Angus G. Dalgleish A Novel Subclass of Thalidomide Analogue with Anti-Solid Tumor Activity in Which Caspase-dependent Apoptosis is Asso-ciated with Altered Expression of bcl-2 Family ProteinsCancer Res., Feb 2003; 63: 593 - 599.

Hiroaki Kawasaki and Kazunari Taira Short hairpin type of dsRNAs that are controlled by tRNAVal promoter significantly induce RNAi-mediated gene silencing in the cytoplasm of human cellsNucleic Acids Res., Jan 2003; 31: 700 - 707.

Peter Y. Hahn, Scott E. Evans, Theodore J. Kottom, Joseph E. Standing, Richard E. Pagano, and Andrew H. Limper Pneumocystis carinii Cell Wall -Glucan Induces Release of Macrophage Inflammatory Protein-2 from Alveolar Epithelial Cells via a Lactosylceramide-mediated MechanismJ. Biol. Chem., Jan 2003; 278: 2043 - 2050.

Akihito Ishigami, Toshiko Fujita, Setsuko Handa, Takuji Shirasawa, Haruhiko Koseki, Tsuneo Kitamura, Nobuyuki Enomoto, Nobuhiro Sato, Tatsuo Shimosawa, and Naoki Maruyama Senescence Marker Protein-30 Knockout Mouse Liver Is Highly Susceptible to Tumor Necrosis Factor-- and Fas-Mediated Apo-ptosisAm. J. Pathol., Oct 2002; 161: 1273 - 1281.

NELSON ARISPE and MICHAEL DOH Plasma membrane cholesterol controls the cytotoxicity of Alzheimer’s disease AßP (1–40) and (1–42) peptidesFASEB J, Oct 2002; 16: 1526 - 1536.

Yuntao Xie, Björn Skytting, Gunnar Nilsson, Alessandra Gas-barri, Karl Haslam, Armando Bartolazzi, Bertha Brodin, Nils Mandahl, and Olle Larsson SYT-SSX Is Critical for Cyclin D1 Expression in Synovial Sar-coma Cells: A Gain of Function of the t(X;18)(p11.2;q11.2) TranslocationCancer Res., Jul 2002; 62: 3861 - 3867.

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Tongyu Lin, Jian Gu, Lidong Zhang, Xuefeng Huang, L. Clifton Stephens, Steven A. Curley, and Bingliang Fang Targeted Expression of Green Fluorescent Protein/Tumor Necrosis Factor-related Apoptosis-inducing Ligand Fusion Protein from Human Telomerase Reverse Transcriptase Promoter Elicits Antitumor Activity without Toxic Effects on Primary Human HepatocytesCancer Res., Jul 2002; 62: 3620 - 3625.

Shu-Fen Wu, Chyan-Jang Lee, Ching-Len Liao, Raymond A. Dwek, Nicole Zitzmann, and Yi-Ling Lin Antiviral Effects of an Iminosugar Derivative on Flavivirus InfectionsJ. Virol., Apr 2002; 76: 3596 - 3604.

Cell Proliferation Reagent WST-1, Cat.-No. 11 644 807 001

Dong Cho Han, Mi-Young Lee, Ki Deok Shin, Sun Bok Jeon, Jung Min Kim, Kwang-Hee Son, Hyoung-Chin Kim, Hwan-Mook Kim, and Byoung-Mog Kwon 2'-Benzoyloxycinnamaldehyde Induces Apoptosis in Human Carcinoma via Reactive Oxygen SpeciesJ. Biol. Chem., Feb 2004; 279: 6911 - 6920.

Chang Han, Jing Leng, A. Jake Demetris, and Tong Wu Cyclooxygenase-2 Promotes Human Cholangiocarcinoma Growth: Evidence for Cyclooxygenase-2-Independent Mecha-nism in Celecoxib-Mediated Induction of p21waf1/cip1 and p27kip1 and Cell Cycle ArrestCancer Res., Feb 2004; 64: 1369 - 1376.

Lei Li, Zhiwei Feng, and Alan G. Porter JNK-dependent Phosphorylation of c-Jun on Serine 63 Medi-ates Nitric Oxide-induced Apoptosis of Neuroblastoma CellsJ. Biol. Chem., Feb 2004; 279: 4058 - 4065.

Alexias Safi, Marie Vandromme, Sabine Caussanel, Laure Valdacci, Dominique Baas, Marc Vidal, Gilbert Brun, Laurent Schaeffer, and Evelyne Goillot Role for the Pleckstrin Homology Domain-Containing Protein CKIP-1 in Phosphatidylinositol 3-Kinase-Regulated Muscle DifferentiationMol. Cell. Biol., Feb 2004; 24: 1245 - 1255.

Elisa Tedeschi, Marta Menegazzi, Ying Yao, Hisanori Suzuki, Ulrich Förstermann, and Hartmut Kleinert Green Tea Inhibits Human Inducible Nitric-Oxide Synthase Expression by Down-Regulating Signal Transducer and Activa-tor of Transcription-1 ActivationMol. Pharmacol., Jan 2004; 65: 111 - 120.

Darshan S. Sappal, A. Kathleen McClendon, James A. Fleming, Vala Thoroddsen, Kelly Connolly, Corinne Reimer, Ronald K. Blackman, Christine E. Bulawa, Neil Osheroff, Peter Charlton, and Laura A. Rudolph-Owen Biological characterization of MLN944: A potent DNA binding agentMol. Cancer Ther., Jan 2004; 3: 47 - 58.

R.J. Aitken, A.L. Ryan, B.J. Curry, and M.A. Baker Multiple forms of redox activity in populations of human sper-matozoaMol. Hum. Reprod., Nov 2003; 9: 645 - 661.

Friederike Herr, Olin D. Liang, Julio Herrero, Uwe Lang, Klaus T. Preissner, Victor K. M. Han, and Marek Zygmunt Possible Angiogenic Roles of Insulin-Like Growth Factor II and Its Receptors in Uterine Vascular Adaptation to PregnancyJ. Clin. Endocrinol. Metab., Oct 2003; 88: 4811 - 4817.

Kai Kraemer, Susanne Fuessel, Uta Schmidt, Matthias Kotzsch, Bernd Schwenzer, Manfred P. Wirth, and Axel Meye Antisense-mediated hTERT Inhibition Specifically Reduces the Growth of Human Bladder Cancer CellsClin. Cancer Res., Sep 2003; 9: 3794 - 3800.

Dinja Oosterhoff, M. Adhiambo Witlox, Victor W. van Beusechem, Hidde J. Haisma, Gerard R. Schaap, Johannes Bras, Frank A. Kruyt, Bonnie Molenaar, Epie Boven, Paul I. J. M. Wuisman, Herbert M. Pinedo, and Winald R. Gerritsen Gene-directed Enzyme Prodrug Therapy for Osteosarcoma: Sensitization to CPT-11 in Vitro and in Vivo by Adenoviral Deliv-ery of a Gene Encoding Secreted Carboxylesterase-2Mol. Cancer Ther., Aug 2003; 2: 765 - 771.

Natalia I. Dmitrieva, Dmitry V. Bulavin, and Maurice B. Burg High NaCl causes Mre11 to leave the nucleus, disrupting DNA damage signaling and repairAm J Physiol Renal Physiol, Aug 2003; 285: 266 - 274.

Shunli Ding, Tatyana Merkulova-Rainon, Zhong Chao Han, and Gérard Tobelem HGF receptor up-regulation contributes to the angiogenic phe-notype of human endothelial cells and promotes angiogenesis in vitroBlood, Jun 2003; 101: 4816 - 4822.

Vania De Arcangelis, Dario Coletti, Marco Conti, Michel Lagarde, Mario Molinaro, Sergio Adamo, Georges Nemoz, and Fabio Naro IGF-I-induced Differentiation of L6 Myogenic Cells Requires the Activity of cAMP-PhosphodiesteraseMol. Biol. Cell, Apr 2003; 14: 1392 - 1404.

Jaime R. Merchan, Barden Chan, Sujata Kale, Lowell E. Schnip-per, and Vikas P. Sukhatme In Vitro and In Vivo Induction of Antiangiogenic Activity by Plas-minogen Activators and CaptoprilJ Natl Cancer Inst, Mar 2003; 95: 388 - 399.

Seongsoo Sohn, Wonhee Hur, Myung Kuk Joe, Ji-Hyun Kim, Zee-Won Lee, Kwon-Soo Ha, and Changwon Kee Expression of Wild-Type and Truncated Myocilins in Trabecular Meshwork Cells: Their Subcellular Localizations and CytotoxicitiesInvest. Ophthalmol. Vis. Sci., Dec 2002; 43: 3680 - 3685.

Yves Langelier, Stéphane Bergeron, Stéphane Chabaud, Julie Lippens, Claire Guilbault, A. Marie-Josée Sasseville, Stéphan Denis, Dick D. Mosser, and Bernard Massie The R1 subunit of herpes simplex virus ribonucleotide reduc-tase protects cells against apoptosis at, or upstream of, caspase-8 activationJ. Gen. Virol., Nov 2002; 83: 2779 - 2789.

Olga N. Ilinskaya, Florian Dreyer, Vladimir A. Mitkevich, Kevin L. Shaw, C. Nick Pace, and Alexander A. Makarov Changing the net charge from negative to positive makes ribo-nuclease Sa cytotoxicProtein Sci., Oct 2002; 11: 2522 - 2525.

Andrea Elsner, Bernd Kreikemeyer, Andrea Braun-Kiewnick, Barbara Spellerberg, Bettina A. Buttaro, and Andreas Podbielski Involvement of Lsp, a Member of the LraI-Lipoprotein Family in Streptococcus pyogenes, in Eukaryotic Cell Adhesion and Inter-nalizationInfect. Immun., Sep 2002; 70: 4859 - 4869.

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CC147Appendix

Zhiwei Feng, Lei Li, Poh Yong Ng, and Alan G. Porter Neuronal Differentiation and Protection from Nitric Oxide-Induced Apoptosis Require c-Jun-Dependent Expression of NCAM140Mol. Cell. Biol., Aug 2002; 22: 5357 - 5366.

ROK HUMAR, FABRICE N. KIEFER, HARTMUT BERNS, THÉRÈSE J. RESINK, and EDOUARD J. BATTEGAY Hypoxia enhances vascular cell proliferation and angiogenesis in vitro via rapamycin (mTOR) -dependent signalingFASEB J, Jun 2002; 16: 771 - 780.

Fabienne Brilot, Wassim Chehadeh, Chantal Charlet-Renard, Henri Martens, Vincent Geenen, and Didier Hober Persistent Infection of Human Thymic Epithelial Cells by Cox-sackievirus B4J. Virol., May 2002; 76: 5260 - 5265.

Haisong Ju, Sandhya Nerurkar, Charles F. Sauermelch, Alan R. Olzinski, Rosanna Mirabile, Dawn Zimmerman, John C. Lee, Jerry Adams, Joseph Sisko, Marinella Berova, and Robert N. Willette Sustained Activation of p38 Mitogen-Activated Protein Kinase Contributes to the Vascular Response to InjuryJ. Pharmacol. Exp. Ther., Apr 2002; 301: 15 - 20.

MOLECULAR BASIS OF CELL AND DEVELOPMENTAL BIOLOGY:Long-Yuan Li, Hsiu-Ming Shih, Mei-Ying Liu, and Jen-Yang ChenThe Cellular Protein PRA1 Modulates the Anti-apoptotic Activity of Epstein-Barr Virus BHRF1, a Homologue of Bcl-2, through Direct InteractionJ. Biol. Chem., Jul 2001; 276: 27354 - 27362.

Chuanhai Guo, Shihui Yu, Alan T. Davis, Huamin Wang, Jeffrey E. Green, and Khalil Ahmed A Potential Role of Nuclear Matrix-associated Protein Kinase CK2 in Protection against Drug-induced Apoptosis in Cancer CellsJ. Biol. Chem., Feb 2001; 276: 5992 - 5999.

Takako Akiyama, Shin Takasawa, Koji Nata, Seiichi Kobayashi, Michiaki Abe, Nausheen J. Shervani, Takayuki Ikeda, Kei Naka-gawa, Michiaki Unno, Seiki Matsuno, and Hiroshi Okamoto Activation of Reg gene, a gene for insulin-producing -cell regeneration: Poly(ADP-ribose) polymerase binds Reg pro-moter and regulates the transcription by autopoly(ADP-ribosyl)ationPNAS, Jan 2001; 98: 48 - 53.

5’-Bromo-2’-deoxy-uridine Labeling and Detection Kit I, Cat.-No. 11 296 736 001

Sandra Bauer, Jürgen Pollheimer, Johannes Hartmann, Peter Husslein, John D. Aplin, and Martin Knöfler Tumor Necrosis Factor- Inhibits Trophoblast Migration through Elevation of Plasminogen Activator Inhibitor-1 in First-Trimester Villous Explant CulturesJ. Clin. Endocrinol. Metab., Feb 2004; 89: 812 - 822.

Igor Oruetxebarria, Francesca Venturini, Tuija Kekarainen, Ada Houweling, Lobke M. P. Zuijderduijn, Adone Mohd-Sarip, Rob-ert G. J. Vries, Rob C. Hoeben, and C. Peter Verrijzer p16INK4a Is Required for hSNF5 Chromatin Remodeler-induced Cellular Senescence in Malignant Rhabdoid Tumor CellsJ. Biol. Chem., Jan 2004; 279: 3807 - 3816.

Yasutoshi Tatsumi, Satoshi Ohta, Hiroshi Kimura, Toshiki Tsurimoto, and Chikashi Obuse The ORC1 Cycle in Human Cells: I. CELL CYCLE-REGULATED OSCILLATION OF HUMAN ORC1J. Biol. Chem., Oct 2003; 278: 41528 - 41534.

Yi Cao, Zhefeng Zhao, Joanna Gruszczynska-Biegala, and Anna Zolkiewska Role of Metalloprotease Disintegrin ADAM12 in Determination of Quiescent Reserve Cells during Myogenic Differentiation In VitroMol. Cell. Biol., Oct 2003; 23: 6725 - 6738.

Jan-Willem Taanman, John R. Muddle, and Ania C. Muntau Mitochondrial DNA depletion can be prevented by dGMP and dAMP supplementation in a resting culture of deoxyguanosine kinase-deficient fibroblastsHum. Mol. Genet., Aug 2003; 12: 1839 - 1845.

Heather J. Evans, Janea K. Sweet, Robert L. Price, Michael Yost, and Richard L. Goodwin Novel 3D culture system for study of cardiac myocyte developmentAm J Physiol Heart Circ Physiol, Jul 2003; 285: 570 - 578.

Sanna Pasonen-Seppänen, Susanna Karvinen, Kari Törrönen, Juha M. T. Hyttinen, Tiina Jokela, Mikko J. Lammi, Markku I. Tammi, and Raija Tammi EGF Upregulates, Whereas TGF-ß Downregulates, the Hyaluro-nan Synthases Has2 and Has3 in Organotypic Keratinocyte Cul-tures: Correlations with Epidermal Proliferation and DifferentiationJ. Invest. Dermatol., Jun 2003; 120: 1038 - 1044.

Eric Julien and Winship Herr Proteolytic processing is necessary to separate and ensure proper cell growth and cytokinesis functions of HCF-1EMBO J., May 2003; 22: 2360 - 2369.

Kaiwen Ma, Keigo Araki, Solachuddin J.A. Ichwan, Tamaki Suganuma, Mimi Tamamori-Adachi, and Masa-Aki Ikeda E2FBP1/DRIL1, an AT-Rich Interaction Domain-Family Tran-scription Factor, Is Regulated by p53Mol. Cancer Res., Apr 2003; 1: 438 - 444.

Yoshiyuki Konishi and Azad Bonni The E2F-Cdc2 Cell-Cycle Pathway Specifically Mediates Activity Deprivation-Induced Apoptosis of Postmitotic NeuronsJ. Neurosci., Mar 2003; 23: 1649 - 1658.

Jean Buteau, Sylvain Foisy, Erik Joly, and Marc Prentki Glucagon-Like Peptide 1 Induces Pancreatic ß-Cell proliferationVia Transactivation of the Epidermal Growth Factor ReceptorDiabetes, Jan 2003; 52: 124 - 132.

Mary Ann Stepp, Heather E. Gibson, Purvi H. Gala, Drina D. Sta. Iglesia, Ahdeah Pajoohesh-Ganji, Sonali Pal-Ghosh, Marcus Brown, Christopher Aquino, Arnold M. Schwartz, Olga Gold-berger, Michael T. Hinkes, and Merton Bernfield Defects in keratinocyte activation during wound healing in the syndecan-1-deficient mouseJ. Cell Sci., Dec 2002; 115: 4517 - 4531.

Xiaodong Shu, Weicheng Wu, Raymond D. Mosteller, and Daniel Broek Sphingosine Kinase Mediates Vascular Endothelial Growth Fac-tor-Induced Activation of Ras and Mitogen-Activated Protein KinasesMol. Cell. Biol., Nov 2002; 22: 7758 - 7768.

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Kirsi Rilla, Mikko J. Lammi, Reijo Sironen, Kari Törrönen, Merja Luukkonen, Vincent C. Hascall, Ronald J. Midura, Mika Hyttinen, Jukka Pelkonen, Markku Tammi, and Raija Tammi Changed lamellipodial extension, adhesion plaques and migra-tion in epidermal keratinocytes containing constitutively expressed sense and antisense hyaluronan synthase 2 (Has2) genesJ. Cell Sci., Sep 2002; 115: 3633 - 3643.

Satoshi Kurosaka, Yasumitsu Nagao, Naojiro Minami, Masayasu Yamada, and Hiroshi Imai Dependence of DNA Synthesis and In Vitro Development of Bovine Nuclear Transfer Embryos on the Stage of the Cell Cycle of Donor Cells and Recipient CytoplastsBiol. Reprod., Aug 2002; 67: 643 - 647.

Michael L. Whitfield, Gavin Sherlock, Alok J. Saldanha, John I. Murray, Catherine A. Ball, Karen E. Alexander, John C. Matese, Charles M. Perou, Myra M. Hurt, Patrick O. Brown, and David Botstein Identification of Genes Periodically Expressed in the Human Cell Cycle and Their Expression in TumorsMol. Biol. Cell, Jun 2002; 13: 1977 - 2000.

Fang Liao, Jacqueline F. Doody, Jay Overholser, Bridget Finnerty, Rajiv Bassi, Yan Wu, Elisabetta Dejana, Paul Kussie, Peter Bohlen, and Daniel J. Hicklin Selective Targeting of Angiogenic Tumor Vasculature by Vascu-lar Endothelial-cadherin Antibody Inhibits Tumor Growth with-out Affecting Vascular PermeabilityCancer Res., May 2002; 62: 2567 - 2575.

Christopher J. McGann, Shannon J. Odelberg, and Mark T. Keating Mammalian myotube dedifferentiation induced by newt regen-eration extractPNAS, Nov 2001; 98: 13699 - 13704.

Salvador Soriano, David E. Kang, Maofu Fu, Richard Pestell, Nathalie Chevallier, Hui Zheng, and Edward H. Koo Presenilin 1 Negatively Regulates ß-Catenin/T Cell Factor/Lym-phoid Enhancer Factor-1 Signaling Independently of ß-Amyloid Precursor Protein and Notch ProcessingJ. Cell Biol., Feb 2001; 152: 785 - 794.

Jacob B. Hansen, Hongbin Zhang, Thomas H. Rasmussen, Ras-mus K. Petersen, Esben N. Flindt, and Karsten Kristiansen Peroxisome Proliferator-activated Receptor (PPAR)-mediated Regulation of Preadipocyte Proliferation and Gene Expression Is Dependent on cAMP SignalingJ. Biol. Chem., Jan 2001; 276: 3175 - 3182.

Hitoshi Komuro, Ellada Yacubova, Elina Yacubova, and Pasko Rakic Mode and Tempo of Tangential Cell Migration in the Cerebellar External Granular LayerJ. Neurosci., Jan 2001; 21: 527 - 540.

STEPHAN J. RESHKIN, ANTONIA BELLIZZI, SANDRA CAL-DEIRA, VALENTINA ALBARANI, ILARIA MALANCHI, MAN-UELA POIGNEE, MARIANNA ALUNNI-FABBRONI, VALERIA CASAVOLA, and MASSIMO TOMMASINO Na+/H+ exchanger-dependent intracellular alkalinization is an early event in malignant transformation and plays an essential role in the development of subsequent transformation-associ-ated phenotypesFASEB J, Nov 2000; 14: 2185 - 2197.

R. Montgomery Gill and Paul A. Hamel Subcellular Compartmentalization of E2F Family Members Is Required for Maintenance of the Postmitotic State in Terminally Differentiated MuscleJ. Cell Biol., Mar 2000; 148: 1187 - 1202.

John Lynch, Eun-Ran Suh, Debra G. Silberg, Steven Rulyak, Nadine Blanchard, and Peter G. Traber The Caudal-related Homeodomain Protein Cdx1 Inhibits Prolif-eration of Intestinal Epithelial Cells by Down-regulation of D-type CyclinsJ. Biol. Chem., Feb 2000; 275: 4499 - 4506.

5’-Bromo-2’-deoxy-uridine Labeling and Detection Kit II, Cat.-No. 11 299 964 001

Kenneth H. H. Wong, Heather D. Wintch, and Mario R. Capecchi Hoxa11 Regulates Stromal Cell Death and Proliferation during Neonatal Uterine DevelopmentMol. Endocrinol., Jan 2004; 18: 184 - 193.

MARIE-THERESE SIMON, ALAIN PAULOIN, GUILLAUME NORMAND, HANH-TU LIEU, HELENE MOULY, GERARD PIV-ERT, FRANÇOISE CARNOT, J. GUILHERME TRALHAO, CHRIS-TIAN BRECHOT, and LAURENCE CHRISTA HIP/PAP stimulates liver regeneration after partial hepatectomy and combines mitogenic and anti-apoptotic functions through the PKA signaling pathwayFASEB J, Aug 2003; 17: 1441 - 1450.

Andrés F. Muro, Anil K. Chauhan, Srecko Gajovic, Alessandra Iaconcig, Fabiola Porro, Giorgio Stanta, and Francisco E. Baralle Regulated splicing of the fibronectin EDA exon is essential for proper skin wound healing and normal lifespanJ. Cell Biol., Jul 2003; 162: 149 - 160.

Yoshikazu Nakamura, Kiyoko Fukami, Haiyan Yu, Kei Takenaka, Yuki Kataoka, Yuji Shirakata, Shin-Ichi Nishikawa, Koji Hashim-oto, Nobuaki Yoshida, and Tadaomi Takenawa Phospholipase C1 is required for skin stem cell lineage commit-mentEMBO J., Jun 2003; 22: 2981 - 2991.

Jijun Chen, Yajun Tu, Cheil Moon, Eiichiro Nagata, and Gabriele V. Ronnett Heme oxygenase-1 and heme oxygenase-2 have distinct roles in the proliferation and survival of olfactory receptor neurons mediated by cGMP and bilirubin, respectivelyJ. Neurochem., Jun 2003; 85: 1247 - 1261.

Ryuichi Yamada, Yoko Mizutani-Koseki, Takanori Hasegawa, Noriko Osumi, Haruhiko Koseki, and Naoki Takahashi Cell-autonomous involvement of Mab21l1 is essential for lens placode developmentDevelopment, May 2003; 130: 1759 - 1770.

Maarit Rossi, Hiroyuki Morita, Raija Sormunen, Sari Airenne, Marjut Kreivi, Ling Wang, Naomi Fukai, Bjorn R. Olsen, Karl Tryggvason, and Raija Soininen Heparan sulfate chains of perlecan are indispensable in the lens capsule but not in the kidneyEMBO J., Jan 2003; 22: 236 - 245.

B. R. Wamhoff, D. K. Bowles, N. J. Dietz, Q. Hu, and M. Sturek Exercise training attenuates coronary smooth muscle pheno-typic modulation and nuclear Ca2+ signalingAm J Physiol Heart Circ Physiol, Dec 2002; 283: 2397 - 2410.

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James J. A. Contos, Isao Ishii, Nobuyuki Fukushima, Marcy A. Kingsbury, Xiaoqin Ye, Shuji Kawamura, Joan Heller Brown, and Jerold Chun Characterization of lpa2 (Edg4) and lpa1/lpa2 (Edg2/Edg4) Lysophosphatidic Acid Receptor Knockout Mice: Signaling Deficits without Obvious Phenotypic Abnormality Attributable to lpa2Mol. Cell. Biol., Oct 2002; 22: 6921 - 6929.

Stacey Rentschler, Jennifer Zander, Kathleen Meyers, David France, Rebecca Levine, George Porter, Scott A. Rivkees, Gre-gory E. Morley, and Glenn I. Fishman Neuregulin-1 promotes formation of the murine cardiac con-duction systemPNAS, Aug 2002; 99: 10464 - 10469.

Laura P. Stabile, Autumn L. Gaither Davis, Christopher T. Gubish, Toni M. Hopkins, James D. Luketich, Neil Christie, Syd-ney Finkelstein, and Jill M. Siegfried Human Non-Small Cell Lung Tumors and Cells Derived from Normal Lung Express Both Estrogen Receptor and ß and Show Biological Responses to EstrogenCancer Res., Apr 2002; 62: 2141 - 2150.

Eleonora Minina, Hans Markus Wenzel, Conny Kreschel, Seth Karp, William Gaffield, Andrew P. McMahon, and Andrea Vortkamp BMP and Ihh/PTHrP signaling interact to coordinate chondro-cyte proliferation and differentiationDevelopment, Nov 2001; 128: 4523 - 4534.

Kamilah Alexander and Philip W. Hinds Requirement for p27KIP1 in Retinoblastoma Protein-Mediated SenescenceMol. Cell. Biol., Jun 2001; 21: 3616 - 3631.

Shu Wakino, Ulrich Kintscher, Sarah Kim, Simon Jackson, Fen Yin, Sunil Nagpal, Roshantha A. S. Chandraratna, Willa A. Hsueh, and Ronald E. Law Retinoids Inhibit Proliferation of Human Coronary Smooth Mus-cle Cells by Modulating Cell Cycle RegulatorsArterioscler. Thromb. Vasc. Biol., May 2001; 21: 746 - 751.

A. Cevik Tufan and Rocky S. Tuan Wnt regulation of limb mesenchymal chondrogenesis is accom-panied by altered N-cadherin-related functionsFASEB J, Apr 2001; 10.1096/fj.00-0784fje.

Cor F. Calkhoven, Christine Müller, and Achim Leutz Translational control of C/EBP and C/EBP isoform expressionGenes & Dev., Aug 2000; 14: 1920 - 1932.

Maria Luiza C. Albuquerque, Christopher M. Waters, Ushma Savla, H. William Schnaper, and Annette S. Flozak Shear stress enhances human endothelial cell wound closure in vitroAm J Physiol Heart Circ Physiol, Jul 2000; 279: 293 - 302.

Stephan Goetze, Ulrich Kintscher, Hiroaki Kawano, Yasuko Kawano, Shu Wakino, Eckart Fleck, Willa A. Hsueh, and Ronald E. Law Tumor Necrosis Factor Inhibits Insulin-induced Mitogenic Signaling in Vascular Smooth Muscle CellsJ. Biol. Chem., Jun 2000; 275: 18279 - 18283.

Charlotte M. Boney, Philip A. Gruppuso, Ronald A. Faris, and A. Raymond Frackelton, Jr. The Critical Role of Shc in Insulin-Like Growth Factor-I-Medi-ated Mitogenesis and Differentiation in 3T3-L1 PreadipocytesMol. Endocrinol., Jun 2000; 14: 805 - 813.

Emmanuelle Charpentier, Robert M. Lavker, Elizabeth Acquista, and Pamela Cowin Plakoglobin Suppresses Epithelial Proliferation and Hair Growth In VivoJ. Cell Biol., Apr 2000; 149: 503 - 520.

Mette Christiansen, Marie Kveiborg, Moustapha Kassem, Brian F. C. Clark, and Suresh I. S. Rattan CBFA1 and Topoisomerase I mRNA Levels Decline During Cel-lular Aging of Human Trabecular OsteoblastsJ. Gerontol. A Biol. Sci. Med. Sci., Apr 2000; 55: 194 - 200.

Birgit Stallmeyer, Heiko Kämpfer, Nicole Kolb, Josef Pfeilschifter, and Stefan Frank The Function of Nitric Oxide in Wound Repair: Inhibition of Inducible Nitric Oxide-Synthase Severely Impairs Wound Reepi-thelializationJ. Invest. Dermatol., Dec 1999; 113: 1090 - 1098.

Jacob George, Dominique Roulot, Victor E. Koteliansky, and D. Montgomery Bissell In vivo inhibition of rat stellate cell activation by soluble trans-forming growth factor type II receptor: A potential new therapy for hepatic fibrosisPNAS, Oct 1999; 96: 12719 - 12724.

ALESSANDRO FATATIS and RICHARD J. MILLER Cell cycle control of PDGF-induced Ca2+ signaling through modulation of sphingolipid metabolismFASEB J, Aug 1999; 13: 1291 - 1301.

Masahiro Hitomi and Dennis W. Stacey Cellular Ras and Cyclin D1 Are Required during Different Cell Cycle Periods in Cycling NIH 3T3 CellsMol. Cell. Biol., Jul 1999; 19: 4623 - 4632.

5’-Bromo-2’-deoxy-uridine Labeling and Detection Kit III, Cat.-No. 11 444 611 001

Hideyuki Sakoda, Yukiko Gotoh, Hideki Katagiri, Mineo Kurokawa, Hiraku Ono, Yukiko Onishi, Motonobu Anai, Takehide Ogihara, Midori Fujishiro, Yasushi Fukushima, Miho Abe, Nobuhiro Shojima, Masatoshi Kikuchi, Yoshitomo Oka, Hisamaru Hirai, and Tomoichiro Asano Differing Roles of Akt and Serum- and Glucocorticoid-regulated Kinase in Glucose Metabolism, DNA Synthesis, and Oncogenic ActivityJ. Biol. Chem., Jul 2003; 278: 25802 - 25807.

Jeremy K. Wong, Hoa H. Le, Attila Zsarnovszky, and Scott M. Belcher Estrogens and ICI182,780 (Faslodex) Modulate Mitosis and Cell Death in Immature Cerebellar Neurons via Rapid Activation of p44/p42 Mitogen-Activated Protein KinaseJ. Neurosci., Jun 2003; 23: 4984 - 4995.

D. Sun, Y. Gong, H. Kojima, G. Wang, E. Ravinsky, M. Zhang, and G. Y. Minuk Increasing cell membrane potential and GABAergic activity inhibits malignant hepatocyte growthAm J Physiol Gastrointest Liver Physiol, Jun 2003; 285: 12 - 19.

Senji Kasahara, Kazuki Ando, Kuniaki Saito, Kenji Sekikawa, Hiroyasu Ito, Tetsuya Ishikawa, Hiroo Ohnishi, Mitsuru Seishima, Shinichi Kakumu, and Hisataka Moriwaki Lack of Tumor Necrosis Factor Alpha Induces Impaired Prolifer-ation of Hepatitis B Virus-Specific Cytotoxic T LymphocytesJ. Virol., Feb 2003; 77: 2469 - 2476.

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Li-Fen Lee, Junlin Guan, Yun Qiu, and Hsing-Jien Kung Neuropeptide-Induced Androgen Independence in Prostate Cancer Cells: Roles of Nonreceptor Tyrosine Kinases Etk/Bmx, Src, and Focal Adhesion KinaseMol. Cell. Biol., Dec 2001; 21: 8385 - 8397.

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Kana Mizuno, Hiroyuki Okamoto, and Takeshi Horio Ultraviolet B Radiation Suppresses Endocytosis, Subsequent Maturation, and Migration Activity of Langerhans Cell-Like Dendritic CellsJ. Invest. Dermatol., Feb 2004; 122: 300 - 306.

Marc-André Raymond, Anik Désormeaux, Patrick Laplante, Normand Vigneault, Janos G. Filep, Karine Landry, Alexey V. Pshezhetsky, and Marie-Josée Hébert Apoptosis of endothelial cells triggers a caspase-dependent anti-apoptotic paracrine loop active on vascular smooth muscle cellsFASEB J, Feb 2004; 10.1096/fj.03-0573fje.

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ENMEI LIU, HELEN K.W. LAW, and YU-LUNG LAU Insulin-Like Growth Factor I Promotes Maturation and Inhibits Apoptosis of Immature Cord Blood Monocyte–Derived Den-dritic Cells through MEK and PI 3-Kinase PathwaysPediatr. Res., Dec 2003; 54: 919 - 925.

Yun-Hee Noh, Kant Matsuda, Young-Kwon Hong, Rainer Kunst-feld, Lucia Riccardi, Manuel Koch, Hajimu Oura, Soheil S. Dadras, Michael Streit, and Michael Detmar An N-Terminal 80 kDa Recombinant Fragment of Human Thrombospondin-2 Inhibits Vascular Endothelial Growth Factor Induced Endothelial Cell Migration In Vitro and Tumor Growth and Angiogenesis In VivoJ. Invest. Dermatol., Dec 2003; 121: 1536 - 1543.

K Yoshida, A Tsutsumi, Y Ohnishi, T Akimoto, H Murata, and T Sumida T cell epitopes of prothrombin in patients with antiphospholipid syndromeAnn. Rheum. Dis, Sep 2003; 62: 905 - 906.

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ENMEI LIU, HELEN K.W. LAW, and YU-LUNG LAU BCG Promotes Cord Blood Monocyte-Derived Dendritic Cell Maturation with Nuclear Rel-B Up-regulation and Cytosolic IB and � DegradationPediatr. Res., Jul 2003; 54: 105 - 112.

Mitsuhiro Matsuo, Hiroaki Sakurai, and Ikuo Saiki ZD1839, a Selective Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor, Shows Antimetastatic Activity Using a Hepato-cellular Carcinoma ModelMol. Cancer Ther., Jun 2003; 2: 557 - 561.

Jason S. Taub, Rishu Guo, L. M. Fredrik Leeb-Lundberg, John F. Madden, and Yehia Daaka Bradykinin Receptor Subtype 1 Expression and Function in Prostate CancerCancer Res., May 2003; 63: 2037 - 2041.

Takao Sakai, Shaohua Li, Denitsa Docheva, Carsten Grashoff, Keiko Sakai, Günter Kostka, Attila Braun, Alexander Pfeifer, Peter D. Yurchenco, and Reinhard Fässler Integrin-linked kinase (ILK) is required for polarizing the epi-blast, cell adhesion, and controlling actin accumulationGenes & Dev., Apr 2003; 17: 926 - 940.

Yvonne E.M. Dommels, Merel M.G. Haring, Nynke G.M. Keestra, Gerrit M. Alink, Peter J. van Bladeren, and Ben van Ommen The role of cyclooxygenase in n-6 and n-3 polyunsaturated fatty acid mediated effects on cell proliferation, PGE2 synthesis and cytotoxicity in human colorectal carcinoma cell linesCarcinogenesis, Mar 2003; 24: 385 - 392.

Rainer Wessely, Ludger Hengst, Birgit Jaschke, Franziska Wegener, Thomas Richter, Raffaella Lupetti, Makarios Paschali-dis, Albert Schömig, Richard Brandl, and Franz-Josef Neumann A central role of interferon regulatory factor-1 for the limitation of neointimal hyperplasiaHum. Mol. Genet., Jan 2003; 12: 177 - 187.

Marc-André Raymond, Luigina Mollica, Normand Vigneault, Anik Désormeaux, John S.D. Chan, Janos G. Filep, and Marie-Josée Hébert Blockade of the apoptotic machinery by cyclosporin A redirects cell death toward necrosis in arterial endothelial cells: regula-tion by reactive oxygen species and cathepsin DFASEB J, Jan 2003; 10.1096/fj.02-0500fje.

Craig H. Selzman, Stephanie A. Miller, Michael A. Zimmerman, Fabia Gamboni-Robertson, Alden H. Harken, and Anirban Banerjee Monocyte chemotactic protein-1 directly induces human vascular smooth muscle proliferationAm J Physiol Heart Circ Physiol, Oct 2002; 283: 1455 - 1461.

Kahoru Nishina, Katsuya Mikawa, Osamu Morikawa, Hidefumi Obara, and R. J. Mason The Effects of Intravenous Anesthetics and Lidocaine on Prolif-eration of Cultured Type II Pneumocytes and Lung FibroblastsAnesth. Analg., Feb 2002; 94: 385 - 388.

Torsten R. Dunkern and Bernd Kaina Cell proliferation and DNA Breaks Are Involved in Ultraviolet Light-induced Apoptosis in Nucleotide Excision Repair-defi-cient Chinese Hamster CellsMol. Biol. Cell, Jan 2002; 13: 348 - 361.

Ferruccio Galbiati, Daniela Volonte', Jun Liu, Franco Capozza, Philippe G. Frank, Liang Zhu, Richard G. Pestell, and Michael P. Lisanti Caveolin-1 Expression Negatively Regulates Cell Cycle Progres-sion by Inducing G0/G1 Arrest via a p53/p21WAF1/Cip1-depen-dent MechanismMol. Biol. Cell, Aug 2001; 12: 2229 - 2244.

Kelly L. Davenpeck, Cezary Marcinkiewicz, Dian Wang, Rodica Niculescu, Yi Shi, Jack L. Martin, and Andrew Zalewski Regional Differences in Integrin Expression : Role of 5ß1 in Regulating Smooth Muscle Cell FunctionsCirc. Res., Feb 2001; 88: 352 - 358.

Masahide Tokunou, Toshiro Niki, Keisuke Eguchi, Sanae Iba, Hitoshi Tsuda, Tesshi Yamada, Yoshihiro Matsuno, Haruhiko Kondo, Yukihito Saitoh, Hiroji Imamura, and Setsuo Hirohashi c-MET Expression in Myofibroblasts : Role in Autocrine Activa-tion and Prognostic Significance in Lung AdenocarcinomaAm. J. Pathol., Apr 2001; 158: 1451 - 1463.

Marie-Claude Amoureux, Bruce A. Cunningham, Gerald M. Edelman, and Kathryn L. Crossin N-CAM Binding Inhibits the Proliferation of Hippocampal Pro-genitor Cells and Promotes Their Differentiation to a Neuronal PhenotypeJ. Neurosci., May 2000; 20: 3631 - 3640.

KD Coutant, AB de Fraissinette, A Cordier, and P Ulrich Modulation of the activity of human monocyte-derived dendritic cells by chemical haptens, a metal allergen, and a staphylococ-cal superantigenToxicol. Sci., Dec 1999; 52: 189 - 198.

In Situ Cell Proliferation Kit, FLUOS, Cat.-No. 11 810 740 001

Juan Carlos Casar, Claudio Cabello-Verrugio, Hugo Olguin, Rebeca Aldunate, Nibaldo C. Inestrosa, and Enrique Brandan Heparan sulfate proteoglycans are increased during skeletal muscle regeneration: requirement of syndecan-3 for successful fiber formationJ. Cell Sci., Jan 2004; 117: 73 - 84.

Iwan Walev, Sebastian Chakrit Bhakdi, Fred Hofmann, Nabil Djonder, Angela Valeva, Klaus Aktories, and Sucharit Bhakdi Delivery of proteins into living cells by reversible membrane permeabilization with streptolysin-OPNAS, Mar 2001; 98: 3185 - 3190.

Amrit Mann, Kai Breuhahn, Peter Schirmacher, Arnd Wilhelmi, Carsten Beyer, Andrea Rosenau, Suat Özbek, Stefan Rose-John, and Manfred Blessing Up- and Down-Regulation of Granulocyte/Macrophage-Colony Stimulating Factor Activity in Murine Skin Increase Susceptibil-ity to Skin Carcinogenesis by Independent MechanismsCancer Res., Mar 2001; 61: 2311 - 2319.

Mario Encinas, Montse Iglesias, Yuhui Liu, Hongyin Wang, Ashraf Muhaisen, Valentin Ceña, Carme Gallego, and Joan X. Comella Sequential Treatment of SH-SY5Y Cells with Retinoic Acid and Brain-Derived Neurotrophic Factor Gives Rise to Fully Differen-tiated, Neurotrophic Factor-Dependent, Human Neuron-Like CellsJ. Neurochem., Sep 2000; 75: 991 - 1003.

Satomi S. Tanaka, Yayoi Toyooka, Ryuko Akasu, Yuko Katoh-Fukui, Yoko Nakahara, Rika Suzuki, Minesuke Yokoyama, and Toshiaki Noce The mouse homolog of Drosophila Vasa is required for the development of male germ cellsGenes & Dev., Apr 2000; 14: 841 - 853.

Qianghua Hu and Sankar N. Maity Stable Expression of a Dominant Negative Mutant of CCAAT Binding Factor/NF-Y in Mouse Fibroblast Cells Resulting in Retardation of Cell Growth and Inhibition of Transcription of Various Cellular GenesJ. Biol. Chem., Feb 2000; 275: 4435 - 4444.

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Allen D. Everett, Jill V. Narron, Tamara Stoops, Hideji Nakamura, and Amy Tucker Hepatoma Derived Growth Factor is a Pulmonary Endothelial Cell Expressed Angiogenic FactorAm J Physiol Lung Cell Mol Physiol, Jan 2004; 10.1152/ajplung.00427.2003.

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Masaaki Hayashi, Richard I. Tapping, Ta-Hsiang Chao, Jeng-Fan Lo, Charles C. King, Young Yang, and Jiing-Dwan Lee BMK1 Mediates Growth Factor-induced Cell Proliferation through Direct Cellular Activation of Serum and Glucocorticoid-inducible KinaseJ. Biol. Chem., Mar 2001; 276: 8631 - 8634.

Thomas F. Westbrook, Don X. Nguyen, Barry R. Thrash, and Dennis J. McCance E7 Abolishes Raf-Induced Arrest via Mislocalization of p21Cip1Mol. Cell. Biol., Oct 2002; 22: 7041 - 7052.

Paola Secchiero, Arianna Gonelli, Claudio Celeghini, Prisco Mirandola, Lia Guidotti, Giuseppe Visani, Silvano Capitani, and Giorgio Zauli Activation of the nitric oxide synthase pathway represents a key component of tumor necrosis factor-related apoptosis-inducing ligand-mediated cytotoxicity on hematologic malignanciesBlood, Oct 2001; 98: 2220 - 2228.

Hanna E. Kleczkowska, Giancarlo Marra, Teresa Lettieri, and Josef Jiricny hMSH3 and hMSH6 interact with PCNA and colocalize with it to replication fociGenes & Dev., Mar 2001; 15: 724 - 736.

Bing Jiang, Amrita Kamat, and Carole R. Mendelson Hypoxia Prevents Induction of Aromatase Expression in Human Trophoblast Cells in Culture: Potential Inhibitory Role of the Hypoxia-Inducible Transcription Factor Mash-2 (Mammalian Achaete-Scute Homologous Protein-2)Mol. Endocrinol., Oct 2000; 14: 1661 - 1673.

ZE Smith and DR Higgs The pattern of replication at a human telomeric region (16p13.3): its relationship to chromosome structure and gene expressionHum. Mol. Genet., Aug 1999; 8: 1373 - 1386.

Teresa Lettieri, Giancarlo Marra, Gabriele Aquilina, Margherita Bignami, Nigel E.A. Crompton, Fabio Palombo, and Josef Jiricny Effect of hMSH6 cDNA expression on the phenotype of mis-match repair-deficient colon cancer cell line HCT15Carcinogenesis, Mar 1999; 20: 373 - 382.

Manho Kim, H-S Lee, Genevieve LaForet, Charmian McIntyre, Eileen J. Martin, Patrick Chang, Tae Wan Kim, M. Williams, P. H. Reddy, Dan Tagle, Frederick M. Boyce, Lisa Won, Alfred Heller, Neil Aronin, and Marian DiFiglia Mutant Huntingtin Expression in Clonal Striatal Cells: Dissocia-tion of Inclusion Formation and Neuronal Survival by Caspase InhibitionJ. Neurosci., Feb 1999; 19: 964 - 973.

References


CC153Appendix

Anti-Bromodeoxyuridine-Peroxidase, Fab fragments formalin grade, Cat.-No. 11 585 860 001

Atsushi Terunuma, Kimberly L. Jackson, Veena Kapoor, William G. Telford, and Jonathan C. Vogel Side Population Keratinocytes Resembling Bone Marrow Side Population Stem Cells Are Distinct From Label-Retaining Kerat-inocyte Stem CellsJ. Invest. Dermatol., Nov 2003; 121: 1095 - 1103.

Neela Patel, Li Sun, Deborah Moshinsky, Hui Chen, Kathleen M. Leahy, Phuong Le, Katherine G. Moss, Xueyan Wang, Audie Rice, Danny Tam, A. Douglas Laird, Xiaoming Yu, Qingling Zhang, Cho Tang, Gerald McMahon, and Anthony Howlett A Selective and Oral Small Molecule Inhibitor of Vascular Epi-thelial Growth Factor Receptor (VEGFR)-2 and VEGFR-1 Inhib-its Neovascularization and Vascular PermeabilityJ. Pharmacol. Exp. Ther., Sep 2003; 306: 838 - 845.

Liang Ma, Jian Liu, Tobey Wu, Maksim Plikus, Ting-Xin Jiang, Qun Bi, Yi-Hsin Liu, Sven Müller-Röver, Heiko Peters, John P. Sundberg, Rob Maxson, Richard L. Maas, and Cheng-Ming Chuong `Cyclic alopecia' in Msx2 mutants: defects in hair cycling and hair shaft differentiationDevelopment, Jan 2003; 130: 379 - 389.

Michael R. Albert, Ruth-Ann Foster, and Jonathan C. Vogel Murine Epidermal Label-Retaining Cells Isolated by Flow Cytometry do not Express the Stem Cell Markers CD34, Sca-1, or Flk-1J. Invest. Dermatol., Oct 2001; 117: 943 - 948.

Tino D. Piscione, Tien Phan, and Norman D. Rosenblum BMP7 controls collecting tubule cell proliferation and apoptosis via Smad1-dependent and -independent pathwaysAm J Physiol Renal Physiol, Jan 2001; 280: 19 - 33.

Martha Campbell-Thompson, Gregory Y. Lauwers, Kristen K. Reyher, Josh Cromwell, and Kathleen T. Shiverick 17ß-Estradiol Modulates Gastroduodenal Preneoplastic Alter-ations in Rats Exposed to the Carcinogen N-Methyl-N'-Nitro-NitrosoguanidineEndocrinology, Oct 1999; 140: 4886 - 4894.

References



3.3 General references

1. Schwartzman, R. A. and Cidlowski, J. A. (1993) Apoptosis: the biochemistry and molecular biology of programmed cell death. Endocrine Rev. 14, 133.

2. Vermes, I. and Haanan, C. (1994) Apoptosis and programmed cell death in health and disease. Adv. Clin. Chem. 31, 177.

3. Berke, G. (1991) Debate: the mechanism of lymphocyte-mediated killing. Lymphocyte-triggered internal target disintegration. Immunol. Today 12, 396.

4. Krähenbühl, O. and Tschopp, J. (1991) Debate: the mechanism of lymphocyte-mediated killing. Perforin-induced pore formation. Immunol. Today 12, 399.

5. Van Furth, R. and Van Zwet, T. L. (1988) Immunocytochemical detection of 5-bromo-2-deoxyuridine incorporation in individual cells. J. Immunol. Methods 108, 45. (CHECK THIS ONE OUT-page number showed as “4”)

6. Cohen, J. J. (1993) Apoptosis. Immunol. Today 14, 126.

7. Savill, J. S. et al. (1989) Macrophage phagocytosis of aging neutrophils in inflammation. Programmed cell death in the neutrophil leads to its recognition by macrophages. J. Clin. Invest. 83, 865.

8. Wyllie, A. H. (1980) Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 284, 555.

9. Leist, M. et al. (1994) Application of the Cell Death Detection ELISA for the Detection of Tumor Necrosis Factor-induced DNA Fragmentation in Murine Models of Inflammatory Organ Failure Biochemica No. 3, 18–20.

10. Fraser, A. and Evan, G. (1996) A license to kill. Cell 85, 781–784.

11. Duke, R. C. (1983) Endogenous endonuclease-induced DNA fragmentation: an early event in cell-mediated cytolysis. Proc. Natl. Acad. Sci. USA 80, 6361.

12. Duke, R. C. & Cohen, J. J. (1986) IL-2 addiction: withdrawal of growth factor activates a suicide program in dependent T cells. Lymphokine Res. 5, 289.

13. Trauth, B. C. et al. (1994) Eur. J. Cell. Biol. 63, 32, Suppl 40.

14. Matzinger, P. (1991) The JAM test. A simple assay for DNA fragmentation and cell death. J. Immunol. Methods 145, 185.

15. Kaeck, M. R. (1993) Alkaline elution analysis of DNA fragmentation induced during apoptosis. Anal. Biochem. 208, 393.

16. Prigent, P. et al. (1993) A safe and rapid method for analyzing apoptosis-induced fragmentation of DNA extracted from tissues or cultured cells. J. Immunol. Methods 160, 139.

17. Huang, P. & Plunkett, W. (1992) A quantitative assay for fragmented DNA in apoptotic cells. Anal. Biochem. 207, 163

18. Bortner , C. D. et al. (1995) Trends Cell Biol. 5, 21.

19. Gold, R. et al. (1994) Differentiation between cellular apoptosis and necrosis by the combined use of in situ tailing and nick translation techniques. Lab. Invest. 71, 219.

20. Sgonc, R. et al. (1994) Simultaneous determination of cell surface antigens and apoptosis. Trends Genet. 10, 41-42.

21. Darzynkiewicz, Z. et al. (1994) Assays of cell viability: discrimination of cells dying by apoptosis. Methods Cell Biol. 41, 15.

22. Darzynkiewicz, Z. et al. (1992) Features of apoptotic cells measured by flow cytometry. Cytometry 13, 795.

23. Duvall, E. et al. (1985) Macrophage recognition of cells undergoing programmed cell death (apoptosis). Immunology 56, 351–358.

References

General references

CC155Appendix

24. Savill, J. S. et al. (1993) Phagocyte recognition of cells undergoing apoptosis. Immunol. Today 14, 131–136.

25. Asch, A. S. et al. (1987) Isolation of the thrombospondin membrane receptor. J. Clin. Invest. 79, 1054–1061.

26. Fadok, V. A. et al. (1992) Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J. Immunol. 148, 2207–2216.

27. Vermes, I. et al. (1995) A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J. Immunol. Methods 184, 39.

28. Homburg, C. H. E. et al. (1995) Human neutrophils lose their surface Fc gamma RIII and acquire Annexin V binding sites during apoptosis in vitro. Blood 85, 532.

29. Verhoven, B. et al. (1995) Mechanisms of phosphatidylserine exposure, a phagocyte recognition signal, on apoptotic T lymphocytes. J. Exp. Med. 182, 1597.

30. Dive, C. et al. (1992) Analysis and discrimination of necrosis and apoptosis (programmed cell death) by multiparameter flow cytometry. Biochem. Biophys. Acta 1133, 275.

31. Schmid, I. et al. (1994) Sensitive method for measuring apoptosis and cell surface phenotype in human thymocytes by flow cytometry. Cytometry 15, 12.

32. Afanasev, V. N. et al. (1986) FEBS Letts. 194, 347.

33. Ormerod, M. G. (1992) Apoptosis in interleukin-3-dependent haemopoietic cells. Quantification by two flow cytometric methods. J. Immunol. Methods 153, 57.

34. Meyaard, L. et al. (1992) Programmed death of T cells in HIV-1 infection. Science 257, 217.

35. Gavrieli, Y. et al. (1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J. Cell. Biol. 119, 493.

36. Manning, F. C. R. and Patierno, S. R. (1996) Apoptosis: inhibitor or instigator of carcinogenesis? Cancer Invest. 14, 455–465.

37. Stewart, B. W. (1994) Mechanisms of apoptosis: integration of genetic, biochemical, and cellular indicators. J. Natl. Cancer Inst. 86, 1286–1296.

38. Ellis, H. M. and Horvitz, H. R. (1986) Genetic control of programmed cell death in the nematode C. elegans. Cell 44, 817–829.

39. Yuan, J. Y. and Horvitz, H. R. (1990) The Caenorhabditis elegans genes ced-3 and ced-4 act cell autonomously to cause programmed cell death. Dev. Biol. 138, 33–41.

40. Hentgartner, M. O., Ellis, R. E. and Horvitz, H. R. (1992) Caenorhabditis elegans gene ced-9 protects cells from programmed cell death. Nature 356, 494–499.

41. Baffy, G. et al. (1993) Apoptosis induced by withdrawal of interleukin-3 (IL-3) from an IL-3-dependent hematopoietic cell line is associated with repartitioning of intracellular calcium and is blocked by enforced Bcl-2 oncoprotein production. J. Biol. Chem. 268, 6511–6519.

42. Miyashita, T. and Reed, J. C. (1993) Bcl-2 oncoprotein blocks chemotherapy-induced apoptosis in a human leukemia cell line. Blood 81, 151–157.

43. Oltvai, Z. N., Milliman, C. L. and Korsmeyer, S. J. (1993) Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell 74, 609–619.

44. Yonish-Rouach, E. et al. (1991) Wild-type p53 induces apoptosis of myeloid leukaemic cells that is inhibited by interleukin-6. Nature 352, 345–347.

45. Bissonnette, R. P. et al. (1992) Apoptotic cell death induced by c-myc is inhibited by bcl-2. Nature 359, 552–554.

46. Wagner, A. J., Small, M. B. and Hay, N. (1993) Myc-mediated apoptosis is blocked by ectopic expression of Bcl-2. Mol. Cell. Biol. 13, 2432–2440.

References

General references


47. Curnow, S. J. (1993) The role of apoptosis in antibody-dependent cellular cytotoxicity. Cancer Immunol. Immunother. 36, 149.

48. Danks, A. M. et al. (1992) Cellular alterations produced by the experimental increase in intracellular calcium and the nature of protective effects from pretreatment with nimodipine. Mol. Brain Res. 16, 168–172.

49. Kolber, M. A. et al. (1988) Measurement of cytotoxicity by target cell release and retention of the fluorescent dye bis-carboxyethyl-carboxyfluorescein (BCECF). J. Immunol. Methods 108, 255–264.

50. Oldham, R. K. et al. (1977) Direct comparison of three isotopic release microtoxicity assays as measures of cell-mediated immunity to Gross virus-induced lymphomas in rats. J. Natl. Cancer Inst. 58, 1061–1067.

51. Decker, T. and Lohmann-Matthes, M.-L. (1988) A quick and simple method for the quantitation of lactate dehydrogenase release in measurements of cellular cytotoxicity and tumor necrosis factor (TNF) activity. J. Immunol. Methods 15, 61–69. (PubMed MedLine shows this as 115)

52. Martin, A. and Clynes, M. (1991) Acid phosphatase: endpoint for in vitro toxicity tests. In Vitro Cell. Dev. Biol. 27A, 183–184.

53. Mosmann, T. R. (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55.

54. Mosmann, T. R. and Fong, T. A. T. (1989) Specific assays for cytokine production by T cells. J. Immunol. Methods 116, 151.

55. Sanderson, C. J. (1981) The mechanism of lymphocyte-mediated cytotoxicity. Biol. Rev. Camb. Philos. Soc. 56, 153.

56. Keilholz, U. et al. (1990) A modified cytotoxicity assay with high sensitivity. Scand. J. Clin. Lab. Invest. 50, 879.

57. Cook, J. A. and Mitchell, J. B. (1989) Viability measurements in mammalian cell system. Anal. Biochem. 179, 1.

58. Roehm, N. W. et al. (1991) An improved colorimetric assay for cell proliferation and viability utilizing the tetrazolium salt XTT. J. Immunol. Methods 142, 257.

59. Slater, T. F. et al. (1963) Biochem. Biophys. Acta 77, 383.

60. Berridge, M. V. and Tan, A. S. (1993) Characterization of the cellular reduction of 3-(4,5-dimethyltiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT): subcellular localization, substrate dependence and involvement of mitchondrial electron transport in MTT reduction. Arch. Biochem. Biophys. 303, 474.

61. Cory, A. H. et al. (1991) Use of an aqueous soluble tetrazolium/formazan assay for cell growth assays in culture. Cancer Commun. 3, 207.

62. Jabbar, S. A. B. et al. (1989) The MTT assay underestimates the growth inhibitory effects of interferons. Br. J. Cancer 60, 523.

63. Scudiero, E. A. et al. (1988) Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines. Cancer Res. 48, 4827.

64. Vistica, D. T. et al. (1991) Tetrazolium-based assays for cellular viability: a critical examination of selected parameters effecting formazan production (published erratum appears in Cancer Res. 1991 Aug 15; 51 (16): 45et). Cancer Res. 51, 2515.

65. Magaud, J. P. et al. (1988) Detection of human white cell proliferative responses by immunoenzymatic measurement of bromodeoxyuridine uptake. J. Immunol. Methods 106, 95.

66. Porstmann et al. (1985) Quanitation of 5-bromo-2-deoxyuridine incorporation into DNA: an enzyme immunoassay for the assessment of the lymphoid cell proliferative response. J. Immunol. Methods 82, 169.

67. Konttinen, Y. T. et al. (1988) An immunoperoxidase-autoradiography double labeling method for analysis of lymphocyte activation markers and DNA synthesis. J. Immunol. Methods 110, 19.

References

General references

CC157Appendix

68. Steel, G. G. (1977) In: Growth Kinetics of Tumours, Clarendon Press, Oxford, UK.

69. Takagi, S. et al. (1993) Detection of 5-bromo-2-deoxyuridine (BrdUrd) incorporation with monoclonal anti-BrdUrd antibody after deoxyribonuclease treatment. Cytometry 14, 640.

70. Gerdes, J. et al. (1984) Cell cycle analysis of a cell proliferation-associated human unclear antigen defined by the monoclonal antibody Ki-67. J. Immunol. 133, 1710.

71. Hall, P. A. and Levison, D. A. (1990) Review: assessment of cell proliferation in histological material. J. Clin. Pathol. 43, 184.

72. Hall, P. A. et al. (1990) Proliferating cell nuclear antigen (PCNA) immunolocalization in paraffin sections: an index of cell proliferation with evidence of deregulated expression in some neoplasms. J. Pathol. 162, 285.

73. Kreipe, H. et al. (1993) Determination of the growth fraction in non-Hodgkin’s lymphomas by monoclonal antibody Ki-S5 directed against a formalin-resistant epitope of the Ki-67 antigen. Am. J. Pathol. 142, 1689.

74. Scott, R. J. et al. (1991) A comparison of immunohistochemical markers of cell proliferation with experimentally determined growth fraction (see comments). J. Pathol. 165, 173.

References

General references


4. General abbreviations

ABTS 2,2’-azino-di-[3-ethylbenzthiazoline-sulfonate (6)]

Ac N-acetyl

ActD actinomycin D

ALT alanine aminotransferase

AP alkaline phosphatase

APAAP alkaline phosphatase anti-alkaline phosphatase

APES aminopropyl-triethoxysilane

BCIP 5-bromo-4-chloro-3-indolyl phosphate

B-CLL chronic lymphocytic leukemia (B-type)

Bio biotin

BrdU 5-bromo-2’-deoxyuridine

BSA bovine serum albumin

CAM campothecin

Con A concanavalin A

cpm counts per minute

CTL cytotoxic T lymphocytes

DAB 3,3’-diaminobenzidine

DES diethylstilbestrol

DX dexamethasone

ELISA enzyme-linked immunosorbent assay

Fab protease-generated antibody fragments

F(ab’)2 protease-generated antibody fragment

FACS fluorescence activated cell sorter

FAQs frequently asked questions

FITC fluorescein isothiocyanate

FLUOS 5(6)-carboxyfluorescein-N-hydroxysuccinimide ester

FSC forward light scatter

G0 resting phase

G1 gap between mitosis and DNA synthesis

G2 gap between DNA synthesis and mitosis

h hour

HMW DNA high molecular weight DNA

HSV herpes simplex virus type I antigen

[3H]-TdR tritiated thymidine (2’-deoxy)

ICE interleukin-1�-converting enzyme

INT 2-[4-iodophenyl]-3-[4-nitrophenyl]-5-phenyltetrazolium chloride

INV-A influenza A virus antigen

INV-B influenza B virus antigen

INV-KA influenza control antigen

ISNT in situ nick translation

kD kilodalton

LAK cells lymphokine-activated killer cells

LDH lactate dehydrogenase

LMW DNA low molecular weight DNA

LSC liquid scintillation counting

M-phase mitosis

MTP microtiter plate

MTT 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide

General abbreviations

CC159Appendix

NBT 4-nitro-blue tetrazolium chloride

NK cells natural killer cells

OKT3 anti-CD3 monoclonal antibody

PARP poly(ADP-ribose) polymerase

PBL peripheral blood lymphocytes

PBS phosphate buffered saline

PFA paraformaldehyde

PHA phytohemagglutinin

PI propidium iodide

PMS phenazine methosulfate

pNA 4-nitranilide

POD peroxidase

PS phosphatidylserine

PVDF polyvinylidene difluoride

PWM pokeweed mitogen

ref. reference

rlu/s relative light units/second

RT room temperature

RUV rubella virus antigen

SA streptavidin

SAC Staphylococcus aureus Cowan I

SN supernatant

SOD superoxide dismutase

S-phase DNA synthesis (replication)

SSC side light scatter

TdR thymidine

TdT terminal deoxynucleotidyltransferase

TMB tetramethylbenzidine

TNF tumor necrosis factor

TRITC tetramethylrhodamine isothiocyanate

TUNEL terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling

WST-1 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate

X-dUTP hapten-labeled deoxyuracil triphosphate

X-dNTP hapten-labeled deoxynucleoside triphosphate

XTT 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide

Z carbobenzoxy



Amino acids

Name 3-letter 1-letter

Alanine Ala A

Arginine Arg R

Asparagine Asn N

Aspartic Acid Asp D

Cysteine Cys C

Glutamic Acid Glu E

Glutamine Gln Q

Glycine Gly G

Histidine His H

Homoserine Hse –

Isoleucine Ile I

Leucine Leu L

Lysine Lys K

Methionine Met M

Methionine sulfoxide Met (O) –

Methionine methylsulfonium Met (S-Me) –

Norleucine Nle –

Phenylalanine Phe F

Proline Pro P

Serine Ser S

Threonine Thr T

Tryptophan Trp W

Tyrosine Tyr Y

Valine Val V

�-aminoisobutyric acid Aib –


CC161Appendix

5. Ordering Guide

* only sold in the US.

Products for Measuring Apoptosis in Cell Populations Cat. No. Pack Size

Apoptotic DNA Ladder Kit 11 835 246 001 1 kit (20 tests)


Cell Death Detection ELISAPLUS, 10x11 774 425 00111 920 685 001

1 kit (96 tests)10 x 96 tests

Cell Death Detection ELISA 11 544 675 001 1 kit (96 tests)

Anti-PARP 11 835 238 001 100 µl

Caspase 3 Activity Assay 12 012 952 001 1 kit (96 tests)

Homogeneous Caspase Assay, fluorimetric 03 005 372 00112 236 869 001

100 tests1000 tests

Products for Measuring Apoptosis in Individual Cells

Cat. No. Pack Size

TUN

EL k

its a

nd r

elat

ed r

eage

nts

In Situ Cell Death Detection Kit, Fluorescein 11 684 795 001 1 kit (50 tests)

In Situ Cell Death Detection Kit, TMR red 12 156 792 001 1 kit (50 tests)

In Situ Cell Death Detection Kit, AP 11 684 809 001 1 kit (50 tests)

In Situ Cell Death Detection Kit, POD 11 684 817 001 1 kit (50 tests)

TUNEL Label Mix 11 767 291 001 3 x 550 µl (30 tests)

TUNEL Enzyme 11 767 305 001 2 x 50 µl (20 tests)

TUNEL POD 11 772 465 001 3.5 ml (70 tests)

TUNEL AP 11 772 457 001 3.5 ml (70 tests)

TUNEL Dilution Buffer 11 966 006 001 2 x 10 ml

DAB substrate, precipitating (POD substrate) 11 718 096 001 1 pack

NBT/BCIP Stock Solution (AP substrate) 11 681 451 001 8 ml

Fast Red Tablets (AP substrate) 11 496 549 001 20 Tablets

Fluo

resc

ence

labe

ls

Propidium iodide solution* 11 348 639 001 20 ml

DAPI 10 236 276 001 10 mg

Ann

exin

-V a

nd r

elat

ed

reag

ents

Annexin-V-FLUOS 11 828 681 001 250 tests

Annexin-V-FLUOS Staining Kit 11 858 777 001 1 kit (50 tests)

Annexin-V-Biotin 11 828 690 001 250 tests

Annexin-V-Alexa 568 03 703 126 001 250 tests

Streptavidin-POD 11 089 153 001 500 U (1 ml)

Streptavidin-AP 11 089 161 001 1000 U (1 ml)

Ant

ibod

ies

M30 CytoDEATH 12 140 322 00112 140 349 001

50 tests250 tests

M30 CytoDEATH, Fluorescein 12 156 857 001 250 tests

Anti-Fas (CD95/Apo-1) 11 922 432 001 100 µg

p53 pan ELISA 11 828 789 001 1 kit (96 tests)

Products for Measuring Cytotoxicity Cat. No. Pack Size

Cytotoxicity Detection Kit (LDH) 11 644 793 001 1 kit (2000 tests)

Cellular DNA Fragmentation ELISA 11 585 045 01 1 kit (500 tests)

Cell Proliferation Kit I (MTT) 11 465 007 001 1 kit (2500 tests)

Cell Proliferation Kit II (XTT) 11 465 015 001 1 kit (2500 tests)

Cell Proliferation Reagent WST-1 11 644 807 001 2500 tests

Ordering Guide


Products for Measuring Cell Proliferation in Cell Populations

Cat. No. Pack Size

Cell Proliferation Kit I (MTT) 11 465 007 001 1 kit (2500 tests)

Cell Proliferation Kit II (XTT) 11 465 015 001 1 kit (2500 tests)

Cell Proliferation Reagent WST-1 11 644 807 001 2500 tests

Cell Proliferation ELISA, BrdU (colorimetric) 11 647 229 001 1 kit (1000 tests)

Cell Proliferation ELISA, BrdU (chemiluminescent) 11 669 915 001 1 kit (1000 tests)

FixDenat 11 758 764 001 4 x 100 ml (2000tests)

BrdU Labeling and Detection Kit III 11 444 611 001 1 kit (1000 tests)

Products for Measuring Cell Proliferation in Individual Cells

Cat. No. Pack Size

In Situ Cell Proliferation Kit, FLUOS 11 810 740 001 1 kit (100 tests)

BrdU Labeling and Detection Kit I 11 296 736 001 1 kit (100 tests)

BrdU Labeling and Detection Kit II 11 299 964 001 1 kit (100 tests)

Anti-BrdU (clone BMC 9318)unlabeled, formalin gradefluorescein-labeled, formalin grade

11 170 376 00111 202 693 001

50 µg (500 µl)50 µg (500 µl)

Anti-BrdU, POD-labeled (clone BMC 6H8), Fab fragments, formalin grade

11 585 860 001 15 U

Additional Products for Cell Death/Cell Proliferation Studies

Cat. No. Pack Size

Calpain inhibitor I 11 086 090 001 25 mg

Calpain inhibitor II 11 086 103 001 25 mg

DNase I, RNase free 10 776 785 001 10 000 units

DNase I, grade I 10 104 132 001 20 000 units

DNase II, grade II 10 104 159 001 100 mg

Interleukin-1�, human, recombinant (E. coli) 11 457 756 001 10 0000 units

Interleukin-1�, mouse recombinant (E. coli) 11 444 590 001 10 0000 units

Nuclease S7 10 107 921 001 15 000 units

Nuclease P1 10 236 225 001 1 mg

Proteinase K, lyophilizate 03 115 836 001 25 mg

Staurosporine 11 055 682 001 500 µg

TNF-�, human, recombinant (E. coli) 11 371 843 001 10 µg(1 000 000 units)

TNF-a, human, recombinant (yeast) 11 088 939 001 10 µg(1 000 000 units)

TNF-�, mouse, recombinant (E. coli) 11 271 156 001 5 µg(2 000 000 units)

TNF-�ELISA, human 11 425 943 001 1 kit (96 tests)

Ordering Guide

CC163Appendix

Trademarks

ABTS is a registered trademark of a Member of the Roche Group

Alexa is a trademark of Molecular Probes, Inc., Eugene, OR, USA

BOBO is a trademark of Molecular Probes, Inc., Eugene, OR, USA

Ordering Guide


6. Index

A“A0” cells ................................................................... 49Acridine orange ....................................................... 49Alkaline elution analysis of DNA ............................ 30Annexin V

-Alexa 568 ........................................................... 44assays for ............................................................. 45binding of phosphatidylserine .......................... 43-Biotin ................................................................ 47-FLUOS .............................................................. 44-FLUOS Staining Kit .......................................... 44

Anti-BrdU ................................................................. 105DNA .................................................................... 15PARP ................................................................... 27

Antibody to, see Anti-Apopain ............................. 123Apoptosis

assays for cell populations ................................. 10assays for individual cells ................................... 32biochemical characteristics of ............................. 2definition of ......................................................... 2difference between cytotoxicity and ................. 58difference between necrosis and ......................... 3hallmark of ......................................................... 10inducers of .......................................................... 11morphological characteristics of ......................... 3overview of ........................................................... 2pathways ............................................................... 5proteases, role of ................................................ 18simultaneous detection of necrosis and ........... 15surface morphology changes during .................. 5

Apoptotic DNA Ladder Kit ..................................... 13Aspartate at proteolysis site ..................................... 18

Bbad gene .................................................................. 123bax .......................................................................... 123BCIP ......................................................................... 42bcl-2 gene ................................................................ 123bcl-xL gene .............................................................. 123bcl-xS gene .............................................................. 123“Beads on a string” .................................................. 11Bisbenzimidazole dye, see Hoechst dye5�-Bromo-2�-deoxy-uridine

Labeling and Detection Kit I ........................... 101Labeling and Detection Kit II .......................... 101Labeling and Detection Kit III .......................... 91labeling of DNA ............................................... 100incorporation assay .......................................... 100release assay ........................................................ 74

Bromo-deoxy-uridine, see 5�-Bromo-2�-deoxy-uridine

CCAM, see CampothecinCampothecin ............................................... 15, 46, 48Caspases ................................................................... 18Caspase 3 Activity Assay ......................................... 22Ced-3 ..................................................................... 123ced-9 gene .............................................................. 123Cell cycle, overview of ............................................. 75Cell death

accidental ............................................................. 2and cytotoxicity ................................................... 2programmed ........................................................ 2

Cell Death Detection ELISAPLUS ............................ 15Cell-mediated cytotoxicity ...................................... 58Cell proliferation

assays for cell populations ................................. 82assays for individual cells ................................ 100assays that use tetrazolium salts ....................... 82ELISA, BrdU (chemiluminescent) ................... 94ELISA, BrdU (colorimetric) ............................. 94Kit I (MTT) ....................................................... 85Kit II (XTT) ....................................................... 86overview of ........................................................ 74Reagent WST-1 .................................................. 87

Cell viability assays, see Cell proliferation assaysCellular

DNA Fragmentation ELISA .............................. 64Ceramide, role in apoptosis .................................. 123Chemiluminescent cell proliferation ELISA .......... 94Chromatin aggregation ............................................. 3c-jun gene ............................................................... 123c-myc gene .............................................................. 123Colorimetric assays

for cytotoxicity ............................................ 61, 64for proliferation ................................................. 94

CPP32 .................................................................... 123Crm A .................................................................... 123Cr release assay, radioactive .................................... 74Cyclin ....................................................................... 75Cysteine proteases ..................................................... 6Cytotoxic T cells ........................................................ 2Cytotoxicity

assays ............................................................ 61, 64cell-mediated ..................................................... 58definition of ....................................................... 58Detection Kit (LDH) ......................................... 61effectors of ......................................................... 58overview of ........................................................ 58

Index

CC165Appendix

DDAB substrate .......................................................... 42Damage/leakage of plasma membrane, assays for . 49DAPI ......................................................................... 49Deoxynucleotidyltransferase, terminal .................. 33Dexamethasone ....................................................... 35DNA cleavage, see DNA fragmentationDNA fragmentation

during apoptosis ............................................ 6, 10DNA fragments, histone-associated ....................... 12DNA end labeling .................................................... 33DNA ladder

appearance of ..................................................... 11assay for .............................................................. 13size of fragments ................................................ 11

DNA polymerase ..................................................... 32DNA synthesis

assays .................................................................. 89Dye

exclusion assays .................................................. 49uptake ................................................................. 50

EEffector cells for inducing cell death ...................... 66ELISA

kits .......................................................... 15, 91, 94End labeling of DNA ............................................... 33Epidermis tissue, in vivo labeling of ..................... 104Ethidium bromide ................................................... 49Exclusion assays, see Dye exclusion assays

FFADD ..................................................................... 124False positive, TUNEL ........................................... 114Fast red ..................................................................... 42FIENA ...................................................................... 22FixDenat ................................................................... 94Fluorochrome staining assays for measuringDNA loss .................................................................. 49Flow cytometric techniques, kits for, see

Annexin V-FLUOS Staining Kit ......................... 44In situ Cell Death Detection Kit, Fluorescein ... 36In situ Cell Proliferation Kit, FLUOS .............. 102

Flow cytometric measurementof Annexin V-stained cells ................................ 44of apoptosis ........................................................ 32of BrdU label .................................................... 103of cell cycle position ........................................ 107of ISNT method ................................................. 33of normal and apoptotic cells ..................... 46, 49of peripheral blood lymphocytes ...................... 34of total DNA .................................................... 103of TUNEL method ............................................ 38

Flow cytometryassays for apoptotic cells ............................. 36, 44

Formazaninsoluble ............................................................ 84soluble ................................................................ 84

fos gene .................................................................. 125Fragmentation of DNA ........................................... 30

GGlucocorticoid receptor ........................................ 125Granzyme .............................................................. 125

HHallmark of apoptosis ............................................ 10HeLa cells .............................................................. 103Histone-associated DNA fragments ....................... 12Hoechst dye ............................................................. 49Homeostasis, loss during cell death ......................... 3Homogeneous Caspases Assay ............................... 25

IICE ............................................................................. 6ICH-I ..................................................................... 127INT .......................................................................... 61Interleukin converting enzyme (ICE) ...................... 6In situ Cell Death Detection Kit

-AP ..................................................................... 39-POD ................................................................. 39-Fluorescein ....................................................... 36

In situ Cell Proliferation Kit-FLUOS ............................................................ 102

In situ nick translation ............................................ 33ISNT method .......................................................... 33

JJAM test ................................................................... 30

LLactate dehydrogenase, see LDHLDH

Cytotoxicity Detection Kit ................................ 61release assay ....................................................... 74

Leakage/damage of plasma membrane, assays for 49LMW DNA .............................................................. 12Lymphokine-activated killer cells .......................... 58

Index


MM30 CytoDEATH .................................................... 19M30 CytoDEATH, Fluorescein ............................... 19Mch2, Mch3, Mch4 ................................................ 127MCL-1 .................................................................... 125Membrane symmetry during apoptosis ................. 43Method selection guide

for apoptosis assay ........................................... 8, 9for cell proliferation assay ........................... 80, 81

Microwave pretreatment for TUNEL ................... 116Mononucleosomes ................................................... 11MORT-1 ................................................................. 125M-phase ................................................................... 76MTT

assay kit .............................................................. 85biochemical basis for reduction of .................... 82cellular basis for reduction of ............................ 82comparison with other tetrazolium salts .......... 84effect of superoxide dismutase on ................... 121structure of ......................................................... 83use in cell proliferation assay ............................ 82use in cytotoxicity assay ..................................... 59

NNatural killer cells ................................................ 2, 58NBT .......................................................................... 42Necrosis

definition of ......................................................... 2difference between apoptosis and ....................... 3difference between cytotoxicity and ................. 58inducers of ............................................................ 3inflammation during ........................................... 3overview of ........................................................... 3secondary ............................................................. 3

NEDD ..................................................................... 125NF-kappa B ............................................................ 125Nick translation ....................................................... 33Nonradioactive assays

for apoptosis ....................................................... 30for cell proliferation ........................................... 98for DNA fragmentation ..................................... 30

Nucleosome quantification ELISA ......................... 30

OOligonucleosomes ................................................... 11

Pp53 pan ELISA ......................................................... 56PARP ........................................................................ 10Peripheral blood lymphocytes

proliferation of .................................................. 97stimulation of .................................................... 96

Phagocytic cells ......................................................... 3Phosphatidylserine .................................................. 43Phospholipid ........................................................... 43Phospholipid-binding protein, see Annexin V .......................................................... 43Plasma membrane

damage during apoptosis .................................... 3damage during necrosis ...................................... 3

Poly-(ADP-ribose) polymerase, see PARPPositive, false, TUNEL .......................................... 114Proliferating cells

assays for .................................................... 85 – 87increased metabolic activity in ......................... 82

Propidium iodideexclusion assay ............................................. 44, 47properties ........................................................... 49

Proteases in apoptosis ............................................. 18Proteinase K pretreatment for TUNEL ................ 116Proto-oncogene ....................................................... 54

QQuestions frequently asked about cell deathassays ...................................................................... 112

RRadioactive assays

for apoptosis ...................................................... 30for cell proliferation .......................................... 98for DNA fragmentation .................................... 30

Ras .......................................................................... 126Reduced metabolic activity, assay for ..................... 68RIP ......................................................................... 126

Index

CC167Appendix

SS-phase ..................................................................... 76Staining of DNA ...................................................... 52Storage of samples for apoptosis assay ................. 112Streptavidin conjugates ........................................... 48“Sub-G1” peak ......................................................... 49Succinate-tetrazolium reductase ............................ 82Surface glycoproteins .............................................. 43Symmetry of membranes during apoptosis ........... 43

TTdR proliferation assay ........................................... 98TdT ........................................................................... 33Terminal deoxynucleotidyl transferase .................. 33Tetrazolium salt

See also MTT, WST-1, XTTmitochondrial reduction anduse in cell proliferation assays ........................... 82

Thymidine release assay, radioactive ...................... 74TRADD .................................................................. 126Transferase, terminal ............................................... 33Trypan blue exclusion assay .................................... 49Two-color assay for dead and viable apoptotic cells ........................................................ 119TUNEL

AP ....................................................................... 42definition of ....................................................... 33diminished staining during DNA counterstaining ................................................ 116dilution buffer .................................................... 42effect of different fixatives on .......................... 115effect of pretreatments on ............................... 116enzyme ............................................................... 42evaluation of, for in situ apoptotic cell identification .................................................... 113false positives in ............................................... 114high background in ......................................... 115improvement of, for in situ apoptotic cellidentification .................................................... 113kits for ........................................................ 36 – 39label .................................................................... 42low labeling in .................................................. 116nonspecific labeling in ..................................... 115no signal in ....................................................... 116optimization of ................................................ 115overview of ......................................................... 32POD .................................................................... 42pretreatments for ............................................. 116protocol for tissues which tend to give falsepositives ............................................................ 114single reagents for .............................................. 42special applications of ..................................... 119specificity of ....................................................... 33tips for avoiding or eliminating potential artifacts in ........................................................ 115

UUptake of dyes by dead cells ................................... 50

VViable cell number .................................................. 78

WWater-insoluble formazan ...................................... 84Water-soluble formazan ......................................... 84WST-1

assay ................................................................... 87biochemical basis for reduction of ................... 82cellular basis for reduction of ........................... 82comparison with other tetrazolium salts ......... 84effect of reducing agents on ............................ 121effect of superoxide dismutase on .................. 121structure of ........................................................ 83use in cell proliferation assay ............................ 82use in cytotoxicity assay .................................... 59

XXTT

assay kit .............................................................. 86biochemical basis for reduction of ................... 82cellular basis for reduction of ........................... 82comparison with other tetrazolium salts ......... 84effect of reducing agents on ............................ 121effect of superoxide dismutase on .................. 121structure of ........................................................ 83use in cell proliferation assay ............................ 82use in cytotoxicity assay .................................... 59

YYAMA .................................................................... 126

Index


Apoptosis on the Internet

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