High Content Analysis:In Vitro Toxicology & Cell-Based Assays

High Content Analysis (HCA) is high throughput, automated

microscopy which provides a rapid means of quantitative

image acquisition and analysis. It has a direct impact in

every area of cellular research and has facilitated the drug

discovery process by allowing detection of multiple parame-

ters in a single assay. Using Millipore’s high-quality reagents

in ready-to-use kits and cell-based assays that incorporate

the power of HCA, researchers learn more about functional

cell biology in many different systems, including one of the

greatest challenges in drug development: in vitro toxicology.

Accelerating the Pace of High ContentAnalysis with the Most ComprehensiveMultiplex Cytotoxicity KitsOne of the bottlenecks in the drug development process is

compound toxicicity and unanticipated adverse clinical

effects during clinical trials. That’s why Millipore, the original

multiplexing leader, is advancing the use of multiparameter

assays in cytotoxicity studies to improve the drug discovery

workflow. Our tools and solutions for research and drug

discovery include a wide array of cell-based assays for

cytotoxicity using HCA.

With our multiplexed assays and measurements at a

range of time points, you obtain improved sensitivity and

specificity for detection of subtle cellular changes indicative

of toxic effects.

Move your research forward with speed and efficiency.

Find out how Millipore can help you better detect the early

indicators of toxicity while simultaneously measuring multiple

biomarkers for cell viability and providing functional, relevant

cellular data.

Data Sheet

Millipore HCA Kits Offer:• Validated antibodies and dyes for high content analysis

that work on all HCA systems

• Fluorescence-based antibody assays that work on any

fluorescence or confocal microscope

• Multiplexed kits with specific control toxins and compounds

included, enabling you to gain functional data on test

compounds

• Validated protocols and recommended cell lines that require

no further assay development

ORDERING INFORMATIONDescription Application Catalogue No.

CellCiphr Cytotoxicity Assay Human HepG2 Cells Cellular Systems Biology (CSB) Kit Hepatotoxicity HCS100Neurotoxicity and Neurite Outgrowth Assay for High Content Screening Neuroscience, Neurotoxicity HCS220BrdU Assay Apoptosis HCS201Mouse primary antibodyCyclin B1 Assay Cell Cycle HCS202Mouse primary antibodyPhospho-Histone H3Ser10 Assay Cell Proliferation, Aurora Kinase Profiling HCS203Mouse primary antibodyPhospho-Histone H3Ser10 Assay Cell Proliferation, Aurora Kinase Profiling HCS204Rabbit primary antibodyKi-67 Assay Cell Cycle Stage Segregation HCS205Rabbit primary antibodyKi-67 Assay Cell Cycle Stage Segregation HCS206Mouse primary antibodyPhospho-Histone H3(Ser 10) and Ki-67 Assay Cell Proliferation, Aurora Kinase Profiling, HCS209

Cell Cycle EvaluationCyclin B1 and Ki-67 Assay Cell Proliferation, Cell Cycle Evaluation HCS210Phospho-Histone H3(Ser 10) and Cyclin B1 Assay Cell Proliferation, Cell Cycle Evaluation HCS211BrdU and Phospho-Histone H3(Ser 10) Assay Apoptosis, Aurora Kinase Profiling, HCS212

Cell Cycle EvaluationBrdU and Ki-67 Assay Apoptosis, Cell Cycle HCS213High Content Screening Toolkit Buffers, reagents for using mouse antibodies HCS214For Use With Mouse Primary AntibodiesHigh Content Screening Toolkit Buffers, reagents for using rabbit antibodies HCS215For Use With Rabbit Primary AntibodiesPhospho-histone H3 and α-Tubulin Assay Cell Cycle Segregation, Cytoskeletal Analysis HCS216p53 DNA Damage HCS223H2A.X DNA Damage, Apoptosis HCS224p53 + H2A.X DNA Damage, Apoptosis HCS225

DRUG DISCOVERY AND DEVELOPMENTMillipore provides tools and technologies to facilitate

discovery and evaluation of new drug candidates that help

you achieve results faster than ever before. Through the

acquisition of Upstate®, Chemicon® and Linco®, we now

offer a suite of products that span the drug discovery

pipeline from target identification to clinical studies. Our

expert team of scientists and engineers understands the

complexity of your discovery and development and can

support you in these challenges. Your drive to discovery

starts with Millipore.

www.millipore.com/offices

Millipore, Chemicon, Linco and Upstate are registered trademarks of Millipore Corporation.

M Logo and Advancing Life Science Together are trademarks of Millipore Corporation.

CellCiphr is a trademark of Cellumen, Inc.

FSC is a registered trademark of the Forrest Stewardship Council.

Lit. No. DS1837EN00 08DD026 Printed in U.S.A.

© 2008 Millipore Corporation, Billerica, MA 01821 U.S.A. All rights reserved.

ADVANCING LIFE SCIENCE TOGETHER™Research. Development. Production.

For more information on HCA products, visit www.millipore.com/hca. For other ways Millipore supports LifeScience research, contact your local Millipore Drug Discovery Specialist.

*CELLCIPHRTM KITFOR HUMAN HEPATOXICITYLiver damage, or hepatotoxicity, is a major factor in the high

failure rate of lead compounds in drug development and the

withdrawal of drugs from market. The cost of losing a lead

drug candidate in

these late stages to

unanticipated toxicity

is considerable.

Therefore, reliable

biomarkers of liver

damage are required

for prediction of these potential causes of drug failure to

reduce cost and accelerate time to market.

Millipore’s new HCA assay—the CellCiphr kit for human

hepatotoxicity—offers the most comprehensive cell-based

cytotoxicity assay available. CellCiphr provides a new way of

prioritizing lead compounds based on cytotoxicity indices,

enabling you to gain greater insight and advance the most

promising lead candidates.

traditional radiolabelled cell proliferation. Together these kits

provide an overview of cell health and viability on exposure

to various compound concentrations during the drug

discovery process.

The most comprehensivecell-based cytotoxicityassay available foraccurately detectingliver damage.

The CellCiphr panel of antibodies specific for profiling humanhepatotoxicity in HepG2 cells.

APPLICATION NOTE

• The CellCiphr Cytotoxicity kit measures 11 parameters over three time points in a 384-well format using

human hepatocytes, HepG2 cells. The 11 parameters in the CellCiphr kit represent the broadest range of

values to give a complete picture of cell health during drug screening. Measurements are made at acute, early

and chronic exposure time points. The kit enables the analysis of up to 20 compounds at one time. It also

contains software to perform a compilation of data into dose-response curves for each compound. This kit

contains HCA-validated antibodies, reagents and protocols, and works on any HCA instrument.

CELL CYCLE ANDCELL PROLIFERATION ASSAYSDisturbance of the cell cycle, particularly inhibition of

cell proliferation, is a sensitive indicator of cytotoxicity.

Measurements of cell proliferation give indications of cell

viability and responsiveness, which are crucial for under-

standing developmental mechanisms and cellular pathology.

With Millipore’s HCA assays, you’re able to assess phospho-

histone H3ser10, Ki-67, cyclin B1 and α-tubulin during the cell

cycle. These targets represent sensitive biomarkers for

detecting disruption of normal cell division. In addition, the

Millipore bromodeoxyuridine (BrdU) kit is an antibody-based

kit that measures cell proliferation, and is an alternative to

A wide range of kits for an overview ofcell health and viability on exposure tovarious compound concentrations duringthe drug discovery process.

DNA damage and

apoptosis. These

include: p53, an

attractive therapeutic

target since its

tumor-suppressor

activity can be

stimulated to eradicate tumor cells; and the histone

protein, H2A.X.

APPLICATION NOTE

• Detection of pH-H3 is a measure of mitotic index—the percentage of cells in M phase—and allows identification

of compounds that inhibit or induce mitosis. Ki-67 antigen is expressed during late G1, S, G2 and M phase of

the cell cycle, while resting, non-cycling cells (G0 phase) lack Ki-67 expression. Cyclin B1 has distinct expression

patterns during prophase, metaphase and anaphase and discriminates between G2, M, and G0/G1/S phases of

the cell cycle. Microtubules, such as α-tubulin, are crucial to maintain cell shape and for intracellular transport.

Disturbance of microtubule dynamics leads to cell cycle arrest at mitosis and may ultimately cause the

induction of apoptosis

Dose Response of A549 and HeLa Cells to Cell-cycle Inhibition AgentsA549 or HeLa cells were treated for the last 4 hours of culture with serial dilutions of either vinblastine sulfate (Chart A, G2/M phase arrest,max. concentration = 1 µM) or etoposide (Chart B, S/G2 phase arrest, max. concentration = 100 µM). Cells were imaged on the GE IN CellAnalyzer 1000 at 10X (10 fields/well) and analyzed using the IN Cell Analyzer 1000 Workstation (3.4) Multi Target Analysis algorithm. Mean± SEM, n = 4.

DNA DAMAGE & APOPTOSIS ASSAYSAssessment of DNA damage during drug development is

essential for detecting any potential genotoxic or apoptotic

effects caused by drug candidate compounds. Using HCA al-

lows DNA damage to be visualized at the same time as

other cellular parameters such as membrane integrity and

cytoskeleton structure, providing greater insight.

Millipore has developed two kits to enable sensitive

detection and assessment of key proteins that regulate

Two important kits thatenable better detectionand assessment of DNAdamage and apoptosisduring drug development.

APPLICATION NOTE

• The tumor suppressor protein p53 is central to many anti-cancer mechanisms, regulating expression of genes

involved in growth arrest, apoptosis, cellular senescence and repair of DNA damage. p53 is an attractive

therapeutic target because its tumor-suppressor activity can be stimulated to eradicate tumor cells; so much

effort has been placed on identifying cellular proteins that modulate p53 function.

• Histone proteins, such as H2A.X protect DNA. Post-translational modifications of histones mediate rapid and

reversible responses to changing cellular signals. When DNA damage (e.g., DNA strand breakage) occurs,

phosphorylated H2A.X accumulates at the site of the damage. This accumulation helps recruit DNA repair

machinery to the site of the breakage. When DNA damage is severe, the cell can undergo apoptosis. Since

this modification occurs before many of the morphological changes are detectable, it is one of the earliest

indicators of apoptosis.

NEUROTOXICITY ASSAYSMajor efforts in CNS research focus on the identification of

compounds that affect the promotion or inhibition of growth

of new cellular processes (neurite outgrowth) from healthy

neurons. That’s because neurite formation and neuronal

regeneration hold

therapeutic promise

for Alzheimer’s and

Parkinson’s diseases,

and other neuro-

degenerative disorders.

Traditional assays for

neurotoxic endpoints

are slow, labor-

intensive and often subjective. Yet Millipore’s HCA neurite

outgrowth kit is different because it enables rapid screening

of multiple parameters in individual cells, such as cell body

count, neurite count and neurite length on a variety of cell

lines and primary cells. The high sensitivity of the assay,

coupled with sensitive neuronal specific reagents, enables

identification of neuronal cell bodies and neurites in

heterogeneous cell populations and is ideal in large scale

screening for inducers and inhibitors of neurite outgrowth.

A neurite kit that offersrapid screening of multipleparameters in individualcells, making it ideal inlarge scale screening forinducers and inhibitorsof neurite outgrowth.

Validation and specificity of primary antibodies for use indevelopment of neurotoxicity biomarkers assays. Rathippocampal astrocytes labeled for α-tubulin (red) andGFAP (green).

SummaryUsing HCA brings a wide variety of benefits to drug discovery

and development, including increased throughput, productivity

and improved data quality. Single cell analysis in multiple

wells can be performed with quantitative analysis on multiple

parameters on each of the cells, greatly improving statistically

significant data sets. Bringing toxicity testing into earlier

stages of drug development and employing HCA methods for

in vitro toxicology assays offers the opportunity for rapid,

cost-effective and sensitive detection of potentially toxic

compounds ahead of in vivo animal testing and expensive

clinical trials. Millipore is at the forefront of providing tools

and solutions for this rapidly growing and exciting field.

Dose Response of NGF-induced NeuriteOutgrowth StimulationPC12 cells were cultured in low serum differentiation mediacontaining serial dilutions of NGF (max. concentration = 1000 ng/mL)for 6 days, replacing media/NGF very 3 days, then fixed and stained.Cells were imaged on the GE IN Cell Analyzer 1000 (3.3) at 20X (5 fields/well) and analyzed using the GE IN Cell Analyzer 1000 Workstation(3.4) Neurite Outgrowth algorithm. (Mean ± SEM, n = 4)

Dose Response of K252a- and Nocodozole-inducedNeurite Outgrowth StimulationPC12 cells were cultured in low serum differentiation media containing100 ng/mL NGF for 6 days, replacing media/NGF every 3 days. Cellsreceived treatment with either serial dilutions of the protein kinaseinhibitor K252a, for the final 3 days of culture (max. concentration =1000 nM), or the microtubule depolymerization agent nocodozole,for the final 4 hours of culture (max. concentration = 35 µM). Cellswere imaged on the GE IN Cell Analyzer 1000 (3.3) at 10X (10 fields/well) and analyzed using the GE IN Cell Analyzer 1000 Workstation(3.4) Neurite Outgrowth algorithm. (Mean ± SEM, n = 4)

Chart A. Chart B.

*Co-developed by Millipore Corp. and Cellumen, Inc.

*CELLCIPHRTM KITFOR HUMAN HEPATOXICITYLiver damage, or hepatotoxicity, is a major factor in the high

failure rate of lead compounds in drug development and the

withdrawal of drugs from market. The cost of losing a lead

drug candidate in

these late stages to

unanticipated toxicity

is considerable.

Therefore, reliable

biomarkers of liver

damage are required

for prediction of these potential causes of drug failure to

reduce cost and accelerate time to market.

Millipore’s new HCA assay—the CellCiphr kit for human

hepatotoxicity—offers the most comprehensive cell-based

cytotoxicity assay available. CellCiphr provides a new way of

prioritizing lead compounds based on cytotoxicity indices,

enabling you to gain greater insight and advance the most

promising lead candidates.

traditional radiolabelled cell proliferation. Together these kits

provide an overview of cell health and viability on exposure

to various compound concentrations during the drug

discovery process.

The most comprehensivecell-based cytotoxicityassay available foraccurately detectingliver damage.

The CellCiphr panel of antibodies specific for profiling humanhepatotoxicity in HepG2 cells.

APPLICATION NOTE

• The CellCiphr Cytotoxicity kit measures 11 parameters over three time points in a 384-well format using

human hepatocytes, HepG2 cells. The 11 parameters in the CellCiphr kit represent the broadest range of

values to give a complete picture of cell health during drug screening. Measurements are made at acute, early

and chronic exposure time points. The kit enables the analysis of up to 20 compounds at one time. It also

contains software to perform a compilation of data into dose-response curves for each compound. This kit

contains HCA-validated antibodies, reagents and protocols, and works on any HCA instrument.

CELL CYCLE ANDCELL PROLIFERATION ASSAYSDisturbance of the cell cycle, particularly inhibition of

cell proliferation, is a sensitive indicator of cytotoxicity.

Measurements of cell proliferation give indications of cell

viability and responsiveness, which are crucial for under-

standing developmental mechanisms and cellular pathology.

With Millipore’s HCA assays, you’re able to assess phospho-

histone H3ser10, Ki-67, cyclin B1 and α-tubulin during the cell

cycle. These targets represent sensitive biomarkers for

detecting disruption of normal cell division. In addition, the

Millipore bromodeoxyuridine (BrdU) kit is an antibody-based

kit that measures cell proliferation, and is an alternative to

A wide range of kits for an overview ofcell health and viability on exposure tovarious compound concentrations duringthe drug discovery process.

DNA damage and

apoptosis. These

include: p53, an

attractive therapeutic

target since its

tumor-suppressor

activity can be

stimulated to eradicate tumor cells; and the histone

protein, H2A.X.

APPLICATION NOTE

• Detection of pH-H3 is a measure of mitotic index—the percentage of cells in M phase—and allows identification

of compounds that inhibit or induce mitosis. Ki-67 antigen is expressed during late G1, S, G2 and M phase of

the cell cycle, while resting, non-cycling cells (G0 phase) lack Ki-67 expression. Cyclin B1 has distinct expression

patterns during prophase, metaphase and anaphase and discriminates between G2, M, and G0/G1/S phases of

the cell cycle. Microtubules, such as α-tubulin, are crucial to maintain cell shape and for intracellular transport.

Disturbance of microtubule dynamics leads to cell cycle arrest at mitosis and may ultimately cause the

induction of apoptosis

Dose Response of A549 and HeLa Cells to Cell-cycle Inhibition AgentsA549 or HeLa cells were treated for the last 4 hours of culture with serial dilutions of either vinblastine sulfate (Chart A, G2/M phase arrest,max. concentration = 1 µM) or etoposide (Chart B, S/G2 phase arrest, max. concentration = 100 µM). Cells were imaged on the GE IN CellAnalyzer 1000 at 10X (10 fields/well) and analyzed using the IN Cell Analyzer 1000 Workstation (3.4) Multi Target Analysis algorithm. Mean± SEM, n = 4.

DNA DAMAGE & APOPTOSIS ASSAYSAssessment of DNA damage during drug development is

essential for detecting any potential genotoxic or apoptotic

effects caused by drug candidate compounds. Using HCA al-

lows DNA damage to be visualized at the same time as

other cellular parameters such as membrane integrity and

cytoskeleton structure, providing greater insight.

Millipore has developed two kits to enable sensitive

detection and assessment of key proteins that regulate

Two important kits thatenable better detectionand assessment of DNAdamage and apoptosisduring drug development.

APPLICATION NOTE

• The tumor suppressor protein p53 is central to many anti-cancer mechanisms, regulating expression of genes

involved in growth arrest, apoptosis, cellular senescence and repair of DNA damage. p53 is an attractive

therapeutic target because its tumor-suppressor activity can be stimulated to eradicate tumor cells; so much

effort has been placed on identifying cellular proteins that modulate p53 function.

• Histone proteins, such as H2A.X protect DNA. Post-translational modifications of histones mediate rapid and

reversible responses to changing cellular signals. When DNA damage (e.g., DNA strand breakage) occurs,

phosphorylated H2A.X accumulates at the site of the damage. This accumulation helps recruit DNA repair

machinery to the site of the breakage. When DNA damage is severe, the cell can undergo apoptosis. Since

this modification occurs before many of the morphological changes are detectable, it is one of the earliest

indicators of apoptosis.

NEUROTOXICITY ASSAYSMajor efforts in CNS research focus on the identification of

compounds that affect the promotion or inhibition of growth

of new cellular processes (neurite outgrowth) from healthy

neurons. That’s because neurite formation and neuronal

regeneration hold

therapeutic promise

for Alzheimer’s and

Parkinson’s diseases,

and other neuro-

degenerative disorders.

Traditional assays for

neurotoxic endpoints

are slow, labor-

intensive and often subjective. Yet Millipore’s HCA neurite

outgrowth kit is different because it enables rapid screening

of multiple parameters in individual cells, such as cell body

count, neurite count and neurite length on a variety of cell

lines and primary cells. The high sensitivity of the assay,

coupled with sensitive neuronal specific reagents, enables

identification of neuronal cell bodies and neurites in

heterogeneous cell populations and is ideal in large scale

screening for inducers and inhibitors of neurite outgrowth.

A neurite kit that offersrapid screening of multipleparameters in individualcells, making it ideal inlarge scale screening forinducers and inhibitorsof neurite outgrowth.

Validation and specificity of primary antibodies for use indevelopment of neurotoxicity biomarkers assays. Rathippocampal astrocytes labeled for α-tubulin (red) andGFAP (green).

SummaryUsing HCA brings a wide variety of benefits to drug discovery

and development, including increased throughput, productivity

and improved data quality. Single cell analysis in multiple

wells can be performed with quantitative analysis on multiple

parameters on each of the cells, greatly improving statistically

significant data sets. Bringing toxicity testing into earlier

stages of drug development and employing HCA methods for

in vitro toxicology assays offers the opportunity for rapid,

cost-effective and sensitive detection of potentially toxic

compounds ahead of in vivo animal testing and expensive

clinical trials. Millipore is at the forefront of providing tools

and solutions for this rapidly growing and exciting field.

Dose Response of NGF-induced NeuriteOutgrowth StimulationPC12 cells were cultured in low serum differentiation mediacontaining serial dilutions of NGF (max. concentration = 1000 ng/mL)for 6 days, replacing media/NGF very 3 days, then fixed and stained.Cells were imaged on the GE IN Cell Analyzer 1000 (3.3) at 20X (5 fields/well) and analyzed using the GE IN Cell Analyzer 1000 Workstation(3.4) Neurite Outgrowth algorithm. (Mean ± SEM, n = 4)

Dose Response of K252a- and Nocodozole-inducedNeurite Outgrowth StimulationPC12 cells were cultured in low serum differentiation media containing100 ng/mL NGF for 6 days, replacing media/NGF every 3 days. Cellsreceived treatment with either serial dilutions of the protein kinaseinhibitor K252a, for the final 3 days of culture (max. concentration =1000 nM), or the microtubule depolymerization agent nocodozole,for the final 4 hours of culture (max. concentration = 35 µM). Cellswere imaged on the GE IN Cell Analyzer 1000 (3.3) at 10X (10 fields/well) and analyzed using the GE IN Cell Analyzer 1000 Workstation(3.4) Neurite Outgrowth algorithm. (Mean ± SEM, n = 4)

Chart A. Chart B.

*Co-developed by Millipore Corp. and Cellumen, Inc.

*CELLCIPHRTM KITFOR HUMAN HEPATOXICITYLiver damage, or hepatotoxicity, is a major factor in the high

failure rate of lead compounds in drug development and the

withdrawal of drugs from market. The cost of losing a lead

drug candidate in

these late stages to

unanticipated toxicity

is considerable.

Therefore, reliable

biomarkers of liver

damage are required

for prediction of these potential causes of drug failure to

reduce cost and accelerate time to market.

Millipore’s new HCA assay—the CellCiphr kit for human

hepatotoxicity—offers the most comprehensive cell-based

cytotoxicity assay available. CellCiphr provides a new way of

prioritizing lead compounds based on cytotoxicity indices,

enabling you to gain greater insight and advance the most

promising lead candidates.

traditional radiolabelled cell proliferation. Together these kits

provide an overview of cell health and viability on exposure

to various compound concentrations during the drug

discovery process.

The most comprehensivecell-based cytotoxicityassay available foraccurately detectingliver damage.

The CellCiphr panel of antibodies specific for profiling humanhepatotoxicity in HepG2 cells.

APPLICATION NOTE

• The CellCiphr Cytotoxicity kit measures 11 parameters over three time points in a 384-well format using

human hepatocytes, HepG2 cells. The 11 parameters in the CellCiphr kit represent the broadest range of

values to give a complete picture of cell health during drug screening. Measurements are made at acute, early

and chronic exposure time points. The kit enables the analysis of up to 20 compounds at one time. It also

contains software to perform a compilation of data into dose-response curves for each compound. This kit

contains HCA-validated antibodies, reagents and protocols, and works on any HCA instrument.

CELL CYCLE ANDCELL PROLIFERATION ASSAYSDisturbance of the cell cycle, particularly inhibition of

cell proliferation, is a sensitive indicator of cytotoxicity.

Measurements of cell proliferation give indications of cell

viability and responsiveness, which are crucial for under-

standing developmental mechanisms and cellular pathology.

With Millipore’s HCA assays, you’re able to assess phospho-

histone H3ser10, Ki-67, cyclin B1 and α-tubulin during the cell

cycle. These targets represent sensitive biomarkers for

detecting disruption of normal cell division. In addition, the

Millipore bromodeoxyuridine (BrdU) kit is an antibody-based

kit that measures cell proliferation, and is an alternative to

A wide range of kits for an overview ofcell health and viability on exposure tovarious compound concentrations duringthe drug discovery process.

DNA damage and

apoptosis. These

include: p53, an

attractive therapeutic

target since its

tumor-suppressor

activity can be

stimulated to eradicate tumor cells; and the histone

protein, H2A.X.

APPLICATION NOTE

• Detection of pH-H3 is a measure of mitotic index—the percentage of cells in M phase—and allows identification

of compounds that inhibit or induce mitosis. Ki-67 antigen is expressed during late G1, S, G2 and M phase of

the cell cycle, while resting, non-cycling cells (G0 phase) lack Ki-67 expression. Cyclin B1 has distinct expression

patterns during prophase, metaphase and anaphase and discriminates between G2, M, and G0/G1/S phases of

the cell cycle. Microtubules, such as α-tubulin, are crucial to maintain cell shape and for intracellular transport.

Disturbance of microtubule dynamics leads to cell cycle arrest at mitosis and may ultimately cause the

induction of apoptosis

Dose Response of A549 and HeLa Cells to Cell-cycle Inhibition AgentsA549 or HeLa cells were treated for the last 4 hours of culture with serial dilutions of either vinblastine sulfate (Chart A, G2/M phase arrest,max. concentration = 1 µM) or etoposide (Chart B, S/G2 phase arrest, max. concentration = 100 µM). Cells were imaged on the GE IN CellAnalyzer 1000 at 10X (10 fields/well) and analyzed using the IN Cell Analyzer 1000 Workstation (3.4) Multi Target Analysis algorithm. Mean± SEM, n = 4.

DNA DAMAGE & APOPTOSIS ASSAYSAssessment of DNA damage during drug development is

essential for detecting any potential genotoxic or apoptotic

effects caused by drug candidate compounds. Using HCA al-

lows DNA damage to be visualized at the same time as

other cellular parameters such as membrane integrity and

cytoskeleton structure, providing greater insight.

Millipore has developed two kits to enable sensitive

detection and assessment of key proteins that regulate

Two important kits thatenable better detectionand assessment of DNAdamage and apoptosisduring drug development.

APPLICATION NOTE

• The tumor suppressor protein p53 is central to many anti-cancer mechanisms, regulating expression of genes

involved in growth arrest, apoptosis, cellular senescence and repair of DNA damage. p53 is an attractive

therapeutic target because its tumor-suppressor activity can be stimulated to eradicate tumor cells; so much

effort has been placed on identifying cellular proteins that modulate p53 function.

• Histone proteins, such as H2A.X protect DNA. Post-translational modifications of histones mediate rapid and

reversible responses to changing cellular signals. When DNA damage (e.g., DNA strand breakage) occurs,

phosphorylated H2A.X accumulates at the site of the damage. This accumulation helps recruit DNA repair

machinery to the site of the breakage. When DNA damage is severe, the cell can undergo apoptosis. Since

this modification occurs before many of the morphological changes are detectable, it is one of the earliest

indicators of apoptosis.

NEUROTOXICITY ASSAYSMajor efforts in CNS research focus on the identification of

compounds that affect the promotion or inhibition of growth

of new cellular processes (neurite outgrowth) from healthy

neurons. That’s because neurite formation and neuronal

regeneration hold

therapeutic promise

for Alzheimer’s and

Parkinson’s diseases,

and other neuro-

degenerative disorders.

Traditional assays for

neurotoxic endpoints

are slow, labor-

intensive and often subjective. Yet Millipore’s HCA neurite

outgrowth kit is different because it enables rapid screening

of multiple parameters in individual cells, such as cell body

count, neurite count and neurite length on a variety of cell

lines and primary cells. The high sensitivity of the assay,

coupled with sensitive neuronal specific reagents, enables

identification of neuronal cell bodies and neurites in

heterogeneous cell populations and is ideal in large scale

screening for inducers and inhibitors of neurite outgrowth.

A neurite kit that offersrapid screening of multipleparameters in individualcells, making it ideal inlarge scale screening forinducers and inhibitorsof neurite outgrowth.

Validation and specificity of primary antibodies for use indevelopment of neurotoxicity biomarkers assays. Rathippocampal astrocytes labeled for α-tubulin (red) andGFAP (green).

SummaryUsing HCA brings a wide variety of benefits to drug discovery

and development, including increased throughput, productivity

and improved data quality. Single cell analysis in multiple

wells can be performed with quantitative analysis on multiple

parameters on each of the cells, greatly improving statistically

significant data sets. Bringing toxicity testing into earlier

stages of drug development and employing HCA methods for

in vitro toxicology assays offers the opportunity for rapid,

cost-effective and sensitive detection of potentially toxic

compounds ahead of in vivo animal testing and expensive

clinical trials. Millipore is at the forefront of providing tools

and solutions for this rapidly growing and exciting field.

Dose Response of NGF-induced NeuriteOutgrowth StimulationPC12 cells were cultured in low serum differentiation mediacontaining serial dilutions of NGF (max. concentration = 1000 ng/mL)for 6 days, replacing media/NGF very 3 days, then fixed and stained.Cells were imaged on the GE IN Cell Analyzer 1000 (3.3) at 20X (5 fields/well) and analyzed using the GE IN Cell Analyzer 1000 Workstation(3.4) Neurite Outgrowth algorithm. (Mean ± SEM, n = 4)

Dose Response of K252a- and Nocodozole-inducedNeurite Outgrowth StimulationPC12 cells were cultured in low serum differentiation media containing100 ng/mL NGF for 6 days, replacing media/NGF every 3 days. Cellsreceived treatment with either serial dilutions of the protein kinaseinhibitor K252a, for the final 3 days of culture (max. concentration =1000 nM), or the microtubule depolymerization agent nocodozole,for the final 4 hours of culture (max. concentration = 35 µM). Cellswere imaged on the GE IN Cell Analyzer 1000 (3.3) at 10X (10 fields/well) and analyzed using the GE IN Cell Analyzer 1000 Workstation(3.4) Neurite Outgrowth algorithm. (Mean ± SEM, n = 4)

Chart A. Chart B.

*Co-developed by Millipore Corp. and Cellumen, Inc.

High Content Analysis:In Vitro Toxicology & Cell-Based Assays

High Content Analysis (HCA) is high throughput, automated

microscopy which provides a rapid means of quantitative

image acquisition and analysis. It has a direct impact in

every area of cellular research and has facilitated the drug

discovery process by allowing detection of multiple parame-

ters in a single assay. Using Millipore’s high-quality reagents

in ready-to-use kits and cell-based assays that incorporate

the power of HCA, researchers learn more about functional

cell biology in many different systems, including one of the

greatest challenges in drug development: in vitro toxicology.

Accelerating the Pace of High ContentAnalysis with the Most ComprehensiveMultiplex Cytotoxicity KitsOne of the bottlenecks in the drug development process is

compound toxicicity and unanticipated adverse clinical

effects during clinical trials. That’s why Millipore, the original

multiplexing leader, is advancing the use of multiparameter

assays in cytotoxicity studies to improve the drug discovery

workflow. Our tools and solutions for research and drug

discovery include a wide array of cell-based assays for

cytotoxicity using HCA.

With our multiplexed assays and measurements at a

range of time points, you obtain improved sensitivity and

specificity for detection of subtle cellular changes indicative

of toxic effects.

Move your research forward with speed and efficiency.

Find out how Millipore can help you better detect the early

indicators of toxicity while simultaneously measuring multiple

biomarkers for cell viability and providing functional, relevant

cellular data.

Data Sheet

Millipore HCA Kits Offer:• Validated antibodies and dyes for high content analysis

that work on all HCA systems

• Fluorescence-based antibody assays that work on any

fluorescence or confocal microscope

• Multiplexed kits with specific control toxins and compounds

included, enabling you to gain functional data on test

compounds

• Validated protocols and recommended cell lines that require

no further assay development

ORDERING INFORMATIONDescription Application Catalogue No.

CellCiphr Cytotoxicity Assay Human HepG2 Cells Cellular Systems Biology (CSB) Kit Hepatotoxicity HCS100Neurotoxicity and Neurite Outgrowth Assay for High Content Screening Neuroscience, Neurotoxicity HCS220BrdU Assay Apoptosis HCS201Mouse primary antibodyCyclin B1 Assay Cell Cycle HCS202Mouse primary antibodyPhospho-Histone H3Ser10 Assay Cell Proliferation, Aurora Kinase Profiling HCS203Mouse primary antibodyPhospho-Histone H3Ser10 Assay Cell Proliferation, Aurora Kinase Profiling HCS204Rabbit primary antibodyKi-67 Assay Cell Cycle Stage Segregation HCS205Rabbit primary antibodyKi-67 Assay Cell Cycle Stage Segregation HCS206Mouse primary antibodyPhospho-Histone H3(Ser 10) and Ki-67 Assay Cell Proliferation, Aurora Kinase Profiling, HCS209

Cell Cycle EvaluationCyclin B1 and Ki-67 Assay Cell Proliferation, Cell Cycle Evaluation HCS210Phospho-Histone H3(Ser 10) and Cyclin B1 Assay Cell Proliferation, Cell Cycle Evaluation HCS211BrdU and Phospho-Histone H3(Ser 10) Assay Apoptosis, Aurora Kinase Profiling, HCS212

Cell Cycle EvaluationBrdU and Ki-67 Assay Apoptosis, Cell Cycle HCS213High Content Screening Toolkit Buffers, reagents for using mouse antibodies HCS214For Use With Mouse Primary AntibodiesHigh Content Screening Toolkit Buffers, reagents for using rabbit antibodies HCS215For Use With Rabbit Primary AntibodiesPhospho-histone H3 and α-Tubulin Assay Cell Cycle Segregation, Cytoskeletal Analysis HCS216p53 DNA Damage HCS223H2A.X DNA Damage, Apoptosis HCS224p53 + H2A.X DNA Damage, Apoptosis HCS225

DRUG DISCOVERY AND DEVELOPMENTMillipore provides tools and technologies to facilitate

discovery and evaluation of new drug candidates that help

you achieve results faster than ever before. Through the

acquisition of Upstate®, Chemicon® and Linco®, we now

offer a suite of products that span the drug discovery

pipeline from target identification to clinical studies. Our

expert team of scientists and engineers understands the

complexity of your discovery and development and can

support you in these challenges. Your drive to discovery

starts with Millipore.

www.millipore.com/offices

Millipore, Chemicon, Linco and Upstate are registered trademarks of Millipore Corporation.

M Logo and Advancing Life Science Together are trademarks of Millipore Corporation.

CellCiphr is a trademark of Cellumen, Inc.

FSC is a registered trademark of the Forrest Stewardship Council.

Lit. No. DS1837EN00 08DD026 Printed in U.S.A.

© 2008 Millipore Corporation, Billerica, MA 01821 U.S.A. All rights reserved.

ADVANCING LIFE SCIENCE TOGETHER™Research. Development. Production.

For more information on HCA products, visit www.millipore.com/hca. For other ways Millipore supports LifeScience research, contact your local Millipore Drug Discovery Specialist.

High Content Analysis:In Vitro Toxicology & Cell-Based Assays

High Content Analysis (HCA) is high throughput, automated

microscopy which provides a rapid means of quantitative

image acquisition and analysis. It has a direct impact in

every area of cellular research and has facilitated the drug

discovery process by allowing detection of multiple parame-

ters in a single assay. Using Millipore’s high-quality reagents

in ready-to-use kits and cell-based assays that incorporate

the power of HCA, researchers learn more about functional

cell biology in many different systems, including one of the

greatest challenges in drug development: in vitro toxicology.

Accelerating the Pace of High ContentAnalysis with the Most ComprehensiveMultiplex Cytotoxicity KitsOne of the bottlenecks in the drug development process is

compound toxicicity and unanticipated adverse clinical

effects during clinical trials. That’s why Millipore, the original

multiplexing leader, is advancing the use of multiparameter

assays in cytotoxicity studies to improve the drug discovery

workflow. Our tools and solutions for research and drug

discovery include a wide array of cell-based assays for

cytotoxicity using HCA.

With our multiplexed assays and measurements at a

range of time points, you obtain improved sensitivity and

specificity for detection of subtle cellular changes indicative

of toxic effects.

Move your research forward with speed and efficiency.

Find out how Millipore can help you better detect the early

indicators of toxicity while simultaneously measuring multiple

biomarkers for cell viability and providing functional, relevant

cellular data.

Data Sheet

Millipore HCA Kits Offer:• Validated antibodies and dyes for high content analysis

that work on all HCA systems

• Fluorescence-based antibody assays that work on any

fluorescence or confocal microscope

• Multiplexed kits with specific control toxins and compounds

included, enabling you to gain functional data on test

compounds

• Validated protocols and recommended cell lines that require

no further assay development

ORDERING INFORMATIONDescription Application Catalogue No.

CellCiphr Cytotoxicity Assay Human HepG2 Cells Cellular Systems Biology (CSB) Kit Hepatotoxicity HCS100Neurotoxicity and Neurite Outgrowth Assay for High Content Screening Neuroscience, Neurotoxicity HCS220BrdU Assay Apoptosis HCS201Mouse primary antibodyCyclin B1 Assay Cell Cycle HCS202Mouse primary antibodyPhospho-Histone H3Ser10 Assay Cell Proliferation, Aurora Kinase Profiling HCS203Mouse primary antibodyPhospho-Histone H3Ser10 Assay Cell Proliferation, Aurora Kinase Profiling HCS204Rabbit primary antibodyKi-67 Assay Cell Cycle Stage Segregation HCS205Rabbit primary antibodyKi-67 Assay Cell Cycle Stage Segregation HCS206Mouse primary antibodyPhospho-Histone H3(Ser 10) and Ki-67 Assay Cell Proliferation, Aurora Kinase Profiling, HCS209

Cell Cycle EvaluationCyclin B1 and Ki-67 Assay Cell Proliferation, Cell Cycle Evaluation HCS210Phospho-Histone H3(Ser 10) and Cyclin B1 Assay Cell Proliferation, Cell Cycle Evaluation HCS211BrdU and Phospho-Histone H3(Ser 10) Assay Apoptosis, Aurora Kinase Profiling, HCS212

Cell Cycle EvaluationBrdU and Ki-67 Assay Apoptosis, Cell Cycle HCS213High Content Screening Toolkit Buffers, reagents for using mouse antibodies HCS214For Use With Mouse Primary AntibodiesHigh Content Screening Toolkit Buffers, reagents for using rabbit antibodies HCS215For Use With Rabbit Primary AntibodiesPhospho-histone H3 and α-Tubulin Assay Cell Cycle Segregation, Cytoskeletal Analysis HCS216p53 DNA Damage HCS223H2A.X DNA Damage, Apoptosis HCS224p53 + H2A.X DNA Damage, Apoptosis HCS225

DRUG DISCOVERY AND DEVELOPMENTMillipore provides tools and technologies to facilitate

discovery and evaluation of new drug candidates that help

you achieve results faster than ever before. Through the

acquisition of Upstate®, Chemicon® and Linco®, we now

offer a suite of products that span the drug discovery

pipeline from target identification to clinical studies. Our

expert team of scientists and engineers understands the

complexity of your discovery and development and can

support you in these challenges. Your drive to discovery

starts with Millipore.

www.millipore.com/offices

Millipore, Chemicon, Linco and Upstate are registered trademarks of Millipore Corporation.

M Logo and Advancing Life Science Together are trademarks of Millipore Corporation.

CellCiphr is a trademark of Cellumen, Inc.

FSC is a registered trademark of the Forrest Stewardship Council.

Lit. No. DS1837EN00 08DD026 Printed in U.S.A.

© 2008 Millipore Corporation, Billerica, MA 01821 U.S.A. All rights reserved.

ADVANCING LIFE SCIENCE TOGETHER™Research. Development. Production.

For more information on HCA products, visit www.millipore.com/hca. For other ways Millipore supports LifeScience research, contact your local Millipore Drug Discovery Specialist.