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Transcript of Cell Biology Assay kits - INTERCHIM: · PDF fileCell Biology - Assays Kits Cell Biology ......

  • Cell Biology - Assays KitsC

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    Apoptosis Cell and Tissue Control SlidesFollowing control slides will help developing familiarity with the methods of in situ apoptosislabeling and to determine if reagents are working optimally.

    Description Cat.# QtyCell Culture Control Slides Q69270 2 u

    Q69271 5 uTissue Control Slides Q69290 2 u

    Q69291 5 u

    See also Antibodies Research Area #3)( Apoptosis, and related Abs) and Last minute Abs(moitochondrial research).See also M-PCR Kits

    Apoptosis

    Technical tipApoptosis is a programmed, cell-autonomousdeath process, known to impact every stage oflife of multicellular eukaryotes. It is involved in avariety of physiological and pathological events,ranging from normal development (embryogrowth, postnatal organ modeling and cellturnover) to diseases and disorders (cancer,organ failure and ageing, andneurodegenerative diseases). Duringapoptosis, the caspases execute thedisassembly of cellular components byproteolytic cleavage of a variety of substrates,such as poly-(ADP ribose) polymerase (PARP),DNA-dependent protein kinase (DNA-PK),topoisomerases, and protein kinase C (PKC).At least ten caspases have been discovered.Some of these caspases identify and cleave aspecific peptide substrate, while othersrecognize the same peptide substrate.

    Selection guide

    For your research Interchim offers a comprehensive line of products :! Membrane assays (AnnexinV, AppolerCentage)

    ! Caspase assays

    ! Mitochondria assays

    Method Advantages DisadvantagesApoptosis membrane alteration : Dye Single apoptotic cell (IHC). Quick and Limited information available on cell(uptake bioassay: APOPercentage). easy to use. Assay can also be membrane composition.

    quantified, using a digital camera or amicroplate colorimeter/fluorimeter.Necrotic cells cannot retain the dye.Several hundred assays can beperformed in one day.

    Apoptosis membrane alteration: Sensitivity Single cell (FCM, IHF). Does not discriminate between apoptotic(Annexin V binding) Confirms the occurrence of and necrotic cells.

    phosphatidylserine flippase inapoptotic cells, and the activity ofinitiator caspases.

    Fluorescence Activated Cell Sorter Good option for suspension cells. The equipment is designed for cell(FACS). counting and cell sorting. Excessive

    physical force, to remove cell clumps, candamage cells. Lower Sensitivity (>100apoptotic cells to discern cluster in asample scan.

    Apoptotic proteases It is possible to select for individual Lysis or fixation of cells (except for(caspases assays) initiator caspases or execution CytoxiLux)Caspase activation does not

    caspases. Sensitivity : Single cell necessarily imply that apoptosis will(with FCM) (IEA or IFA: occur.>usually 1 x 105 cells) Detection and measurement of specific caspaseactivity.

    Enzymatic end labelling of DNA Can be completed within 3 hours. Fixation of cells. Subject to false positivesstrand breaks (TdT TUNEL: TACS) Fluorescence microscopy or FACS. from necrotic cells and risk of high

    Sensitivity. Single cell background from some viable cells.Collection times can be critical, earlycollection and DNA fragmentation may notyet be extensive, or later and DNAfragmentation can be excessive.

    DNA fragmentation Photographic evidence of large DNA Can be difficult to produce a nucleosome(BET/Electrophoresis : Commet Assay) fragmentsSingle cell ladder as further DNAfragmentation can

    (with Comet Assay) occur during preparation. Necrotic cellsalso generate DNA fragmentsSensitivity. 1 x 106 cells

  • Cell Biology

    Cell Biology - Assays Kits

    e-mail [email protected] Visit our website : www.interchim.com!

    E.157

    Membrane apoptosis events

    Labeled Annexin V

    Apoptosis

    Exposure of phosphatidyl serine

    One of the first identifiable events during apoptosis is the flipping of phosphatidyl serine(PS) on the cell membrane, resulting in exposure of PS on the cell surface. Theanticoagulant annexin V binds specifically to PS in a calcium-dependant event. PStranslocation to the cell surface precedes nuclear breakdown, DNA fragmentation, andthe appearance of most apoptosis-associated molecules, making annexin V binding amarker of apoptosis early-stage.Annexin V is available conjugated with following labels by an optimized chemistry to ensureproper PS binfing.

    Performance of Annexin V-FITC and Annexin V-Fluoprobes488 with Confocal Scanning Laser Microscopy.The Annexin V-Fluoprobes 488 is superior to the FITCprobe, both in quantum yield and in resistance to bleaching.

    Description Cat.# QtyAnnexin V - FluoProbes 488 FP-BH4140 500 lfor confocal microscopy 494/519 nmAnnexin V - FluoProbes 488 FP-BH9390 100 testsfor flow cytometry 494/519 nmAnnexin V - FITC FP-M19652 250 l494/518 nmAnnexin V - R-phycoerythrin (RPE) FP-AH191A 50 tests496, 546, 565/578 nmAnnexin V - Allo-phycocyanin (APC) FP-AK194A 50 tests650/660 nmAnnexin V - X - Biotin FP-M1969A 500 e

    Related products :30 fluorescents streptavidins can be chosen. see page A350Anti - Annexin V - FITC R51380 100 tests494/518 nmAnnexin V, 10x binding buffer BU2080 1.7 ml

    Comparison of the performances of Annexin V- FluoProbes 448 by flow cytometryand Annexin V-FITC.

    The Jurkat cells are incubated with anti-Fas antibody for 3 hours to activate apoptosis.The activated cell suspension is then mixed with Annexin V- FluoProbes 488 or AnnexinV-FITC and further incubated for 15 minutes.

    Figure 2 shows the fluorescence intensities histograms of the cells incubated with annexinV-FluoProbes 488. Table lists the Mean Fluorescence Intensities of the viable cells (M1)and the apoptotic cells (M2) and the ratio of M2/M1 with annexin V- FluoProbes 488 orannexin V-FITC.

    Figure 2 : Histogram of the cells incubated withannexin V-FP488

    Associated product :Prodium iodie Iodide FP-36774A 10 ml see page E123.7.AAD FP-132303 1 mg see page E126 used for dead cellidentification.

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    TACS Annexin V Kitsdetection of apoptotic cells

    Features :! Fast, sensitive detection of apoptotic cells.

    ! Includes propidium iodide for discriminating apoptotic and necrotic (or lateapoptotic) cells.

    ! Annexin V-Biotin provides flexibility in fluorophore choice.

    TACSTM Annexin V Kits allow rapid, specific, and quantitative identification of apoptosis inindividual cells. They use annexin V conjugated to either FITC or to biotin for the detectionof cell surface changes during apoptosis. The annexin V conjugates are supplied with anoptimized Binding Buffer and propidium iodide. Propidium iodide may be used on unfixedsamples to determine the population of cells that have lost membrane integrity, an indicationof late apoptosis or necrosis.

    Description Cat.# QtyTACS Annexin V Kits 372810 100 tests

    372811 250 tests

    Related products :Several complementatry probes are described in section E :

    Description Cat.# QtyPI see page E123 FP-36774A 10 ml @ 1 mg/ml7-AAD is used for dead cell identification See page E126 FP-132303 1 mg

    Flow Cytometry analysis of WEHI 7.1 cells labeled withAnnexin V-Biotin and detected by streptavidin-FITC.WEHI 7.1 cells treated with 25 m etoposide for two hourswith an overnight recovery produce a peak approximatelylog of 103 in the fluorescence channel 1 (FL1) (samples 2and 3). Healthy WEHI 7.1 cells produce a peak less thanlog 101 in the FL1 channel which is similar to unlabeledcells (samples 4.5 and 1 respectively). Analysis was testedusing two different populations of cells (samples 1,2,4 and3,5 respectively).

    Apoptosis

    APOPercentage APOPTOSIS AssayThe APOPercentage Assay is a detection and MEASUREMENT system to monitor theincidence of apoptosis in mammalian anchorage-dependent cells during in vitro culture.

    Apoptotic cells take up the APOPercentage dye, as shown in the pictures below.

    Following dye uptake, the level of apoptosis can be quantified colorimetrically,fluorometrically or by analytical digital photomicroscopy (ADP).

    ! Assay time : 1 hour

    ! Assay sensitivity : a single apoptotic cell.

    ! Compatible with CDD camera znd microplate readers

    Description Cat.# QtyAPOPercentage Apoptosis Assay kits :Trial size Kit (3 x 96 wells; microwell format) R47770 250 testsStandard Assay Kit (6 x 96 wells; microwell format) R47771 500 tests

    Components of the # R47770 kit :APOPercentage Dye reagent (5 ml) in phosphate buffered saline (sterile vial)APOPercentage Dye Reference Standard (10 M/10 ml), (sterile vial)APOPercentage Dye Release reagent (150 ml)Gelatin matrix forming solution (0.4%/100 ml / sterile)

    2 hr 4hr 6hrDigital images of CHO cells treated with 10 mMcycloheximide, to induce apoptosis after 2, 4 and 6h.

  • Cell Biology

    Cell Biology - Assays Kits

    e-mail [email protected] Visit our website : www.interchim.com!

    E.159

    ApoptosisBiochemicals for apoptosis research

    Caspase inhibitor kitFeatures :

    ! Synthetic peptides coupled to fluoromethylketone (FMK) which irreversibly bindsthe caspase.

    ! High concentrations of inhibitor to reduce risks of DMSO toxicity.

    ! Each kit is provided with a negative control and a general caspase inhibitor.

    ! Application range from 0.2 to 200 M.

    Description Cat.# QtyCaspase 3 Inhibitor Kit Q70200 20 lCaspase 8 Inhibitor Kit Q70220 20 lCaspase 9 Inhibitor Kit Q70240 20 lCaspase 3,8,9 Inhibitor Kit