Apoptosis and Cell Proliferation (522915), страница 17
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Itshows no cross-reactivity with BrdU.E The conjugated antibody (Anti-BrdUPOD, Fab fragments) will bind toBrdU-labeled DNA after the DNA ispartially denatured. The antibody specifically recognizes 5-bromo-2’-deoxyuridine. The antibody conjugate showsno cross-reactivity with any endoge-56nous cellular components such as thymidine or uridine.E The ELISA specifically detects BrdUlabeled DNA fragments in culture supernatant and cytoplasm. The ELISAcan detect BrdU-labeled DNA fromany species, so the assay is not speciesrestricted.Can be used to assay:P Culture supernatant and cytoplasmicfractions (lysates) of cells whose DNAhave been metabolically prelabeled withBrdU (e.g.
cell lines and other in vitroproliferating cells). Thus, only cellswhich proliferate in vitro can be used.Kit contents1. Anti-DNA antibody (clone M-CA-33)2. Anti-BrdU-POD, Fab fragments (cloneBMG 6H8)3. Coating buffer4. Washing buffer5. Incubation buffer6. Substrate solution7. BrdU labeling reagent8. Adhesive cover foilsTypical results: See Figures 38 and 39.++––BrdU incorporation (A 405 nm )1.20.9Other applications: For more examples ofhow the Cellular DNA FragmentationELISA can be used in the lab, see Appendix, page 120.CAM, SNCAM, lys.CAM, SNCAM, lys.0.60.310.00123456789Time (h)G Figure 38: Kinetics of camptothecin (CAM)induced cell death in HL60 cells. Cells were prelabeledwith BrdU overnight. Then, cells (1 x 104/well) wereincubated either in the presence of 200 ng/ml CAM(P, m) or without CAM (P, m) for 1–8 h.
Supernatant(100 µl/well) was removed, then cells were lysed andboth supernatant (P, P) and lysate (m, m) 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.2.absorbance (A 405 nm )1.61.20.80.40.00.1110Cell Death – Apoptosis and NecrosisCytotoxicity Assay MethodsAssays that measure plasma membrane leakage100E/TG Figure 39: Kinetics of cytotoxic T lymphocytemediated cytotoxicity in P815 target cells quantifiedwith the Cellular DNA Fragmentation ELISA. 2 x 104BrdU-labeled target cells/well were incubated with CTLsat different effector-to-target ratios (E/T) for varyingtimes. After incubation, culture supernatant samples(100 µl/well) were assayed for DNA fragments.
1 h (P),2 h (P), 4 h (m), and 6 h (m).57Assays that measure metabolic activity1.3.2.2 Assays that measure metabolicactivityLiving (metabolically active) cells reducetetrazolium salts to colored formazan compounds; dead cells do not. Thus, tetrazolium salt-based colorimetric assays detectviable cells exclusively. Because they aresensitive, these assays can readily be performed in a microtiter plate (MTP) withrelatively few cells.1Since a cytotoxic factor will reduce the rateof tetrazolium salt cleavage by a populationof cells, these metabolic activity assays arefrequently used to measure factor-inducedcytotoxicity or cell necrosis53, 54. Applications include:P Assessment of growth-inhibitory or cytotoxic effects of physiological mediators (Figure 40).P Analysis of the cytotoxic and cytostaticeffects of potential anti-cancer andother drugs (Figure 41).P Cell Proliferation Reagent WST-1,Cat.
No. 1 644 807, a modified tetrazolium salt that can be cleaved by metabolically active cells to a water-solubleformazan, which may be directly quantitated with an ELISA plate reader (for amore detailed description of this reagent, see page 75 in this guide).Note: Since proliferating cells are metabolically more active than non-proliferating(resting) cells, these tetrazolium salt-basedassays are also frequently used to measurecell activation and proliferation. For a fulldiscussion of this application, see Section2.2.1.1 on page 70 of this guide.For a more complete discussion of the principles behind these metabolic assays, seethe topic, “Biochemical and cellular basisof cell proliferation assays that use tetrazolium salts“ (Appendix, page 113) in thisguide.P Analysis of cytopathic effects of virusesand screening of compounds with potential anti-viral activity.P Screening of antibodies for growth-inhibiting potential.Roche Molecular Biochemicals offers threeMTP-based metabolic activity assays.
Allmay be used to assay factor-induced cytotoxicity or necrosis. They are:P Cell Proliferation Kit I (MTT), Cat.No. 1 465 007, in which metabolicallyactive cells cleave the tetrazolium saltMTT to a water-insoluble formazanthat can be solubilized and quantitatedwith an ELISA plate reader (for a moredetailed description of this kit, see page73 in this guide).P Cell Proliferation Kit II (XTT), Cat.No.
1 465 015, in which metabolicallyactive cells cleave the modified tetrazolium salt XTT to a water-soluble formazan, which may be directly quantitatedwith an ELISA plate reader (for a moredetailed description of this kit, see page74 in this guide).581.11.00.9Absorbance (A 492 nm /A 690 nm )Cell Death – Apoptosis and NecrosisCytotoxicity Assay Methods0.80.70.60.50.40.30.20.10.00.11101001000hTNF – a [pg/ml]G Figure 40: Measurement of the cytotoxic effectsof human tumor necrosis factor alpha (hTNF-a) onthe mouse fibrosarcoma cell line WEHI-164.
Cells inculture (106 cells/ml) were preincubated with actinomycin C (1 µg/ml) for 3 h. Aliquots of these pretreated cellswere transferred to a microtiter plate (5 x 104 cells/well)and incubated with actinomycin C and various amountsof hTNF-alpha for 24 h. Cellular response to TNF wasmeasured with the Boehringer Mannheim Cell Proliferation Kit II (XTT) and plotted against TNF concentration.Result: Under the assay conditions, 50% of theWEHI-164 cells were killed by a TNF concentrationof 35–40 pg/ml.Parameter analyzedMetabolic ActivityAbsorbance2.5DNA SynthesisBAwithout2.52.02.01.51.51.01.0151050.50.5MTTXTTWSTBrdU [3H]-TdR[ 3 H]-thymidine incorporation (cpm u 10 -3 )with actinomycin DF Figure 41: Differentiation of cytotoxic andcytostatic effects of actinomycin D. A549 cells wereadded to a microtiter plate (104 cells/well) and incubatedwith (m) or without (m) actinomycin D (10 ng/ml)for 20 h.Graph A: Some aliquots of actinomycin-treated cellswere assayed for cytotoxic effects (changes in metabolicactivity).
These cells were assayed with either the CellProliferation 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 cellswere assayed for cytostatic effects (suppression of DNAsynthesis). These cells were incubated with either nonradioactive bromodeoxyuridine (BrdU) or tritiated thymidine ([3H]-TdR).
Incorporation of BrdU into DNA wasdetermined with the Cell Proliferation ELISA, BrdU(colorimetric). Incorporation of [3H]-TdR into DNA wasdetermined 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).Cell Death – Apoptosis and NecrosisCytotoxicity Assay MethodsAssays that measure metabolic activity159Summary of methods for studying cytotoxicitySummary of methods for studying cytotoxicity1.3.2.3 Summary of methods for studying cytotoxicity.Method/Roche Molecular Biochemicals productLabelParameteranalyzedAssay principleAdvantagesLimitations[51Cr] Release Assay55[51Cr] prelabelDamage/ leakage ofplasma membraneP Viable cells are incubated with Na2[51Cr]O4, which binds tightlyto most intracellular proteins (prelabeling).P After washing, cells are incubated with cytotoxic agent.
Duringthis period, labeled proteins are released into the culturesupernatant (SN) due to plasma membrane damage.P The radioactivity in the SN is determined with a gammacounter.P Labeling of proteins by [51Cr] generally independent of the rate ofprotein synthesisP Generally not restricted to target cell type: non-proliferating or slowturn-over populations can be studiedP Quantitative measurement over a large logarithmic rangeP Measurement of cell death in mixed cell populations; may be used toquantitate cell-mediated cytotoxicityP Radioactive isotopeP Requires prelabeling and extensive washing of the target cellsP High spontaneous release: assay limited to cytotoxic events causinghigh cell lysis over short period of time (2–5 h)P 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 prelabelDamage/ leakage ofplasma membraneand DNA fragmentationP Cells proliferating in vitro are incubated with [3H] TdR, which isincorporated into the genomic DNA.P After they are washed, cells are incubated with a cytotoxicagent.
During this period, [3H] labeled DNA is released into theculture SN due to plasma membrane damage.P The radioactivity in the SN and in the pellet is determined witha scintillation b-counter.P Quantitative measurement over a large logarithmic rangeP Low spontaneous release: cytotoxic events causing low cell lysis overa prolonged period of time (8–24 h) can be studiedP Measurement of cell death in mixed cell populations; may be used toquantitate cell-mediated cytotoxicityP Radioactive isotopeP Requires prelabeling and extensive washing of the target cellsP Limited to target cells proliferating in vitroDNA Release Assay, nonradioactiveBrdU prelabelDamage/leakage ofplasma membraneand DNA fragmentationP Cells proliferating in vitro are incubated with BrdU, which isincorporated into the genomic DNA.P Cells are incubated with a cytotoxic agent or non-labeledeffector cells (for cell-mediated cytotoxicity).P During this period, BrdU-labeled DNA is released into thecytoplasm of apoptotic cells or into the culture SN due toplasma membrane leakage of damaged cells.P The BrdU-labeled DNA in the SN or cytoplasm is determinedwith an enzyme linked immunosorbent assay.PPPPPSensitive (103–104 cells/test required)Labeled cells do not have to be washedOptimal for microplate formatNon-radioactiveMeasurement of cell death in mixed cell populations; may be used toquantitate cell-mediated cytotoxicityP Possible to measure apoptosis and necrosis in parallelP Prelabeling of the target cells requiredP Can only assay target cells proliferating in vitroP Narrow range of quantitative measurement (only one order ofmagnitude)noneDamage/leakage ofplasma membraneP Target cells are incubated with cytotoxic agent.
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