Apoptosis and Cell Proliferation (522915), страница 21
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ECulture cells in a MTP for a certain period of time (37°C)Kit I (MTT)Kit II (XTT)WST-1Prepare XTT labeling mixtureAdd MTT labeling reagentAdd XTT labeling mixtureAdd Cell Proliferation Reagent WST-1Incubate cells (0.5–4 h (37°C)Add solubilized solution and incubate(1–24 h (37°C)Measure absorbance using an ELISA plate reader(2 min, RT)76Methods for studying cell proliferation and viability in cell populationsAssays that measure DNA synthesis2.2.1.2 Assays that measure DNAsynthesisDuring cell proliferation the DNA has tobe replicated before the cell is devided intotwo daughter cells.This close association between DNA synthesis and cell doubling (Figure 51) makesthe measurement of DNA synthesis veryattractive for assessing cell proliferation.
IfParent cell in G 1 phasemonoclonal antibodies directed againstBrdU64. The use of BrdU for such proliferation assays circumvents the disadvantages associated with the radioactive compound [3H]-TdR.The first report of this technique involvedthe extraction and partial purification ofDNA from BrdU-labeled proliferatingcells, followed by an enzyme immunoassayin a separate assay71. Because this methodParent cell in S phaseDaughter cellsNucleusCell Proliferation and ViabilityCell divisionDNA Replication(DNA Synthesis)(Mitosis)DNAG Figure 51: Cell proliferation, a close association between DNA synthesis and cell doubling.labeled DNA precursors are added to thecell culture, cells that are about to divide incorporate the labeled nucleotide into theirDNA.
Traditionally, those assays involvethe use of radiolabeled nucleosides, particularly tritiated thymidine ([3H]-TdR).The amount of [3H]-TdR incorporatedinto the cellular DNA is quantitated by liquid scintillation counting (LSC)63, 70.Experiments have shown that the thymidine analogue 5-bromo-2’-deoxy-uridine(BrdU) is incorporated into cellular DNAlike thymidine (Figure 52). The incorporated BrdU could be detected by a quantitative cellular enzyme immunoassay usingwas relatively laborious, the entire BrdUbased procedure was adapted to a 96 wellMTP72.
This adaptation required no harvesting of the cells; the complete assay fromthe start of the microculture to data analysis by an ELISA plate reader was performed in the same MTP (Figure 53).2Figure 52: Molecular structure ofthymidine and BrdU. HThymidine(5-methyluracil-2’-deoxyribose)5-Bromo-2’-deoxyuridine(5-Bromouracil-2’-deoxyribose)OH3C4’H5362OH3’OHBr45’HOCH 2OH2’H15’NHOCH 21’H4NHO4’H5362NH1NOH3’OHH2’HO1’H77Methods for studying cell proliferation and viability in cell populationsAssays that measure DNA synthesisF Figure 53: Measurement of DNA[ 3 H]-TdR incorporation assayLabelingBrdU incorporation assay+ [ 3 H]-TdR ( )+ BrdU ( )Harvestingcells on filtersFixation anddenaturation+ Scintillationfluid+ Anti-BrdU POD ( )Cell Proliferation and ViabilityIncubation(2 – 24 h)+ SubstrateMeasurement20LSCELISA-readerRoche Molecular Biochemicals offers threekits that use the convenient BrdU-basedassay and the MTP format.
The BrdULabeling and Detection Kit III is a firstgeneration assay. The colorimetric and chemiluminescence Cell Proliferation ELISAs,are second generation assays that offerfewer steps, a faster assay, and greater sensitivity than the first generation assay(Table 15). These three kits are describedon the following pages.78synthesis using modified nucleotides[3H]-TdR and BrdU.Methods for studying cell proliferation and viability in cell populationsAssays that measure DNA synthesisBrdU Labeling and Detection Kit IIICat.
No. 1 444 6111000 testsType1st generation ELISA with colorimetric detectionUseful forQuantitation of DNA synthesis during cell activation and proliferationSampleAdherent or suspension cell culturesMethodIncubation of cells with BrdU, followed by partial digestion of DNA andimmunodetection of incorporated BrdU labelTime2.5–6 h (+ cell labeling)Test principle: The assay is a cellular immunoassay which uses a mouse monoclonal antibody directed against BrdU (Figure54 and Flow Chart 18).Fixed cells with partiallydegraded BrdU-labeled DNANucleaseAnti-BrdU-PODfab fragmentsCell Proliferation and ViabilitySignificance of kit: The BrdU Labeling andDetection Kit III measures cell proliferation by quantitating BrdU incorporatedinto the newly synthesized DNA of replicating cells.
It offers a nonradioactive alternative to the [3H]-thymidine-based cellproliferation assay.ABTS ®substrateLabel cells with BrdU (2–18 h, 37°C)G Figure 54: How the BrdU Labeling and Detection Kit III works.Suspension cellsAdherent cellsRemove labeling medium Remove labeling mediumusing a cannulaby inverting the MTPAir dry cells (180 min,60°C)Add fixative and incubate (30 min, –20°C)Wash MTP 3 times (15 min, RT)Add Nuclease and incubate (30 min, 37°C)Wash MTP 3 times (15 min, RT)Add Anti BrdU-POD and incubate (30 min, 37°C)Wash MTP 3 times (15 min, RT)Add substrate and incubate (2–30 min, RT)Measure absorbance using an ELISA plate reader(2 min, RT)Note: This kit belongs to the first generationof kits used to measure DNA synthesis.
Thesame assay procedure has been optimizedand improved in the second generation CellProliferation ELISA, BrdU (colorimetric)kit (see Table 17).2Sensitivity: 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 proliferating cells in a certain samplestrongly depends on the amount of BrdUincorporated into the cells and thus on thelabeling period. In most cases, detection requires a labeling period of 2 to 4 h.Specificity: The antibody conjugate (AntiBrdU-POD, Fab fragments) will bind toBrdU-labeled DNA after the DNA is denatured. The antibody specifically recognizes 5-bromo-2’-deoxyuridine; it showsno cross-reactivity with any endogenouscellular components such as thymidine oruridine.F Flow Chart 18: Assay procedure, BrdU Labeling andDetection Kit III.79Methods for studying cell proliferation and viability in cell populationsAssays that measure DNA synthesisCan be used to assay:P Adherent cells as well as cells cultured insuspension in 96-well microtiter plates(e.g.
cell lines, activated peripheralblood lymphocytes and other in vitroproliferating cells).Other applications: For examples of howthe BrdU Labeling and Detection Kit IIIcan be used in the lab, see Appendix, page125.Cell Proliferation and ViabilityKit contents1. BrdU labeling reagent (1000 x), sterile2.
Anti-BrdU-POD Fab fragments3. Incubation buffer (ready-to-use)4. Washing buffer (10 x)5. Nucleases6. Substrate buffer7. ABTS® substrate tablets8. Substrate enhancerTable 15: Improvements of the assayprocedure used by the Cell ProliferationELISA, BrdU (colorimetric) and CellProliferation ELISA, BrdU(chemiluminescence) described on thefollowing pages. E280ParameterBrdU Labeling and Detection Kit III Cell Proliferation ELISA BrdU (colorimetric)Cell Proliferation ELISA, BrdU (chemiluminescence)Incubationsteps32Washing steps3–41Workingsolutions6 (4 included in the kit)4 (all included in the kit)Assay time2.5–6 h1.5–2.5 hIncubationtemperatures–20°C: FixationRT: Substrate reaction37°C: Nuclease treatment60°C: Air dryingFor Cell Proliferation ELISA, BrdU (colorimetric) eachstep at RTMeasuringrangeAbsorbance: 0.1–2.5 U (factor 25)Same as BrdU Kit IIIFor Cell Proliferation ELISA, BrdU (chemiluminescence):rlu/s: 103–106 (factor 1000)SensitivityAlmost as sensitive as [3H]-TdRAs sensitive as [3H]-TdRMethods for studying cell proliferation and viability in cell populationsAssays that measure DNA synthesisCell Proliferation ELISA, BrdU (colorimetric)Cat.
No. 1 647 2291000 testsCell Proliferation ELISA, BrdU (chemiluminescence)1000 testsType2nd generation ELISAs with colorimetric or chemiluminescent detectionUseful forQuantitation of DNA synthesis during cell activation and proliferationSamplesAdherent or suspension cell culturesMethodIncubation of cells with BrdU, followed by immunodetection of incorporatedBrdU labelTime1.5–2.5 h (+ cell labeling)Note: These two kits belong to the second,improved generation of kits for measuringDNA synthesis (see Table 16).Significance of the kits: The two Cell Proliferation ELISAs measure cell proliferation by quantitating BrdU incorporatedinto the newly synthesized DNA of replicating cells.
They offer a nonradioactive alternative to the [3H]-thymidine-based cellproliferation assay with comparable sensitivity.Test principle: The assay is a cellular immunoassay which uses a mouse monoclonal antibody directed against BrdU. Theprocedure (Figure 55, Flow Chart 19) involves:1Culturing the cells in a 96-well microtiterplate and pulse-labeling them withBrdU. Only proliferating cells incorporate BrdU into their DNA.2Fixing the cells with FixDenat solution.This FixDenat solution also denaturesthe genomic DNA, exposing the incorporated BrdU to immunodetection.3Locating the BrdU label in the DNAwith a peroxidase-conjugated antiBrdU antibody (anti-BrdU-POD).4Quantitating the bound anti-BrdUPOD with a peroxidase substrate. TMBis used as a substrate in the Cell Proliferation, BrdU (colorimetric). Luminol/4-iodophenol is used as a substrate inthe Cell Proliferation, BrdU (chemiluminescence).Cell Proliferation and ViabilityCat.
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