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1、CellDeathDetectionELISAPLUSCat.No.1177442500196testsCat.No.1192068500110x96testsType：One-stepsandwichELISA,colorimetricSamples：Celllysates,cellculturesupernatants,serum,orplasmaMethod：Celllysis,followedbyimmunochemicaldeterminationofhistone-com-plexedDNAfragme
2、ntsinamicroplatewell(Note:Fordetectionofnecrosis,histone-complexedDNAfragmentsaredetecteddirectlyintheculturesupernatant,withoutcelllysis)Time：Approximate3h(afterinductionofapoptosis)Significanceofkit:Usethiskitforrelativequantificationofhistone-complexedDNAfr
3、agments(mono-andoligonucleosomes)outofthecytoplasmofcellsaftertheinductionofapoptosisorwhenreleasedfromnecroticcells.Sincetheassaydoesnotrequireprelabelingofcells,itcandetectinternucleosomaldegradationofgenomicDNAduringapoptosisevenincellsthatdonotproliferatei
4、nvitro(forexample,freshlyisolatedtumorcells).Theantibodiesusedintheassayarenotspecies-specific,sothekitmaybeusedtoassaycellsfromawidevarietyofspecies(see“Otherapplications”inthisarticle).Testprinciple:Theassayusesanone-stepsandwichimmunoassaytodetectnucleosome
5、s.Theprocedure(Figure7andFlowChart2)involves:1.Incubatingcellsinamicroplatewell(forinstance,104humancellsin200μlculture)withanagentthatinducescelldeath(forexample,campothecin(CAM)).Aftertheincubation,thecellsarepelletedbycentrifugationandthesupernatantis(conta
6、iningDNAfromnecroticcellsthatleakedthroughthemembraneduringincubation)discarded.2.Resuspendingandincubatingcellsinlysisbuffer.Afterlysis,intactnucleiarepelletedbycentrifugation.3.Transferringanaliquotofthesupernatanttoastreptavidin-coatedwellofamicroplate.4.Bi
7、ndingnucleosomesinthesupernatantwithtwomonoclonalantibodies,anti-histone(biotin-labeled)andanti-DNA(peroxidase-conjugated).Antibody-nucleo-somecomplexesareboundtothemicroplatebythestreptavidin.5.Washingtheimmobilizedantibody-histonecomplexesthreetimestoremovec
8、ellcomponentsthatarenotimmunoreactive.6.Incubatingsamplewithperoxidasesubstrate(ABTS).7.Determiningtheamountofcoloredproduct(andthus,ofimmobilizedantibody-histonecomplexes)spectrop
