ThefragmentationofgenomicDNAbycellularnucleasesduringthelaterstagesofapoptosisisalsooneofthemosteasilymeasuredfeaturesofapoptoticcells.NucleaseactivitygeneratesDNAfragmentsrangingfrom~300bpto50bpinlength,resultinginatypicalDNA"˜laddering"™appearancewhenanalyzedbyagarosegelelectrophoresis.Thesefragmentshaveexposed3"™-hydroxyl(OH)endswhichcanbelabeledwithbromolateddeoxyuridinetriphosphates(Br-dUTP).Anenzyme,terminaldeoxynucleotidyltransferase(TdT),isusedtocatalyzethetemplate-independentadditionofBr-dUTPtothe3"™-OHendsofdoubleorsinglestrandedDNA.ThismethodisoftencalledTUNEL(terminaldeoxynucleotidyltransferasedUTPnickendlabeling)orendlabeling.SiteswheretheBr-dUTPisincorporatedcanthenbedetectedwithanantibodyspecifictoBrdU.
WiththeAPO-BrdUKit,cellsarelabeledwithBr-dUTPusingthemethoddescribed,andthensitesofincorporationaredetectedthroughstainingwithaFITCanti-BrdUantibody.Samplescanthenbeanalyzedviaflowcytometry.Samplesthatareapoptoticwillstainbrightlywiththeanti-BrdUantibodyduetothesubstantialnumberofexposed3"™-OHsites,whilecellsthatarenon-apoptoticwillnothaveincorporatedsignificantamountsofBr-dUTPandwillstaindimly.
TheAPO-BrdUKitisshippedinonecontainerandconsistsoftwopackages.Uponarrivaloneshouldbestoredat2-8Candtheotherat-20C.
LiXandDarzynkiewiczZ.1995.CellProlif.28(11):571-579.
LiX,TraganosF,MelamedMRandDarzynkiewiczZ.1995.Cytometry.20(2):172-180.
TomeiLDandCopeFD,eds.1991.CurrentCommunicationsinCellandMolecularBIOLOGy.3:47-60.ColdSpringsHarbor,NY.
ArendsMJ,MorrisRGandWyllieAH.1990.AmJPathol.136(3):593-608.
DarzynkiewiczZ,CrissmanHAandRobinsonJR,eds.1994.MethodsinCellBiology:FlowCytometrySecondEdition.41:15-38.AcademicPressInc.
EschenfeldtWH,PuskasRSandBergerSL.1987.MethodsEnzymol.152:337-342.
DarzynkiewiczZ,BrunoS,DelBinoG,GorczycaW,HotzMA,LassotaPandTraganosF.1992.Cytometry.13(8):795-808.
