BD-FlowCytometry-DNA-Staining-Protocol

BDBiosciencesTechnicalResourcesPage1December2011DNAStainingProtocolforFlowCytometryForResearchUseOnly.Notforuseindiagnosticortherapeuticprocedures.bdbiosciences.com23-13851-00MaterialsandReagentsFullNameShortNameCatalogNumberPBSBDCytofix™fixationbuffer(optional,foralternativefixationstep)FixationBuffer554655Ethanol,70–80%,storedat-20°CBDPharmingen™stainbuffer,orequivalent,1XPBS,2%FBS,0.1%NaN3(pH7.1–7.4)StainBuffer554656BDPharmingen™PI/RNasestainingbuffer(forPI/RNasestaining)PI/RNaseStainingBuffer550825BDPharmingen™7-AAD(7-Amino-ActinomycinD)stainingsolution(for7-AADstaining)7-AADStainingSolution559925ProceduralNoteDNAdyes,especiallypropidiumiodide(PI),canbesticky.Afterrunningthesamples,cleantheflowcytometerbeforethenextuseaccordingtheinstructionsprovidedinthecytometerUser’sGuide.Fixedcellscanbeusedupto12monthsafterfixing(storedin70to80%ethanolat-20°C).Procedure1.Pelletthecellsandwashthembyadding30to40mLofPBS.2.Centrifugethecellsat1,000rpmfor10minutesandaspiratethesupernatant.3.[Optional]FixthecellswithFixationBufferfor15to30minat4°C.4.Whilevortexing(toloosenthepellet),add5mLofcold70to80%ethanoldropbydropintothecellpellet.Incubateat-20°Cfor2hoursminimum.5.Washtwicetoremovetheethanol.Performthefirstwashin1XPBSandthesecondwashinStainBuffer.6.Centrifugethecellsfor10minutesat1,000to1,500rpmandaspiratethesupernatant.7.Tostain,use106cellspertestanddooneofthefollowing:ForPI/RNasestaining,resuspendthecellsin0.5mLofPI/RNaseStainingBuffer.For7-AADstaining,resuspendthecellsin0.1mLofStainBufferandadd20μLof7-AADStainingSolution.8.Incubate15minutesatroomtemperature.9.Storetubesat4°Cprotectedfromlightpriortoanalyzing.Analyzeontheflowcytometerwithin1hour.