FlowCytometry–BestProtocols®Page1of5StainingCellSurfaceAntigensforFlowCytometryResearchUseOnlyRevised09-11-2013ProvidedasacourtesybyeBioscience,AnAffymetrixCompany•Copyright©2000-2013eBioscience,Inc.Tel:888.999.1371or858.642.2058•Fax:858.642.2046•www.ebioscience.com•info@ebioscience.comProtocolA:CellSuspensionsProtocolB:HumanLysedWholeBloodIntroductionFlowcytometryisausefultoolforsimultaneouslymeasuringmultiplephysicalpropertiesofindividualparticles(suchascells).Cellspasssingle-filethroughalaserbeam.Aseachcellpassesthroughthelaserbeam,thecytometerrecordshowthecellorparticlescattersincidentlaserlightandemitsfluorescence.Usingthisflowcytometricanalysisprotocol,onecanperformasimultaneousanalysisofsurfacemoleculesatthesingle-celllevel.GeneralNotes1.Foroptimalperformanceoffluorochromeconjugatedantibodies,storevialsat2-8°Cinthedark.Donotfreeze.2.Priortouse,quickspintheantibodyvialtorecoverthemaximumvolume.Wedonotrecommendvortexingtheantibodyvial.3.Exceptwherenotedintheprotocol,allstainingshouldbedoneat2-8°Cwithminimalexposuretolight.4.Ifyoudoneedtostoreyoursamplesthatwerestainedwithantibodiesconjugatedtoorganicfluorochromes,werecommendyoucompleteyourstainingprotocolandfixyoursampleswithICFixationBuffer(cat.00-8222)(100µLofsamplewith100µLofICFixationBuffer)or2mLof1-stepFix/LyseSolution(cat.00-5333).Cellscanbestoredinthesebuffersforupto3daysinthedarkat2-8°C.WehaveobservedminimalimpactonbrightnessorFRETefficiency/compensationwhenusingtheICFixation(cat.00-8222)or1-stepFix/LyseSolution(cat.00-5333).DifferencesinfixationbufferqualitycanaffectfluorochromebrightnessorFRETefficiency.Fixationoftandemdyes,suchasAPC-eFluor®780andPE-Cy7,doesnotsignificantlyincreasetheamountofcompensationrequiredfromtheAPCorPEdetector,respectively.Somegeneralizationsregardingfluorophoreperformanceafterfixationcanbemade,butclonespecificperformanceshouldbedeterminedempirically.UsefulwebsitesMarioRoederer'sHomePage(http://www.drmr.com/compensation/index.html)MarioRoedererisakeyopinionl...
