staining-cell-surface-antigens-for-flow-cytometry.pdfVIP免费

FlowCytometry–BestProtocols®Page1of4StainingCellSurfaceAntigensforFlowCytometryResearchUseOnlyRevised10-5-2010ProvidedasacourtesybyeBioscience,Inc.Copyright©2000-2010eBioscience,Inc.Tel:888.999.1371or858.642.2058•Fax:858.642.2046•www.ebioscience.com•info@ebioscience.comProtocolA:CellSuspensionsProtocolB:HumanLysedWholeBloodIntroductionFlowcytometryisausefultoolforsimultaneouslymeasuringmultiplephysicalpropertiesofindividualparticles(suchascells).Cellspasssingle-filethroughalaserbeam.Aseachcellpassesthroughthelaserbeam,thecytometerrecordshowthecellorparticlescattersincidentlaserlightandemitsfluorescence.Usingthisflowcytometricanalysisprotocol,onecanperformasimultaneousanalysisofsurfacemoleculesatthesingle-celllevel.GeneralNotes1.Foroptimalperformanceoffluorochromeconjugatedantibodies,storevialsat4°Cinthedark.Donotfreeze.2.Priortouse,quickspintheantibodyvialtorecoverthemaximumvolume.Wedonotrecommendvortexingtheantibodyvial.3.Exceptwherenotedintheprotocol,allstainingshouldbedoneoniceorat4°Cwithminimalexposuretolight.4.ModificationsrelevanttostainingwitheFluor®nanocrystal(NC)reagentsarenotedinthegeneralprotocolbyboldprint.UsefulwebsitesMarioRoederer'sHomePage(http://www.drmr.com/compensation/index.html)MarioRoedererisakeyopinionleaderinthefieldofflowcytometry.PurdueUniversityCytometryLaboratories(http://www.cyto.purdue.edu/index.htm)FlowCytometrybasedpublicforummaintainedbythePurdueUniversity.ProtocolA:CellsuspensionsMaterials12x75mmroundbottomtesttubesor96-wellroundbottommicrotiterplatesPrimaryantibodies(directlyconjugatedorpurified)Secondaryreagents,ifnecessary(forindirectstaining)AffinityPurifiedAnti-MouseCD16/32(eBioscienceCat.No.14-0161)AffinityPurifiedHumanFcγR-BindingInhibitor(eBioscienceCat.No.14-9161)eBioscience®FlowCytometryStainingBuffer(eBioscienceCat.No.00-4222)eFluor®NCFlowCytometryStainingBuffer(eBioscienceCat.No.00-3222)7-AADViabilityStainingSolution(eBioscienceCat.No.00-6993)orPropidiumIodideStainingSolution(eBioscienc...