staining-intracellular-antigens-for-flow-cytometry.pdfVIP免费

FlowCytometry–BestProtocols®Page1of5StainingIntracellularAntigensforFlowCytometryResearchUseOnlyRevised10-5-2010ProvidedasacourtesybyeBioscience,Inc.Copyright©2000-2010eBioscience,Inc.Tel:888.999.1371or858.642.2058•Fax:858.642.2046•www.ebioscience.com•info@ebioscience.comProtocolA:Two-stepprotocolforintracellular(cytoplasmic)proteinsProtocolB:One-stepprotocolforintracellular(nuclear)proteinsIntroductionAmodificationofthebasicimmunofluorescentstainingandflowcytometricanalysisprotocolcanbeusedforthesimultaneousanalysisofsurfacemoleculesandintracellularantigensatthesingle-celllevel.Inthisprotocol,cellsarefirststainedforsurfaceantigensasinthesurfaceantigenstainingprotocol,thenfixedwithformaldehydetostabilizethecellmembraneandpermeabilizedwiththedetergentsaponintoallowanti-cytokineantibodiestostainintracellularly.Invitrostimulationofcellsisusuallyrequiredfordetectionofcytokinesbyflowcytometrysincecytokinelevelsaretypicallytoolowinrestingcells.Stimulationofcellswiththeappropriatereagentwilldependonthecelltypeandtheexperimentalconditions.Forexample,tostimulateTcellstoproduceIFN-γ,TNF-α,IL-2,andIL-4,acombinationofPMA(aphorbolester,proteinkinaseCactivator)andIonomycin(acalciumionophore)oranti-CD3antibodiescanbeused.ToinduceIL-6,IL-10orTNF-αproductionbymonocytes,stimulationwithlipopolysaccharide(LPS)canbeused.Note:Theoptimalstimulationconditionforinductionofagivencytokineisvariableandmustbedeterminedempirically.Forexample,thebesttimefordetectionofIL-6-producingcellsbyhumanLPS-activatedmonocytesis6hours,whereasdetectionofIL-10requiresstimulationforatleast24hours.IncontrasttodetectionofsecretedcytokinesbyELISA,itisnecessarytoblocksecretionofcytokineswithproteintransportinhibitors,suchasMonensinorBrefeldinA,duringthelastfewhoursofthestimulationfordetectionofintracellularcytokinesbyflowcytometry.Itisadvisedthatinvestigatorsevaluatetheuseandefficacyofdifferentproteintransportinhibitorsintheirspecificassaysystem.Thefixationandpermeabilizationbuffersusedforintracellu...