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FlowCytometry–BestProtocols®Page1of4RBCLysisResearchUseOnlyRevised10-4-2010ProvidedasacourtesybyeBioscience,Inc.Copyright©2000-2010eBioscience,Inc.Tel:888.999.1371or858.642.2058•Fax:858.642.2046•www.ebioscience.com•info@ebioscience.comProtocolA:RBCLysisofMouseSplenocytesProtocolB:RBCLysisofMouseBloodProtocolC:RBCLysisofHumanPeripheralBloodProtocolD:LysisofHumanBloodforFlowCytometricAnalysisIntroductionPriortousinglymphoidtissuecellsuspensionsforflowcytometricanalysisand/orforinvitrofunctionalassays,itisrecommendedtoremoveredbloodcells.TheeBioscience1XRBCLysisBuffer(Cat.No.00-4333)isformulatedforoptimallysisoferythrocytesinsinglecellsuspensionsofmousetissuessuchasspleenandhumanperipheralblood.Thebuffercontainsammoniumchloride,whichlysesredcellswithminimaleffectonlymphocytes.Thisstepisnotnecessarywhenworkingwithmousethymusandlymphnodes.Whenusinghumanperipheralbloodforflowcytometricanalysis,aredcelllysingstepisincorporatedintothestainingprotocol.Forlysinghumanbloodinbulk,a10-20Xvolumeof1XRBClysisbuffertototalbloodisrecommended;however,analternativemethodforremovalofredbloodcellswhenworkingwithhumanbloodistouseisolationofPBMCbyFicoll-Hypaque™.ProtocolA:RBCLysisofMouseSplenocytesMaterials1XPBS:o80.0gNaCl80.0gNaClo11.6gNa2HPO4o2.0gKH2PO4o2.0gKCloDIH20upto10.0LopHto7.0eBioscience1XRBCLysisBuffer(Cat.No.00-4333)50mlconicaltubesInstrumentsPipettesandpipettorsCentrifugeHemacytometerandmicroscopeExperimentDuration20minutesFlowCytometry–BestProtocols®Page2of4RBCLysisResearchUseOnlyRevised10-4-2010ProvidedasacourtesybyeBioscience,Inc.Copyright©2000-2010eBioscience,Inc.Tel:888.999.1371or858.642.2058•Fax:858.642.2046•www.ebioscience.com•info@ebioscience.comExperimentalProcedure1.Harvestmousespleenandprepareasinglecellsuspension.2.Pelletthecellsbycentrifugation(300-400xg)at4°Candaspiratethesupernatant.3.Resuspendthepelletin5mL/spleenofLysisBuffer.4.Incubateatroomtemperaturefor4-5minuteswithoccasionalshaking(wehaveperformedthissteponicesuccessful...