cell-preparation-for-flow-cytometry(1).pdfVIP免费

FlowCytometry–BestProtocols®Page1of3CellPreparationforFlowCytometryResearchUseOnlyRevised10-4-2010ProvidedasacourtesybyeBioscience,Inc.Copyright©2000-2010eBioscience,Inc.Tel:888.999.1371or858.642.2058•Fax:858.642.2046•www.ebioscience.com•info@ebioscience.comProtocolA:CellPreparationofTissueCultureCellsProtocolB:CellPreparationfromLymphoidTissueProtocolC:CellPreparationfromNon-LymphoidTissueProtocolD:IsolationofPBMCfromwholebloodIntroductionThesearethestandardprotocolsusedateBioscienceforthepreparationofvariouscelltypestobeusedforflowcytometricanalysis.Thisinformationistoserveasaguideasindividualinvestigatorsmayneedtooptimizeprotocolsfortheirparticularcelltype.GeneralNotes1.Singlecellsuspensionsarerequiredforoptimalstainingofsamplesforflowcytometry.2.Thenarrowboresofthesampleinjectionneedleandtubingonaflowcytometerwillbeeasilycloggedbyaggregatedcellsanddebris.3.Preparationofsinglecellsuspensionsfromsolidtissuerequiresmechanicaldissociationand/orenzymaticdigestionforoptimalrecoveryofcellsfromthetissue.Conditionsandenzymerequirementsfordigestionofthetissueofinterestwillneedtobedeterminedempirically.UsefulwebsitesWorthingtonTissueDissociationGuide(http://www.tissuedissociation.com)TheWorthingtonTissueDissociationGuideprovidesausefulsummaryandguideofthevariousmethodsthatcanbeusedfortissuedissociation.ProtocolA:CellPreparationofTissueCultureCellsMaterialsAccutase™EnzymeCellDetachmentMedium(eBioscienceCat.No.00-4555)eBioscience®FlowCytometryStainingBuffer(eBioscienceCat.No.00-4222)15or50mLconicalcentrifugetubesExperimentalProcedure1.Forcellsthatgrowinsuspension,decantthecellsintoaconicalcentrifugetubeandperformacellcountandviabilityanalysis.ProceedtoStep3.2.Foradherentcellslines,werecommendusingAccutase™EnzymeCellDetachmentMediumtodetachcellsfromculturedishanddissociatecellaggregates.Placecellsintoaconicalcentrifugetubeandperformacellcountandviabilityanalysis.ProceedtoStep3.(AccutaseisaregisteredtrademarkofInnovativeCellTechnologies,SanDiego,CA.)F...