1. Collectblood(75microliters)into1mlPBScontaining5microMEDTA(10microlitersof0.5Mstock)andmiximmediatelytopreventclotting.Keeptubesonice.
2. RemoveRBCsfromsamples.Thiscanbeaccomplishedbyseveralmeans.AtTheJacksonLaboratory,RBCsarelysedusingeitherGey"ssolutionorabufferedammoniumchloride(ACK)solution.(Alternatively,Becton-Dickinsonsellsaproductcalled"FACSlysisbuffer"thatisusedafterthestainingprotocoltolyseRBCsandfixthecells.)
3. Cellsshouldbewashed2-3xwithFACSbuffer(PBSsupplementedwitheither1%BSAor5%FBSandcontaining0.05%NaN₃).SUSPendthepelletfromthefinalwashin50microlitersFACSbuffer(ormoreifmorethanoneanalysisistobedoneonasinglesample-uptothreeseparatestainingreactionscanbesetupfromasinglesample).
4. Add50microlitersofcellsuspensionto10microlitersofantibodysolutionandmixgently.Youwillneedtodeterminetheproperconcentrationforeachantibodyused.
5. Incubatefor30minutesonice.
6. Washcells2-3xwithFACSbufferandsuspendin200-300microlitersFACSbufferforanalysis.
7. Forlive/deaddiscrimination,add10microliterspropidiumiodide(PI)solution(stock=10micrograms/ml).Iffixingcellsbeforeanalysis,donotaddPI.

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