• IsolatecellsanddissociatetosinglecellsUSPension(canuseGibcoCellDissociationBuffer,AccutaseorTrypsin)
• Washwith10%FBS/DMEM:F12
• ForsurfaceMarkers,resuspendinappropriatevolumeof10%FBS/DMEM:F12andkeeponice.
• Forintracellularmarkers,resuspendin1ml4%paraformaldehyde/PBSandincubatefor10minatroomtemperature
• Washwith10%FBS/DMEM:F12
• Resuspendin0.1%TritonX-100/PBSandincubatefor15minonice
• Washwith10%FBS/DMEM:F12
• Resuspendinanappropriatevolumeof10%FBS/DMEM:F12andkeeponice
• Add50μlcellsuspensionto50μl10%FBS/DMEM:F12containingappropriateamountofantibodytogivethefinalconcentrationlistedbelowandincubatefor30minto1hour
• Washtwicewith10%FBS/DMEM:F12
• Resuspendin100μlof10%FBS/DMEM:F12containingsecondaryantibody(AlexaFluor488conjugate)at1:100
• Incubatefor30min
• Washtwicewith10%FBS/DMEM:F12
• Resuspendinappropriatevolumeof10%FBS/DMEM:F12

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