FACS Analysis of ES Cells
来自 : 蚂蚁淘
- 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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