I wonder if I can thaw a frozen PBMCs from nitrogen phase, wash them and surface or fix/perm and internally immunostain them and test them with flow cytometry? I tried to do surface staining on frozen/thawed/wahsed PBMCs, but they all were dead or (not recovered).
Does any one have a tested protocol?.
Yes it is possible to thaw PBMC and immunostain them in all possible ways. The thawing and freezing protocol is critical. Do a trypan blue staining after thawing of frozen samples to check the viablity of cells. The immunostaining protocols are same for frozen/thawed samples and fresh samples. If you see large clumps while thawing of PBMCs, you may need to do DNase treatment.
best wishes,
Thanks Farid and Shann for your answers. I'm going to test this soon and get back to you. Do you recommend specific supplements to be added to RPMI for PBMCs. Another thing is, should the viability of PBMCs be >90% once I thaw them and trypan blue them?If viability is less than 80%, should I discard the cells?
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