We are receiving blood samples that sometimes we can't make to stain them freshly for FACS. Is it better to keep them ON in RPMI+FBS10%, cold PBS (4ºC), or cold methanol? We perform both intracelullar and membrane staining, thank you!!!
We keep the isolated PBMCs in PBS at 4°C overnight, adding FCS or BSA might also be helpful. Or you can fix the cells in BD Cytofix and store them in PBS.
Thank you both! Melanie, have you ever performed membrane staining after BD-Cytofix fixation?
Normally, we perform membrane staining prior to fixation and intracellular staining after fixation and Triton-X treatment. But I think it should also work to stain membrane-bound proteins after fixation, but we have never tried.
How long are you looking to store the cells for before FACS? Normally I keep my cells in PBS at 4 degrees when the other samples are being prepared. But we normally perform FACS on them on the same day.
I'm not sure about this, but I thought for mouse PBMCs it is okay to store them in PBS at 4°C (for whatsoever reason), but for human ones I would rather recommend to "store" them in RPMI (10% FCS) in the incubator, especially if you stain them the next day. Some markers are downregulated when the cells are exposed to low temperatures, I suppose it could also be that some are upregulated, so I would keep the conditions as close to "normal" as possible.
And for the staining, I do it like Melanie does it, first membrane staining, then fixing and permeabilization and afterwards the intracellular staining.
It might also work differently, but I guess some of your membrane antigens/markers might be masked by the fixation...
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