I am doing Flow-cytomteric staining of PBMCs for extra- and intra-cellular markers. I have read that I can stop the protocol after fixing and permealising the cells (fix-perm step), but I am just wondering which buffer could I use to store my cells to carry on next day? would PBS be enough for this purpose?
Thanks
Fixing the ells with paraformaldehyde cross links the proteins and their structure is retained. However storage in paraformeldehyde for longer period is not recommended. After fixing, the cells should be washed and stored in PBS or FACS buffer (PBS+1%FBS+ 0.02%Azide) at 4 degree Celsius. Next day wash the cells and incubate with antobodies.
If your permeabilization reagent is saponin, it must be kept with the cells at least until the intracellular staining is done. Removal of saponin before intracellular staining will result in poor staining because the pore forming action of saponin is reversible.
See https://www.researchgate.net/post/Is_saponin_permeabilization_reversible
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Thanks all for sharing your experience
Michel, I am sorry I didn't share what buffer I am using. For the BD buffer I am using might be quiet different from Saponin as in the FAQ sheet attached here, they suggest to keep the cells overnight in 1x D-PBS.
https://www.bdbiosciences.com/documents/Pharmingen-Transcription-Factor-Buffer-Set-Protocol.pdf
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