We would like to examine cytokine release as well as MAPK phosphorylation in PBMCs and I would like to be sure the PBMCs will not be stressed and therefore activated by the RBC lyis step.
Thanks to all!
I have not had a problem with this, at least in CD4 and CD8 cells. We use Pharm Lyse from BD towards the end of our standard PBMC prep from buffy coats. We freeze the PBMC and rest them overnight after thawing prior to any stimulation according to this paper http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0076215. My day 0 cytokine levels by flow (with golgi block reagents) is essentially zero.
you can use lysis buffer that is laboratory prepared with the lowest effect on PBMCs,
composition: ammonium chloride, sodium EDITA, sodium bicarbonate.
I have used this method in invitro antigen presentation of tumor associated antigen
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