Hi Tania, Mike, Komal and Holden,
I'm frightened that there is a little misunderstanding: Tania, if I'm correct you stimulate with LPS in vivo, and then collect your cells for cytometry analysis ? If you don't see a shift either with LPS stimulation or with PMA-Iono stimulation, this culd be due to some identifiable issues:
- How long do you stimulate in vivo with LPS ? I ask because if this stimulation is too short, you may not elicit a sustained proi-nflammatory phenotype in your immune cells, so you won't see any effect of LPS stimulation vs. unstimulated state, at least regarding cytokines secretory activity, once you isolate cells and stimulate them + inhibit exocytosis. Didi you try analysis of cells from LPS treated vs untreated animals directly, without PMA-Iono + GolgiPlug ?
- I think you do, but do you stimulate cells ex-vivo with MPA-iono + GolgiPlug ? And if yes, do you do it at least for 4h in order to let cytokines accumulate in secretory cells ? Because in my hands, I was able to see cytokines without PMA-iono + brefeldinA/monencine only in some rare inflammatory PBMC in humans, and even in this case only in some inflammatory CD4+ cells regarding only some specific cytokines (which, I think, are abundant enough to be visualized without long and artificial accumulation). I have no experience with unstimulated cytokines analysis in mouse yet.
Hope it will help
Regards
Olivier