I'm measuring the phosphorylation status of different proteins (e.g. NFKB, ERK, AKT1, p38 etc) using phosphoflow cytometry.
The protocol for surface and intracellular staining works well, all the antibodies are also working well. However the basal phosphorylation of my PBMCs is quite high in all phosphoproteins that I have been measuring and I can't see a clear distinction of unstimulated and stimulated (PMI+ION, TNFa etc) conditions.
I have tried to rest the cells for 2 or 4 h in RPMI media at 37C, 5% CO2, but the basal phopho level was still high.
Any tips on how to reduce basal phosphorylation?
Please have a look in the example attached. The x-axe represents a photoprotein X, each double line represents different antibody dilutions. The grey histogram is unstained cells, the blue histograms are unstimulated cells (basal phospho) and the red histograms are stimulated (PMA+ION) cells.