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.
Dear Paulo,
I actually do not have experience of dealing with any of these markers NFKB, ERK, AKT1, p38, but i am doing different phospho-proteins experiments.
i would suggest you to decrease your stimulation time by time gradient experiments. Try starting your time gradient from 20 minutes to 2 hr at different time intervel (20 mins, 30 mins, 45 mins, 60 mins, 90 mins, 120 mins). As in my experiments of phospho-proteins, maximum stimulation time with concern cytokine is no more than 30 minutes.
PMA (no more than 50 ng/mL) and Inomycin (1-2 uM) should be used (as i am using PMA/Inomycin in my other experiments). i found cyto-toxicity when used highr concentrations.
I hope your time gradient experiment will give you best results.
Do tell us when you resolve this issue.
All the best.
Dear Paulo,
I actually do not have experience of dealing with any of these markers NFKB, ERK, AKT1, p38, but i am doing different phospho-proteins experiments.
i would suggest you to decrease your stimulation time by time gradient experiments. Try starting your time gradient from 20 minutes to 2 hr at different time intervel (20 mins, 30 mins, 45 mins, 60 mins, 90 mins, 120 mins). As in my experiments of phospho-proteins, maximum stimulation time with concern cytokine is no more than 30 minutes.
PMA (no more than 50 ng/mL) and Inomycin (1-2 uM) should be used (as i am using PMA/Inomycin in my other experiments). i found cyto-toxicity when used highr concentrations.
I hope your time gradient experiment will give you best results.
Do tell us when you resolve this issue.
All the best.
Thanks Jitendra. Sorry i didn't mentioned but yes I have used PMI 50 ng/mL and Ionomycin 50 ng/mL. In fact the best time to measure this phosphoprotein is after 15 min so I guess I still need to have a low basal phospho prior stimulation. Cheers.
Dear Paulo,
May I ask you about the graph (or difference in autofluoresce) of Cells only versus Unstimulated.
Possibly if there is vide difference b/w cells and Unstimulated, it may be caused either due to high residual activity or high concentration of antibody.
Blue histograms are "unstimulated" cells while red histograms are "stimulated" cells, e.g. I have stained both unstimulated and stimulated with antibody 1:20 dilution.
Well, it seems unlikely, as you can see both unstimulated and stimulated have the same staining pattern (two picks of negative and positive cells), even with low antibody concentration (1:320).
I found one solution though. Resting the cells in normal RPMI (5-10% FBS) overnight (16h) prior to the experiment seems to work pure well to reduce the basal phospho signal in some publications. I'll try that, and I'll post the result here. Cheers.
Depends what cell population you are interested in within PBMC. I have found that monocytes tend to have high basal phosphorylation in cRPMI, whereas T cells don't seem to have this issue
Indeed Melanie, I have observed the same phenomenon. Still lymphocytes have high basal phophos when I compare unstimulated vs stimulated cells.
Hmm I always do an overnight rest in cRPMI so that should help. You can also try lowering the amount of serum (1%) or using serum-free media like Immunocult T cell expansion media
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