Hello Dino.

Often calcium ionophore stock solution causes cell damage, leading to death before cell fixation. It would be caused by the solvent of the stock solution which is toxic to the cells. Therefore the initial concentration of the stock solution should be high enough to be diluted under 0.1% at the final concentration in the cell treatment. If you were diluting 1mg/ml solvent, that is about 0.1%. It is also critical the way you add to the cells. Avoid local cell damage when you add it to the cells.

Regards.

How are you measuring fold change? When doing PhosphoFlow, you don't tend to see massive shift in histograms. It is usually very subtle and sometimes, you see small cell populations expressing the phosphorylated protein. This is picked up better when looking at dot plots. Depending on how big this population is, you might see a tiny peak in histograms.

What I would recommend is to try longer incubation times. When looking at cell signaling, I have seen signals go up until 3 hours, stabilize at 6 hours and then drop. So maybe try stimulating the cells for 30 mins-6 hours and then stain them. You could also test multiple stimulant doses (20-100 ng/mL PMA + 100-1000 ng/mL ionomycin).

One last thing: If you still fail to see a signal, it might be worthwhile to use alternate fix/perm strategies. Some work better for certain intracellular signaling proteins compared to others. I haven't worked with CREB, to recommend one.

Hi Sang,

Thank you for your help. We have diluted our ionomycin in DMSO at a conc of 1mg/mL, I’ve been using 1ug/mL so 1ul in 1mL of media giving me 0.001%. The PMA is also in DMSO but is being diluted first 1 in 100 (PBS) then a further 1 in 10 before use (which is something like 0.001% again). I can’t see the amount of solvent being the issue. I normally make the dilutions and add them to a stock of culture media before mixing with my cells for incubation.

Thank you!

Hi Julie,

It’s a fairly simple flow experiment with only one colour so I use FSC and SSC to differentiate between lymphocytes and monocytes / other. I gate on the lymphocytes. I have been looking at histograms, I’ll check out the dot plot data to see if it looks any clearer, thank you!. I was just hoping to see a distinct shift like I observed on the BD website for the pCREB PE antibody.

The literature seems to be set on the 15 min mark for pCREB but I will try a time course and a few different PMA and ionomycin concs.

I’ve tried Perm I and II for the protocol and Perm II is the better of the two, I’ll investigate to see what the other options are for fixation, I know that Perm III is supposed to be quite harsh so I’m reticent to use it when looking at phospho expression, but I may end up looking at that if I don’t see anything!

Thank you for your suggestions!

I am working with pCREB both in human and murine samples, so I hope I can help. Your stimulation time is correct, I do see a difference after 15 min but the optimal stimulation time for pCREB is 30' at 37C.

In case of buffers for fix or perm - I've used exactly the same ones you are using and they worked fine. In my protocol I am stimulating only with PMA at conc. 100 ng/ml. Try also the forskolin which is a direct AC activator [10 uM]. As Julie suggested the changes are usually subtele and 1.5 fold increase in phosphorylation is quite good.

Wish you luck!