I'm trying to evaluate the contribution of PI3K to S6 phosphorylation using PIK294 to inhibit p110deta after TCR stimulation. I'm wondering if 30 minutes stimulation with cells in incubator would be ideal? I've tried shorter time points and stained pS6 to check on flow cytometry but no signal was identified. Thanks for any help and suggestions.
May be do a time kinetics 10mins, 30mins, 1hr, 3hrs, 6hrs.
However TCR stimulation is quick and longer timepoints will not give more information. You may try checking on western blot as its more reliable. With FACS you need a special protocol (ICS) to stain the intracellular kinases.
Thanks. I’ve already done western blot, which worked. However, Facs will provide me with information on pS6 in specific cells such as T CD4+ and T CD8+ due to multiple staining. I tried 5, 10, and 15 minutes with no success by Facs...
I think 30 min. stimulation is shorter than usual. Perhaps you should perform a pilot study trying several times intervals using the same stimulation protocol until you find the incubation time after which there is no increase in signal.
Thank you all. Heads up that I’ve performed a time course including 5, 15, 30 minutes, 1 and 2 hours and it did not work. I decided to fix using paraformaldehyde immediately after 30 minutes stimulation and I could see S6 phosphorylation. I guess the dephosphorylation occurs very fast. This said, it seems fixing after stimulation may be advised as this strategy provided me with an efficient signal solving the problem.Thanks again!
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