i try to measure pNFkB and NFATc1 after the Stimulation with PHA for 30-60-90-120min , by Flow cytometry , but It does not work and it does not find a Signal of phosphorylation .
anyone please can help me . thanks
Hi Michael
How is your permeabilization protocol? Are you making your own solution or using a commercial one?
How is your flow? Are you staining any other markers or only the transcription factors? Which fluorochrome is your Abs conjuated to?
Hi Andre
I used perm Buffer III from BD,
i stimulate cells next lyse/fixation Buffer BD, next perpermeabilization , staining for CD3/CD4/CD8/ anti NFATc1 AAlexa Fluor 647 from sant Cruz or NFkB Alexa Fluor 647 BD (staining 1h). next i Assay with FACScanto
Michael
Regarding your flow:
Did you use a AF647 FMO to gate your events? Did you eliminate dead cells using 7AAD? DId you permeabilize your samples on ice?
Regarding your experiment:
Is your PHA working? (Have you tried measuring IL2 for example?)
I never used PHA alone, I used PHA and PMA for western blot analysis. Have you tried other combinations or TNFalpha stimulus?
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