I am wondering if I stimulate T cells in vitro with anti CD3/CD28, how long will the s6 ribosomal protein be phosphorylated. I would like to look at ps6 after 4 days or stimulation to correlate with a specific phenotype, but am getting baseline levels when looking at ps6 by flow cytometry.
Hello, as with any phosphorylated protein, phospho S6 will not be active after such a long time period. The AKT/mTOR pathway is very fluid and is activated and inhibited by numerous proteins. You're getting baseline readings because when you want to see phosphorylation, it's because you're looking for activity. Cells will not be upregulated for 4 days after a stimulation. Try a shorter incubation time, such as 30 mins to 2h. If it's because you need the cells confluent, grow them and then do the stimulation when the cells are ready. Especially if you're just looking for a phenotype after stimulation. Good luck.
I agree with Kristy Howell . Signaling happens way before you start seeing phenotypes. We used to measure NFkB/AKT/MAPK signaling in macrophages and we used to do time kinetics starting at 10 minutes post stimulation going up to 24 hours. The differences were more obvious in the first 6 hours and were gone by 24 hours which was when we started observing phenotypic differences. You could try a preliminary time course with 0-6 hours and every 8 hours after that up until 4 days. You need to have unstimulated control cells too so you could have a comparison.
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