I've been working with bEnd.3 and sEnd.1 cells which are endothelial cell lines from the periphery (sEnd1.s) and the brain (bEnd.1s). They are immortalised using middle T antigen.
I need to serum starve the cells as my project looks at insulin and acetylcholine cell signalling, both of which signal through the PI3K pathway.
After many different serum starves (0hr, 30min, 1hr, 2hr, 3 hr, 4 hr, 6hr, 8hr, 16hr, 25hr and also 1% SS for 8,16,24hrs) the cells just do not go down to basal state and have consistently high levels of pPKB s473 and peNOS s1177 (downstream target of PKB in endothelial cells).
I suspect the cells are consistently active due to the middle T antigen which I have read can stimulate cell growth pathways even in the absence of serum.
However, I was wondering if anyone had any experience with starving these cells and found anything different? Also, if anyone has any experience wth middle T antigen inhibition in cell lines and whether this would be an avenue to consider?
Thanks in advance :)
Hi Burkitkan
I use DMEM high glucose, with sodium pyruvate. Then I add 1% p/s and 2mM L-glut.
However, I have also tried starving the sEnd.1s in a Krebs-like solution as well and it still didn't work.
Best wishes
Beth
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