hi all, it has been a big hurdle for me working with T cell lines (probably true for other lines too) to get a batch of cell, if not zero but atleast, near to zero activation level. after defrosting a new batch, once the cells are in culture, whenever I am doing a anti-phospho tyrosine blot, I am getting all many phosphorylated protein which are hardly expected to see in resting cells.
someone suggested me to use serum free medium for atleast 24 hrs, but did not help.
if you have guessed the reason for such constitutive high activation please share. any idea how to get them resting?
thanks:))
-nath.
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