Cells dying in serum-free medium

John Boswell knight at grafton.dartmouth.edu
Sun Sep 26 11:50:02 EST 1993


Hi,
	Our lab is new to cell culture, and we are having a bit of a problem.
We have a cell line (HEK) containing a plasmid encoding a protein we wish
to overexpress.  The cells *do* express the protein, as we detect it via
Western blot and activity assays.  The problem is that though the cells
do quite nicely in DMEM with fetal calf serum (also with pen/strep), when
we switch to serum-free medium the cells last a day or two at most, then
detach and die. :(  The serum free medium has no fcs (obviously), and we
also add trypsin inhibitor, insulin, transferrin and bacitracin.  Otherwise
it is the same as with serum.  We've increased the insulin and transferrin levelto see if that was the problem, but they had no effect.  We can't leave the
trypsin inhibitor out, as our protein would be chewed up.  So, that leaves
bacitracin.  We aren't sure *why* bacitracin is used (as the medium also
has pen/strep).  However, we are reticent to leave it out or increase
it, as it may be used to somehow induce the plasmid to express our protein
(or is that nonsense?)
	Anyway, does someone know if we are doing something stupid, based
on the above quick description?  I know debugging (so to speak) such things
is difficult with all the things that can go wrong, but maybe we are doing
(or not doing) something that is obvious to someone with more experience.
	Well, thanks a lot in advance,
-John Boswell

--
Dr. John Boswell	 			knight at grafton.dartmouth.edu 	
Oregon Graduate Institute, Portland, OR		503-690-1086



More information about the Methods mailing list