Cells dying in serum-free medium

macdouga at wren.sunnybrook.utoronto.ca macdouga at wren.sunnybrook.utoronto.ca
Mon Sep 27 00:52:39 EST 1993


In a previous article, knight at grafton.dartmouth.edu (John  Boswell) wrote:
> 
>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



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