Cells dying in serum-free medium

Antje Haase antje at RT1.MENZIES.SU.EDU.AU
Mon Sep 27 15:34:02 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
> 
> 
Hi John,

I'm not at all surprised that your cells are dying. FCS provides a lot of
components that cells need, it is not just the bit of insulin or transferrin.
Even a bad batch of serum can kill the cells - for cryptic reasons. You can
keep them for 12 hrs, even a day, some  tough cells for 2 days, without
serum. If you need the serumfree conditions for longer use the synthetic
"serum" that Gibco, for instance, sells. It is just as expensive as FCS and
has everything to keep cells happy. 

Good luck

Antje

__________________________________________
Antje Haase
Menzies School of Health Research
Australia
email: antje at menzies.su.edu.au
___________________________________________














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