Ascorbate and HEPES

lowy at lowy at
Wed Mar 23 06:52:14 EST 1994

In Article <1994Mar22.153654.13920 at>
przemko at (Przemko) writes:
>I have a question concerning tissue culture.
>First of all bicarb buffer that is so commonly used is in fact 
>quite lousy (as opposed to HEPES) at physiological pH. So, why
>do we still use bicarb and not HEPES. I know that some people use
>but it appears to be not a SOP.
>Secondly, ascorbate is known to be rather unstable. In fact it 

 Do I add? How much? Will the decomposition product
>screw me up? I asked various technical services at various companies
I agree with the points made by gallin
Also HEPES is cytotoxic to some cell lines and many primaries. A way around
this is to use other "Good's" buffers w/ or w/o HEPES - but all a lower
concentration (say 5-10 mM) but the if overlaping buffer regions are used
then the buffering capacity is not reduced verus 25 mM HEPES and the buffering
range is actually extended. The same stratigie can be used for salines.
Another reason to include some bicarb even in phsiological salines is that many
ion transport processes including those linked to pHi regulation are bicarb
linked/dependent. For short half life nutrients (glutamine is another culprite)
I've generally added 50-100% of the orginal formualtion on the assumption that,
a) with a few day 1/2 life most has gone away, b) some is better than none
and double shouldn't be too harmful - but best check the orginal cell/media
culture discriptions.

Joel Lowy				The usual disclaimers.
Physiology Dept.
AFRRI, 8901 Wisconsin Ave., Bethesda, MD 20889-5603, USA 				 	
lowy at
301-295-1790  FAX 301-295-0313

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