Ascorbate and HEPES

Warren Gallin wgallin at
Tue Mar 22 13:59:01 EST 1994

In Article <1994Mar22.153654.13920 at>, przemko at
(Przemko) wrote:
>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 
>appears that, in solution, it will be good for a day or two. On
>the other hand medium such as alpha MEM contains ascorbate. Companies
>guarantee this medium for a year or so which is fine exept that if I 
>would like to add ascorbate (because I work on e.g. collagen) then
>the problem begins. Do I add? How much? Will the decomposition product
>screw me up? I asked various technical services at various companies
>but NOBODY gave me a decent answer (well that is how we do it...).
>Any pointers?

The reason for using bicarbonate is twofold: 1) bicarbonate is a necessary
nutrient for many cell lines, and 2) most T.C. incubators use a
bicarbonate/CO2 buffering system.  HEPES won't buffer for crap in a 5% or
10% CO2 atmosphere, it will be driven to a very acidic pH.  Usually what you
want to do is have HEPES plus bicarbonate; the bicarbonate will buffer in
the CO2 atmosphere, the HEPES will help resist the alkalinization of the
medium when you have it out of the incubator for whatever manipulations you
may be doing.
   As for the ascorbate, I haven't a clue.  Good luck trying to find out,
but I would suggest looking at the papers that describe the development of
the medium.  Eagle did a ton of work on this and published what the
requirements were.
Warren Gallin,
Department of Zoology, University of Alberta
wgallin at

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