HEPES is a so-called Good's buffer:

http://en.wikipedia.org/wiki/Good's_buffers

So in principle it is cell impermeant and biochemically inert. Of course, in practice it can be involved in some radical reactions, particularly in response to light.

It's not uncommon to find media formulated with up to 25mM HEPES; in fact, that's one of the most common concentrations, for example for DMEM with HEPES.

Of course, it's impossible to say for sure what effect changing the HEPES concentration will have on your cells and in your assay, so you'll have to test it. However, the same would be true with just about any media formulation change.

Just make sure that if you are adding HEPES to existing media, that you adjust the pH afterwards -- it should not change much (HEPES pKa is 7.55), but it's worth double checking.

@Zineb

Most of the cells have bicarbonate (HCO3-)dependent and bicarbonate independent pH regulatory mechanisms. sodium hydrogen exchangers (NHEs) are one of the important bicarbonate independent mechanism in cells and NHE1 is ubiquitously expressed. When you try culturing cells explicitly in 25mM HEPES instead of a combination of 10 mM HEPES along with bicarbonate, bicarbonate dependent pH regulatory mechanisms are significantly compromised . Thus the answer to your question is cells remain happier in the low HEPES medium.

Further to Peter S's comment "make sure to adjust the pH afterwards", there are some issues with using strong acid or alkali to adjust the pH of a culture medium.

- First, pH depends on temperature. If you adjust to a given pH at room temperature, the pH will fall on warming to 37C.

- Second, if the medium is to be gassed with CO2, that can change the pH drastically.

Adding extra HEPES might have one benefit, of stabilizing the pH for longer with a cell type that grows rapidly and tends to acidify the medium with lactate. The same can be achieved by using more bicarbonate combined with more CO2.

But in order to make sure you then incubate at pH 7.4 (or whatever your target pH), you can do as follows. All this is before adding any serum:

1. Add the extra buffer to the medium, sterilely of course.

2. Take (say) a 20-ml sample and gas it fully, either by bubbling from a cylinder of 10% or 5% CO2 (whatever you will use in the incubator), or by leaving the tube in the incubator overnight with loose top. It's best to use the bubbling method - a good cell culture lab should have such a cylinder around to re-gas medium that loses its CO2 on storage.

3. Place tube in 37C waterbath in a rack. Measure the pH at 37C (quickly, before CO2 evolves), and adjust with strong acid or alkali as needed, by adding measured amounts (and noting these).

4. Re-gas again, and re-adjust again with more acid etc if needed. Repeat this until gassing makes no significant difference.

5. Now add up the total amount of acid/alkali added. You can now add this in one step to the whole bottle of medium (best to check with another smaller sample first). Fully gas the medium, and you're ready to go.