I have read discussions on which buffer is better to use in order to regulate pH of the cells.. I use mammalian cell line for which I use Gibco's DMEM formulation.. Most of the forums mentioned that in CO2 environment, bicarbonate can be used where as in the absence of CO2 HEPES is required to regulate pH. They also mention that when HEPES based buffers are used, it is required to maintain cells in sealed flask, although I do not get the purpose of it. Is it due to the phototoxicity of HEPES where it can generate ROS? or is there any other reason?
Hi Ishwarya,
It is perfectly fine to use HEPES in in DMEM containing bicarbonate. In fact the combination results in superior buffering capacity. I have also used HEPES in "open" tissue culture plates with no observable problem.
If you don't mind me asking, where did you get your information?
Cells produce CO2 and require some CO2 for growth. For a low density culture of cells buffered with HEPES, you can stopper the flask and the cells can "recycle" the CO2 they produce. I don't think it's a big problem with high density cultures. When buffered with bicarbonate, they can use CO2 produced from the bicarbonate and do no require the vessel to be sealed. That is what I've gathered from the abstract of the article I've linked. Let me know if you think I've missed the mark on this.
http://www.sciencedirect.com/science/article/pii/0014482774903498
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