How can I calculate the quatity of HEPES needed for my medium (ATCC DMEM 30-2002)?
(Endconcentration x Endvolume)/ Stock concentration= Volume of Stock needed
Both concentrations need to bee the same unit.
I am curious to know, Why do you want to add HEPES to the medium? The medium formulation and the sodium bicarbonate we provide along with serum are sufficient to maintain pH and osmolarity of cells. I tried once with HEPES to maintain higher pH. But increased sodium bicarbonate amount served me well than HEPES.
DMEM uses a sodium bicarbonate buffer system (3.7 g/L), and therefore requires a 5–10% CO2 environment to maintain physiological pH.
Adding HEPES to DMEM will change the osmolarity. If you want HEPES in media to maintain cells without CO2, you need to reduce salt (NaCl) in the media. Separate formulations are available for this purpose.
i am new in this field. i'm afraid if we run out of CO2 gas, adding HEPES in DMEM would help. i'm using ATCC DMEM 30-2002. so, i would like to know the volume of HEPES needed. if i need to reduce salt (NaCl) in the media, can i know the process?thank you for the answer.
Dear Wan
You can add 25mM HEPES to your DMEM media which will prevent pH fluctuation, no need to change Nacl concentration. This will support your cell for some time without CO2. 3.7gm of sodium bicarbonate is required and cell need to be cultured in Co2 environment. If you want to avoid co2 completely you can use L15 media which uses HEPES buffer system. But all cells will not grow. We use L15 media for MDA-MB231 cells.
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