Hi All,
I need to ask the experts for clarification on HEPES buffer.
We intend to incubate (30-60 min, undecided as of yet) EaHy cell line with varying concentrations of glucose, possibly up to 30 mM.
After reading a number of papers where they have performed similar studies, I am confused as to which composition of HEPES buffer we should order/use.
One paper suggested the following; 25mM Hepes, 125mM NaCl, 3mM KCl, 1.28mM CaCl2, 1.1mM MgCl, but I can't confirm if that would be appropriate for our study.
Has anyone any experience with this?
Any help is much appreciated!
Hi Cristopher, HEPES is inert when it comes to cell glucose studies. It is charged at physiological pH and will not cross the cell membrane.
HEPES has a structure similar to taurine, but that is a strictly intracellular molecule and has no effects on glucose metabolism as far as I know.
If you are uncomfortable using HEPES, you can use phosphate or carbonate buffers.
I have used HEPES buffer for handling mouse embryos or human embryos when they are outside the carbon dioxide incubator for some manipulations,
HEPES Use in Cell Culture
The most commonly used buffering system for media is bicarbonate. This is due to its nutritional benefits despite the reduced buffering capacity at physiological pH. Addition of HEPES provides supplemental buffering to cell culture medium at pH 7.2 through 7.6.
The level of HEPES in cell culture media may vary from 10mM to 25mM. The most commonly used concentration is 25mM.
HEPES has no nutritional benefit to cells. It is added to the media solely for extra buffering capacity when cell culture requires extended periods of manipulation outside of a CO2 incubator.
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