HEPES use in cell culture

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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.  In GIBCO DMEM it is at a concentration of 25mM.  In GIBCO DMEM/F-12 it is at a 15mM.  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.


In the process of cell culture, HEPES buffer is often indispensable. So, how to prepare and use HEPES buffer?

HEPES buffer is a disadvantage. If HEPES is added in the open culture conditions, it can prevent the increase of pH oxidation in the medium, thus maintaining PH at about 7.0.

HEPES molecular weight is 238.31, chemical name: 4-hydroxyethyl piperazine ethylsulfonic acid, molecular formula: C8H18N2O4S. HEPES is commonly used as a biological buffer, pH buffer range: 6.8-8.2, used in biochemical diagnostic kit, DNA/RNA extraction kit and PCR diagnostic kit. It has no toxic effect on cells. It is a hydrogen ion buffer, which can control the constant pH range for a long time. The final concentration was 10-50 mmol/L.