HEPES Buffer Solution (1 M)

HEPES (N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid) is a commonly used zwitterionic buffer with a pKa of 7.3 at 37 °C. HEPES should be used in addition to, not instead of, sodium bicarbonate, as it is important to maintain sufficient bicarbonate in the medium for nutritional purposes. HEPES may be added to cell culture media at a final concentration of 10mM to 25 mM to provide additional buffering capacity if required. Lower concentrations are usually not adequate to control fluctuations in pH, and higher concentrations are often toxic. Since the buffering capacity of HEPES is independent of the CO₂ concentration, it is an ideal buffer for maintaining the pH of cultures when cell cultures require extended periods of manipulation outside of a CO₂ incubator. HEPES buffered media are resistant to rapid, drastic pH changes, but will not prevent pH shifts entirely. Since high levels of HEPES can cause cytotoxicity, concentrations of this organic buffer should be reduced if toxicity is apparent for a specific cell line or primary cell culture.