HEPES (N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid) is a zwitterionic organic chemical buffering agent commonly used in cell culture media. The addition of 10–25 mM HEPES provides extra buffering capacity when cell culture requires extended periods of manipulation outside of a CO2 incubator. HEPES is a good buffering choice for many cell culture systems because it is membrane impermeable, has limited effect on biochemical reactions, is chemically and enzymatically stable, and has very low visible and UV light absorbance.