HEPES (N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid) is a zwitterionic organic chemical buffering agent commonly used in cell culture media. It maintains the pH in a range of 6.7–8.6 and the addition of 10 to 25mM HEPES provides extra buffering capacity when cell culture requires extended periods of manipulation outside of a CO2 incubator. HEPES is suitable for many cell culture systems because it is membrane impermeable, has limited effect on biochemical reactions, is chemically and enzymatically stable. HEPES Buffer Solution is prepared in cell culture grade water.