HEPES buffer (N-2-hydroxyethylpiperazin-N '-2-ethanesulfonic acid) is a commonly used zwitterionic buffer, with the advantage that it does not bind to magnesium, calcium, manganese or copper ions. Its pKa at 37 ℃ is 7.3, suitable for cell culture. When used together with sodium bicarbonate, HEPES can maintain the bicarbonate in the cell culture medium unchanged, thereby maintaining the nutrients in the culture medium. The final concentration of HEPES used in cell culture medium is 10 mM to 25 mM to provide additional buffering capacity. Lower concentrations are usually not sufficient to control pH fluctuations, while higher concentrations are usually toxic. Due to the fact that the buffering capacity of HEPES is independent of CO2 concentration, it is an ideal buffer incubator for maintaining the pH value of cell cultures when they require long-term operation outside of CO2. The HEPES buffer medium can resist rapid and drastic pH changes, but it will not completely prevent pH changes. Due to the cytotoxicity caused by high levels of HEPES, if there is significant toxicity to specific cell lines or primary cell cultures, the concentration of this organic buffer should be reduced, attention must be paid to maintaining the osmotic pressure concentration of weight molecules in the culture medium, and toxicity to specific cell lines needs to be evaluated. HEPES usually exists in the form of powder, as its configuration is simple and can be prepared and used immediately. In solid form, it has advantages in both preservation and transportation. If HEPES is exposed to light for 3 hours after being configured as a solution, it will produce toxicity and cannot be used again. If you pass the filter membrane first and store it in a four degree refrigerator, it can be temporarily stored, but if there are flocs, it cannot be used. The powder state can be sealed and kept away from light for a long time without deterioration.