When should HEPES be used in cell culture media?
HEPES (4-hydroxyethylpiperazine ethanesulfonic acid) is a commonly used zwitterionic buffer with a pKa of 7.3 at 37°C. In addition to sodium bicarbonate in the medium, we should also use HEPES, because it is important to maintain sufficient bicarbonate in the medium to achieve nutritional goals. If desired, HEPES can be added to the cell culture medium at a final concentration of 10 mM to 25 mM to provide additional buffering capacity. Lower concentrations are usually not enough to control pH fluctuations, while higher concentrations are usually toxic.
Since the buffering capacity of HEPES has nothing to do with CO2 concentration, it is an ideal buffer incubator to maintain culture pH when cell culture requires long-term operation outside of CO2. HEPES buffer medium can resist sharp pH changes, but it cannot completely prevent pH changes. Since high levels of HEPES can cause cytotoxicity, if it is significantly toxic to specific cell lines or primary cell cultures, the concentration of this organic buffer should be reduced.
Why use HEPES?
Because the growth and metabolism of cells in culture will cause dramatic changes in the environmental pH of the culture medium, in order to maintain an environment that can stably culture cells, it is necessary to add an appropriate amount of certain biological buffers. However, there are so many biological buffers and alternatives on the market that can be used for cell culture, why use HEPES?
Generally, the best culture environment for most cells is pH 7.2-pH 7.4, and the buffer range of HEPES is just within pH 6.8-pH 8.2, which is beneficial for cell culture. Compared with other buffers (such as PBS (phosphate buffered saline) and TRIS), HEPES has higher stability in maintaining the pH of the cell culture medium. This is why HEPES is widely used in cell culture, tissue culture, protein purification and extraction, immunoprecipitation, cell lysis, live cell imaging and other biological/biochemical research.
What are the benefits of using HEPES?
HEPES acts as a buffer for enzymes under low temperature conditions
Generally, the use condition of biological buffer is 25 degrees Celsius at room temperature, or higher temperature (according to the optimal pH range of the enzyme), but sometimes it is necessary to experiment under low temperature conditions, and some buffers are obviously not suitable for use due to the influence of low temperature. The decomposition ability of HEPES can increase with the increase of temperature, and decrease with the decrease of temperature. However, compared with other buffers, its decomposition constant does not change much with temperature, which makes HEPES buffers better at low temperatures. A buffer that maintains the structure and function of the enzyme.
HEPES has no toxic side effects on cells
HEPES buffer can control a constant pH range for a long time. Low-concentration HEPES has no toxic effect on cells. It is often used in cell culture. For example, HEPES can be added to the culture solution or virus preservation solution of various bacteria and fungi to maintain cells, The pH environment required by bacteria and viruses.