HEPES is an important biochemical preparation in biochemical industry. It is considered to be one of the important buffers in the field of biological research. The chemical name of HEPES is: N-hydroxyethylpiperazine-1-ethanesulfonic acid, the character is white crystalline powder , Is a weak acid, often used as zwitterionic buffer and pharmaceutical intermediate in organic chemical industry. It is often selected as a buffer for cell culture because of its advantages:
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1. The decomposition capacity of HEPES can be enhanced with the increase of temperature and weakened with the decrease of temperature, but compared with other buffers, its decomposition constant does not change much with temperature, which makes HEPES buffer become A buffer that better maintains the structure and function of the enzyme. The buffer can control a constant pH range for a longer period of time and has no toxic effect on cells.
2. The pH required for most cells is 7.2-7.4, HEPES has a good buffer capacity in this range, but the optimal pH value varies with the type of cell cultured. Fibroblasts prefer higher pH (7.4-7.7), while subcultured cell lines require acidic pH (7.0-7.4). Most culture medium relies on sodium bicarbonate (NaHCO3) and CO2 system for buffering. The CO2 concentration in the gas phase should be balanced with the sodium bicarbonate concentration in the culture medium. In this case, the cell culture bottle cap should not be screwed too tight to ensure gas exchange. However, after storage for a certain period of time, the pH of the buffer system will increase with the CO2 volatilization. At this time, the culture solution quickly becomes purple when the cell is subcultured and blown, making the cells cultured with this culture medium difficult to survive. Even if it survives, the activity It is also very low, and the proliferation rate is very slow. Adding HEPES to the culture solution can avoid this problem. HEPES can be used as a buffer to replace bicarbonate to eliminate the limitation of the high-concentration CO2 culture environment. The final concentration of HEPES buffer used is generally 10-25mM.
How to use HEPES:
1. HEPES can be directly added to the prepared culture solution at the required concentration, and then sterilized by filtration. Add 2.38 grams of HEPES per 1000ml of culture solution, adjust the pH to 7.2 with lN NaOH after dissolution, filter and sterilize and use At this time, the use concentration of HEPES is 10 mmol/L.
2. It can also be prepared into 100 x storage solution (l mol/L). Before use, 99ml of culture solution is added to lml storage solution, and the final application concentration is still 10mmol/L. l mol/L (100 x) HEPES stock solution preparation method: dissolve 23.8g HEPES in 90ml double-distilled water, adjust pH to 7.5-8.0 with 1N NaOH, then dilute to 100ml with water, filter and sterilize, and dispense vials ( 2ml/bottle), store at 4℃ or -20℃.
Desheng Biochemical reminded that when used as a buffer for cell culture, HEPES in the culture medium exposed to visible light for more than 3h will produce hydrogen peroxide that is toxic to the cells. It is recommended that the culture medium be stored in the dark.
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