Using HEPES buffered medium is common in live cell microscopy experiments to effect maintenance of pH in the absence of the 5% CO2 atmosphere required by bicarbonate buffered media. Without perfusion of CO2 enriched atmosphere, DMEM (and other bicarbonate buffered media) containing 3.7g/L of NaHCO3 undergoes a substantial (0.3 pH Units) increase in pH in as little as 15 minutes. After 30 minutes of exposure to air ( 0.04% CO2) the pH of the media can rise to 7.8, out of the so-called physiological range and continues to increase to >8 within an hour of air exposure (BioTechniques 27:292-294 August 1999).
To alleviate pH changes in the absence of 5% CO2, cell culture media is commonly supplemented with 10-25 mM N-2-Hydroxyethylpiperazine-N’-2-ethanesulfonic Acid (HEPES). Although this achieves pH homeostasis, an unwanted side effect of the HEPES addition is the light induced production of Hydrogen peroxide ONLY in the presence of HEPES. This happens at light doses equivalent to those found in tissue culture hoods with fluorescent lamps. It is thought that HEPES is acting as a catalyst for the production of peroxides from riboflavins or other compounds in the culture medium (Spierenburg et al. Phototoxicity of N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid-buffered culture media for human leukemic cell lines. Cancer Res (1984) vol. 44 (5) pp. 2253-4).
Inclusion of 2mM NaPyruvate in the culture medium eliminates the toxic effect of light completely from HEPES containing medium.