The growth medium controls the pH of the culture and buffers the cells in culture against changes in the pH. Usually, this buffering is achieved by including an organic (e.g., HEPES) or CO2-bicarbonate based buffer. Because the pH of the medium is dependent on the delicate balance of dissolved carbon dioxide (CO2) and bicarbonate (HCO3–), changes in the atmospheric CO2 can alter the pH of the medium. Therefore, it is necessary to use exogenous CO2 when using media buffered with a CO2-bicarbonate based buffer, especially if the cells are cultured in open dishes or transformed cell lines are cultured at high concentrations. While most researchers usually use 5 – 7% CO2 in air, 4 – 10% CO2 is common for most cell culture experiments. However, each medium has a recommended CO2 tension and bicarbonate concentration to achieve the correct pH and osmolality; refer to the media manufacturer’s instructions for more information.