Concentration of the medium/buffer solution, and the toxicity to the cells.

In live-cell culture, pH disturbances are an inescapable consequence of metabolism and there is a general tendency for media to undergo acidification, the extent of which is also a function of medium buffering capacity.

The primary initiation for the addition of HEPES in culture media is to maintain the cells environment in optimum pH. This is very important especially when you are spending a lot of time working with the cells outside of the incubator because this would results in pH change.

Do note that sodium bicarbonate is another buffer used to stabilize the pH of the culture media. However, HEPES (Cell culture grade) should not be used in lieu of sodium bicarbonate as the culture media will become more acidic than anticipated. The reason so is because normal bicarbonate buffer is CO₂ dependent whereas HEPES (Cell culture grade) is CO₂ independent. This is why the phenol red (pH indicator) in the media changes color over time if you just leave the media out in room air versus in the incubator.

One important question to take into consideration is that what type of primary cells are you culturing? Some primary cells (fibroblasts) are very easy to culture and do not require many supplements to media, while others (t-cells for example) are very picky and require highly buffered and supplemented conditions.

The recommended final working concentration of HEPES in cell culture media usually ranged from 10mM to 25mM. Higher concentration may results in toxicity to cells under long incubation period.

The 9 best biological buffers for cell culture

Why choose HOPAX?

As a professional biological buffer manufacturer, HOPAX produces high-quality HEPES and also provides HEPES cell culture grades that pass cytotoxicity testing. In addition, we can also provide special testing services, such as bioburden, endotoxin, heavy metals, and DNase/RNase/protease activity testing, etc., as per your applications. In any case, HOPAX is absolutely the best choice for your purchase of HEPES!

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