HEPES is a zwitterionic buffer that holds pH in the physiological range without depending on dissolved CO2, which keeps cultures stable outside the incubator. It is normally used at 10 to 25 mM alongside bicarbonate rather than replacing it. Higher concentrations are cytotoxic to some lines and increase phototoxicity during fluorescence imaging.
HEPES is a buffer added to cell culture media to keep the pH stable, especially when cells spend time outside a CO2 incubator. It is a “good” biological buffer that resists pH swings across the range cells care about. This guide explains what HEPES is, how it works, how to use it, and what to watch for.
Key Facts
- Type: Zwitterionic “good” biological buffer
- Holds pH: ~7.2 to 7.4, independent of CO2
- Typical use: 10 to 25 mM added to media
- Note: Protect from strong light; avoid high concentrations
What is HEPES?
HEPES is a synthetic zwitterionic buffer that holds pH steady in the physiological range, roughly pH 7.2 to 7.4. Unlike the bicarbonate buffer in most media, HEPES does not depend on a CO2 atmosphere, so it keeps media from drifting toward alkaline pH when cultures are handled on the bench.
Why use HEPES in cell culture?
Most media use a sodium bicarbonate buffer, which only holds the correct pH inside a CO2 incubator. As soon as you take cells out, CO2 escapes and the pH rises. Adding HEPES gives an extra layer of buffering that keeps pH stable during longer manipulations, such as imaging, cell counting, or lengthy setup steps. It is usually used together with the standard bicarbonate buffer rather than replacing it.
How to use HEPES
HEPES is typically added to media at a final concentration of about 10 to 25 mM, either as a component of the medium or from a concentrated stock solution. Use the lowest concentration that keeps pH stable for your workflow. Because HEPES does not replace nutrients or the bicarbonate system, cultures are still usually kept in a CO2 incubator for growth.
What to watch for
HEPES is generally well tolerated, but a few points are worth knowing. At higher concentrations it can be toxic to some sensitive cells, and HEPES-containing media exposed to light can generate reactive species that may harm cells, so protect it from strong light. Use only as much as your workflow needs.
Buffer solutions are normally sterilized by passing them through a 0.22 micron filter before they are added to culture.
Order from PurMa Biologics
PurMa Biologics manufactures a HEPES Stock Buffer Solution in the USA, backed by a Certificate of Analysis. See our HEPES Stock Buffer Solution or browse the full range of cell culture buffers. You can also read our related guide on L-15, a CO2-independent medium. Questions or need a quote? Contact our team.
