I am adding 25mM HEPES buffer to EGM2 media to culture HUVECs. I initially buffer the solution to 6.9 (I started at 7.1 but I lowered it a bit to compensate for the pH rising), but in just 24 hours the pH rises to 7.5 and my cells die. The media contains bicarbonate, which I presume is responsible for the pH change, but most protocols recommend that 25 mM should have enough buffering capacity to regulate this. Has anyone encountered this problem before?
What you report is quite unusual, because HEPES pH is stable over temperature changes. Have you calibrated your pHmeter before bringing your pH to 6.9?
If you did it, I would suggest two controls: prepare some HEPES buffer at room temperature 25 mM pH 7.0, then (a) mix it with the EGM2 medium (keep the same ratio you would use in your cell cultures) and keep the mixture over night at 37 °C; (b) put it at 37°C overnight as it is. Then the next day measure the pH.
If both tubes have the same initial pH, then it is the metabolism of your cells that is highly perturbing the medium.
If both (a) and (b) have changed, then use HEPES from a different supplier, since no changes should occur with temperature.
If (a) has changed but not (b) then the EGM2 medium should be used with a different buffer (check the supplier's manual and trouble shooting).
I hope this helps
Hi Joshua Hall . It could be something else in the HEPES besides the pH that is killing the cells.
Is the HEPES you are using tissue culture grade? What about the H2O? Is the HEPES solution sterile filtered or autoclaved?
@Adriana Erica Miele yes I’ve done that previously and found that the HEPES media in a CO2 controlled environment does not change pH, while the HEPES media in an incubator with no CO2 changes from 6.9 to 7.5
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