The cells are peripheral blood monocytes whose media can be changed every 3-4 days. We have found that the medium pH changes rapidly, so adjusting the pH only when medium is changed won't be enough. How can I be sure that the pH in the cell culture (in 96-well plates) will be e.g. 7.0 for the whole 2 week period?
Hi Elina, silly question but you aren't using a media with a buffer in it are you such as Bi-carbonate or HEPES? If you are this maybe the reason (if growing cells in CO2 the, the CO2 will dissolve also dissolve into the media and lower the pH so you may have to put the media in the incubator and allow it to equilibrate before measuring it's pH or adding HCl to the desired pH).
Hi Elina!
I tried several approaches for acidifying cell culture media for the following publication:
Rajamäki et al JBC 2013 Extracellular acidosis is a novel danger signal alerting innate immunity via the NLRP3 inflammasome.
I was aiming to compare the pH values 7.5 / 7.0 / 6.5 / 6.0, and really the only way was to lower the bicarbonate concentration of regular RPMI formulation from the ~24 mM to 5 mM, and to replace this with 20 mM PIPES buffer that has a buffering range more suitable to the desired pH range (a custom media ordered from Athena Enzyme Systems). As Dr. Eagle in his ancient studies found, PIPES buffer was well-tolerated by the cells (macrophages) and by this formulation, we achieved stable acidosis in the CO2 incubator at least up to 24 h.
Now I really tried to adjust both regular DMEM and RPMI with a simple addition of sterile HCl solution in our lab, but the pH kept shifting FOREVER especially in DMEM with even higher bicarbonate concetration. Of course you are aiming only at a very mild acidosis of pH 7.0, so you just might make it with RPMI, if you try to add some extra buffering perhaps (e.g. 10 or 20 mM PIPES; further nontoxic options presented in Eagle's article).
2 weeks is a long time, but if you change the medium e.g. every 2 days, I think it is can be done. Of course you will unavoidably have some slow shifting of the medium pH due to cellular metabolism towards acidosis, but the shift caused by equilibration of the bicarbonate system was a much bigger problem in my experience. Some monitoring of the pH on culture plates in the incubator, with and without cells, will be required to ensure proper performance before starting the experiments.
Hope this was helpful, best of luck with your experiments! Don't hesitate to drop me a message if you want to ask further details.
Thank you so much for the helpful answers!
I am currently using DMem, so maybe I could try the RPMI medium you suggested. I started a new cell culture yesterday, and I try to change the medium now every other day, and see how it will affect the cells.
I prepared the media yesterday and adjusted the pH, and added 25 mM HEPES. I will check today what pH they have, and maybe shift to your suggested media if with DMEM it doesn't work out.
Thank you also for your article, it will be very helpful for my studies!
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