I am incubating cells in RPMI in a CO2 incubator at 37C and 80% RH. The phenol red started as a pink color and after 24 hours in the incubator it has faded. The color is more or less the same, just less bright. Is that normal?
When you work with your cells, your cell culture medium is exposed to atmospheric CO2 concentration which is a lot lower than what you have in the incubator. This leads to an increase in the pH and phenol red appear to be pinkish. When you put the cells in the incubator CO2 dissolves into the medium and the pH decreases and the color is redder and less intense.
(see https://en.wikipedia.org/wiki/Phenol_red#/media/File:Phenol_red_pH_6,0_-_8,0.jpg )
It should not be pink to start with, the pink color indicates elevated pH, so you should check that first, is the faded color the same shade of pink? Could you add some photos?
It is a normal event that depends largely on temperature and pH.
Cell culture medium with phenol red appears usually in red when the optimum temperature is maintained. When you keep it outside for long time while performing assay, it turns into pink. But after incubated at 37C (5% CO2), the color revert back to red. In following days once cells starts to metabolize the contents, the pH drops and the medium looks yellow.
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