Hi all,
So my CO2 incubator broke and I have added hepes buffer to maintain my HL-60 cells which are in RPMI 1640 medium with 10% FBS. However the medium turns purple within a couple of hours. I have varied the hepes concentration from 10mM to 35mM but it does not seem to be working. Has anyone experienced this problem or know what the problem is?
Any help would be much appreciated.
The media is turning alkaline due to the excessive loss of CO2 to the atmosphere, RPMI 1640 uses a bicarbonate buffer to maintain the pH at close to neutrality, for this neutral pH to be maintained the atmospheric CO2 concentration must be ~5%. The bicarbonate buffer has 3 components, bicarbonate, dissolved carbonic acid in equilibrium with dissolve carbon dioxide, and a CO2 atmosphere in equilibrium with the dissolve CO2 in the media. Thus the buffering pH at equilibrium (it can take a while to reach this) is dependent on the CO2 concentration in the atmosphere. When acid is generated in the media it is neutralized by the bicarbonate generating carbonic acid which intern produces increased CO2 which equilibrates with the atmosphere (is lost to the atmosphere). This results in the net loss of acid and the maintenance of the pH until the bicarbonate is nearly exhausted. If base were somehow to be generated in the media or was added to the media it would be neutralized by the carbonic acid and more CO2 would form carbonic acid to restore the CO2 carbonic acid equilibrium, then more carbon dioxide from the atmosphere would dissolve to replace the lost CO2.
When there is essentially no CO2 in the atmosphere above the media (as with your broken incubator) CO2 is lost to the atmosphere and the concentration of dissolved CO2 drops in the media, since this is in equilibrium with the concentration of dissolved carbonic acid this too drops making the media more alkaline, as it is the ratio of carbonic acid to bicarbonate that sets the pH.
HL60 cells exhibit a very strong Warburg effect (the tendency of cancer cells to use a high flux through glycolytic/anaerobic energy pathways irrespective of the availability of oxygen) they produce large amounts of lactic acid but consume relatively little oxygen. A work around could be to tightly seal the tissue culture flasks to prevent CO2 escape (plus the addition of your extra HEPES) and occasionally vent the flask, especially if the media shows a sign of yellowing. There is 11 mM glucose in RPMI 1640 and so a lot of lactic acid can be generated (2 per glucose) by the HL60 cells so it could be a challenge to smoothly buffer this media a high cell densities in the absence of a functional incubator.
