Adjusting pH of RPMI/ bicarbonate containing media - (Nov/05/2012 )
I'm attempting to do an apoptosis assay using RPMI as my culture medium, whilst it doesn't contain HEPES it does contain Na-bicarbonate, which buffers it to pH ~7.4. the problem I'm having is that after a few hours (about 8-9) the pH has been reasserted from my desired pH, and I want to do incubations of up to 24 hours.
The problem I'm having is that I want my medium to contain bicarbonate, but I don't want it to buffer the medium. Is there a way/ alternative media to include bicarb without it doing this?
RossM on Mon Nov 5 17:32:17 2012 said:
the problem I'm having is that after a few hours (about 8-9) the pH has been reasserted from my desired pH, and I want to do incubations of up to 24 hours.
Is this problem in the incubator or bottles out on the bench/in the fridge? If in the incubator - check the CO2 content, the bicarb buffering system requires CO2 to keep the pH stable at around 7.4.
RPMI has been formulated for use in 5% CO2 atmosphere.
Carbonate-bicarbonate is stable at pH about 9.6-10. At so low pH is quite close to the pKa1 of carbonate system (6.37) so you are losing bicarbonate as CO2, that's why the pH changes
The incubator is set to 5% Co2 at 37C. I've found on the bench that it stays stable at pH 7.4, and when left out in the open will go to that. I can adjust the pH and leave it in sealed containers and it will maintain the pH.
I'm only doing slight adjustments as well, to pH 6.7 and 7.
Would a sealed system be required do you think then to maintain the pH? Or is it not possible to make medium with HCO3- in it that won't rebufferer itself?
Usually when medium (with bicarb) is left out on the bench, the pH will rise (turns purple if using phenol red) as the medium loses CO2 to the air. Putting it into the incubator will return it to a normal pH.
How much bicarb did you add per litre?
Lowering the pH even more it will increase the CO2 lose due the pKa1 of the carbonate.
I am using RPMI right now, the recipe says 2 g/l. I'm trying to copy an experiment right now basically from a paper I erad, though they didn't say how or whether they kept the pH, but just modified the pH using isotonic HCl, they didn't say much more than that unfortunately, apart from acidic pH's were kept in a 7% CO2 environment (something I don't have access to right now).
I've considered maybe trying to seal the plates I've been using (96-well plates) with parafilm to see if they would be able to maintain the pH better.
2 g/l should be fine for 5% CO2. Are you sure that you are making medium up from bicarbonate FREE medium?
I'm not making the medium up myself as it is right now, but simply using bought RPMI. What I am doing is adding HCl to change the pH of it. I found that when open at room temperature it stayed stable around 7.4. I made some media at pH 7 and 6.7. I tested yesterday by putting samples of all these in the incubator (37C 5% CO2) for 24hrs and found that the pH 6.7 raised to 7.1, and 7 raised to 7.2 andf the pH 7.4 raised to 7.5 (though that wasn't so much a problem for me.)
I think it's this gradual change over time that is stopping me from being able to copy the experiment I read of in the paper.
Did you measure pH before or after sterile filtration? I saw a protocol indicating to adjust to a pH slightly lower than the final desired as filtering raised the pH about 0.1-0.3
Much more detailed preparation:
RossM on Wed Nov 7 09:58:18 2012 said:
That's because of the disociation rate, if you don't have enough pCO2 you won't be able to make stable acidic medium using bicarbonate. I really think that it will be impossible without increasing the CO2 concentration