5 replies to this topic

[Radar]

Hello everybody, I hope not everybody is on holiday...

We are starting in our cell culture lab, and from time to time basic questions arise. Right now we are preparing a SH-SY5Y culture, but the question is a generic one.

We have realized that depending on the paper, researchers use one or another media for the same cell lines and similar experiments. Usually in the Material and Methods section the %CO2 is not mentioned. Each media has its own bicarbonate concentration and thus its own optimal % CO2 (e.g. EMEM for 5% and DMEM for 10%), but from some suppliers we may have DMDM with lower amount of bicarbonate and vice versa, so no % may be a priori excluded.

My question is: If the bicarbonate concentration and the %CO2 of the incubator are harmonized, the pH should be the desired 7.4, right? In this case, is the CO2 atmospheric content a relevant factor for the cell behaviour?? As far as I understand, in a pH 7.4 media the content of CO2/bicarbonate should be the same, regardless the atmospheric CO2 concentration...

Did I explain my question clearly enough?  

I will appreciate any explanation, either theorical or practical ones.

Thank you!!

[gfischer]

I'd start with the conditions recommended by whomever you got the cells from.  The ATCC is usually a good resource too.  If you have multiple incubators, you could also just try different CO₂ concentrations and see what works best.  Even with the same media, a different CO₂ concentration might be better.  I worked with a myeloma line a few years back that wouldn't grow at 5%, even though that's what ATCC recommended.  When we upped the concentration to 10%, they grew perfectly.

[Radar]

Thanks for the answer. That is what I use to do, to follow the cell source indications at least till we have expanded the cells enough we can allow us to do some experiments with the culture conditions. I want just a bit more of background information.

My question is a bit broader, based on these different protocols for similar experiments. In your example of myeloma line, did you change the recommended media according to the %CO2 or did you use the same media all the time? If you did the second one, then the pH was different and the responsible for the different grow pattern; if not, it was the total %CO2 the responsible one, and that would be the answer to my general question: if CO2 per se plays a role.

Thanks again

[bob1]

The bicarbonate concentration is dependent on the %CO₂, the atmospheric content forces the reaction away from the HCO₃^- towards the H⁺, keeping the pH buffered correctly. If there is too much bicarbonate in the medium for the %CO₂, the medium will have a higher pH (leave a small amount of medium exposed to air for a short while, with phenol red as an indicator it will go purple indicating basic conditions).  This also accounts for the colour change you see when you approach the bottom of a bottle, which then corrects itself when put in an incubator.

It is entirely possible to grow cells at the "wrong" %CO₂, the pH isn't too different, usually a difference of about 0.2-0.4 pH units between 5 and 10%

[Shree]

hii all...i have a similar query posted by radar...but m still not clear with d xplainations given so far..hence want to continue d thread...m still not understanding d logic behind use of surplus co2, relation between co2 & bicarbonate (added in media)

[bob1]

The bicarbonate acts as a buffer.  If you don't understand that principle, then you should have a look for a basic chemistry text book, which will explain the principles well.