Sodium Pyruvate - What is it's use in (NIH 3T3) cell culture? (Apr/28/2010 )
A colleague of mine was using NIH 3T3 cell media Invitrogen DMEM 10564-029 but he left and we are trying to figure out what components were in the media. In the media formulations on Invitrogen's website, the DMEM excludes sodium pyruvate but the recommended concentration of sodium pyruvate is 0.11 g/L according to ATCC's website.
What is the effect of sodium pyruvate on cells when it is missing from the media? He was having a lot of detachment and cell death, would this be a possible cause?
In addition, if anyone can answer this, what exactly does sodium bicarbonate do? I know it is a buffer, but in the ATCC 3T3 media, there is less (1.5 g/L) without HEPES and in the Invitrogen media there is more (3.7 g/L) with 5.958 ~6 g/L of HEPES. When there's more bicarbonate, does there also have to be more HEPES? I also know that less bicarbonate is used for 5% CO2 incubators, which is what we have.
Any help is appreciated.
I'll partially answer my own question. "The inclusion of pyruvate in the medium enables cells to increase their endogenous production of C)2, making them independent of exogenous CO2, as well as HCO3-." (Culture of Animal Cells. Wiley, 5th Edition.)
The cell death is most likely due to the selection markers used to stably transfect the cell line with the dF CFTR.
I could be very wrong about this, but as I recall, sodium pyruvate is also a buffer similar to sodium bicarbonate. Some cells prefer to be in sodium pyruvate vs being cultured in sodium bicarbonate.
DMEM comes in many types of flavors depending on whether or not sodium pyruvate is wanted/ needed. If you know that your cells to require sodium pyruvate, then I would simply add it to the media at the appropriate concentration. I have cultured NIH 3T3 and have always used media containing both sodium pyruvate and L-glutamine. If it missing, then the cells are not receiving the proper buffer in the media and will most likely die as a result of the missing buffer.
I would follow the ATCC guidelines and try it with the recommended media. All things being equal, you'll find that the cells will behave much better and reproducibly.
Hope that helps!
Hi,can we prepare sodium pyruvate stock solution ourselves and then add the filtered one to the media as a supplement?or it will increase the risk of contamination? one more question does it induce stress to the cells if it is added in the second day cells being in the normal media?
You can add the pyruvate as 0.2um filtered solution, so long as your sterile technique is OK, it should not increase the risk of contamination.
It is always best to culture your cells in the required components. If you do not do this, then you run the risk of having the cell behaviours change subtly - which can then affect your results, and additionally causes laboratory selection for cells that can tolerate those conditions.
A day may not make much difference, though it is very hard to tell. You will need to decide for yourself on this one - talk to your supervisor.