I'm culturing primary mouse astrocytes with mutant p53 in DMEM/F12 + 10% FBS. After a few days in culture, the media starts to rapidly turn yellow and the cells die. The cells are mycoplasma negative, roughly 60-80% confluent, the media is clear (no obvious contamination) and the incubator is fine (37C and 5% CO2). Any idea what the problem could be? I've been testing my cells with the mycoplasma test kit from ATCC which is supposed to be one of the most sensitive and I'm the only one in the lab with this issue.
Supplement the media with 1% antibiotic. It ll reduce possible contamination. Make sure the hood and chamber is clean with less things inside.
If contamination is not a problem most possibly changing pH of the medium by a particular metabolite of your cells is the issue here. Check whether you are adding all constituents of the cell culture medium in exact concentration, as well as check pH of the medium before and after the color change. I suggest you may need to subculture the cells before medium color change.
add 1% (PS) Penicillin-streptomycin solution. It will prevent from any bacterial contamination. I have experienced that problem too. 5ml of PS for 500 ml of DMEM. Good luck. http://cellgro.com/products/microbiology-and-molecular-biology/antibiotics-antimycotics/penicillin-streptomycin-solution-100x.html
Hi, I would like to suggest you to check the pH of the medium before and after color change. And, what is your subsequent passage duration? Also, try to put up a check plate so that you can conclude the issue with the medium if any.
Hope this helps
If the media is turbid, it could be a problem with bacterical contamination. I am in accord with Enkhbaatar. Try to add 5 ml PS (penicillin/streptomicin) to the medium.
May be your having high numbers of white cells from blood. Add more media volume 24 hs after seeding cells . Another option is to eliminate blood cells with a percoll gradient.
Are you able to culture other types, e.g. cell lines? I wonder if perhaps the primary astrocytes are very sensitive to atmospheric oxygen concentrations (about 21%). I suspect that they experience a much lower O2 partial pressure in vivo (about 3-5% perhaps) and possibly they can't keep up with the oxidative stress that atmospheric O2 puts on them. I culture primary cells as well, and find that if I put them in 21% O2, the media changes to yellow almost immediately (no metabolites are produced that quickly) and the cells grow, just not very well. Culturing in 3-5% O2 gets rid of that problem in my experience. Perhaps you have a tri-gas incubator set up in your lab already -- if not, Airgas does offer 5% O2, 5% CO2, 90% N2 mixes for about $140 if I recall correctly. You'd have to hook that up to an airtight container within your incubator. But if it makes your cells grow well, it'd be worth it.
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