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[biouser]

My MNFS60 cells are dying suddenly after overnight incubation . I propagate cells in RPMI 1640 + 10% FBS + 1% Penstrep + 0.05 mM 2-Mercaptoethanol + 62 ng/ml MCSF as recommended by the cell line provider. I have tried varying different batch of media,  different incubators, vessels, ordered different MNFS60 batch etc. All the chemicals are newly ordered. Mercaptoethanol dilutions are prepared fresh. My incubators were validated and checked for temperature and CO2 just 2 days before the cell death. Materials are stored at recommended temperature. Important point that I noticed was-  the alliquote of cells used for cell count was left at room temperature by mistake. The cells in it were viable next day. It did not contain mercaptoethanol and MCSF. Where as the cells that I incubated the same day, died!! My incubators always show a slight colour change in the media (orangish yellow) with and without cells. Please help!!

[bob1]

Could be contamination, probably a slow growing bacterium, maybe mycoplasma. If you have a significant color change then it will not be mycoplasma as these are obligate intracellular parasites. Media often show a minor change in color when placed in the incubator as they adjust to the CO2 and buffer correctly. RPMI should be reddish-orange at the correct pH.

 

How are you assessing the cell death? If it is just that the cells lost attachment, then it could be a number of things including that your reagents have gone off (most likely the M-CSF).