I've been working with the mouse thymoma line, EL4, and have had a couple occasions in which the cells have suddenly and completely died off. Is it known if these cells have a senescence limit that they reach at high passage number? Are there any other culture parameters that they are extremely sensitive to? (ie. cell density too high/low, gas exchange non-optimal, etc. I've been using RPMI1640 supplemented with pyruvate, glutamine, beta-mercapto, HEPES, FBS, pen/strep. I put them into a T-75 flask resting horizontally with 20 mL media in it...cells were seeded at 600,000/mL. There are no obvious signs of contamination, the incubator is holding 37 °C, 5% CO2, and the RH pan has been topped up regularly. Any ideas or tips are appreciated
Thanks Richard, I have not tested for it but this is my next troubleshooting step for sure. However, I was under the impression that while mycoplasma can have unpredictable effects on cell cycle and metabolism, it's usually not toxic. Am I wrong about this?
It would be good to always freeze this kind of cell lines after 3 or 4 passages and then use them. I am not completely aware whether cell lines have a senescence limit but from my observation, it certainly does look like it.
Looks like the issue was mycoplasma. I tested and they were positive. Going back to old stocks, they tested negative and seem to be growing well
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