I 'm currently doing some experiments with A9 mouse cells, growing them in 175cm² cell culture flasks and seeding them in 96-well plates. Recently i've seen that many cells seem to burst, after sowing in 96 wells and incubation at 36 degrees they did not grow any further. Is this an apoptotic phenotype? This happens spontaneously, not in all of the previously split vessels, some still have the 'normal' phenotype. I think these cells seems to have a problem with attachment and/or growth since they don’t grow in 96 well plates anymore. I already checked the media ( DMEM 5%FBS), vessels, incubator (36°C; 4,5% CO2; 92%rH), plates, tips, possibilities of contamination (mycoplasma and bacteria), timespan of trypsin. I'm working with high quality, multiple tested reagents ( even FBS is gamma-irritated) and the reagents are tested for the presence of viru
Nothing seems to cause this alteration
Does anybody know whats wrong with my cells, or any advice what I should check?
Thanks for your help!
Hi Melanie,
I never worked with A9 mouse cells, thus, I don't know the common phenotype of this cell line. But looking at your cell monolayers looks that you have a syncytial cell formation due to a viral infection, that may come from the cell line contamination, or from FBS contaminated with BVD, or other virus. I've a cell line that I have purchased and known to be infected by BVD that looks exactly the same.
If the stock of your cell line is contaminated is very difficult to get ride of the virus. I would recommend to discard the cells and begin experiments with lower passages of the A9 cells. Be very careful when purchasing FBS, always check if FBS is tested as virus free, namely BVD.
All the best for your research.
Isabel
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