I have a lentiviral transfection protocol on 293T cells that has been working for years, but for the past few months, my 293T cells starts de-attaching from the plate. I finally figured out it was the issue with my BGS serum so we switched to DMEM+10% FBS and they starts growing normally again and I noticed cells tend to adhere better in FBS media. Well, this time, they starts dying after transfection with lentival constructs at 5ug of plasmid. I changed media overnight after transfection and was bring very gentle. I have been using the same protocol for a few years now, and I can't seem to figure out what is the issue of them not growing well this time. Any advice would be appreciated!
Test your cells for mycoplasma contamination. It can really screw up your cells.
Another thing might be your DNA preps. Has there been any change to how your DNA is being prepared? Carryover contaminants from plasmid prep can have a large impact on cell health.
Finally, your media might benefit from the addition of HEPES buffer. Virus production can really drop the pH of a culture quickly because of metabolic activity. We've found that the basic bicarbonate buffering in DMEM doesn't cut it.
I usually use DMEM + FBS 10% too, but coat dishes for LV-production with Poly-L-ornithin for better adherence. However, it also works fine without and I do not think it will fix your problem. I don't know what you use for transfection - I use
Polyethylenimine which works fine. Maybe conc. of your stock/aliq. recently changed?
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