Hello every one, I have lots of problems with transfection of human normal foreskin fibroblast with lipofectamine 2000. I have used several concentrations and ratios but either the transfection efficiency is low or the cell toxicity is very high. I have even changed the upper opti-MEM transfection medium just 1h after transfection but my cells seem to become vacuolated 24h prior to transfection. I will be appreciated if someone could help me.
Have you checked for mycoplasma contamination? And yes high ratios of DNA and lipid can be cytotoxic as well.
Be aware of the following issues:
- transfection cell medium is free of antibiotics
- FBS is below 0,5%. Higher concentrations decrease the efficiency
- your cells may be sensitive to the medium change and opti-MEM is not always optimal indeed))
- What kind of growth medium do you use? In our practice 1 day pri transfection we place our cells in antibiotics free growth medium with 0.5% FBS and 12-24 hours after transfection we change medium to the normal FBS even without antibiotics.
- check the compatibility between your cell line and the type of transfectam that you use. In case of low compatibility you may be recommended to increase the ammount of transfectam
Good luck!
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