I am working with a new cell line, MRC9s lung fibroblasts, and for RNAi experiments Lipofectamine RNAimax works great! BUT for transfecting plasmid DNA Lipofectamine 3000 (low and high volumes as suggested by manufacturer), Lipofetamine 2000 and electroporation seem to be toxic to the cells. Does anyone have a good protocol for these specific cell lines?
Note: My plasmid has no mutations and ORF is fine.
Expression works well (as tested in other cell lines).
Tested reagents without DNA thus eliminating toxicity arising from plasmid, and tested empty vector eliminating toxicity arising from gene.
Tested incubation of complexes from 4-24 hours (before changing media) and see the same toxicity albeit slightly less at 4hours.
Tested incubation with OPTIMEM (serum free media) only for 4-24hrs with the same toxicity.
"Toxicity" cells begin to detach, aggregate, cell death visible via microscope.
What about the cell density at the day of transfection? Cells resist better the transfection-induced stress when they are dense.
You could also give a try to calcium-phosphate transfection, it's easy to prepare cost effective and sometimes it can rescue transfections which do not work with "regular" commercial transfection reagents (my own experience).
Cyrille,
Yes I always plate at 1.5x10^5 cells/mL at 2mL volume per well (6 well plate) surface area 9cm^2. Which next day they look 85%-95% confluent. After transfection and changing media confluency drops to about 50%-75% with "sickly" looking cells. I will give calcium-phosphate transfection a try! Thank you.
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