I am currently trying to transfect NBTII cells (wt) with lipofectamine 2000 in Opti-MEM. I tried several things already; such as splitting them 24h or 48h after transfection (I always changed the media after overnight and used pen strep free media with only 10%FBS), doing transfection in 6 well or 24 well plate, using NaCl or DMEM instead of Opti-MEM. However all failed. I do see the mCherry signal 24h later but cells die after 48h. Did anybody face a similar problem? Is there an easier way to transfect these cells?
If you see mCherry expression and the cells then die - it could be that they are too strongly over-expressing the protein.
Have you tried titrating the amounts of DNA or lipofectamine you use for transfection?
Alternatively - are you sure your DNA is completely free of possible endotoxins? CAn you transfect other cell lines with no issues?
Hi,
Sarah is right, if you see expression in a considerable number of cells, your transfection has worked well.
Overexpression is a good explanation for what you observe, consider using less DNA and / or transfection mix as well as a weaker promoter for driving expression (e.g. EF1).
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