I am currently trying to transfect EMT6 cells using Neon Transfection. A majority of the cells die within 24 hours of transfection, and the remaining cells show very low expression of GFP. I have tried several settings as well as plasmid concentrations. Anyone with previous experience in using the Neon system on these cells?
In my opinion, main problem might be caused by the plasmids. How do you get your plasmids? Are they pure enough for applications? Other problems can occur during antibiotic application for selection of plasmid containing cells. Maybe you can create dilutions of the antibiotic and give them to cells as gradients to check the GFP signals. Therefore , you can also optimize antibiotic concentration according to your cells. Because not all cells types have same metabolic rate , plasmid processing may take time to produce both GFP and plasmid resistance protein.
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