I am trying to carry out Linear polyethyleneimine (PEI) mediated transfection on HEK293T cells. I also tried the same with NIH3T3 cells. I am using a 3:1 PEI to DNA ratio. However, I find my cells to be dying upon the addition of the transfection media. I am having difficulty understanding where the mistake is being made.
For HEK cells, the transfection media should be removed after 3-4 hours. Also, the PEI/DNA complexes should be formed in serum & antibiotic free media. Once the complexes form (~20-30 minutes) you can add media with serum (antibiotic free though) and then add to the cells. The cells become stressed in the transfection media and the presences of antibiotics increase the stress.
Simran Tolani Hello dear, can you share with us if you have solved this problem?
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