I have been transfecting Huh 7 and Hek 293T cell lines using PEI (2:1). I usually do it later in the day around 4pm and then leave it overnight change the media around 8am the next morning and i check for gfp expression to asses transfection efficiency. thus far after repeating three times i have seen GFP only on the outskirts of the well and nothing in the middle. At first try my well was abit overcrowded so perhaps that couldve caused cell death but 2nd and 3rd time there was no over crowding. also i dont think it could be toxicity from pei because why are the outskirts cells well?
any suggestions or tips, please and thank you