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why is my cells dying in the centre of the wells after transfections

Cell culture Hek 293 T transfections huh 7

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#1 KhaleesiDany



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Posted 13 July 2015 - 01:16 AM



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

#2 Curtis


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Posted 14 July 2015 - 12:25 AM

Usually all dead cells, unrelated to their location, accumulate in the center of the well. This is a common problem. I advise you to double check the outskirts and the center. I don't think the cells in the center show less GFP. 


Also, in order to avoid accumulation of the transfection reagent and plasmids in the middle of the well, instead of swirling the plate, move it north to south, east to west like a +, few times and it will dissolve. 

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