Hello, everyone. I am working with HEK 293 cells. I have been transiently transfecting them with 8mg DNA in 500micL serum free media. I use 1:10 DNA/PEI ratio, such that I have been adding 80micL PEI. Although I haven't deviated from this protocol, recently my HEK 293 cells have started detaching during the night of transfection (I change the serum free media+DNA/PEI with fresh fully supplemented DMEM media, 4-5 hours after the start of the transfection). Any suggestions? Thanks a bunch. Peace
They detach when they die, you can check for cell death by flow or trypane staining. PEI is pretty toxic, try lipofection (Lipofectamine) or calcium phosphate.
thanks a lot. I am currently trying to optimize DNA:PEI ratio. Also, I am trying to transfect them only when they reach 70% confluence.
I think that 8 ug is to much DNA for cells and that could be the reason why they die... it is best to check cell death first by propidium iodide or some other way and then try to optimize DNA, DNA:PEI ratio...
Thanks for the answer. I guess increasing the confluence at the point of transfection works alright so far.
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