Hi,
I am trying to transfect my vector (about 10 kb) into the HEK293 T by reverse transfection method. I have a big problem with this cell line because it is semi suspend. When I want to change the media 6 hours after transfection, the cells detached easily, then they become multilayer, finally, the transfection rate goes down. what do you think about it?
Do you know an adherent cell line for high-efficiency transfection?
I would appreciate it if you have any suggestions for overcoming my problem.
Does it need to be human? If not, NIH 3T3 cells work well.
What's the timeline of your experiment between the initial plating of your cells and the changing of the media?
Hello
I already used HEK293T in a previous lab and it was adherent. No need to trypsinised the cells, only flushing but when the medium was carrefully removed the cells remained in the wells. I had the idea that it was HEK293 (without T antigen) that was non adherent cells.
Try CHO. It's extremely easy to work with and sticks like super glue.
Here's a trick for the HEK media change I once used. Get a small perfume bottle, like with a mist spray nozzle. It needs to be sterilizable in some way. Usually alcohol will do fine. You can use it to carefully spray a mist of medium on to the cells, without causing an force on them at all. It takes some time to transfer enough medium in that way, and also takes some practice. But works :)
Ragan Pitner ,
yes, I have to use a human cell line for transfection.
about your question: I change the media 6 hours after transfection.
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