Hello!
I've been trying to transfect EO771 using lipofectamine 2000, but despite changing the transfection media after 6 hours of incubation I have a high rate of cell death. My untransfected control well has the same media changes, and I don't see any cell death on that control.
I'm using 2.5 micrograms of DNA and 10 microlitres of lipofectamine per well for a range that goes from 1.5 to 2.5x10e5 cells plated in a 6-well. So far the best condition has been to plate 2.5x10e5 cells, but I got a very low transfection efficiency (around 10%), which is insufficient for the following experiment.
Has anybody a protocol to transfect these cells with high efficiency? Or do you have some advice on what I could improve to avoid the high rate of cell death?
Regards.
Hi Paula,
I haven't worked with this specific cell line but it looks to me like you could try reducing the amount of Lipofectamine you use per well. I have transfected a murine adherent cell line for which I use 2ul of Lipofectamine per 1ug of DNA and that has worked well for both over-expression plasmids and silencing plasmids. I generally transfect the cells for 8 hours.I have also used half the amount of DNA per well in a 6-well dish and have had satisfactory silencing/over-expression. You could try a range of Lipofectamine to DNA ratios to find what would work well for your cells. Hope this helps!
Thank you Bhavana Kayyar. I'm doubtful about using less DNA since my transfection efficiency is lower than needed, but I will try different amounts of lipofectamine.
Regards!
Dear Paula Gonzalez
Check this article if helpful.
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