I am growing my cells in a 6-well plate and carry out the transfection 24h later when they have reached about 80% confluency.

I am using Optimem media with not antibiotics in it.

I use 1μg DNA to 3μl Lipofectamine 2000. In the beginning, I would leave my cells within the same media for 24h and then check for any transfected cells but that seems to be too much for them and they end up dead.

Now, I just change the media after 4-6 hours but still no transfected cells.

Any ideas?