I am currently having problems with my transfections and request help. I use lipofectamine 3000 to transfect Hek 293 cells in 6 wells plates. For the last transfection, I transfected at 60% confluency. Also, I used 2500 ng of DNA per well, 5µl of P3000 per DNA, 5µl of Lipofectamine 3000, and DMEM with P/s + L-glu instead of OMEM. However, after 48 hours of transfections majority of the cells were lifted off the plate. However, even though the cells detached from the plate and little pellets remained after they were centrifuged, the lysate showed protein expression but not the medium (shown from western western analysis). But the problem is that I need protein to also express in the medium. Even when I incubated the transfected cells for 72 hours and used OMEM, there still isn't protein expression shown in the medium.
Any advice or recommendations will be greatly appreciated.