Transfecting primary cells with plasmid - (Jun/10/2010 )
I have been trying for quite some time now to transfect my cells, which are primary vascular rendotelial cells, with a plasmid.
The system I use is Lipofectamin 2000. I transfect the cells and I see the signal in the cells (GFP linked), I propagate the cells,
split them, freeze them down and then thaw them. All this is fine but the problem starts when I thaw them.
For some reason the signal is there in the first couple of hours and then it diminishes rapidly, as if the cells do not express the plasmid any more.
Has anybody seen sth like this before?
have you compared this with cells that are not frozen? What is the name of the plasmid you are using?
my boss says that cells degrade GFP inside the host cell specially when the cells are dying.