Dear All
I have done a number of trials to overexpress catalase in an adherent human cancer cell line. I have used different combinations of DNA/lipofectamine ratios per well in a six-well plates (2ug:2uL, 2ug/1uL, 1ug/1uL, and 1ug/2uL). I performed transfection in OPTI-MEM medium and I changed it with a complete medium without antibiotics 7 hrs after the transfection process. The problem I am having is that 24 hrs after transfection I found a proportion of dead cells floating. Since the aim of catalase transfection was to investigate whether ROS generation is responsible for the death induced by my treatment, such a proportion of floating dead cells complicate results interpretation.
what should be done to avoid such cell death 24 hrs after transfection?
comments are welcome
thanks

Hi,

I had a similar problem with Lipofectamine and it turned out that my cells were not at a high enough confluency. I started out using cells at 60% confluency and had high cell death but then I upped the seed number to give me 80% confluency at the time of transfection which solved the problem.

At what confluency do you tranfect your cells currently?

P

i used 50% confluency because i need to treat the transfected cells for an additional 24hr. so if i used 80% confluency at the time of transfection, the cells will be confluent during drug treatment and they will be arrested by contact inhibition, especially if you know that my treatment induces growth arrest and i want to study the involvement of ROS generation into drug treatment-induced growth arrest.
my question now is if used 80% confluency at the time of transfection, can i re-seed the transfected cells at a lower confluency 24hr after transfection for drug treatment experiment? I mean performing transfection at 12-well plate with 80% confluency then 24 hrs after transfection harvest the cells and re-plate in 6-cm dish for example.