Background: I recently seeded HEK cells on a poly-L-lysine coated plate and used those for transfection. My vectors are backsplicing vectors with the ZKSCAN introns which generates circular RNAs so it takes a while for the GFP signal to be observable with a microscope, even for my most active IRES of interest (more active than EMCV and comparable to c-myc 5'UTR). Most papers, like this one:Article Engineering highly efficient backsplicing and translation of...
grow cells for 4 to 5 days. However, I found that cells would become more confluent, acidify the media too fast and die. Then, I might lose the GFP-expressing cells. I tried changing media everyday when cells reach high confluency, but the media always turn very yellow the next day. If I seed fewer cells, then they may become too sensitive to the transfection, as I have noticed especially for the backsplicing vectors. Coating the plate with poly-L-lysine did help tremendously to prevent cell death after transfection, but after 2 days cells begin to die.
Question: So for experiments that require longer incubation/treatment periods, what do people do to maintain cell health at high/100% confluency?
