I'm using two lentiviral vectors to infect HEK293 cells, the first virus has a hygromycin resistance gene, and the cells survive 100 ug/ml hygromycin selection. I then infect the cells with the second virus, which has a puromycin resistance gene, but the cells do not survive 0.5 ug/ml puromycin selection.

I've performed antibiotic kill curves, and these concentrations are the lowest I can use that kill all non-resistance cells.

The HEK293 cells were growing under puromycin selection and reached confluence after 2 days, so I trypsinised them and passaged them into a new flask containing new 0.5 ug/ml puromycin-containing media to continue the puromycin selection, but they were all dead the next morning, even though they were growing previously.

Should I avoid trypsinising cells under puromycin selection? I didn't have this problem when I infected them with the first virus containing the hygromycin resistance gene. Any suggestions or possible mistakes I'm making would be very much appreciated. The vectors I'm using do not have any fluorescence markers so I can't use that as a way to check for successful transfection.