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death of transfected cells after first passage in G418 medium - (Sep/02/2013 )



I wondered if anyone had similar problem or maybe could suggest what went wrong. I transfected my cells (SW756 and CaSki) with plasmid containing luciferase gene and G418 resistance gene. Before the transfection I made the kill curve to get the right concentration of the G418 for selection of my transfected cells (result: 500ug for CaSki and 1200ug for SW756). I transfected my cells on 19.08 and 24hrs later changed for selective media. The transfections worked, my positive controls expressed GFP proteins. I changed the selective media every 3 to 4 days, transfected cells were dividing and looked healthy (my control, untransfected cells died in about a week) and last Friday (30.08) after 11 days, I decided to split the SW756 cells because they were very confluent. I trypsinised them and passaged them (1/10) into selective media. Unfortunately, I discovered today that most of my cells died and I am not sure why.

Now I wondered whether I should have split the cells in regular media and replace with selective media the next day when the cells are more confluent? Or at least reduce the G418 concentration after passage? Any ideas? I would be very grateful for any suggestions to help me avoid the same situation when I decide to split my CaSki cells.






You need to ensure that the cells don't get above about 70% confluent as G418 only really works on rapidly dividing cells - once they get too confluent growth rate slows and the resistance plasmid is likely to be lost.