Screening stably transfected cells? so little? - (Apr/28/2005 )

Hello, it is me again. I am trying the establish the cell line stably transfected with some drug receptors. I did transient transfection in HEK 293 cells first. And I got very good transfection efficiency when I tested with Ca++ imaging, like 20 to 40%. The plasmid has Geneticin resistance. However, when I applied G418 for a week, I didnt see any sign of a single clony. If 20% cells get transfected, why didnt I see them?

BTW, for HEK 293 cells, I use 600ug/ml Geneticin (solution) from Invitrogen.

Thanks !!!!

hi
i did have to wait for two weeks to see clones in my experiments. so i assume it's a short time before you willsee your clones.
Regarding G418 concentration, i use 400µg/ml for 293 cells. I was told not to get over 500µg/ml. Maybe 600 is quite strong and reduce the cell division speed of your cells?...

You should titrate your G418 on normal 293 cells to find the proper killing dose and use that.

Thanks guys!

I did a little search. It was saying that the good dose for G418 is when you see massive cell death on the third day. I saw a lot of cells died on the third day in 600ug/ml group (HEK 293T cells) and even after a week, I still saw fairly amount of cells in 400ug/ml group. So I decied to use 600ug/ml. Somebody even suggest that I may use 800ug/ml to kill a lot and then switch 400ug/ml to maintain growing. I dont know if this makes senses to your guys.

BTW, I see my little clones under the scope now, not very good looking. But they are there. I still see a lot of cell debris in the medium or on the plate bottom. How to get rid of them? Do I have to leave my little babies with them forever?

thanks again!

Now that you can see some little clones, there should be no harm in removing the debris-containing media and replacing with fresh geneticin media to keep the little guys growing.