Puromycin Killing curve -How to determinate the right concentration by killing curve

[wangleipurdue]

Dear all,

I need to select stable transfected MCF10A by Puromycin. In order to determine the right concentration to use, I am doing a killing curve with non trasfected MCF10 A cells using Puromycin concentration at 0.125 µg/ml,0.25 µg/ml, 0.5 µg/ml,1 µg/ml and 2µg/ml.I started the puromycin treatment from day2 after plating cells (the confluence is almost 60 to 70%). The 2 µg/ml starts to kill cells on day2 after treatment (day4 after plating), and on day4 after treatment (day6 after plating), all of cells die. The 1 µg/ml starts to kill cells on day3 after treatment (day5 after plating), and on day4 after treatment (day6 after plating), most of cells die. The 0.5µg/ml starts to kill cell at the same time as 1µg/ml, it just does not kill as many as cells, so on day4 after treatment (day6 after plating), the confluence is 50% (the control is more than90%). The 0.125 and 0.25 µg/ml does not kill cells at all.

Which concentration do you think is safe for me to use to get stable cell line or should I do one more killing curve by using some concentrations between 0.25 and 0.5 µg/ml, please?

Thanks.

Lei