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

Puromycin Killing curve -How to determinate the right concentration by killing curve - Cell Biology and Cell Culture

Puromycin Killing curve -How to determinate the right concentration by killing curve - Cell Biology Forum. Cell Culture Forum. Post and ask questions about cell culturing, cell lysis, cell transfection, cell growth, and cell biology.


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Old 06-09-2009, 04:08 AM
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Default Puromycin Killing curve -How to determinate the right concentration by killing curve



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 g/ml,0.25 g/ml, 0.5 g/ml,1 g/ml and 2g/ml.I started the puromycin treatment from day2 after plating cells (the confluence is almost 60 to 70%). The 2 g/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 g/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 0.5g/ml starts to kill cell at the same time as 1g/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 g/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 g/ml, please?

Thanks.

Lei
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