I am looking for any papers that mentioned about using puromycin in cell selection after transfection. I already got the optimized of MTX and G418. Then I started cell selection with puromycin at 1ug/mL, then gradually increased puromycin concentration to 2, 4, 8, 12, 16 ug/mL. Also the expression of interest antibody had promising band in western. Should I go further to do cell selection with higher puromycin, eg 24 and 36 ug/mL ?
I would suggest to determine the optimal concentration by doing a kill curve: Just seed some cells in a 6-well, at different concentrations of your antibiotic, observe for about a wekk. Use the lowest concentration that managed to kill your cells after one week.
And I agree with Andrea, gradually increasing the antibiotics will probably lead to resistance.
I am also treating puromycin for my cell selection. I think it depends on your cell line, because in my case, using mouse ovarian cancer (ID8 cell), I can only go up to 5 ug/mL (in 6-well plate setting) and beyond that, my transfected cells also die. So, I usually treat them below 5 ug/mL.
But if your cell line, can be resistant at up to 16 ug/ml, you can go higher.
The concentration of antibiotic depends on the cell line that you use. However, I would suggest one thing. Instead to increase gradually the conc of antibiotic, I prefer to split the cells in different plates (or wells) and give them different conc of antibiotic since the beginning (I mean, when you start the selection). This because if you gradually increase the antibiotic conc, you may risk to make the cells resistent to the drug and in that case you're just adding more antibiotic for nothing. But, once again, all depends on the cell line that you use.
I would suggest to determine the optimal concentration by doing a kill curve: Just seed some cells in a 6-well, at different concentrations of your antibiotic, observe for about a wekk. Use the lowest concentration that managed to kill your cells after one week.
And I agree with Andrea, gradually increasing the antibiotics will probably lead to resistance.
I agree with Andrea and Sophie, with the exception that I would like to double the concentration of selecting drug respect to the dose that kill my cell population (to minimize the risk of selecting spontaneous resistances).
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