Hello Antra ..

It would be difficult to answer your question. Cell lines respond differently to puromycin selection. In mammalian cells, the optimal level of puromycin is typically around 1µg/ml. But I suggest that you determine the optimal puromycin concentration for your cell line before initiating your experiment.

You may follow the protocol provided below.

1. On day 1, plate the oral carcinoma cells in ten 60mm petri dish and grow at 37°C, 5% CO2 overnight.

2. On day 2, when the cells are approximately 80-85% confluent, add puromycin (diluted in culture media) at a final concentration of 1-10μg/mL in 1μg/mL increments to the cells and label the petri dish (1-10).

3. On day 3, examine the cells each day and change to fresh puromycin-containing media every other day.

4. The minimum concentration of puromycin that results in complete cell death after three to five days is the concentration that should be used for selection in your experiments. If you wish, you may repeat this titration with finer increments of puromycin to determine a more precise optimal puromycin concentration.

I am also attaching another protocol which will help.

https://goldbio.com/documents/1094/Titration+Kill+Curve+Protocol.pdf

Best.