Incubate your cells with Puro for at least 24-48 hours, carefully monitor the cells daily for cell death, only positive cells should survive. Depending on the Puro concentration this may take longer. As soon as a (significant) proportion of your cells has died, replace the medium not adding Puro and let the cells recover. You might do a second round or Puro selection.

Then you need to confirm that your gene of interest has been knocked out and depending on your research question you may either us the cell bulk or continue with single cell cloning.

Thank you very much for your response Sabine Strehl

Hello Innocent Agida

Good day to you.

Before your actual experiment, you will have to first perform a pilot experiment namely, an antibiotic (puromycin) kill curve. This experiment will help you to determine the optimal concentration of puromycin that you will be using to select the positive clones.

An antibiotic kill curve is a dose response experiment in which mammalian cells are subjected to increasing amounts of selection antibiotic (i.e., puromycin) to determine the minimum concentration of an antibiotic that can kill all the cells in a specific period of time (say over the course of 2 to 7 days). This is a crucial experiment which you should perform before using a selection antibiotic (puromycin) to kill non-transduced cells and select the positive clones.

For puromycin, the optimal concentration is the lowest concentration that kills 100% of non-transduced cells and shows maximal survival of transduced cells in 48-72 hours. Please note that the optimal concentration will be cell type dependent.

For the puromycin kill curve experiment, you may refer to the protocol given below.

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

Then later you perform your experiment using the optimal concentration of puromycin which you have determined from the above pilot experiment to select the positive clones.

You could follow the below protocol:

1. After having infected the cells with the desired lentivirus construct, gently aspirate the media from the cells.

2. Add complete media to the wells containing puromycin at the optimal concentration which you have already selected from the pilot experiment. This will be the beginning of the selection process.

3. Observe the dish every day to ensure that the non-transduced cells in the well are dying. Perform regular fluid changes and monitor the growth of cells.

Please note: Depending on the efficiency of your transduction, you will see different degrees of cell death upon antibiotic selection. It is important to monitor these cells regularly and replace the cell media. Cell death in culture may adversely affect the surviving cells in culture. So, it is important to do regular media changes and maintain optimal growth conditions for the surviving cells. Even in the absence of cell death, the cell media should be changed every 2-3 days to maintain the dose of puromycin, which may not be stable at 37 °C.

4. The antibiotic selection should last at least as long as it takes for the non-transduced cells to completely die. After that, the cells may undergo additional selection while the population expands. At this time, you may reduce the concentration of the antibiotic in culture or remove the antibiotic entirely. If the antibiotic is reduced or removed from culture, check the cells regularly to confirm whether your gene of interest has been knocked out.

5. Once the individual wells become confluent, expand into larger vessels. For instance, a confluent well of a 6-well dish can be expanded into a 10 cm dish. A confluent 10 cm dish can be expanded into two 75 cm^2 flasks and so on and so forth.

6. Once you have sufficient positive clones growing and have been sufficiently expanded, you may prepare cell stocks. You may also harvest these cells to test for protein expression (i.e., whether your gene of interest has been knocked out).

Good Luck!