Hi,
I am culturing HepG2 cells and using lentivirus to knock down the genes. After the knockdown, the cells are screened with puromycin (1ug/mL). I now have a problem with many black dots appearing, as shown in figure 1. Some dots are floating while the others are attached to the dish. Moreover, they are not swimming. I find if using a normal medium without puromycin, no dots appear, but they appear after adding puromycin for 1 day or 2 days. I think puromycin may be the problem. If so, I cannot use puromycin to select the transfected cell lines.
Anyone can help me? I have been confused about this problem for a long time.
There are many discussions about the mysterious dots in cell culture online. You could checkout the researchgate as well. IMO, they are an indication of poor health of the cell. This could be due to puromycin selection or the effect of the Knockdown, which may not be obvious before selection. I would suggest try to use the lowest concentration of puromycin for selection, which kills all non-transduced cell in about a week time, rather than using high concentration of puromycin which kills non-transduced cells in 1 to 2 days. A puromycin kill curve assay is needed in order to find an optimal puromycin concentration for your cell.
Wenxiu Li I would suggest to lower the puromycin concentration to the level that could kill all the wildtype cell in one week rather than 3 days. Or try multiple rounds of treatment: apply puromycin for 1 day, wash away dead cells and change into normal media and let the remaining cells recover and grow. Passage once confluent and do the second round treatment. Repeat the procedure until you did not see more cell death ( I guess three rounds should be enough).
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