I am making stable cell line of HepG2 cells after transducing them with shRNA-Lenti virus. I have done a kill curve and used 2 ug/mL of Puromycin for the selection pressure. I can identify now resistant colonies. Today is the 7th day of Puro selection, and I noticed that all my un-transfected cells have died now when used the same 2ug/mL of Puro.

Now, I must expand these polyclonal-resistant colonies. They are now in 10 cm plates. There are various protocols, and I just dont know which one should work best for me now. My intention is to do now cell-based assays, but also want to make stock of my stable transfected cells by expanding and freezing them.

The simplest method to me looks like I should now trypsinize them and expand these colonies by doing a lower dilution (1:5, or 1:10), and still use low conc. of Puro (0.5 or 1 ug/mL) to keep maintaining the selection pressure. I just don't know when to remove the selection pressure. Also, other people noticed that their stably transfected cells kept dying even if they use low conc. of Puro to maintain selection pressure.

There are other ways of doing it by doing single clone isolation and expansion by trying a limiting dilution method/using clonal rings/trypsin disc. Do anyone has any detailed protocol of doing this. Also, HepG2 cells doesn't grow when plated at very low density. So, I am really confused how to proceed form here. Please help.