Some Thoughts On Creating Stable Cell-Lines

Creating stable cell-lines can be a straightforward or tricky process. In this post I will share some techniques or quirks, which I found worked when creating stable cell-lines.

* Use cells with low passage number – Thaw out your cell stocks^1,2 and start with “new” cells with low passage number.

* Culture cells in antibiotic-free media – By cutting out penicillin/streptomycin, I found increases in my transfection efficiencies. It also avoids any possibility of the penicillin/streptomycin interfering with the selection antibiotic during the selection process.

* Make the selection media fresh – Have aliquots of your selection antibiotic at a higher concentration and dilute it into fresh media each time you need to do a media change.

* Change the selection media daily – After transfection, change the selection media daily up until you reach the limiting dilution stage. It may seem excessive or wasteful but I have found that by having freshly made selection media and a daily media change ensures that the untransfected cells are effectively killed off.

* Have high concentrations of your antibiotics aliquoted – Freeze-thaws and heat-cool cycles can affect the efficacy of some antibiotics so to avoid any issues calculate the approximate amount that you will need for selection and aliquot small amounts into separate tubes.