How to Produce a Stable Cell Line

Biochemistry, Molecular Biology, and Cell Biology Protocols >> How to Produce a Stable Cell Line

1. Perform a kill curve with various concentrations of the antibiotic to determine the amount of antibiotic required to kill the cells after one or two weeks.
2. Perform a transfection as normally, and then maintain the cells in the selection agent for at least two weeks.
3. Change the media every few days with new media containing selection agent.
4. After two weeks or more, clone out single colonies of cells by diluting them such that there are fewer than 1 cells per well on average (use a 96-well plate).
5. Wait a few weeks, and check that they have formed colonies under the microscope.
6. Expand the desired clones of cells, and assay for protein expression either by immunofluorescence or western blot.
7. Select the clones that have low, medium, and high expression, and freeze down a batch of the cells.
8. Selection pressure should be maintained as much as possible.