How to Produce a Stable Cell Line

  1. Perform a kill curve with various concentrations of antibiotics to determine the amount of antibiotics needed to kill the cells after one or two weeks.
  2. Do transfection as normal, and then maintain the cells in a selection agent for at least two weeks.
  3. Change the media every few days with new medium containing a selection agent.
  4. After two weeks or more, a single colony of clones out of cells by diluting them so that there is less than 1 cell per well on average (using a 96-well plate).
  5. Wait a few weeks, and check that they have formed colonies under the microscope.
  6. Extending the desired clone cells, and testing for protein expression either by immunofluorescence or western blot.
  7. Select the clones that have low, medium, and high expression, and below freezing cell batches.
  8. Selection pressure should be maintained as much as possible.