ES cells and feeders - (Aug/04/2006 )
I made some homozygous ES cells which do not produce certain protein. Now I will do western but I need to get rid of feeder cells since they will contaminate my western results.
What I did was I trpsinized them, replate on collagen treated wells and after 15 or 30 min replate once more. The idea is feeders will attach first , es cells later. It worked to an extend but not completely. Anybody has an idea how to get rid of feeder cells completely?
FACS - flourescent cell sorting... but you will need a flourescent antibody to a specific external marker for your ES cells, (which the feeder cells do NOT have, or vice versa) and a cell sorting facility available. This is best method for nearly total separation and publishable results.
Of course if you pick an antibody to the ES cells, you will then have antibody proteins contaminating your western, so best to get an antibody specific for the feeder cells, separate the two by FACS, and you will then have unmolested ES cells to use.