ICC Cell Adherence Issue - (May/04/2010 )

Hi,

I'm trying to perform an ICC protocol but an having an issue with cell adherence. 786-O cells are grown in a 24 well plate. Each well has a sterile glass cover slip in the bottom. The cells are seeded directly into the well and allowed to grow over the weekend for fixation the following Monday. The beginning of the protocol that I have been using follows.

1. Remove media and wash 2x with 1X DPBS for 5 min/wash
2. Fix with 4% PFA for 15 min at RT
3. Wash 3x with ice cold 1X DPBS for 5 min/wash

The problem is that by the end of step 3 there are very few to no cells remaining on the cover slip. I have also noticed that the cells tend to become more rounded in appearance following the first or second wash in step 1.

Any help would be greatly appreciated!

Thanks!

Try coating the coverslips in an adherent material such as collagen (gelatin), or poly d-lysine.