any tips for detecting phospho-ptotein in fixed adherent cells by flow cytometry - (Jul/24/2007 )

I can successfully detect phospho-protein in fixed/permeabalized suspension cells by flow cytometry, however, I have trouble using the same method on adherent cells. Any suggestion?

There are a few potential areas of problem here and to really get at the problem you are going to have to supply a bit more info. Are you fixing/permeabilizing the adherent cells the same as your cell suspension? Some antibodies will only work with specific fixations. Are these the same cells or at least are the two from the same species? Some antibodies are very species specific. On the odd side, are you using glass or plastic for the adherent cells? I actually have an antibody that looks great on glass but has such a high background on plastic that I can't detect the specific staining I see on glass.

The only other thing that comes to mind is that flow cytometry is a much more sensitive system. You use a computer/photomultiplier to detect staining which is much more sensitive than your eyes and a microscope.

How do you harvest the adherent cells?