Cells are lost after fixation - (Nov/30/2007 )

After fixing Hepg2 cells in 4-well cells i've found that nearly all cells have dissapeared. The cells were > 90% confluent to start with. That's the procedure:
Cells were washed 3x with PBS (no Ca nor Mg, would that make a big difference?). They were fixed after that with 4% paraformaldehyde for 15 min. Washed 3x PBS (no Ca nor Mg). blocked with 10% goat serum 0.1 % saponin. Then antibodies were added. A colleague has also has problems fixing his cells but didn't lose all cells. What am i doing wrong? Is it true that Hepg2 are difficult to fix?

Check the cells after each step to know which step is responsible.

I would guess PBS with calcium and magnesium should solve it. Make your PFA and all other solutions for immuno staining with this PBS.

You never use PBS with phosphate, Ca and Mg. I sent you an e-mail that you should respond to.

Is it on purpose that you keep your cells that confluent (I haven't worked with those cells before)? As I know from other cell lines that they detach easier when they are to confluent.

The cells are very happy with washing with PBS. I know that from culturing, trypsinazing or spiltting them but its always done with PBS no Ca or Mg. My colleagues has also suggested PBSCM. I will test the fixation with less confluent cells and with PBSCM and see.

not exactly. You only exposed cells briefly in PBS w/o Ca, Mg.

It might not be from Ca or Mg and it could be. I've been reading emphasis on fixing at 50-70-% confluency. So, that could be the reason.

and, maybe you should treat the fixed cells more gently during washing steps?

You have many procedural problems.