How to reduce cell clumping for immunocytostaining - (Sep/03/2005 )

Hi,

I have been off-late doing lots of immunocytochem with HEK-293T cells. I find that the quality of my slides seem to be dropping suddenly for unknown reasons. The cells on the slide seem too clumped and I really cant do good imaging because of this.

I basically fix my cells by 4%PFA for 10mins @RT
0.1% tritonX for 30mins RT

Can any one suggest to me tricks I can try to reduce clumping of cells.

Regds,
anand

I find that with my HepG2 cells I need to squeeze them through a small needle to get them to unclump.
I'm not quite sure how applicable that is to HEK cells (I've heard they take a long time to attach to surfaces), but I suppose if you do this and then avoid jostling or moving the cells during their attachment phase, it might help.