primary cortical neuronal culture--how to avoid cell clumping? - (Sep/10/2006 )

I do corical cultures from rat fetuses. So far, my cells are not consistent. Sometimes they spread out individually (great), but sometimes they clump up as clusters. It looks like the coating problem. I coated with poly-l-lysine hydrobromide (high mol. w. pll with enhanced solubility) at 20 ug/ml (higher conc. appeared to harm cells) overnight and washed with water twice. Some people mentioned laminin coating on top of that, I tried (10 ug/ml in media, 1hr, washed with media, and dried) but had more cell clustering.

The other detail is, I culture cells on elastic sillicone membranes. With the same coating conditions, I always get consistent cell spreading on tissue culture plates (just PLL coating). Would the elasticity somehow play a role for cells to clump? or anyone knows of problems of PLL coating on sillicone membranes?

Can anyone give suggestion about a coating technique that is consistent with cell spreading (not clumping)?

Any suggestion is welcome!

Min

Sometimes plating cells in a low density can help.