cells dont attach hard enough to 96 well - (Feb/28/2011 )

Hi everyone and hope you can help me.
I have immortalized cells that grow rapidly in a flask and are attached strongly. For an XTT assay I grow them in 96 well. I seed 3000 cells per well. After 24 hr I wash them with PBS and change medium. I notice a lot of the cells (majority)are washed away during this process. Cells remain in the center and near the walls.. In the beginning they spred evenly. Could you advice me what can I try to attach them better?
Thank you so much

What sort of cells? Cells do tend to aggregate/grow more on the edges of wells and there isn't anything I have come across to prevent this. YOu could try washing them more gently by adding the PBS very slowly down the side of the well.

You could try coating the plate with gelatine (collagen), poly-L-lysine, fibronectin...

I agree, the type of cell matters a lot in these type of detachment issues. I was wondering if the same cells remain attached to the flask 24 h after seeding when you wash them?
Also, I heard people use PBS with Ca++ Mg++ to wash gentle cells, you can read the relevant discussion here http://www.protocol-online.org/forums/topic/13294-wash-l929-cells-with-dpbs-with-or-wout-ca-mg/

good luck.

If your cells is not dependent on specific growth factors and your assay is not too sensitive, you can even try skipping the PBS washing step. Just aspirate you medium and directly add fresh warm medium. It might help tilting your plate and gently add your medium to the wall of the well and then gently tilt back the plate to submerge your cells.