cell floating in 96-well plate - (Jul/04/2009 )

Hi all, recently I have encountered a problem on culturing RAW264.7 in 96-well plate. I need to do a fluorometric experiment.

here is my protocol:

Day 1: seed 2x105 cell into 96-well plate. Maintain it O/N in DMEM (no serum).

Day 2: remove medium (i tried using a pump or pepitte), add back fresh DMEM (no serum).

The problem is at step 2, the cells start to detach once I added the medium. I cannot do any washing in the downstream steps.

I don't think there is a problem with the coating. I routinely use the one from Corning and try plates from other labs which they have no probelm.

What else could cause the problem? (e.g. passage number? the cell is at 24th passage)

Thx

Lack of serum is probably the problem. If your cell line has a limited proliferative ability, that could be a problem too - pretty easy to test, just get up some more of an earlier passage.