I've done soft agar assay for several times in past in various formats. For the first time, I am doing it in 6-well plate format (by titrating everything down based on the surface area differences between different plates).
However, now I came across a bizzare problem. I can see many cells adhered to the plate within a few days of cell plating and growing in monolayer instead of forming typical three-dimensional colonies. I know I had no problem in pouring the agar. These are breast cancer cells. Bottom layer is 2.5 ml of 0.5% Agar Noble (in DMEM) per well and top agar is 1 ml of 0.33% Agar Noble per well (with 25,000 to 100,000 cells in MEGM).
Please help me out here if you guys have any thoughts, tips and tricks.