since this is my first post, please excuse any mistakes.
I am trying to set up a Soft-Agar-Assay for MCF-7 and T47D-cells. I use Agarose DNA Pure Grade in a .5% Bottomlayer and 0.35% Toplayer (containing cells) on 12-well plates. I managed to get the cells (usually 25000 or 50000 per well) to grow and form colonies, but after a couple of days, the just die. I change medium every three days. I've tried several things in order to optimize the protocoll I am using, but nothing helped so far. I know, that T47D cells grow poorely in Soft-Agar, but MCF-7s should do better. The colonies growing are to small to get reproducable results by counting after staining with crystal violette.
So the question is, does anyone has encountered similar problems and is able to offer a solution?
Thank you in advance!