For now i've tried in 6 well plates coated at 0.6 % agar (1,5ml) and cells at 3 different concentration (20 000, 50 000 and 100 000 cells per well) in 0.4% agar (1,5 ml). I used media 2x + 20% FBS so the cells are happy in media +10% FBS final (as they normally grows in). I got 5-10 colonies after 14 days in the 20 000 cells. I've stained with crystal violet 0,05% for 1h and wash a bit with water but that wash doesn't seem to matter.
I've tried again but this time in 96 well plate as already published eith those cells. I've used the same concentration of agar and put 100ul of coating agar and 100ul of cells in agar (2000 cells per well). and incubated for 11 days and got nothing.
When I'm aspirating a part of the media or the stain do I aspirate the colonies? I'm far from touching the agar layer thought. I'm using selec agar from sigma, should I use another type of agar? Basically I don't know what i'm doing good or wrong here. Tks for your help.