The volume of RPMI depends on the size of the plate you are using.  For a 6-well plate, I generally use 3mL media so that the cells are evenly plated and not subjected to the mensicus effect pushing the cells towards the walls.  The number of cells to plate depends on the cell line and treatment condition prior to plating and must be determined independently.

Hey Bindeshwar,

When you're doing a clonogenic assay, the goal is to see single discrete colonies while also having a reliable amount on the plate for analysis. You're not supposed to count colonies that are growing on top of each other or ones that are attached as there is no way of knowing if those are true colonies or if they're a singe colony that got split.

Often, the limitation is the size of the vessel, not really the amount of cells. I use the minimum amount of media which covers the plate and I usually add liquid beside the wells outside to prevent dehydration. For 6 well, I use 1 mL, for 96 well, I use 100 uL.

I suggest you dose-find the concentration of cells that you need (and refer to literature using your cell line). For 6-well plates, I use 1000 cells/well and my cell lines range from 30% clonogenic to 70% clonogenic.