I have a few questions pertaining to soft agar assay. I would appreciate anyone's help.

1) my cells of interest are mammary epithelial cells infected with virus.
They are serum-free cells. I am not sure what kind of medium to use in the bottom layer since many protocols use DMEM with or without serum.

2) What kind of agar should I use? LMP or other. Difco agar noble seems popular? Any insights.

3) Some protocols use two layers others use 3 layers (cells in middle layer)
Any advice on this?

Any general points or even protocols would be great. Thanks a lot!!!

I have a lot of experience withe hematopoietic colony assays in soft agarose.

I use Low Melting Temperature Agarose (not agar). This way you can keep it in the hood while you work in diluted form (0.36-0.5% is what I've used as the final concentration fro top and bottom layers). I make up 10X stock in sterile water (yes water) and autoclave (short liquids cycle). Then I aliquot about 50ml per bottle. To use I microwave just enough to get it to boil, mix and then let it cool to 37°C in a water bath. I dump the last 10ml or so of a bottle as it usually has gone through a number of cycles and lost some of its water content.

I don't know why one would use three layers, maybe to squeeze the cells into a thin plane. I've use double layers for a couple purposes. One is that cells in the top layer have no chance to adhere to the plastic and grow under the agarose. Two, the bottom layer lets you expand the volume of the culture without having your cells distributed throughout a thick layer of agarose making it hard to see them under the microscope.

Single layers work ok (e.g 1ml in a 35mm2 dish) if your not worried about cells sticking to the plate surface.

I can't answer your media requirements, because I don't know the cells your working with. However, top and bottom layers should contain the same medium, it all equilibrates across the layers anyway. So just use what is recommended for your cells.

Thanks a lot for your help and feedback.