DMEM/F-12, no phenol red

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Use of 6-well plate with an ultra-low attachment surface Seeding of 10x103 sorted cells When seeding of sorted cells, the plate should be left undisturbed for 5 days at 37°C

Protocol tips
Use of 6-well plate with an ultra-low attachment surface Seeding of 10x103 sorted cells When seeding of sorted cells, the plate should be left undisturbed for 5 days at 37°C

Publication protocol

For in vivo mammosphere assay, primary tumors were surgically removed from orthotopic mice, and chopped into small chunks by blade. The tumor chunks were further dissociated into single cells by passing through a 25G needle (6 strokes). mCherry-labeled tumor cells were isolated from stroma cells by using FACS sorting. Sorted tumor cells at a density of 10x103 cells were resuspended in 2 mL of mammosphere media and cultured in a 6-well plate with an ultra-low attachment surface (Costar). The plate was left undisturbed for five days in a 37°C incubator. Mammospheres were counted and measured using an inverted Axiovert 40 CFL microscope at 200X magnification.

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