Matrigel and Mammosphere Assays for Cancer Stem Cells

In the Matrigel assay, cells were seeded at 2500 cells/well in 200 μL of media atop of 40 μL of corning Matrigel matrix in 8-well slide chamber and pictures were taken on Day 7. U0126, EGF, or vehicle controls were added directly into media and incubated for the 7-days assay duration. In the mammosphere assay cells were seeded and cultured as previously described [9 (link)]. Briefly, cells were trypsinized and passed through a 40 μm cell strainer (BD, Franklin Lakes, New Jersey, USA) and seeded into ultra-low adherent plates (Corning, NY, USA) in MammoCult media (StemCell Technologies, Vancouver, BC, Canada) as per manufacturer's instructions. Mammosphere formed after 7 days were collected by centrifugation at 1000 rpm. For serial passaging, obtained mammosphere were dissociated into single cells and seeded again into ultra-low adherent plates (Corning, NY, USA). Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay (#G3580, Promega, Madison, WI, USA) according to the manufacturer's instructions.