Sphere formation assays were performed as previously described (17 (link)). In brief, cells were seeded in 24-well ultra-low attachment plates at 1 x 103 cells/well in 500 µl of MammoCult® Human Medium Kit (Catalog # 05620, Stem Cell Technologies, Vancouver, Canada). Spheres were counted between 7–10 days after plating, using Gel Count TM—Oxford OPTRONIX version 1.03. To subculture the tumorspheres for secondary and tertiary generation, supernatant was removed and 1 mL of pre-warmed Trypsin-EDTA (0.25%) (Stem Cell Technologies) was used to dissociate the tumorspheres. Pellets were resuspended in MammoCult Human Medium and viable cells were counted according to the manufacturer’s instructions. Similar cell densities were plated to form subsequent generations.
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Tumorsphere Formation and Subculturing
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Variable analysis
independent variables
- Cell seeding density (1 x 10^3 cells/well)
- Sphere formation and counting
- Volume of medium (500 µl)
- Culture vessel (24-well ultra-low attachment plates)
- Culture medium (MammoCult® Human Medium Kit)
- Trypsin-EDTA (0.25%) for passaging
- No explicit mention of positive or negative controls
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