Tumor spheres were formed under nonadherent and serum-free conditions as previously described for stem cell-selective conditions.25 (link),26 (link) Briefly, cells were resuspended in 0.8% methylcellulose-based serum-free medium (Stem Cell Technologies, Vancouver, Canada) supplemented with 20 ng/mL epidermal growth factor (BD Biosciences, San Jose, CA), 20 ng/mL basic fibroblast growth factor, and 4 mg/mL insulin (Sigma-Aldrich, St. Louis, MO) and seeded into 24-well ultralow-attachment-surface plates at 5 × 103 cells/well in 0.5 mL of medium. Cells were cultured for 2 weeks and examined by light microscopy. Only spheres exceeding 50 μm in diameter were scored as positive in five random fields per well. A minimum of three independent tumor sphere assays was performed for each experiment.
Protocol detail
Tumor Sphere Assay for Cancer Stem Cells
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methylcellulose-based serum-free medium
epidermal growth factor
basic fibroblast growth factor
insulin
Assays
Basic fibroblast growth factor
Cells
Epidermal growth factor
Insulin
Light microscopy
Methylcellulose
Serum
Stem cell
Tumor
Corresponding Organization :
Other organizations : Mahidol University, National Institute for Occupational Safety and Health
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Variable analysis
independent variables
- Nonadherent and serum-free conditions for tumor sphere formation
- Formation of tumor spheres with diameter exceeding 50 μm
- Cells were seeded at 5 × 10^3 cells/well in 0.5 mL of medium
- Cells were cultured for 2 weeks
- No positive or negative controls were explicitly mentioned in the input protocol.
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