Tumor Sphere Assay for Stem Cells

Tumor sphere assay was performed under non-adherent and serum-free conditions as previously described as stem cell-selective conditions.^{25 (link),46 (link)} Briefly, cells were suspended in 0.8% methylcellulose-based medium (Stem Cell Technologies, Vancouver, Canada) supplemented with 20 ng/mL epidermal growth factor (BD Biosciences, Franklin Lakes, NJ), basic fibroblast growth factor and 4 mg/mL insulin (Sigma-Aldrich, St Louis, MO) and plated at 5×10³ cells in ultralow adherent 24-well plates (Corning, Corning, NY). Cells were cultured for two or three weeks and visualized under a light microscope.

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Luanpitpong S., Li J., Manke A., Brundage K., Ellis E., McLaughlin S.L., Angsutararux P., Chanthra N., Voronkova M., Chen Y.C., Wang L., Chanvorachote P., Pei M., Issaragrisil S, & Rojanasakul Y. (2015). SLUG is required for SOX9 stabilization and functions to promote cancer stem cells and metastasis in human lung carcinoma. Oncogene, 35(22), 2824-2833.

Publication 2015

methylcellulose-based medium epidermal growth factor basic fibroblast growth factor insulin ultralow adherent 24-well plates

Assay Basic fibroblast growth factor Cells Epidermal growth factor Insulin Methylcellulose Microscope light Serum Stem cell Tumor

Corresponding Organization : Mahidol University

Other organizations : West Virginia University, Alderson Broaddus University, National Institute for Occupational Safety and Health, Chulalongkorn University

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Variable analysis

independent variables

Cell suspension in 0.8% methylcellulose-based medium
Addition of 20 ng/mL epidermal growth factor
Addition of basic fibroblast growth factor
Addition of 4 mg/mL insulin
Plating cells at 5x10^3 cells in ultralow adherent 24-well plates

dependent variables

Tumor sphere formation

control variables

Non-adherent and serum-free conditions

controls

Positive control: Tumor sphere formation under stem cell-selective conditions (referenced in publications 25 and 46)

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