Dmem f12 1 1

A total of 500 single viable cells (TVM-A12 and TVM-A12-CD133+) were seeded into 48-well tissue culture plates coated with 0.5 mg/ml of poly-2-hydroxyethyl methacrylate (Poly-HEMA, Sigma) in a 500 μl of serum-free DMEM/F12 (1:1) (Sigma) basal medium supplemented with L-glutamine (2 mM), Penicillin-Streptomycin (100 mg/ml), 20 ng/ml human epidermal growth factor (EGF) 20 ng/ml, human fibroblast growth factor-2 (FGF-2) (ProSpec, Rehovot, Israel), and 1:50 B-27 supplement (Gibco_, Life Technologies, Carlsbad, CA, USA) and cultured at 37 °C in a humidified 5% CO₂ for 10 days to form melanospheres. For serial passage, these melanospheres were counted using a manually prepared “quadrant grid” under microscopic observation, harvested and centrifuged at 1000 rpm for 5 min, trypsinized to dissociate in to single cell, counted and viable cells reseeded in the Poly-HEMA coated 48-well plates for subsequent passages.

Three human breast cancer cell lines (MCF7, SkBr3, and SUM159 PT) and one human nontumorigenic epithelial breast cell line (MCF10A), purchased from EACC or Elabscience, Austria, were used in this study. MCF7 is an estrogen receptor (ER)- and progesterone receptor (PR)-positive cell line, SkBr3 is an HER2-positive cell line, and SUM159 PT is a triple-negative (ER-, PR-, and HER2-negative) cell line. Three tumor cell lines were grown in DMEM (Sigma Aldrich, St. Louis, MO, USA) supplemented with 10% FCS (fetal calf serum, Sigma Aldrich), while MCF10A was grown in DMEM:F12 1:1 (Sigma Aldrich) supplemented with 10% FCS, 20 ng/mL EGF, 10 µg/mL insulin, and 100 ng/mL cholera toxin in a humidified atmosphere with 5% CO2 at 37 °C.