Isolation and Characterization of Melanoma Stem Cells

Melanoma stem cells and non-stem cells were sorted from MDA-MB-435 cells in our laboratory, which were performed as previously described^{14 (link)}. Briefly, the top 1% ALDH1-positive cells were sorted from MDA-MB-435 cells with flow cytometry. As reported, one marker is not enough to fully characterize melanoma stem cells^{15 (link)}. To confirm the sorted melanoma stem cells, the tumorsphere formation assay and tumorigenesis in nude mice of ALDH1-positive cells were conducted. All the data demonstrated that the sorted ALDH1-positive cells were melanoma stem cells^{14 (link)}. Melanoma non-stem cells were cultured in Leibovitz’s L-15 medium (Sigma, USA) supplemented with 10% fetal bovine serum at 37 °C with 100% humidified atmosphere. Melanoma stem cells were grown in DMEM/F-12 medium (Invitrogen, USA) supplemented with 20 ng/mL epidermal growth factor (MedChemExpress, USA), 10 ng/mL basic fibroblast growth factor (MedChemExpress), 5 μg/mL of insulin (MedChemExpress), and 2% of B-27 (Invitrogen) at 37 °C in a humidified atmosphere with 5% CO₂.