Metastatic Melanoma Cell Line Cultivation

The metastatic human melanoma cell lines MeWo, C32, A2058, SK-Mel-28, SH-4, A375, Hs 852.T, the human erythroleukemia cell line K562, the B cell leukemia NALM-18, and the primary/metastatic pair WM-115/WM-266–4 (derived from the same patient) were purchased from the American Type Culture Collection (ATCC, Virginia, USA). The primary T1 and the metastatic melanoma G1 cell lines, derived from the same patient, were obtained from the Institute Gustave Roussy (Villejuif, France). The abovementioned cell lines were expanded in RPMI-1640 medium (Euroclone, Italy) supplemented with 2 mM L-glutamine (Euroclone), 1% streptomycin and penicillin (Euroclone), and 10% fetal bovine serum (FBS; Thermo-Fisher Scientific, Massachusetts, USA). In some experiments, melanoma cell lines were treated for 36 h with 1 ng/mL of interferon alpha (IFN-α) (Merck, Canada) and 1 ng/ml of anti-CD40 monoclonal antibodies (mAb) (clone 5C3 - Functional Grade, Thermo-Fisher Scientific). In other experiments, WM-115, WM-266–4, T1, G1, A2058, and SK-Mel-28 melanoma cells were treated with 100 ng/mL of phorbol-12-myristate-13-acetate (PMA, Sigma-Aldrich) for 3 h as previously described (28 (link)). All cell lines were confirmed as mycoplasma-negative by reverse transcription PCR (RT-PCR) (Eurofins, Luxembourg).