Wm1341d

A375 and Hs294T cell lines were obtained from the American Type Culture Collection (LGC Standards, Lomianki, Poland), WM1341D and WM9 cell lines were from Rockland Immunochemicals, Inc. (Limerick, PA, USA), whereas SK-MEL-28 were bought at the Cell Lines ServiceGmbH (CLS, Eppelheim, Germany). All cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco), 100 U/mL penicillin (Gibco), 100 µg/mL streptomycin and 2 mM l-glutamine (Gibco) (complete medium) at 37 °C in a humidified atmosphere containing 5% CO₂. All cells were subcultured twice per week in Mycoplasma-free conditions.

A375 and Hs294T cell lines were obtained from the American Type Culture Collection (LGC Standards, Lomianki, Poland), WM1341D and WM9 cell lines were from Rockland Immunochemicals, Inc. (Limerick, PA, USA), whereas SK-MEL-28 were bought at CLS Cell Lines Service GmbH (Eppelheim, Germany). Cell lines were subcultured twice a week. Some experiments were conducted on PMA- and LPA-stimulated cells. Before stimulations, cells were starved for 24 h in medium without FBS (fetal bovine serum). Cells were stimulated with 1 µM LPA for 10 min or with 100 nanomolar (nM) PMA for 5 min at 37 °C in 5% CO₂.

Cancer-associated fibroblasts were differentiated from normal human dermal fibroblasts (Lonza) using four melanoma cell lines: A375 and Hs294T obtained from the American Type Culture Collection and WM1341D and WM9 cell lines purchased from Rockland Immunochemicals, Inc. Fibroblasts were cultured in FBM (Fibroblast Growth Basal Medium, Lonza) cell culture medium (supplemented with FGM™-2 SingleQuots™ from Lonza), whereas melanoma cells were grown in DMEM (Dulbecco’s Modified Eagle Medium) medium containing 4.5 g/l glucose and 1.5 g/l NaHCO₃ supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, and antibiotics (10,000 U/ml penicillin, 10 mg/ml streptomycin, 25 µg/ml amphotericin B). Cells were cultured in 25 cm² tissue culture flasks (VWR) at 37 °C in 5%CO₂/95% humidified air and passaged twice a week using 0.25% trypsin/0.05% EDTA solution (IITD PAN, Wroclaw, Poland).