Culturing Melanoma and Melanocyte Cells

Malignant human melanoma cells (A375, LOX-IMVI, G-361) from ATCC (Manassas, VA, USA) were cultured in RPMI medium (10% FBS and 2 mM L-glutamine) or McCoy’s 5a medium (10% FBS), respectively. A375 cells resistant to the BRAF^V600E-kinase inhibitor vemurafenib (A375R) were generated by continuous selective culture (>12 weeks) employing increasing concentrations of vemurafenib (0.1–5 µM) with subsequent maintenance culture (5 µM) following a published standard procedure [36 (link),37 (link)]. Primary human epidermal melanocytes (adult skin, lightly pigmented: HEMa-LP from Life Technologies, Grand Island, NY, USA; abbreviated HEMa) were cultured using Medium 154 medium supplemented with HMGS-2 growth supplement. HEMa cells were passaged using recombinant trypsin/EDTA and defined trypsin inhibitor. Human diploid dermal fibroblasts (Hs27, ATCC) were cultured as described by us before [14 (link)]. Cells were maintained at 37 °C in 5% CO₂, 95% air in a humidified incubator.