Metastatic Melanoma Cell Viability Assay

The viability of cells isolated from metastatic malignant melanoma tissues treated with 1,25(OH)₂D₃, vemurafenib or both was assessed using sulforhodamine B (SRB) as described previously [17 (link),30 (link),79 (link)]. The cells were seeded in 96-well plates at a density of 2000 of cells per well and left overnight. Subsequently, the cells were treated simultaneously with serial dilutions of vemurafenib (3, 15–200 nM) and 1,25(OH)₂D₃ at a 100 nM or 1 µM concentration for 72 h. The cells were fixed with 10% trichloroacetic acid for 1 h at 4 °C, washed with distilled water and stained for 15 min with 0.4% SRB solution (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) in 1 % acetic acid. SRB dye was solubilised using a solution of 10 mM buffered Tris Base (pH 10.5), and absorbance was measured at 570 nm using an Epoch spectrophotometer (BioTek, Winooski, USA). The relative IC50 value was calculated as described previously [30 (link)] as the mid-point between no inhibition and the maximum observed decrease in proliferation (n = 6).