Melanoma Cell Cycle Analysis by Flow Cytometry

The cell cycle status was analysed with flow cytometry, based on quantification of DNA content, as described previously [21 (link)]. Next, A375 and RPMI7951 melanoma cells were treated with 1,25(OH)₂D₃ at 100 nM concentration, CPL304110 or AZD4547 at 5 µM concentration or with a combination of vitamin D with the selected FGFR inhibitor for 24 or 48 h. Trypsinized human malignant melanoma cells, as well as cells from the culture medium, were fixed together in 70% ethanol for 24–48 h at 4 °C. Following treatment with ribonuclease, the DNA was stained with propidium iodide (PI; Sigma Aldrich; Merck KGaA) for 30 min at 37 °C. The fluorescence of the PI-stained cells was measured by flow cytometry (FACSCalibur™; Becton, Dickinson and Company, Franklin, Lakes, NJ, USA). The results were analysed using the CellQuest™ Pro Software version 6.0 (Becton, Dickinson and Company) and expressed as a percentage of cells with DNA content corresponding to apoptotic/necrotic cells (subG1 fraction) or cells in G1, S and G2/M phases of the cycle.