Draq7 dye

To generate WM164 melanoma cell lines stably expressing the FUCCI constructs, mKO2‐hCdt1 (30–120) and mAG‐hGem (1–110) (Sakaue‐Sawano et al, 2008) were subcloned into a replication‐defective, self‐inactivating lentiviral expression vector system and the lentivirus was produced by co‐transfection of human embryonic kidney 293T cells. High‐titre viral solutions for mKO2‐hCdt1 (30/120) and mAG‐hGem (1/110) were prepared and used for co‐transduction into melanoma cell lines and subclones were generated by single‐cell sorting (Haass et al, 2014). Cells were seeded in multiwell tissue culture plates, and time‐lapse microscopy was performed using an Olympus IX‐81 inverted fluorescence microscope equipped with an incubation chamber at 37°C and 5% CO₂. Images were taken at intervals of 15 min using 10× objective. Cells were treated with 160 nM dabrafenib, 80 nM trametinib or equivalent DMSO volumes. DRAQ7 dye (Beckman Coulter) was added to all conditions to a final concentration of 3 μM to track cell death. Cells were monitored and the occurrence of cell cycle phases as well as cell death recorded.

To determine the ratio of EGFP-positive cells, mES cells transfected with the EGFP gene by recombinase or integrase were collected, stained with DRAQ7 dye (Beckman Coulter, USA) to remove inactive cells, and then analyzed by a Galios flow cytometer (Beckman Coulter).