Lrs fortessa

Cells were incubated at 37 °C with complete RMPI in all cell cycle experiments for 72 h unless stated otherwise. Parental SNU-475 cells were incubated with RMPI alone or with 1.5, 3, and 6 μM R428 at 37 °C for 24, 48, and 72 h. Cell cycle analysis was performed as described briefly herein. First, cells were trypsinized and collected by centrifugation. Cell pellets were then rinsed and resuspended in PBS and fixed with ice-cold ethanol, added dropwise while vortexing the samples to achieve 70% final concentration. Cells were initially kept on ice for 15 min and then at −20 °C overnight. The next day, fixed cells were rinsed with PBS and resuspended in PI staining solution containing 50 µg/mL PI, 1 mg/mL RNAase A, 0.05% Triton X. Staining was performed at 37 °C for 45 min. PI staining was evaluated by BD LRS Fortessa (BD Bioscience, San Jose, CA, USA). Analysis was performed by FlowJo v10 (Flowjo, OH, USA) with Watson Pragmatic algorithm.

Cells from the murine studies were analyzed using a BD LRSFortessa or BD LRSFortessa X20, and the forward scatter (FSC) files were afterwards manually gated with FlowJo v10 (Tree Star). The graphical visualizations were done using GraphPad Prism v8.3.0. The type of statistical test performed together with the exact P value is indicated in the individual figure legends. A P value below 0.05 was considered significant.

CHL-1 or 624-mel cells were seeded at a density of 70,000 cells per well in a 12-well plate. After overnight incubation, cells were treated with different cell death inducers at indicated concentrations. The percentage of FBS in the culture medium for this assay was lowered to 2.5%. After 24, 48 h or 72 h, viable, dying and death cells were harvested. After washing the cells in PBS, single cells were stained in 50 µL of a 1/4000 dilution of Fixable Viability Dye eFluor780 (ThermoFisher Scientific, 65-0865-1B) in PBS for 20 min at room temperature in the dark. At the end of the incubation time, cells were washed with FACS buffer. Afterwards, cells were resuspended in 400 µL Krebs–Ringer solution (125 mM NaCl, 5 mM KCl, 1 mM MgSO4, 0.7 mM K₂HPO₄, 6 mM glucose and 2 mM CaCl2, 25 mM HEPES, pH 7.4) and were incubated for 5 min at room temperature. Cells were pelleted and resuspended in 300 µL quinacrine (0.125 µM; Sigma-Aldrich, 69-05-6; diluted in Krebs–Ringer solution) and incubated for 30 min at 37 °C and in the dark. After incubation, cells were washed and resuspended in FACS buffer. Cells were immediately analyzed using flow cytometry (BD LRS Fortessa). Flow cytometric data were analyzed with FlowLogic software (Miltenyi Biotec, Version 7.3).