2.9. Cell cycle distribution analysis

Flow cytometry analysis of cell cycle distribution by propidium iodide staining was conducted as previously published [29] with some adjustments. In brief, NCI-H508 cells were subjected to test agents for 72 h. Subsequently, cells were washed with PBS, harvested and fixed with 70% ice-cold ethanol. Subsequently, cells were washed with ice-cold PBS twice and treated for 5 min at room temperature with 100 μg/mL of DNase free RNase A in PBS containing 0.1% v/v Triton-X 100. Alterward, cells were stained with 20 μg/mL propidium iodide in PBS containing 0.1% v/v Triton-X 100 while protected from light for 15 min at room temperature. Cells were then analyzed with a flow cytometer (Attune NxT, Thermo Fischer). Data analysis was performed using Attune NxT Software (Thermo Fischer). All experiments were conducted in triplicate.