Cell Cycle Analysis by Flow Cytometry

Cellular DNA content was determined by flow cytometry using the propidium iodide (PI)-labeling method as described previously [23 (link)]. Briefly, B16F10 cells were seeded at a density of 4 × 10⁵ cells/dish in 10 cm dishes, and the cell cycle was synchronized by the addition of double thymidine (3 mM) for 16 h. Cell cycle-synchronized cells were then washed with PBS and re-stimulated to enter the G1 phase together by the addition of fresh medium, which also contained various concentrations of CoQ₀ (0-20 μM). Cells were harvested at 24 h, and the cell cycle analysis was performed using a FAC-Scan cytometry assay kit (BD Biosciences, San Jose, CA, USA) equipped with a single argon ion laser (488 nm). The DNA content of 1×10⁴ cells/analysis was monitored using the FACScalibur system. Cell cycle profiles were analyzed with ModFit software (Verity Software House, Topsham, ME, USA).