Reversible and effective cell cycle synchronization method for studying stage-specific processes.

The cell cycle is a crucial process for cell proliferation, differentiation, and development. Numerous genes and proteins play pivotal roles at specific cell cycle stages to ensure precise regulation of these events. Understanding the stage-specific regulations of the cell cycle requires the accumulation of cell populations at desired cell cycle stages, typically achieved through cell cycle synchronization using kinase and protein inhibitors. However, suboptimal concentrations of these inhibitors can result in inefficiencies, irreversibility, and unintended cellular defects. In this study, we have optimized effective and reversible cell cycle synchronization protocols for human RPE1 cells by combining high-precision cell cycle identification techniques with high-temporal resolution live-cell imaging. These reproducible synchronization methods offer powerful tools for dissecting cell cycle stage-specific regulatory mechanisms.