Synchronization of Cultured Cells to G1, S, G2, and M Phases by Double Thymidine Block

The typical cell cycle in eukaryotes is composed of four phases including the G1, S, G2, and M phases. G1, S, and G2 together are called interphase. Cell synchronization is a process that brings cultured cells at different stages of the cell cycle to the same phase. For many experiments, it is desirable to have a population of cells that are traversing the cell cycle synchronously, as it allows population-wide data to be collected rather than relying solely on single-cell experiments. While there are various drugs that can be used to arrest the cell at each specific phase of the cell cycle, they may cause undesired side effects. Here, we describe a protocol to synchronize cells to each cell cycle phase by using only one chemical: thymidine. Non-synchronized cells are synchronized to early S phase by a double thymidine block. The release of the double thymidine block allows the cells to progress through the cell cycle in a synchronized pace. By collecting the cells at various time intervals following the release of double thymidine block, we are able to harvest cells synchronized to the G2, M, and G1 phases. This synchronization can be assessed by various methods, including flow cytometry to examine the DNA content, Western blotting to examine the expression of various cell phase-specific markers, and microscopy to examine the morphology of the chromosome.