Wang Richard C, Wang Zhixiang
Johns Hopkins School of Medicine, Baltimore, MD, USA.
Department of Medical Genetics, University of Alberta, Edmonton, AB, Canada.
Methods Mol Biol. 2022;2579:61-71. doi: 10.1007/978-1-0716-2736-5_5.
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.
真核生物典型的细胞周期由四个阶段组成，包括G1期、S期、G2期和M期。G1期、S期和G2期统称为间期。细胞同步化是一个将处于细胞周期不同阶段的培养细胞带到同一阶段的过程。对于许多实验来说，拥有一群同步进行细胞周期的细胞是很有必要的，因为这样可以收集群体水平的数据，而不是仅仅依赖单细胞实验。虽然有各种药物可用于将细胞阻滞在细胞周期的每个特定阶段，但它们可能会引起不良副作用。在这里，我们描述一种仅使用一种化学物质——胸腺嘧啶核苷，使细胞同步到每个细胞周期阶段的方法。通过双胸腺嘧啶核苷阻滞将非同步化细胞同步到早期S期。解除双胸腺嘧啶核苷阻滞可使细胞以同步的速度进行细胞周期进程。在解除双胸腺嘧啶核苷阻滞后的不同时间间隔收集细胞，我们能够收获同步到G2期、M期和G1期的细胞。这种同步化可以通过多种方法进行评估，包括用流式细胞术检测DNA含量、用蛋白质免疫印迹法检测各种细胞阶段特异性标志物的表达以及用显微镜检查染色体形态。
