ABSTRACT

The precise control of the cell cycle G2 phase to Mitosis (M phase) transition is central for cell fate determination. The commonly used methods for assessing G2 to M phase progression are based on synchronizing cells and involve perturbation of the natural cell cycle progression. Additionally, these methods are often time-consuming and labor-intensive. Here, we report a flow cytometry-based method that offers a kinetic analysis of G2 to M phase progression in asynchronous cells using nocodazole, 5-Ethynyl-2´-deoxyuridine staining, and histone H3 serine 28 phosphorylation (pH3) staining. Nocodazole is used to collect mitotic cells and prevent their progression into G1, at the same time EdU is added for use as a dump channel during analysis. The remaining cells can then be identified as either G1 or G2/M based on their DNA content. Finally, G2 and M phase cells can be separated based on a mitotic marker, phosphorylation of ser28 on histone H3. While developed to assay G2/M phase progression, this method also resolves G1/S phase progression with no additional steps other than analysis. Compared to double thymidine block, this method does not require extended pre-treatments and is compatible with a greater variety of cell lines, while at the same time offering enhanced consistency and temporal resolution.

摘要

细胞周期 G2 期向有丝分裂（M 期）转变的精确控制对于确定细胞命运至关重要。评估 G2 到 M 期进展的常用方法是基于同步细胞并涉及对自然细胞周期进展的扰动。此外，这些方法通常既费时又费力。在这里，我们报告了一种基于流式细胞术的方法，该方法使用诺考达唑、5-Ethynyl-2´-脱氧尿苷染色和组蛋白 H3 丝氨酸 28 磷酸化 (pH3) 染色对异步细胞中 G2 到 M 期进展进行动力学分析。诺考达唑用于收集有丝分裂细胞并防止其进展为 G1，同时添加 EdU 用作分析期间的转储通道。然后可以根据其 DNA 含量将剩余的细胞识别为 G1 或 G2/M。最后，G2 期和 M 期细胞可以基于有丝分裂标记物，即组蛋白 H3 上 ser28 的磷酸化来分离。虽然开发用于分析 G2/M 期进展，但该方法还解决了 G1/S 期进展，除了分析之外没有其他步骤。与双胸苷阻滞相比，这种方法不需要延长的预处理，并且与更多种类的细胞系兼容，同时提供增强的一致性和时间分辨率。