Amin Mohammed A, Varma Dileep
Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University.
Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University;
J Vis Exp. 2017 Dec 5(130):56513. doi: 10.3791/56513.
Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The synchronization of cell populations at specific stages of the cell cycle has been found to be very useful in such experimental endeavors. Synchronization of cells by treatment with chemicals that are relatively less toxic can be advantageous over the use of pharmacological inhibitory drugs for the study of consequent cell cycle events and to obtain specific enrichment of selected mitotic stages. Here, we describe the protocol for synchronizing human cells at different stages of the cell cycle, including both in S phase and M phase with a double thymidine block and release procedure for studying the functionality of mitotic proteins in chromosome alignment and segregation. This protocol has been extremely useful for studying the mitotic roles of multifunctional proteins which possess established interphase functions. In our case, the mitotic role of Cdt1, a protein critical for replication origin licensing in G1 phase, can be studied effectively only when G2/M-specific Cdt1 can be depleted. We describe the detailed protocol for depletion of G2/M-specific Cdt1 using double thymidine synchronization. We also explain the protocol of cell fixation, and live cell imaging using high resolution confocal microscopy after thymidine release. The method is also useful for analyzing the function of mitotic proteins under both physiological and perturbed conditions such as for Hec1, a component of the Ndc80 complex, as it enables one to obtain large sample sizes of mitotic cells for fixed and live cell analysis as we show here.
以阶段依赖的方式研究细胞周期的各种调控事件，能让人清楚地了解细胞生长和分裂。已发现将细胞群体同步于细胞周期的特定阶段在这类实验中非常有用。与使用药理学抑制药物相比，用毒性相对较小的化学物质处理来同步细胞，对于研究后续的细胞周期事件以及获得特定有丝分裂阶段的特异性富集可能更具优势。在此，我们描述了在细胞周期不同阶段同步人类细胞的方案，包括通过双胸腺嘧啶核苷阻断和释放程序使细胞同步于S期和M期，以研究有丝分裂蛋白在染色体排列和分离中的功能。该方案对于研究具有已确定的间期功能的多功能蛋白的有丝分裂作用极为有用。在我们的研究中，只有当G2/M特异性的Cdt1能够被耗尽时，才能有效地研究G1期复制起点许可关键蛋白Cdt1的有丝分裂作用。我们描述了使用双胸腺嘧啶核苷同步化耗尽G2/M特异性Cdt1的详细方案。我们还解释了细胞固定的方案，以及在胸腺嘧啶核苷释放后使用高分辨率共聚焦显微镜进行活细胞成像的方案。该方法对于分析在生理和受干扰条件下有丝分裂蛋白的功能也很有用，例如对于Ndc80复合体的一个组分Hec1，因为正如我们在此所示，它能让人获得大量用于固定和活细胞分析的有丝分裂细胞样本。
