细胞周期是导致细胞复制的一系列事件。当在含血清的培养基中以低细胞密度铺板时,培养的细胞开始增殖,通过细胞周期的四个阶段:G1,S,有丝分裂是细胞周期最活跃的时期,涉及几乎所有细胞组件的重大重组。有丝分裂进一步分为前期,前中期,中期,后期,和发展,它们可以通过蛋白质标记和/或在荧光显微镜下比较它们的染色体形态来容易地彼此区分。通过这些有丝分裂亚期的细胞周期进程受到复杂分子机制的严格调控。细胞与有丝分裂亚期的同步对于理解这些分子机制很重要。这里,我们描述了一个将Hela细胞同步到前中期的协议,中期,和后期/末期。在这个协议中,Hela细胞首先通过双胸苷阻滞同步到早期S期。在释放块之后,用诺考达唑处理细胞,MG132和blebbistatin在前中期逮捕他们,中期,和后期/末期,分别。使用蛋白质印迹和荧光显微镜评估成功的同步。
The cell cycle is a series of events leading to cell replication. When plated at low cell densities in serum-containing medium, cultured cells start to proliferate, moving through the four phases of the cell cycle: G1, S, G2, and M. Mitosis is the most dynamic period of the cell cycle, involving a major reorganization of virtually all cell components. Mitosis is further divided into prophase, prometaphase, metaphase, anaphase, and
telophase, which can be easily distinguished from one another by protein markers and/or comparing their chromosome morphology under fluorescence microscope. The progression of the cell cycle through these mitotic subphases is tightly regulated by complicated molecular mechanisms. Synchronization of cells to the mitotic subphases is important for understanding these molecular mechanisms. Here, we describe a protocol to synchronize Hela cells to prometaphase, metaphase, and anaphase/
telophase. In this protocol, Hela cells are first synchronized to the early S phase by a double thymidine block. Following the release of the block, the cells are treated with nocodazole, MG132, and blebbistatin to arrest them at prometaphase, metaphase, and anaphase/
telophase, respectively. Successful synchronization is assessed using Western blot and fluorescence microscopy.