Dulla Kalyan, Santamaria Anna
Department of Molecular Diagnostics, Philips Research, Eindhoven, The Netherlands.
Methods Mol Biol. 2011;761:65-74. doi: 10.1007/978-1-61779-182-6_4.
Understanding cell growth and cell division involves the study of regulatory events that occur in a cell cycle phase-dependent manner. Studies analyzing cell cycle regulatory mechanisms and cell cycle progression invariably require synchronization of cell populations at specific cell cycle stages. Several methods have been established to synchronize cells, including serum deprivation, contact inhibition, centrifugal elutriation, and drug-dependent synchronization. Despite potential adverse cellular consequences of synchronizing cells by pharmacological agents, drug-dependent methods can be advantageous when studying later cell cycle events to ensure specific enrichment at selected mitotic stages. This chapter describes protocols used in our laboratory for isolating mitotic mammalian cells in a large-scale manner. In particular, we discuss the technical aspects of adherent or suspension cell isolation, the methods necessary to enrich cells at different mitotic stages and the optimized culture conditions.
了解细胞生长和细胞分裂涉及对以细胞周期阶段依赖性方式发生的调控事件的研究。分析细胞周期调控机制和细胞周期进程的研究总是需要将细胞群体同步于特定的细胞周期阶段。已经建立了几种使细胞同步化的方法，包括血清饥饿、接触抑制、离心淘洗和药物依赖性同步化。尽管通过药物试剂使细胞同步化可能会对细胞产生潜在的不良后果，但在研究后期细胞周期事件时，药物依赖性方法可能具有优势，以确保在选定的有丝分裂阶段进行特异性富集。本章描述了我们实验室用于大规模分离有丝分裂哺乳动物细胞的方案。特别是，我们讨论了贴壁或悬浮细胞分离的技术方面、在不同有丝分裂阶段富集细胞所需的方法以及优化的培养条件。
