Liu Yongqiang, Nan Bei, Niu Junhua, Kapler Geoffrey M, Gao Shan
Institute of Evolution and Marine Biodiversity, Ocean University of China, Qingdao, China.
Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao, China.
Front Cell Dev Biol. 2021 Apr 20;9:664418. doi: 10.3389/fcell.2021.664418. eCollection 2021.
Cell synchronization is a powerful tool to understand cell cycle events and its regulatory mechanisms. Counter-flow centrifugal elutriation (CCE) is a more generally desirable method to synchronize cells because it does not significantly alter cell behavior and/or cell cycle progression, however, adjusting specific parameters in a cell type/equipment-dependent manner can be challenging. In this paper, we used the unicellular eukaryotic model organism, as a testing system for optimizing CCE workflow. Firstly, flow cytometry conditions were identified that reduced nuclei adhesion and improved the assessment of cell cycle stage. We then systematically examined how to achieve the optimal conditions for three critical factors affecting the outcome of CCE, including loading flow rate, collection flow rate and collection volume. Using our optimized workflow, we obtained a large population of highly synchronous G1-phase as measured by 5-ethynyl-2'-deoxyuridine (EdU) incorporation into nascent DNA strands, bulk DNA content changes by flow cytometry, and cell cycle progression by light microscopy. This detailed protocol can be easily adapted to synchronize other eukaryotic cells.
细胞同步化是了解细胞周期事件及其调控机制的有力工具。逆流离心淘析（CCE）是一种更普遍适用的细胞同步化方法，因为它不会显著改变细胞行为和/或细胞周期进程，然而，以依赖细胞类型/设备的方式调整特定参数可能具有挑战性。在本文中，我们使用单细胞真核模式生物作为优化CCE工作流程的测试系统。首先，确定了流式细胞术条件，该条件可减少细胞核黏附并改善细胞周期阶段的评估。然后，我们系统地研究了如何为影响CCE结果的三个关键因素实现最佳条件，包括加载流速、收集流速和收集体积。使用我们优化的工作流程，通过将5-乙炔基-2'-脱氧尿苷（EdU）掺入新生DNA链、流式细胞术检测的总体DNA含量变化以及光学显微镜观察的细胞周期进程，我们获得了大量高度同步的G1期细胞。这个详细的方案可以很容易地适用于同步其他真核细胞。
