Diermeier-Daucher Simone, Brockhoff Gero
Department of Gynaecology and Obstetrics, University of Regensburg, Regensburg, Germany.
Curr Protoc Cell Biol. 2010 Sep;Chapter 8:Unit 8.6.1-23. doi: 10.1002/0471143030.cb0806s48.
Dynamic proliferation assessment via flow cytometry is legitimately supposed to be the most powerful tool for recording cell cycle kinetics in-vitro. The preeminent feature is a single cell-based multi-informative analysis by temporal high-resolution. Flow cytometric approaches are based on labeling of proliferating cells via thymidine substitution by a base analog (e.g., 5-bromo-2'-deoxyuridine, BrdU) that is added to cell cultures either for a short period of time (pulse labeling) or continuously until cell harvesting. This unit describes the alternative use of the thymidine analog 5-ethynyl-2'-deoxyuridine (EdU) in place of BrdU for three different applications: (1) dynamic proliferation assessment by EdU pulse cell labeling; (2) the same approach as (1) but in combination with live/dead cell discrimination; and (3) dynamic cell cycle analysis based on continuous cell labeling with EdU and Hoechst fluorochrome quenching. In contrast to the detection of BrdU incorporation, EdU-positive cells can be identified by taking advantage of click chemistry, which facilitates a simplified and fast cell preparation. Further analysis options but also limitations of the utilization of EdU are discussed.
通过流式细胞术进行动态增殖评估理应是体外记录细胞周期动力学的最强大工具。其卓越特性是基于单个细胞的高分辨率多信息分析。流式细胞术方法基于通过碱基类似物（如5-溴-2'-脱氧尿苷，BrdU）替代胸苷对增殖细胞进行标记，该碱基类似物可在短时间内添加到细胞培养物中（脉冲标记），或持续添加直至收获细胞。本单元描述了胸腺嘧啶类似物5-乙炔基-2'-脱氧尿苷（EdU）替代BrdU的三种不同应用：（1）通过EdU脉冲细胞标记进行动态增殖评估；（2）与（1）相同的方法，但结合活/死细胞鉴别；（3）基于用EdU连续细胞标记和Hoechst荧光淬灭的动态细胞周期分析。与检测BrdU掺入不同，EdU阳性细胞可利用点击化学进行鉴定，这有助于简化和快速的细胞制备。还讨论了EdU利用的进一步分析选项及局限性。
