Radnai Laszlo, Stremel Rebecca F, Vaissiere Thomas, Lin Li, Cameron Michael, Martin William H, Rumbaugh Gavin, Kamenecka Theodore M, Griffin Patrick R, Miller Courtney A
Department of Molecular Medicine, The Scripps Research Institute, Jupiter, FL 33458, USA.
Department of Neuroscience, The Scripps Research Institute, Jupiter, FL 33458, USA.
J Biol Methods. 2020 Sep 17;7(3):e136. doi: 10.14440/jbm.2020.335. eCollection 2020.
Cytokinesis is the last step of mitotic cell division that separates the cytoplasm of dividing cells. Small molecule inhibitors targeting either the elements of the regulatory pathways controlling cytokinesis, or the terminal effectors have been of interest as potential drug candidates for the treatment of various diseases. Here we present a detailed protocol for a cell-based cytokinesis assay that can be used for the discovery of novel cytokinesis inhibitors. The assay is performed in a 96-well plate format in 48 h. Living cells, nuclei and nuclei of dead cells are identified by a single staining step using three fluorescent dyes, followed by rapid live cell imaging. The primary signal is the nuclei-to-cell ratio (NCR). In the presence of cytokinesis inhibitors, this ratio increases over time, as the ratio of multinucleated cells increases in the population. The ratio of dead nuclei to total nuclei provides a simultaneous measure of cytotoxicity. A screening window coefficient (`) of 0.65 indicates that the assay is suitable for screening purposes, as the positive and negative controls are well-separated. EC values can be reliably determined in a single 96-well plate by using only six different compound concentrations, enabling the testing of 4 compounds per plate. An excellent test-retest reliability ( = 0.998) was found for EC values covering a ~1500-fold range of potencies. Established small molecule inhibitors of cytokinesis operating direct action on actin dynamics or nonmuscle myosin II are used to demonstrate the robustness, simplicity and flexibility of the assay.
胞质分裂是有丝分裂细胞分裂的最后一步，它将正在分裂的细胞的细胞质分开。靶向控制胞质分裂的调控途径元件或终末效应器的小分子抑制剂作为治疗各种疾病的潜在候选药物受到关注。在此，我们提供了一种基于细胞的胞质分裂检测的详细方案，该方案可用于发现新型胞质分裂抑制剂。该检测在96孔板中进行，为期48小时。使用三种荧光染料通过单一染色步骤识别活细胞、细胞核和死细胞的细胞核，随后进行快速活细胞成像。主要信号是核与细胞比率（NCR）。在存在胞质分裂抑制剂的情况下，随着群体中多核细胞的比率增加，该比率会随时间增加。死细胞核与总细胞核的比率可同时衡量细胞毒性。筛选窗口系数（`）为0.65表明该检测适用于筛选目的，因为阳性和阴性对照区分良好。通过仅使用六种不同的化合物浓度，就可以在单个96孔板中可靠地确定EC值，从而每板能够测试4种化合物。对于涵盖约1500倍效力范围的EC值，发现了出色的重测可靠性（ = 0.998）。已建立的对肌动蛋白动力学或非肌肉肌球蛋白II具有直接作用的小分子胞质分裂抑制剂用于证明该检测的稳健性、简单性和灵活性。
