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Laszlo Radnai

Rebecca F. Stremel

Thomas Vaissiere

Li Lin

Michael Cameron

William H. Martin

Gavin Rumbaugh

Theodore M. Kamenecka

Patrick R. Griffin

Courtney Miller
The Scripps Research Institute
United States

Associate Professor

Department of Molecular Medicine

Department of Neuroscience

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Home > Vol 7, No 3 (2020) > Radnai

DOI: http://dx.doi.org/10.14440/jbm.2020.335

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A simple and robust cell-based assay for the discovery of novel cytokinesis inhibitors

Laszlo Radnai, Rebecca F. Stremel, Thomas Vaissiere, Li Lin, Michael Cameron, William H. Martin, Gavin Rumbaugh, Theodore M. Kamenecka, Patrick R. Griffin, Courtney Miller

Abstract

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 (Z`) of 0.65 indicates that the assay is suitable for screening purposes, as the positive and negative controls are well-separated. EC50 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 (R2 = 0.998) was found for EC50 values covering a ~1500-fold range of potencies. Established small molecule inhibitors of cytokinesis operating via direct action on actin dynamics or nonmuscle myosin II are used to demonstrate the robustness, simplicity and flexibility of the assay.

Graphical Abstract

graphical abstract

Keywords

drug discovery, actin cytoskeleton, screening, multinucleated, cytotoxicity
Citation: Radnai L, Stremel RF, Vaissiere T, Lin L, Cameron M, Martin WH, Rumbaugh G, Kamenecka TM, Griffin PR, Miller C. A simple and robust cell-based assay for the discovery of novel cytokinesis inhibitors. J Biol Methods 2020;7(3):e136. doi: 10.14440/jbm.2020.335


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