Assessing Cell Fusion and Cytokinesis Failure as Mechanisms of Clone 9 Hepatocyte Multinucleation In Vitro

In this in vitro model of hepatocyte multinucleation, separate cultures of rat Clone 9 cells are labeled with either red or green cell tracker dyes (Red Cell Tracker CMPTX or Vybrant CFDA SE Cell Tracer), plated together in mixed‐color colonies, and treated with positive or negative control agents for 4 days. The fluorescent dyes become cell‐impermeant after entering cells and are not transferred to adjacent cells in a population, but are inherited by daughter cells after fusion. The mixed‐color cultures are then evaluated microscopically for multinucleation and analysis of the underlying mechanism (cell fusion/cytokinesis). Multinucleated cells containing only one dye have undergone cytokinesis failure, whereas dual‐labeled multinucleated cells have resulted from fusion. Curr. Protoc. Toxicol. 53:14.9.1‐14.9.17. © 2012 by John Wiley & Sons, Inc.