Live-cell imaging of mitosis and migration

Cells in complete DMEM minus phenol red were plated in Ibidi µ-slide eight-chamber glass-bottom culture slides precoated with poly-d-lysine (5 µg/cm²). Cells were imaged by phase contrast at intervals of 5 min for 16 h on a Leica DMi8 inverted microscope set on a 63×/1.4 magnification oil objective using a Leica DFC9000 GT digital camera and LASX software. 10 regions of the culture chamber were imaged and analyzed per cell line, with >45 cells analyzed for all lines. Normal mitotic events were defined by cells balling up and then splitting into two daughter cells, whereas abnormal mitotic events were determined by the following criteria: multipolar mitosis (cell splits or attempts to split into >2 cells); mitotic slippage and failed cytokinesis (cell balls up but then flattens without splitting into daughter cells, or attempts to split but then fails), bi/multinucleation (cells undergo mitosis and daughter cells have more than one nucleus), or bi/multinucleate recovery (cells with more than one nucleus ball up and split into two or more cells with daughter cells having one or more nuclei).