Live cell imaging and analysis

DIC microscopy was used to visualize mitotic duration and outcome. These experiments were performed on a Zeiss Cell Observer inverted wide field microscope, with 20 × 0.8 NA air objective, at 37 °C, 10% CO₂ and atmospheric oxygen. Images were captured every 6 min for a duration up to 65 h using an Axiocam 506 monochromatic camera (Zeiss) and Zen software (Zeiss). FUCCI live cell imaging was conducted on the same instrument, using the same imaging duration and protocol, with the addition of a Zeiss HXP 120C mercury short-arc lamp and compatible filter cubes to obtain fluorescent images. Quantitative live cell imaging of mitotic chromosome dynamics in HT1080 6TG H2B-mCherry cultures and derivatives was done using either combined DIC and fluorescent imaging on the Zeiss Cell Observer described above with a 40 × 0.95 NA plan-Apochromat air objective; or on a Zeiss Cell Observer SD spinning disk confocal microscope imaged using a 561 nm, 50 mW solid state excitation laser and appropriate filter sets with a 40×, 1.3 NA objective and an Evolve Delta camera (Photometrics). For these experiments, cells were cultured at 37 °C, 10% CO₂ and atmospheric oxygen and images captured every 3 min for 60 h. For all movies, mitotic duration was scored by eye and calculated from nuclear envelope breakdown until cytokinesis or mitotic death. FUCCI Movies were scored by eye, for G1 (red) and S/G2 (green) by colour, and for mitotic entry, duration and outcome by cell morphology. Chromosome dynamics in HT1080 6TG-mCherry H2B were scored by eye. All live cell imaging analysis was done using Zen software.