For live-cell imaging, cells were seeded in 8-well plates (μ-Slide 8 well, Ibidi). Imaging of mCherry-Tubulin and GFP-Spindly was performed over 7 Z-slices separated by 200 nm every 5-15 seconds at 1x1 binning on an Andor CSU-W1 spinning disk (50 µm disk) with 100x 1.45 NA oil objective lens (Nikon). 488 and 561 nm laser were used for sample excitation and images were acquired using Andor iXon-888 EMCCD camera. Emission filters were 525nm-50 bandpass for GFP and 655nm-150 bandpass for mCherry. Cells were kept at 37°C and 5% CO2 using a cage incubator and Boldline temperature/CO2 controller (OKO-Lab).
For mitotic progression experiments, chromatin was visualized with siR-DNA (Spirochrome). Eight hours later after thymidine release, Cpd-5 was added and filming was immediately started, taking 8 z-slices separated by 2 μm every 4 minutes at 2x2 binning on a Nikon Ti-E motorized microscope equipped with a Zyla 4.2Mpx sCMOS camera (Andor) and 40x 1.3 NA objective lens (Nikon). Fluorescence excitation was done using Spectra X LED illumination system (Lumencor) and Chroma-ET filtersets. Cells were kept at 37°C and 5% CO2 using a cage incubator and Boldline temperature/CO2 controller (OKO-Lab). Maximal intensity projections were performed and scored for chromosome missegregations. Only cells showing all chromosomes aligned before Cpd-5 addition were selected for analysis.
For mitotic progression experiments, chromatin was visualized with siR-DNA (Spirochrome). Eight hours later after thymidine release, Cpd-5 was added and filming was immediately started, taking 8 z-slices separated by 2 μm every 4 minutes at 2x2 binning on a Nikon Ti-E motorized microscope equipped with a Zyla 4.2Mpx sCMOS camera (Andor) and 40x 1.3 NA objective lens (Nikon). Fluorescence excitation was done using Spectra X LED illumination system (Lumencor) and Chroma-ET filtersets. Cells were kept at 37°C and 5% CO2 using a cage incubator and Boldline temperature/CO2 controller (OKO-Lab). Maximal intensity projections were performed and scored for chromosome missegregations. Only cells showing all chromosomes aligned before Cpd-5 addition were selected for analysis.