Orca er

Transfected cells were loaded with 5 μM Fura-2-AM (340/380 nm) or 5 μM Fluo4-AM (488 nm) to measure cytosolic Ca²⁺ and 5 μM X-Rhod1 AM (562 nm) to measure mitochondrial Ca²⁺ levels, in HBSS (Sigma). Dyes were incubated for 20 min and washed three times prior experiments. For all the experiments with Thapsigargin HBSS was removed prior the beginning of the recordings and replaced with a Ca²⁺ free saline. Fluorescence measurements were obtained either using a cooled charge-coupled device (CCD) camera (Hamamatsu, Orca ER) or 710 Zeiss Confocal system.

For fluorescence imaging, cells were imaged in 8-well chamber slides (ibidi) in Leibovitz L-15 media with a heated 37°C chamber or in DMEM (no phenol red) with a heated 37°C chamber in 5% CO₂. Images were taken every 4 minutes with either a 20x /0.4 NA air objective using a Zeiss Axio Observer 7 with a CMOS Orca flash 4.0 camera at 4x4 binning or a 40x/1.3 NA oil objective using a DV Elite system equipped with Photometrics CascadeII:1024 EMCCD camera at 4x4 binning. For brightfield imaging, cells were imaged in a 24-well plate in DMEM in a heated chamber (37°C and 5% CO₂) with a 10x/0.5 NA objective using a Hamamatsu ORCA-ER camera at 2x2 binning on a Zeiss Axiovert 200M, controlled by Micro-manager software (open source: https://micro-manager.org/) or with a 20x/0.4 NA air objective using a Zeiss Axio Observer 7 as detailed above. Mitotic exit was defined by cells flattening down in the presence of nocodazole and MPS1 inhibitor. In assays where both recombinant BUBR1 and KNL1 are expressed in cells, cells were selected for quantification based on high levels of Turq2 as an indication of successful transient transfection of Turq2-BUBR1 constructs into cells stably expressing YFP-tagged KNL1 constructs.

GFP and RFP epifluorescence in transgenic animals was visualized either on a Leica DMRA2 compound microscope, where the images were captured by a Hamamatsu Orca-ER camera using the OPENLAB software, or on a Zeiss LSM 710 confocal microscope. Subsequent image analysis was performed using ImageJ and Photoshop CC.