Stage top humidified incubation chamber

Cell lines used have CRISPR/spCas9 and sgRNA stably integrated, but spCas9 expression is only induced upon doxycycline addition before each experiment, allowing for genetic manipulation of essential mitotic genes. For imaging, cells were plated on 35 mm #1.5 coverslip glass-bottomed dishes coated with poly-D-lysine (MatTek) and imaged in a stage-top humidified incubation chamber (Tokai Hit, Fujinomiya-shi, Japan) maintained at 30 °C and 5% CO₂. To label mitochondria, 25 nM MitoTracker Red (Sigma) was added to cell media for 30 min and washed out before imaging. For some experiments, 100 nM siR-tubulin dye (Cytoskeleton, Inc.) was added 2 h prior to imaging to label microtubules, along with 10 μM verapamil (Cytoskeleton, Inc.). Under these conditions, there was no detected defect in spindle appearance or microtubule dynamics. As described in [21 (link)], cells were imaged using an Eclipse Ti-E inverted microscope (Nikon Instruments) with a Yokogawa CSU-X1 spinning disk confocal operated by MetaMorph (7.7.8.0; Molecular Devices), with either a 100X 1.45 Ph3 oil objective (Figure 3), a 60X 1.4 Ph3 oil objective (Figures 1–2), or a 20X 0.5 Ph1 air objective (Figure 4), and with an Andor iXon3 camera.