Cy sc007

For live imaging, cells were plated in Willco Wells 35 mm dishes (Willco Wells HBST-3522), ibidi 2 well imaging chambers (ibidi 80287) with DIC lid (ibidi 80055); or ibidi 8 well imaging chambers (ibidi 80827). Samples were imaged in a CO2-, temperature-, and humidity-controlled Tokai Hit Stage Top Incubator. Objectives were also heated to 37°C. For CO2-controlled imaging, the imaging media used was Fluorobrite DMEM (Gibco A1896701) supplemented with 10% FBS. U2OS GFP-Sec61β/H2B-mCherry mitotic cells were imaged using a custom aluminum stage insert heated to 37°C with heating tape and temperature monitored using a Physitemp thermistor (BAT7001H) and probe (IT-18), with objective heating and using 140 mM NaCl, 2.5 mM KC, 1.8 mM CaCl₂, 1.0 mM MgCl₂, 20 mM HEPES, 15 mM glucose, pH 7.4 as the live cell imaging solution. When indicated, cells were treated with 1 μM SiR-DNA (Cytoskeleton, Inc. CY-SC007) for 1 hour prior to imaging and kept in SiR-DNA-containing live imaging media during imaging. When indicated, cells were treated with 1 μM ER Tracker Green (Invitrogen E34251) for 30 min and washed out prior to imaging, and cells were imaged for a maximum of 2 hours after treatment.