Imaging Polycomb Bodies in Live Cells

To image Polycomb bodies in live cells, PCGF2-HaloTag SCC1^DEG cells were plated on a gelatinised 35 mm Petri dish, 14 mm Microwell 1.5 coverglass dishes (MatTek) at least 5 hours prior to imaging. Cells were labeled with 500 nm Halo-JF549 (Grimm et al., 2015 (link)) for 15 min at 37°C, followed by three washes, changing medium to Fluorobrite DMEM (Thermo Fisher Scientific) for imaging, which was supplemented as described for general ESC culture above. Cells were incubated for a further 30 min in supplemented Fluorobrite DMEM with 10 μg/mL Hoechst 33258 (Thermo Fisher Scientific) at 37°C and washed once more before imaging. Imaging was performed with an IX81 Olympus microscope connected to a Spinning Disk Confocal system (UltraView VoX PerkinElmer) using an EMCCD camera (ImagEM, Hamamatsu Photonics) in a 37°C heated, humidified, CO₂-controlled chamber. Z stacks were acquired using a PlanApo 100x/1.4 N.A. oil-immersion objective heated to 37°C, using Volocity software (PerkinElmer). PCGF2-HaloTag-JF549 was imaged with a 561 nm laser at 1.25 s exposure at 15% laser power, SCC1-AID-GFP with a 488 nm laser at 1 s exposure at 40% laser power, while Hoechst was imaged with a 405 nm laser at 250 ms exposure at 20% laser power. Z stacks were acquired at 150 nm intervals. A total of at least 28 cells were imaged per condition in two biological replicates.