Visualizing Polycomb Bodies in Live Cells

To image Polycomb bodies in live cells, HaloTag-SUZ12;PRC1^CPM or RING1B-HaloTag;PRC1^CPM cells were plated on gelatinised 35 mm Petri dish, 14 mm Microwell 1.5 coverglass dishes (MatTek, #P35G-1.5-14-C) at least 5 h before imaging. Prior to imaging, cells were labeled with 500 nm JF₅₄₉ (Grimm et al., 2017 (link)) for 15 min at 37°C, followed by 3 washes, changing medium to Fluorobrite DMEM (Thermo Fisher Scientific) 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. Cells were imaged on 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 100x PlanApo NA 1.40 oil-immersion objective heated to 37°C, using Volocity software (PerkinElmer). HaloTag-JF₅₄₉ was imaged with a 561 nm laser at 1.25 s exposure at 15% 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.