Time-lapse Imaging of Osteoclast Resorption

Time-lapse recordings were done as described previously^{27 (link)}. Cortical bovine bone slices (0.4 mm; BoneSlices.com, Jelling, Denmark) were labeled with rhodamine fluorescent dye (ThermoFisher) as previously described^{27 (link)}. Mature OCs were detached with accutase (Biowest BW, France), harvested by centrifugation (500 g for 5 min), and resuspended in αMEM containing 10% FCS, 25 ng/ml M-CSF, and 25 ng/ml RANKL. For control and lower inhibitor concentrations cells from 3 to 5 donors were used and for the highest inhibitor concentrations (T06 1 µM and ODN 50 nM) 2 donors were used. For each condition and for each donor 3–5 bone slices were used and cells were seeded at a density of 100,000 cells per bone slice in a 96-well plate. In order to label F-actin in living OCs, 100 nM SiR-actin (excitation at 652 nm; emission at 674 nm) and 10 μM verapamil (both supplied by Spirochrome, Stein am Rhein, Switzerland) were added and incubated for 5 h at 37 °C in 5% CO₂ in a humidified chamber. Inhibitors (ODN and tanshinone IIA sulfonate (T06), which block CatK activity, were added to the plates. Subsequently, bone slices were transferred to Nunc Lab-Tek™ II Chambered Coverglass (ThermoFisher Scientific) wells in medium containing M-CSF, RANKL, SiRactin, and verapamil (with or without inhibitors) as described above. Time-lapse images were made using an Olympus Fluoview FV10i microscope (Olympus Corporation, Shinjuku, Tokyo, Japan) at 5% CO₂ and 37 °C, with a 10 × objective lens with a confocal aperture of 2.0 corresponding to a z-plane depth of 20.2 μm. The initial focus was set to the bone surface. Recordings were made for a period of 72 h taking images every 7 or 21 min (at least 3 recording areas per bone slice). Neither SiR-actin nor verapamil affected the extent of resorption. We analyzed between 50 and 150 OCs per condition on 3–5 bone slices per donor-derived OCs.