Quantifying Bone Resorption Using Osteoclasts

For bone resorption assays, BMDMs or pre-osteoclasts were seeded and cultured on bovine cortical bone slices (DT-1BON1000-96; Immunodiagnostic Systems) with 20 ng/ml M-CSF and 30 ng/ml RANKL (R&D Systems; Wu et al., 2017 (link); Zhang et al., 2018 (link); Zhu et al., 2020 (link)), in the presence or absence of galectin-3 (8259-GA; R&D Systems), galectin-3C (10110-GA; R&D Systems), GCS-100 (La Jolla Pharmaceutical), RAP (4480-LR; R&D Systems), an anti–galectin-3 blocking antibody (sc-32790L; Santa Cruz), or an anti-Lrp1 blocking antibody (MA1-27198; Thermo Fisher Scientific; Chen et al., 2015 (link); Demotte et al., 2010 (link); John et al., 2003 (link); Moxon et al., 2015 (link); Seguin et al., 2017 (link)). After the indicated culture period, bone samples were sonicated in PBS, stained with 20 μg/ml WGA-lectin (L3892; Sigma-Aldrich) for 45 min and then incubated with DAB tablets (D4418; Sigma-Aldrich) for 15 min. Image J software was used to quantify the resorbed area. The concentration of the CTX-I was measured using the CrossLaps for Culture CTX-I ELISA kit (AC-07F1; Immunodiagnostic Systems) according to the manufacturer’s instructions.