Crosslaps for culture ctx 1 elisa kit

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.

For bone resorption assays, BMDMs or preosteoclasts were seeded and cultured on bovine cortical bone slices (Immunodiagnostic Systems) in the presence of M‐CSF and RANKL, as described previously (Wu et al, 2017 (link); Zhang et al, 2018 (link); Zhu et al, 2020 (link)). After the indicated culture period, bone slices were then incubated in 0.5 N NaOH for 30 s, and the cells scraped off using a cotton swab. Bone samples were sonicated in PBS, stained with 20 μg/ml WGA‐lectin (Sigma‐Aldrich) for 45 min and then incubated with 3,3′‐diaminobenzidine (DAB) tablets (Sigma‐Aldrich) for 15 min. The area resorbed was determined using Image J software. To analyze bone resorption pit depth, three‐dimensional profiles of resorption pits were characterized using a reflective confocal laser scanning microscope (RCLSM; Leica SP8). Quantitative analysis of resorption pit depth was performed using Imaris software (Bitplane). The concentration of the CTX‐I was measured using the CrossLaps for Culture CTX‐I ELISA kit (Immunodiagnostic Systems) according to the manufacturer's instructions.