Wga lectin

Osteoclast formation was induced as previously described (Cai et al., 2020 ; Zhao et al., 2020 ). Briefly, BM, spleen, or CD11b⁺Ly6C^hiLy6G⁺ cells were seeded in 24-well plates at a density of 1–2×10⁵ cells/well and cultured in α−10 medium (α-MEM, 10% FCS, 1x PenStrep) in the presence of RANKL (100 ng/ml) and 10% M-CSF for 5–7 days. Cells were then fixed and stained for TRAP activity. TRAP⁺ multinucleated cells (MNCs) were counted as mature osteoclasts. Osteoclast function were assessed by F-actin ring formation and in vitro bone resorptive ability. For F-actin ring staining, differentiated cells were fixed, permeabilized, and then staining with rhodamine phalloidin (Molecular Probes, Eugene, OR). Images were obtained with a fluorescence microscope with the Leica Texas Red filter (Nikon Eclipse TE2000-E, Japan). For in vitro bone resorption assay, MDSCs were inoculated onto bovine cortical bone slices plated in 24-well culture plates, and osteoclast differentiation was induced as described above. Bone slices were then sonicated in PBS, and soaked in 0.3% H₂O₂ for 30 min. Bone resorption pits were visualized by wheat germ agglutinin (WGA)-lectin (Sigma-Aldrich) staining. The percentage of bone resorption area on bone slices was analyzed using ImageJ analysis software (NIH). All experiments were done in triplicate.

Bone resorption activity was assessed as described (29 (link)–31 (link)). For scanning electron microscopy analysis, a consistent number of MBM cells were cultured on bovine cortical bone slices in 24-well plates stimulated by RANKL/M-CSF for 48 hours. 0.25 M ammonium hydroxide and mechanical agitation were utilized to remove cells adhering to the bone slices when the bone slices were harvested on day 6. Scanning electron microscopy (SEM) of bone slices was done using a Philips 515 SEM (Department of Materials Science and Engineering, UAB). Bone resorption pits were analyzed by WGA stain as described (32 (link)) with peroxidase-conjugated WGA-lectin (Sigma-Aldrich, L-3892) and DAB Peroxidase (HRP) Substrate Kit (Vector laboratories, SK-4100). The assays were performed in triplicate and presented as a representative observing area from each assay. Data quantification was achieved by measuring the percentage of the areas resorbed in three random resorption sites using ImageJ software from the National Institute of Health.