Texas red filter

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.