2.5. In vitro osteoclast differentiation

One × 10⁵ BM derived MDSCs were seed into 96-well plates and cultured in α-minimum essential medium (αMEM) supplemented with 10% FBS, 25 ng/mL recombinant murine macrophage-colony stimulating factor (M-CSF) (R&D Systems, UK) and 50 ng/mL receptor activator of NF-κB ligand (RANKL) (R&D Systems, Minneapolis, MN, USA), in the presence of increasing concentration of recombinant EDIL3 (R&D systems, Abingdon, UK). Media was replaced every 3 days and cells were maintained for up to 15 days for differentiation. Tartrate-resistant acid phosphatase (TRAP) staining was performed using TRAP kit (Sigma-Aldrich, St Louis, Missouri, USA) followed the manufacturer's procedure. TRAP positive cells were counted in each well (cells containing more than 3 nuclei were considered as TRAP positive cells). And Nine sections were counted in each separate experiment. Slides were viewed using a Nikon Eclipse Ni-E microscope. For some assays, MDSCs were pre-treated with antibody against CD11b or CD11a (Biolegend, San Diego, CA) or isotype control (Biolegend, San Diego, CA) (10 µg/ml).