Preosteoclasts, prepared like the BMDMs, were plated in 96-well plates (7,000 cells per well) in standard medium supplemented with 20 ng/ml M-CSF and 50 ng/ml Receptor Activator of Nuclear Factor Kappa-B Ligand (RANKL) (R&D Systems, Minneapolis, MN, USA). On the 3rd day (after 48 h), the medium was replaced by the CM of BMDM, supplemented with RANKL and M-CSF. Where indicated, we also added 2 μg/ml of neutralizing antibodies (Ab) against IL1β (Kineret, anakinra, SOBI, Stockholm, Sweden), IL6-receptor (Actemra, tocilizumab, Roche, San Francisco, CA, USA), or TNFα (Humira, adalimumab, AbbVie Inc., Chicago, IL, USA). These neutralizing Ab, effective in both humans and mice (15 (link)–19 (link)), are referred to as anti-IL1β, IL6, and TNFα Ab, respectively. On the 4th day, cells were stained using a TRAP kit (Sigma-Aldrich, St. Louis, MO, USA), and multinucleated (>3 nuclei) TRAP-positive cells were defined as osteoclasts. Images were acquired at an original magnification of × 4 (Evos FLC, Life Technologies, MS, USA). The number of osteoclasts and the total osteoclast area were measured using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
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