The impact of anti-CD38 Ab on OC differentiation from CD14-purified monocytes ex vivo. (A) CD38 was examined by immunoblotting in indicated cells. (B) CD38 expression was examined by flow cytometry during osteoclastogenesis. (C) Purified CD14⁺ monocytes were cultured with RANKL and M-CSF in 10% FBS RPMI 1640 medium for 7 days, followed by the addition of anti-CD38 mAb (SAR, 1, 10, 100 μg/mL) into the medium for an additional 7 days. At day 14, CD38 expression on OCs was examined by flow cytometry and immunoblotting. (D-E) CD14⁺ monocytes were cultured with RANKL/M-CSF in 10% RPMI medium for 7 days, and then SAR (0.1, 1 μg/mL) or IFN-γ (20 IU/mL) were added for an additional 7 days followed by TRAP staining to determine TRAP⁺ MNC ( >3 nuclei). (F-G) After CD14⁺ monocytes were stimulated with RANKL and M-CSF for 14 days, SAR, activated autologous T cells (Tact), and IDO inhibitor were added. After 3 days, cytotoxicity was evaluated by measuring LDH activity in supernatants. OCs were stained with Annexin V/PI and analyzed by flow cytometry. MNC, multinucleated cells; PI, propidium iodide.