Human igg1

The methods for the in vitro TRAP⁺ osteoclast assays were essentially as described.35 Bone marrow cells were obtained from two femurs of 8‐week‐old male C57BL/6 WT mice, processed and plated on a 96‐well plate at a density of 2 × 10⁴ cells per plate, in 200 μL per well of complete DMEM with 10% foetal calf serum and 50 ng mL⁻¹ of human recombinant CSF‐1 (R&D Systems, Minneapolis, MN, USA). After 48 h at 37°C, media were replenished with complete DMEM, 50 ng mL⁻¹ human recombinant CSF‐1 plus 200 ng mL⁻¹ of soluble mouse RANKL (Miltenyi Biotec, Auburn, CA, USA) and various concentrations in a serial dilution fashion of either anti‐RANKL mAb (IK22.5 rat IgG2a), anti‐RANKL/PD‐1 BsAb (human IgG1 D265A) or isotype control (human IgG1 Fc). Media and appropriate reagents were replenished every 48 h, and after 7 days, cells were in situ stained with TRAP and TRAP⁺ multinucleated (more than three nuclei) cells were enumerated under the pathology microscope.