Anti rankl ab

OCLs and pre-OCLs induced from peripheral monocytes were stimulated using 1 ng ml⁻¹ lipopolysaccharide (LPS) (Sigma-Aldrich, St. Louis, MO, USA), 5 μg ml⁻¹ anti-RANKL Ab (R&D Systems) or 5 μg ml⁻¹ anti-Mouse IgG2B κ isotype control Ab (BD BioSciences, San Diego, CA, USA), with GolgiStop protein transport inhibitor (BD BioSciences), and incubated for 5 h at 37°C. Cells were then stained for 15 min at 4°C with fluorescent antibody RANK-Alexa Fluor (AF)-488 (Novus, Littleton, CO, USA). Next, cells were fixed for 30 min at 26°C using Cytofix/Cytoperm™ fixation kit (BD) and stained for 30 min at 4°C with fluorescent antibodies: IL-8-PE (BioLegend), PE Mouse IgG2b κ isotype control (BD BioSciences) and cathepsin K-PE (Abcam, Tokyo, Japan). Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich). Finally, cells were photographed using a BZ-X710-All-in-One Fluorescence Microscope (Keyence, Osaka, Japan).

CD14⁺ cells were cultured for 10 days in 96-well flat-bottom plates under the following conditions: (i) 50 ng ml⁻¹ M-CSF (BioLegend, San Diego, CA, USA) and 125 ng ml⁻¹ RANKL (BioLegend); (ii) 50 ng ml⁻¹ M-CSF and 5 µg ml⁻¹ anti-RANKL Ab (R&D Systems, Minneapolis, MN, USA) with or without 10 ng ml⁻¹ IL-8 (PeproTech, Rocky Hill, New Jersey, USA); and (iii) 50 ng ml⁻¹ M-CSF, 125 ng ml⁻¹ RANKL and 50 ng ml⁻¹ TNF-α (BioLegend) with or without 3 ng ml⁻¹ FK506. After culture, the number of OCLs per well was counted for each culture condition. Ten days after culture, cells were washed with phosphate-buffered saline (PBS), supplied with fresh media and cultured again overnight under the following conditions: (i) no medication, (ii) anti-RANKL Ab (5 µg ml⁻¹) and (iii) anti-RANKL Ab (5 µg ml⁻¹) and FK506 (3 ng ml⁻¹). Media were collected from overnight cultures and cryopreserved at −30°C.