Rankl

To obtain osteoclast precursor cells, bone marrow mononuclear cells (BMMCs) were collected from tibias and femurs of C57BL/6 mice and were cultured in the wells of a 96-well plate (3.0 × 10⁵ cells/well) or 24-well plate (1.0 × 10⁶ cells/well) with a minimum essential medium-α (MEM-α) supplemented with 10% fetal bovine serum (FBS), 100 µg/mL streptomycin and 100 U/mL penicillin as well as 292 μg/mL L-glutamine and 50 ng/mL M-CSF (BioLegend) at 37 °C in humidified air with 5% CO₂ for 3 days. Subsequently, the OC precursors were differentiated to osteoclasts using 50 ng/mL RANKL (BioLegend, San Diego, CA, USA).

Bone marrow was collected from femurs and tibias of 8-week-old, male C3H/He mice (Japan SLC) and cultured for 3 days at a density of 5 × 10⁶ cells/100-mm dish in MEMα (FUJIFILM Wako, Osaka, Japan) supplemented with 10% FBS and 100 ng/mL of macrophage colony-stimulating factor (M-CSF; BioLegend, San Diego, CA, USA). To induce osteoclast precursor cells (OPCs), the cells were cultured for additional 3 days at 5 × 10⁵ cells/100-mm dish. For osteoclastogenesis, OPCs were cultured for 5 days at 1 × 10⁵ cells/12-well plate in MEMα with 10% FBS, 20 ng/mL M-CSF, and 50 ng/mL of receptor activator of NF-kappaB ligand (RANKL; Biolegend). To determine effects of SEVs, 1 × 10¹⁰ particles/mL or indicated doses of SEVs were added to the medium at the beginning of osteoclastogenesis. To determine the effects of miRNA, 60 pmol of miRNA mimic transfection control with Dy547 (miR-Ctrl) or hsa-miR-146a-5p mimic (Horizon Discovery, Cambridgeshire, United Kingdom) was transfected into OPCs using Lipofectamine RNAiMAX (Thermo Fisher Scientific) at the beginning of osteoclastogenesis. Cellular growth was evaluated using WST-8 assay reagent (Nacalai Tesque, Kyoto, Japan). TRAP staining was performed after 5 days of osteoclastogenesis by using an Acid Phosphatase Leukocyte Kit (Merck), and was evaluated using microscopy BZ-X710 and BZ-X Analyzer.

Osteoclastogenesis assay was performed with RAW 264.7 cells (1.5*10⁴ cells per well in a 24-well plate) that were incubated with 30 ng/ml RANKL (Peprotech, Israel) for 5 days. RANKL-treated cells were also treated with Zol and/or Dex, recombinant mouse IFN-β (0.25 or 2.5 ng/ml, Biolegend), or anti-IFN-β antibodies (2 μg/ml, Abcam, United Kingdom) for the first 2 days of incubation and then washed. RANKL was supplemented after washing.

Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation (density: 1.077 g/mL; PanBiotech, Aidenbach, Germany) and washed twice with DPBS (Gibco).
Cells were then resuspended in alphaMEM Glutamax (Gibco, Life technologies, Darmstadt, Germany) supplemented with 10% FCS, 100 U/mL penicillin (Life technologies, Darmstadt, Germany), 10 μg/mL streptomycin (Life technologies), and 25 ng/mL MCSF (BioLegend, Amsterdam The Netherlands). The cells were seeded at specific concentrations, depending on the culture plate used (1.5 × 10⁶ cells/well in 6 well plates, 0.15 × 10⁶ cells/well in 48 well plates, and 0.05 × 10⁶ cells/well in 96 well plates) and cultured at 37 °C, 5% CO₂. After 24 h, the medium was replaced with a differentiation medium containing alphaMEM Glutamax (Gibco, Life technologies, Darmstadt, Germany) supplemented with 10% FCS, 100 U/mL penicillin (Life technologies), 10 μg/mL streptomycin (Life technologies, Darmstadt, Germany), 25 ng/mL MCSF, and 50 ng/mL RANKL (BioLegend, Amsterdam The Netherlands). As an osteoclast differentiation control, cells were cultured without RANKL. The cells were cultured for 14 days, and the medium was replaced twice a week.