Mouse bone marrow cells were plated at densities of 1.2×105 and 1.0×106 cells into 12-well and 60-mm dishes, respectively, and cultured with 10 ng/ml macrophage colony-stimulating factor (M-CSF; PeproTech, Inc., Rocky Hill, NJ, USA) at 37°C for 3 days. The surface-attached cells were used as osteoclast precursors (22 (link)). These cells were cultured with 10 ng/ml M-CSF and 50 ng/ml RANKL (PeproTech, Inc.). A total of 5–20 µM guanabenz (R&D Systems, Inc., Minneapolis, MN, USA) or 10–20 µM xylazine (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was applied at the same time point as RANKL; 10–20 µM clonidine (Sigma-Aldrich; Merck KGaA) was administered with RANKL or 1 day after RANKL administration. After a 60-h treatment with RANKL at 37°C, cells were fixed in 10% formalin neutral buffer solution at room temperature and stained with TRAP for 1 h at 37°C. The number of TRAP-positive cells containing three or more nuclei was determined. All positive cells in each well were counted using a light microscope (magnification, ×100; Zeiss AG, Oberkochen, Germany).
RAW264.7 cells were plated at 1.0×105 cells into 60-mm dishes and cultured with 25 ng/ml RANKL in the presence or absence of 5–20 µM guanabenz, 10–20 µM clonidine or 10–20 µM xylazine with or without 10–20 µM yohimbine or 10–20 µM idazoxan (Sigma-Aldrich; Merck KGaA) at 37°C for 2–4 days for qPCR analysis.
RAW264.7 cells were plated at 1.0×105 cells into 60-mm dishes and cultured with 25 ng/ml RANKL in the presence or absence of 5–20 µM guanabenz, 10–20 µM clonidine or 10–20 µM xylazine with or without 10–20 µM yohimbine or 10–20 µM idazoxan (Sigma-Aldrich; Merck KGaA) at 37°C for 2–4 days for qPCR analysis.
