48 well culture plates

The wells of 48-well culture plates (Becton Dickinson Labware, Lincoln Park, NJ, USA) were filled with 500 µl of α-MEM (Gibco-BRL, Grand Island, NY, USA) supplemented with 50 µg/ml streptomycin and 50 U/ml penicillin (Penicillin–Streptomycin Solution [×100], Wako, Osaka, Japan) containing 10% fetal bovine serum (Biosera; Nuaillé, France) (CM). Then, D-WMTA or Na-WMTA were placed on the bottoms of wells (one disc per well). Wells filled with CM without WMTA discs (No WMTA) were prepared as controls. All plates were maintained at 37 °C in a humidified atmosphere of 5% CO₂ and 95% air. Media was collected on days 1, 7, 14, and 28 (n = 3 discs per time point). The amount of Ca²⁺ release was assessed with a QuantiChrom Calcium Assay Kit (Bio Assay Systems, Hayward, CA, USA), then measured in accordance with the manufacturer’s instructions using a microplate reader at an absorbance of 590 nm. The pH level was measured by Twin pH Meter II LQUA twin (Horiba Advanced Techno, Kyoto, Japan).

Suspension of bone marrow cells was prepared as described above. After counting, cells were plated at a density of 7.5×10⁵ cells/0.5 mL/well in 48-well culture plates (Falcon), in α-MEM with 1% penicillin and streptomicin and 10% dialyzed FBS, supplemented with 20 ng/mL of RANKL and M-CSF (R&D). Medium was changed at day 3, and at day 4, cells were rinsed in PBS and lysed in TRIreagent (Ambion) for RNA isolation. Differentiation into osteoclasts was confirmed by TRAP staining (Sigma-Aldrich) and counting under microscope.
In some experiments, osteoclasts were treated with 5HT (Sigma-Aldrich) in final concentration of 50 nM, insulin (Eli Lilly) in final concentration of 1 μM, LX1032, in final concentration of 5 nM, or their combination (n = 3 wells/treatment group). Substances were added from the beginning of the culture.

The NO production measurements were performed as described previously [8 (link),33 (link),35 (link)]. Briefly, BMMs (1 × 10⁶ cells/well, n = 4 per group) were seeded into 48-well culture plates (Falcon) to prepare the seven experimental groups (control (only BMMs), 0.1 μg/mL of LPS, 1 μg/mL of MP, and 0.5, 1, 10, and 100 μg/mL of TEGO). After 24 h, NO products accumulated in the supernatant were detected using a Griess reagent system (Promega, Madison, WI, USA). Briefly, the supernatant and sulfanilamide were mixed in equal amounts. Each group was measured at a wavelength of 548 nm using a microplate absorbance reader (Bio-Rad, Hercules, CA, USA). The NO concentration in the supernatant fluid was measured with a standard curve generated with sodium nitrite.