Elisa plate reader

We seeded 1 × 10⁴ cells into each well of 96-well black-bottom plates and incubated them with PMA for 24 h. Then, the cells were pretreated with the indicated concentrations of atorvastatin, simvastatin, or mevastatin for 24 h and stimulated with MSU crystals (0.3 mg/mL) for 24 h. After MSU treatment, the medium was removed and incubated with 10 μM 2′,7′-dichlorofluorescein diacetate (H₂DCF-DA, Thermo, Rockford, IL, USA) for 20 min at 37 °C in the dark. The cells were washed with phosphate-buffered saline, and ROS levels were measured at an excitation wavelength of 485 nm and emission wavelength of 535 nm using an ELISA plate reader (BMG Lab Technologies, Offenburg, Germany).

Bone resorption activity was evaluated using a bone resorption assay kit (COSMO Bio, Tokyo, Japan). Briefly, cells seeded on calcium phosphate-coated plates (1 × 10⁴ cells/well) were cultured with sRANKL (100 ng/mL) and MSU crystals (0.1 mg/mL) for 6 days and further treated with diverse concentrations of CKD-WID (0.5, 1, and 3 µM) for 4 days. Culture supernatants were placed into a black flat-bottom 96-well microplate, and resorption assay buffer was added. ELISA plate reader (BMG Lab Technologies, Offenburg, Germany) with 485 nm excitation and 520 nm emission was applied to measure the fluorescence intensity.
To examine bone resorption, the medium was removed from wells, and cells were added with 5% sodium hypochlorite for 5 min. After washing twice with PBS, wells were dried, and the resorption pits were photographed using a microscope (Olympus, Tokyo, Japan).

2 x 10⁴ viable hippocampal cells per well were plated onto a Poly-L-Lysine (Sigma Aldrich, Deisenhofen, Germany) coated 96-well plate and incubated with different CORT concentrations (0.1 μM, 1 μM, 10 μM, 100 μM, 1000 μM) (Sigma Aldrich, Deisenhofen, Germany) or vehicle for 24 h at 37°C/ 5% CO₂. CORT was first dissolved in Ethanol (EtOH, 60%) and then further diluted to the appropriate concentrations with saline. Vehicle consisted of saline containing comparable amounts of EtOH as used in the respective CORT stimulation condition. The cell viability of the hippocampal cells was determined 24 h later with a commercially available colorimetric assay (Cell Titer AQ_ueous One Solution Cell Proliferation Assay Promega, Mannheim, Germany), by measuring the absorbance at a wavelength of 485 nm by using an ELISA plate reader (BMG Labtech GmbH, Ortenberg, Germany). To account for differences in background activity, the absorbance of wells containing only medium for a given treatment was subtracted from the corresponding wells containing cells.