Csr bra 48kit

The bone resorption assay kit (CSR-BRA-48KIT; COSMOBIO, Tokyo, Japan) was used to check resorption activity of osteoclast cells according to the manufacturer’s protocol. Briefly, Raw264.7 cells (5 × 10³ cells/well) were seeded into calcium phosphate (CaP)-coated 48-well culture plate. Additional collagen type I coating was performed using 50 μg/mL collagen type I (3447-020-01; R&D System, Minneapolis, MN, USA) to mimic a bone biomimetic surface. Raw264.7 cells were cultured at 37 °C and 5% CO₂ in DMEM containing 10% FBS. After 24 h, the medium was changed to complete α-MEM medium without phenol-red and then pretreated with a vehicle (DMSO) and citreoviridin (0.5 and 1μg/mL) for 1 h. Subsequently, RANKL (100 ng/mL) was added in the medium to induce an osteoclast differentiation. RANKL and citreoviridin were re-treated every 2 days for 6 days. On day 6, the cells were washed with PBS and treated with 5% sodium hypochlorite for 5 min to remove the cells. Then, the plate was washed with D.W. and dried. Osteoclast resorbing areas were captured using a digital camera (Olympus) attached to microscope (Olympus) and the pit areas obtained from 20 different regions by 4 independent experiments were measured by NIH ImageJ 1.52a software.

The bone resorption assay was conducted using a bone resorption assay kit (CSR-BRA-48KIT; COSMOBIO, Tokyo, Japan), following the manufacturer’s instructions with slight modifications. For this assay, Raw264.7 cells (4 × 10³ cells/well) were seeded onto a calcium phosphate (CaP) and collagen-type I-coated 48-well culture plate. A 50 μg/mL coating of collagen type I (3447-020-01; R&D System, Minneapolis, MN, USA) was applied to create a bone biomimetic surface. The cells were then cultured at 37 °C with 5% CO₂ in DMEM supplemented with 10% FBS. After 24 h, the cells were pre-treated with DMSO (vehicle) and varying concentrations of EN (10 μg/mL and 20 μg/mL) in α-MEM medium for 1 h. RANKL (100 ng/mL) was added to the medium to induce osteoclast differentiation over a period of 7 days. At the end of the culture period, cells were rinsed with PBS and treated with 5% sodium hypochlorite to detach the cells. Following this, the plate was washed with distilled water (D.W.) and allowed to dry. The area of bone resorption was imaged using a digital camera (Olympus) attached to a microscope (Olympus). The extent of the resorptive pits was quantified using NIH ImageJ 1.52a software.