Bone resorption assay kit csr bra 48kit

The bone resorbing activity of the osteoclast cells was measured by Bone resorption assay kit (CSR-BRA-48KIT; COSMOBIO, Tokyo, Japan). Raw264.7 cells were seeded onto calcium phosphate (CaP)-coated 48-well culture plate at density of 5 × 10³ cells per wells. Raw264.7 cells were cultured at 37 °C and 5% CO₂ in DMEM containing 10% FBS. Subsequently, DMSO, ILG, and ILG derivatives (5 and 10 μM/mL) were added into each well for 1 h before RANKL (100 ng/mL) treatment as an inducer of osteoclast differentiation in phenol red-free α-MEM containing 10% FBS. All reagents were retreated every 2 days for 6 days. On day 6, the conditioned medium was discarded and 5% sodium hypochlorite was treated into each well for 5 min to remove the cells. After the plate was washed with D.W. and dried. The pit area, resorbed area by osteoclast cell, was captured using a digital camera (Olympus) attached to microscope (Olympus). The pit areas were measured by NIH ImageJ 1.52a software.

The assay was performed in accordance with a typical assay procedure of Bone Resorption Assay Kit (CSR-BRA-48KIT, Cosmo Bio Co., LTD). The kit contains 48-well plates pre-coated with carbonate apatite (CaP). Prior to cell seeding, each well in the plate was coated with fluoresceinamine-labelled chondroitin sulphate (FACS) for 2 h. RAW264.7 cells (1 × 10⁴ cells per well) were used for the differentiation process with RANKL (30 ng/ml), TGF-β1 (10 ng/ml) and/or inhibitors. Plates were incubated at a humidity of 5% CO₂ for 5 days at 37°C. On day 5, the conditioned medium (100 μl) was moved into a 96-well black plates. For each well, the bone resorption assay buffer (50 μl) was later added. A plate shaker was used for mixing. The fluorescence was measured according to the previously set parameters. The media were aspirated on day 5 to analyze pit formation. 5% sodium hypochlorite (100 μl) was added for 5 min. The wells dried at room temperature from 3 to 5 h after washing with distilled water. The pit areas were visualized under bright-field using a microscope. The Image J software analyzed the images. A microplate reader (Tecan Spark, Switzerland) was used to read the fluorescence signal produced.