Osteo assay surface well

RAW 264.7 cells (1 × 105 cells/well) were seeded on Corning® Osteo Assay Surface well (Corning, NY, USA) followed by addition of different concentrations of fucoidan and RANKL (50 ng/mL) for 6 days. Then, cells were removed with 1 n NaOH for 20 min. The resorption pit areas were measured by Metamorph imaging analysis (Metamorph Imaging System, Universal Imaging Corp., Downingtown, PA, USA) and the area was expressed as the percentage of the area of RANKL-treated alone group.

RAW 264.7 cells (1 × 10³ cells/well) were cultured on a Corning osteo assay surface well (Corning, NY, USA). After 24 h of incubation, cells were refreshed with α-MEM medium containing 5% FBS and treated or untreated with RANKL (50 ng/mL) and the indicated AD. The cells were maintained for 7 days at 37 °C in a humidified incubator containing 5% CO₂. The medium was changed every day. Subsequently, the medium was removed, cells were washed twice with PBS. Cells were detached by 5% sodium hypochlorite for 5 min, washed with PBS, and dried. The surface of each well was visualized using a microscope (Olympus, Tokyo, Japan) and measured with an ImageJ program.

The area of bone resorption by osteoclasts was measured using osteo assay surface well (Corning, NY, USA). For bone resorption analysis, RAW264.7 cells were cultured in a 96-osteo assay surface well at a concentration of 2 × 10³ cell/well, and then simultaneously treated with 50 ng/mL of RANKL and LGS and ODE. After incubation for 7 days, the medium was removed and washed twice with phosphate-buffered saline (PBS), then treated with 5% sodium hypochlorite (Sigma-Aldrich, Saint Louis, MO, USA) for 5 min to remove cells, and the bone resorption area was photographed using an optical microscope. The bone resorption area of each well was measured by the Image J (National Institutes of Health, Massachusetts, MD, USA) program. Thereafter, the value was then percentage (%) of the only RANKL treatment group and compared with each of the resorbed area.