2.5. TRAP Assay and Measurements

BMMs were seeded at 1.2 × 10⁵ cells on 48-well culture plates and incubated with an osteoclast differentiation culture medium containing GNPs or SNPs. The negative control group (NC) was incubated without RANKL, and osteoclast differentiation was induced in the positive control group (PC) by M-CSF and RANKL. The osteoclasts were fixed by soaking in 4% paraformaldehyde for 15 min and washing three times with DPBS. The cells were placed in 0.1% Triton X-100 for 10 min at room temperature and washed three times with DPBS. Fixed cells were stained at 37 °C in the dark using the TRAP staining kit (Takara). After staining, TRAP-positive ⁽⁺⁾ cells were stained red. The stained cells were evaluated using a light microscope (Olympus IX71). The region of interests (ROIs, 1200 × 1000 μm, n = 4) were randomly designated. An area 1200 × 1000 μm in size was set as 100% and the stained area with TRAP⁺ was evaluated by the Image J program (National Institutes of Health (NIH), Bethesda, MD, USA).