Acid phosphatase leucocyte trap kit

RAW 264.7 cells (5–7 × 10³ cells/well) were seeded in 24‐well plates and treated with RANKL or LPS in the absence or presence of TH‐848 (0.1, 0.2, 0.3 μM), TH‐644 (10, 15 or 20 μM), exogenous PGE₂ (0.1, 1 μM) or Celecoxib (1 μM). After 3 days, the culture medium containing the relevant reagents (indicated above) was changed and the cells were allowed to differentiate for additional 1 day and then fixed in 4% paraformaldehyde (Histolab, Gothenburg, Sweden). Tartrate‐resistant acid phosphatase (TRAP) staining was performed with the commercial acid phosphatase leucocyte (TRAP) Kit (Sigma‐Aldrich) according to the manufacturer's instruction. Multinucleated TRAP‐positive cells with ≥3 nuclei were defined as osteoclasts. Osteoclasts were counted under a light microscope by two independent observers. Mean numbers of osteoclasts from three independent experiments were used in the calculations for the half maximal inhibitory concentration (IC₅₀), determined by interpolation from the plots of percent inhibition versus concentration of the compounds.

After being co-cultured for 7 days, the DFCs/MNCs were stained for TRAP using Acid Phosphatase, Leucocyte (TRAP) Kit (Sigma, USA). The number of TRAP-positive multinucleated cells (≥3 multinuclear) was counted and compared among Clcn7-shRNA group, negative-shRNA group and control group. After 14 days co-culture of DFCs/MNCs on dentin slices (4 mm × 4 mm × 200 μm), the number of resorptive lacunae of each dentine slice was counted under the scanning electron microscopy (SEM). The areas of the lacunas were measured with NIH Image J software (NIH, USA) for at least 50 lacunas each group.