Acid phosphatase kit 387 a

Osteoclast formation was measured by quantifying cells that were positively stained with TRAP (Acid Phosphatase Kit 387-A; Sigma-Aldrich, St. Louis, MO). Briefly, specimens were fixed for 15 mins and then stained with naphthol AS-BI phosphate and a tartrate solution for 1 h at 37 °C, followed by counterstaining with a hematoxylin solution. Control cells are positively stained red; therefore, TRAP-positive multinuclear cells with three or more nuclei were regarded as osteoclasts and were counted under an inverted-phase contrast microscope. We also used a higher concentration of 1 M tartrate (for a final concentration of 20 mM), instead of the tartrate solution provided in the kit to suppress background phosphatase activity. By increasing the tartrate concentration, the staining of the control cells is suppressed, and the RANKL-treated cells remain positively stained. The total number of TRAP-positive cells and the number of nuclei per TRAP-positive cell in each well were counted. The morphological features of osteoclasts were also photographed as our previously described^{32 (link)}.

Tartrate-resistant acid phosphatase (TRAP) staining of mature osteoclasts was done as previously described [10 (link)]. Briefly, cells were stained with TRAP (Acid Phosphatase Kit 387-A; Sigma-Aldrich, St. Louis, MO) for 30 s, naphthol AS-BI phosphate and tartrate solution for 1 h at 37 °C and then counterstained with a hematoxylin solution. In order to control phosphatase activity that may occur in the background, we opted to use a greater dilution of 1 M tartrate (final 20 mM) and not the tartrate supplied as part of the kit [10 (link)]. Intensifying the tartrate dilution suppressed control cells’ staining to allow the RPR and RANKL/M-CSF-treated cells to be positive. The total of TRAP-positive cells and nuclei per TRAP-positive cell in every individual well were respectively counted, followed by the photographing of the morphological features of the osteoclasts.

TRAP-positive cells and soluble TRAP activity were assessed in RANKL-induced differentiation cultures by Acid Phosphatase Kit 387-A, (Sigma Aldrich, St. Louis, MO, USA) according to the manufacturer’s protocol. Briefly, 3 × 10³ RAW 264.7 cells per well (1×10⁴ cells/cm²) were allowed to adhere overnight to 96-well plates. The supernatant was then removed, and cells were stimulated for 5 days with different amounts of CsPE (40 to 320 μg/mL) in the presence of RANKL 100 ng/mL to induce differentiation of RAW 264.7 macrophages into osteoclasts. The culture medium containing stimuli was changed every two days. After 5 days of incubation, TRAP activity was revealed at both the culture medium and cell levels. Thirty microliters of harvested supernatants were added to 170 μL of TRAP reaction buffer, and, after 2.5-h of incubation at 37 °C, absorbance at 540 nm was measured using a UV/visible spectrophotometer (TECAN, Thermo Fisher Scientific, Waltham, MA, USA). Adherent cells were fixed and stained by TRAP reagent at 37 °C for 1 h protected from light. Images of TRAP-positive cells were captured under a bright-field light microscope (EVOS M5000, Thermo Fisher Scientific, Waltham, MA, USA).