Tartrate solution

Tartrate resistant acid phosphatase (TRAP) as a measure for osteoclastic differentiation was measured in cell supernatants. 10 µl supernatant or p-nitrophenol standard was incubated with 90 µl p-nitrophenyl phosphate buffer (1 mg/ml p-nitrophenyl phosphate disodium hexahydrate (71768, Sigma-Aldrich), 0.1 M sodium acetate, 0.1% triton X-100 and 30 µl/ml tartrate solution (3873, Sigma-Aldrich) in PBS) in 96-wells assay plates for 90 min at 37°C. To stop the reaction, 100 µl 0.3 M NaOH was added. Absorbance was read at 405 nm using a plate reader (Synergy™ HTX, Biotek) and absorbance values were converted to TRAP activity (converted p-nitrophenyl phosphate in nmol/ml/min) using standard curve absorbance values.

To estimate the number of osteoclasts during OTM, tartrate-resistant acid phosphatase (TRAP) staining was performed. Sections were incubated in acetate buffer containing naphthol AS-BI phosphoric acid solution, Fast Garnet GBC base solution, sodium solution, and tartrate solution (Sigma-Aldrich, St. Louis, MO), and then counterstained with hematoxylin. The number of TRAP-positive multinucleated cells containing more than three nuclei was determined by microscopy. Three sections of the mesial side of the palatal root at 40, 80, and 120 µm from the bifurcation in each mouse were evaluated and four biological replicates were analyzed, and the number of osteoclasts was expressed as the average of these three sections. UPM Axio Phot2 (Carl-Zeiss, Jena, Germany) was used for TRAP staining.

Cell supernatants were collected after 5 days of differentiation, and TRAP activity was measured with a colorimetric assay. In short, p-nitrophenyl phosphate (New England Biolabs, Ipswich, MA, USA) was diluted in buffer containing 420 mM acetic acid (Sigma-Aldrich) and 160 mM tartrate solution (Merck, Kenilworth, NJ, USA) and added 1:1 to culture supernatant. After 1 hour, the reaction was stopped with 0.5 M NaOH (Sigma-Aldrich), and the absorbance at 405 nm was determined using a spectrophotometric plate reader (Bio-Rad Laboratories, Hercules, CA, USA).