N5000

To prepare the TRAP (tartrate resistant acid phosphatase) staining solution, 2 mg of phosphate disodium salt (N-5000, Sigma-Aldrich, St. Louis, MO, USA) was first dissolved in 200 μl of H₂O_dd. Acetate buffer was prepared by mixing 35.2 ml 0.2 M sodium acetate solution, 14.8 ml 0.2 M acetic acid solution and 50 ml H₂O_d. TRAP buffer pH 5.0 was then freshly prepared with 10 ml of acetate buffer, 2 ml of 0.3 M sodium tartrate, 200 μl naphthol AS-MX phosphate (10 mg/mL; N5000, Sigma-Aldrich, St. Louis, MO, USA), 20 μl Triton X‑100 (T8787, Sigma-Aldrich, St. Louis, MO, USA) and 7.78 ml H₂O_dd. TRAP buffer was preheated at 37 °C and then 0.3 mg/ml Fast Red Violet LB Stain (F-3381, Sigma-Aldrich, St. Louis, MO, USA) were dissolved in TRAP buffer. The TRAP staining solution was preheated at 37 °C until use. Cells were washed with prewarmed PBS (14190-094, Gibco™, Carlsbad, CA, USA) and fixed with 10% glutaraldehyde (G-5882, Sigma-Aldrich, St. Louis, MO, USA) at 37 °C for 15 min. After washing the cells twice with PBS, TRAP staining solution was added and incubated for 10 min at 37 °C. Then TRAP staining solution was removed and stained cells were washed with PBS. TRAP-positive cells (red) were counted using an Olympus IX50 microscope (Olympus, Shinjuku, Japan).