*4.4. Osteoclast Formation*

Then, 3 <sup>×</sup> <sup>10</sup><sup>5</sup> purified human CD14<sup>+</sup> monocytes were seeded in 24-well plates containing a-MEM with FBS (10%, *v*/*v*; Invitrogen, Waltham, MA, USA) and M-CSF (20 ng/mL; PeproTech, Rocky Hill, NJ, USA) for three days. RANKL (100 ng/mL; PeproTech, Rocky Hill, NJ, USA) and TNF-α (100 ng/mL; PeproTech, Rocky Hill, NJ, USA) were added to induce osteoclast differentiation [11]. The osteoclasts were stained with tartrate-resistant acid phosphatase (TRAP) at day 13 using the Acid Phosphate Leukocyte Kit (Sigma, St. Louis, MO, USA) according to the manufacturer's instructions. TRAP-stained cells containing three or more nuclei were defined as osteoclasts [30]. We measured the number of osteoclasts from four randomly selected higher power fields (HPF) (200×) per quadrant of the well. Four HPF were chosen from each quadrant. The number of osteoclasts was counted from the average value from 16 HPFs.
