α mem medium

Human and murine osteoclast precursor cells (OCP) isolation and osteoclast differentiation were performed as described previously [37 (link), 38 (link)]. Human peripheral blood monocytes (PBMC) were isolated from the blood of healthy donors using Ficoll gradient centrifugation (17–1440-03; GE Healthcare). Monocytes were purified from PBMCs using CD14-positive magnetic beads (130-050-201; Miltenyi Biotec) according to the manufacturer’s instructions. Briefly, human peripheral blood mononuclear cells (PBMC)/CD14 + cells were cultured in 20 ng/mL human M-CSF (300 − 25; Peprotech) in α-MEM medium (Gibco) including 10% FBS (Gibco) and 1% penicillin/streptomycin (Gibco) for 2 days to generate osteoclast precursor cells (OCPs). Then, OCPs were incubated with 20 ng/mL M-CSF and 40 ng/mL RANKL (310-01; Peprotech) for an additional 6 days. Culture medium was replenished every 3 days. Murine bone marrow cells were obtained from femurs and tibias of curdlan-injected SKG mice and were treated with ACK lysis buffer (A10492-01; Gibco) to remove RBCs and cultured on dishes with 10 ng/mL murine M-CSF (315-02; Peprotech) for 1 day. Then, suspension cells were collected and further cultured with 20 ng/mL murine M-CSF for 1 day, followed by treatment with 50 ng/mL M-CSF and 100 ng/mL RANKL (315 − 11; Peprotech) for 4 days. The culture medium was replaced every 3 days.
At the end of the culture period, mature osteoclasts were fixed in 10% formalin and stained for acid phosphatase 5, tartrate resistant (TRAP) using a commercial kit (PMC-AK04-COS; CosmoBio) according to the manufacturer’s protocol. TRAP + osteoclasts (more than three nuclei) were counted. To detect F-actin ring formation, cells were fixed and permeabilized with 0.1% Triton X-100 and then incubated with FITC-conjugated phalloidin (P5282; Sigma- Aldrich) for 60 min at 37 °C. Images of stained cells were collected under a microscope equipped with a camera (Ti-U; Nikon, Tokyo, Japan).