Osteoclast Differentiation from Human and Mouse Precursors

Human CD14⁺ cells were incubated with 20 ng/ml of M-CSF for one day to generate OCPs. For human osteoclastogenesis assays, cells were added to 96 well plates in triplicate at a seeding density of 5 ×10⁴ cells per well. Osteoclast precursors were incubated with 20 ng/ml of M-CSF and 40 ng/ml of human soluble RANKL for an additional 5 days in α-MEM supplemented with 10 % FBS. Cytokines were replenished every 3 days. On day 6, cells were fixed and stained for TRAP using the Acid Phosphatase Leukocyte diagnostic kit (Sigma) as recommended by the manufacturer. Multinucleated (greater than 3 nuclei) TRAP-positive osteoclasts were counted in triplicate wells. For mouse osteoclastogenesis, bone marrow (BM) cells were flushed from femurs of mice and cultured with murine M-CSF (20 ng/ml, Peprotech) on petri dishes in α-MEM supplemented with 10% FBS after lysis of RBCs using ACK lysis buffer (Cambrex, Walkersville, MD). Then, the non-adherent cell population was recovered on the next day and cultured with murine M-CSF (20 ng/ml) for an additional 3 days. We defined this cell population as mouse OCPs. For murine osteoclastogenesis assays, we plated 2 ×10⁴ OCPs per well in triplicate wells on a 96 well plate and added M-CSF and RANKL (100 ng/ml) for an additional 6 days, with exchange of fresh media every 3 days. For detection of actin ring formation, cells were fixed, permeabilized with 0.1% Triton X-100, and incubated with fluorescein isothiocyanate-phalloidin in a humidified chamber for 45 min at 37 °C. After rinsing in PBS, cells were imaged using a Zeiss Axioplan microscope (Zeiss, Germany) with an attached Leica DC 200 digital camera (Leica, Switzerland).