Rankl

Bone marrow was extracted from mouse limbs, and monocytes were collected. The red blood cells were lysed, and the remaining cells were washed with PBS and filtered through a strainer. The cells were centrifuged and cultured in 60 mm dishes for 1 day. Cells in suspension were counted, and 3×10⁵ cells were incubated in α-MEM containing 10% FBS and 10 ng/mL M-CSF (PeproTech, Inc, Rocky Hill, NJ, USA) for 48 hr, followed by incubation for 72 hr with 30 ng/mL RANKL (PeproTech, Inc.) and 10 ng/mL M-CSF plus eupatilin at concentrations of 0, 1, 10, and 100 nM and 1, 2, 5, and 10 µM. After 72 hr, RANKL, M-CSF, and eupatilin were again added. The formation of multinuclear cells was observed through a microscope. Prior to becoming apoptotic, the cells were stained with tartrate-resistant acid phosphatase (TRAP) solution.

RAW 264.7 murine monocyte/macrophage cells were obtained from the American Type Culture Collection (ATCC, USA). The cells were cultured in α-minimal essential medium (α-MEM; Invitrogen, USA) containing 10% fetal bovine serum (FBS), 2 mM/L L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37℃ in a humidified atmosphere of 5% CO₂. After culturing, the cells were seeded in 6-well plates (1.5 × 10⁴ cells/mL) and incubated for 24 h. The medium was replaced with serum-free α-MEM plus 25 ng/mL M-CSF and 30 ng/mL RANKL (PeproTech, USA), and the cells were cultivated for an additional 48 h. For the OPG treatment groups, 0, 10, 20, 50, or 100 ng/mL OPG (PeproTech) were added to the cultured cells treated with M-CSF + RANKL, and the cells were cultured for another 30 min.

Mononuclear cells obtained from the peripheral blood of one healthy and one affected (III‐9 in family 1) individual were plated in 24‐well plates at approximately 2 × 10⁵ cells/mL in alpha‐MEM supplemented with 10% FBS and penicillin/streptomycin in the presence of macrophage colony‐stimulating factor (M‐CSF; 30 ng/mL; Peprotech, Rocky Hill, NJ, USA) and RANKL (50 ng/mL; Peprotech). Osteoclastic differentiation was usually monitored for 5 days while periodically changing the medium and fixing and staining for TRAP activity.
Bone marrow cells obtained from WT and Pfn1^+/mut mouse femurs and tibias were plated in 25‐cm culture bottles at approximately 5 × 10⁶ cells/mL in alpha‐MEM supplemented with 10% FBS and penicillin/streptomycin. After 24 hours of incubation at 37°C, 5% CO₂, nonadherent bone marrow cells were replated on 24‐well plates at 5 × 10⁴ cells/well. For the osteoclastogenesis assay, bone marrow cells were cultured for 72 hours in the presence of M‐CSF (30 ng/mL; Peprotech); the medium was then changed to medium containing both receptor activator of NF‐κB ligand (RANKL; 50 ng/mL; Peprotech) and M‐CSF. Osteoclastic differentiation was usually monitored for 5 days while periodically changing the medium. The cells were fixed and stained for TRAP activity.