Gst rankl

Osteoclastogenesis in vitro was studied by plating bone marrow-derived macrophages (BMDMs) from 8-week-old mice in culture dishes containing α-MEM (GIBCO) with 10% FBS (MP Biomedicals). After incubation for 8 hr, nonadherent cells were collected, and cells were seeded (3×10⁵ cells/dish) in 6 cm suspension dishes containing α-MEM with 10 ng/ml M-CSF (R&D Systems). After 2 days (about 80% confluent), adherent cells were used as osteoclast precursor cells after washing out the nonadherent cells. Cells were cultured in the presence of 10 ng/ml M-CSF and 235 ng/ml GST-RANKL (Oriental yeast, Japan) for 60 hr to generate osteoclast like cells.

Primary murine calvarial cells were isolated from the calvariae of neonates, as previously described [14 (link)]. To generate osteoclasts, bone marrow cells were co-cultured for 6‒7 d with calvarial cells in minimal essential media (α-MEM) containing penicillin/streptomycin (100 units and 100 μg/ml, respectively), 2 mM L-glutamine, and 10% fetal bovine serum with or without baricitinib (Chemscene, Monmouth Junction, NJ) in the presence of 10⁻⁸ M 1,25D₃ and 10⁻⁶ M PGE₂ (both; Wako, Osaka, Japan).
Mice were sacrificed by cervical dislocation, and bone marrow cells were collected by flushing the tibia. Bone marrow macrophages were obtained from cultures treated with M-CSF (50 ng ml^-1) for 3 d and subsequently cultured with or without baricitinib in the presence of M-CSF and GST-RANKL (200 ng ml^-1; Oriental Yeast, Tokyo, Japan) for 3‒4 d. These cultures were fixed with 4% paraformaldehyde in PBS and stained for tartrate-resistant acid phosphatase (TRAP) activity, as previously described [14 (link)]. TRAP-positive multinuclear cells (TRAP⁺ MNC; more than three nuclei) were counted as osteoclasts.

RAW264.7 cells were seeded in NICO‐1 at a density of 7.5 × 10³ cells per well or seeded in 96‐well plates at a density of 4.0 × 10³ cells per well. Recombinant mouse sRANKL (GST‐RANKL; Oriental Yeast Co., Tokyo, Japan) was administered at a concentration of 50 ng·mL⁻¹ along with seeding of RAW264.7 cells. On Day 5, the cells were stained with tartrate‐resistant acid phosphatase (TRAP) using a TRAP Staining Kit (Cosmo Bio, Tokyo, Japan). TRAP‐positive multinuclear cells containing three or more nuclei were counted as osteoclasts under a light microscope.