Srankl

Bone marrow cells were isolated from both femurs, tibias, and pelvic bones by cutting the epiphyses, flushing the bones with αMEM media using a 26G needle, and passing through a 70μm cell strainer. Red blood cells were removed using RBC Lysis Buffer (Invitrogen). The bone marrow cells were then resuspended in αMEM media containing 10% FBS and 1% penicillin-streptomycin and kept on ice. Cells were stained for surface markers as described above (methodssection 2.7.) and were sorted using a BD^™ FACS Aria II SORP. Sufficient numbers of osteoclast precursor Population VI were acquired from each mouse and were used for in vitro osteoclast formation assays. Equal number of cells (1.2×10⁴ cells/well) were seeded in a 48-well dish in αMEM media containing 10% FBS, 1% penicillin-streptomycin, 30 ng/ml M-CSF (Peprotech; #315–02), and 50 ng/ml sRANKL (R&D Systems; #462-TEC) for 3 and 5 days at 37°C in 5% CO₂. Medium was refreshed every other day. The cells were stained with TRAP (Sigma) and the number of TRAP+ mature osteoclasts (≥3 nuclei) per well were quantified after 3 and 5 days of culture. No distinction was made between large and small osteoclasts. Two wells per biological replicate were used for osteoclast formation assays.

Isolated BMMs were seeded at a density of 8 × 10⁴ cells/well of a 24-well plate and in the presence of either 30 ng/mL M-CSF (Peprotech; #315–02) alone or M-CSF (30 ng/mL) and 50 ng/mL sRANKL (R&D Systems; #462-TEC) for 3 and 5 days at 37°C in 5% CO₂. Total RNA was isolated from cells using TRIzol (Invitrogen) according to the manufacturer’s instructions. First-strand cDNA was synthesized with oligo(dT) and random primers using qScript cDNA SuperMix (Quantabio). All qRT-PCR were performed on an Analytik Jena qTower³ G Real-Time PCR Detection System using Applied Biosystems PowerUp SYBR Green master mix. Amplicon authenticity was confirmed by melt curve analysis. Primer sequences are provided in Supplementary Table 1 and β-actin was used as the normalization control. The data were analyzed for fold change using the ΔΔCT method.