Mil 11

Primary calvarial bone cells from 3- to 5-day-old C57Bl/6N mice were isolated by collagenase digestion. For each cell isolation experiment, 8–14 dissected calvariae were initially washed in PBS and incubated in 5 mL 4 mM EDTA in PBS at 37°C, 600 rpm rotation table for two sequential 10 min digestions. Thereafter, the calvariae were incubated with 180 U/mL Collagenase type II (Worthington Biochemical Corporation, Lakewood, NJ, USA) in PBS for 10 sequential 10 min, 37°C, 600 rpm, 5 mL digestions. The last five collagenase fractions were pooled and centrifuged. Primary calvarial osteoblasts were cultured in complete α-MEM medium for 3–5 days in a T75 flask prior to re-seeding at the start of the experiments.
Isolated calvarial osteoblasts were seeded at a density of 20,000 cells/cm² in osteogenic media and were treated with mRANKL (Lys158-Asp316), mOSM, hOSM, msIL6R, mIL6, mNP (CT-2), mIL31, mIL27, hCNTF, mCT-1, mIL-11 (R&D Systems, Minneapolis, MN, USA) or mLIF (Sigma-Aldrich) for 3, 6, 24 or 48h in 37°C, 5% CO₂ and then harvested in RLT buffer (Qiagen, Hilden, Germany) containing 1% β-mercapto ethanol (Sigma-Aldrich) and stored at −80°C for gene expression analysis.