Isolation and Culture of Murine Osteoblasts

The mouse pre-osteoblastic cell line MC3T3-E1 was obtained from the RIKEN Cell Bank (Tsukuba, Japan) and the cells were maintained in a growth medium (α-modified minimal essential medium (α-MEM, Gibco, Rockville, MD, USA) supplemented with 10% fetal bovine serum (Gibco) and 1% antibiotic-antimycotic reagent (Gibco)). Primary osteoblasts were isolated from mouse calvaria as previously described [39 (link)]. Briefly, 4-5 pup ICR mice were sacrificed by decapitation, and the calvariae were dissected from the mice. The calvariae were trimmed and subjected to collagenase II (Sigma, St. Louis, MO, USA) for 2 h at 37 °C. The first digestion was discarded, and the second digestion was neutralized with a growth medium followed by filtration using a Falcon^® 40 μm cell strainer. Mouse primary monocytes were isolated from bone marrow cells in the femur of a nine-week-old mouse as previously described [40 (link)]. Isolated primary monocytes were maintained in the growth medium containing 50 ng/mL of macrophage-colony stimulating factor (M-CSF; PeproTech, Cranbury, NJ, USA). All cells were maintained at 37 °C in a 5% CO₂ incubator.