Cxcl10 neutralizing antibody

BMCs were isolated from wild-type mice and 200 000 cells per well were added on top of primary osteocyte-enriched fractions or primary osteoblasts cultivated in co-culture medium (α-MEM supplemented with 10 nM 1,25-dihydroxyvitamin D3 (Sigma-Aldrich) and 20 ng/ml M-CSF). For pit assay, bone slices (Immunodiagnostic Systems, Tyne and Wear, UK) were used. Cells were removed from bone slices with 1 M ammonium hydroxide overnight and the resorption pits were visualized using 0.5% (w/v in H₂O) toluidine blue.
For conditioned medium-transfer experiments, conditioned medium from Men1-deficient primary osteocyte-enriched fractions or control primary osteocyte-enriched fractions was diluted 1:1 in co-culture medium.
For neutralizing antibody experiments, CXCL10-neutralizing antibody (20 ng/ml; R&D Systems) or IgG was added to co-cultures of wild-type BMCs with Men1-deficient primary osteocyte-enriched fractions in co-culture medium.
The medium was changed every 3 days. After 9 days, cells were stained for TRAP activity.

Mouse CXCL10-neutralizing antibody (R&D Systems) or IgG (R&D Systems) was delivered in the space between the subcutaneous tissue and the periosteum over the sagittal suture of the skull. Before the injection, all animals were anesthetized. One dose of CXCL10-neutralizing antibody (100 μg/mouse) or IgG (100 μg/mouse), each in a 100 μl volume, was injected subcutaneously. Mice were killed 5 days after the injection.