Osteoprotegerin opg

Cell layers were lysed by ultrasonication at 40V for 15 seconds/well (VCX 130; Vibra-Cell, Newtown, CT). The QuantiFluor* dsDNA system (Promega, Madison, WI) was used to determine total DNA content by fluorescence. Alkaline phosphatase activity was measured in the cell layer lysates by the release of para-nitrophenol from para-nitrophenyl phosphate (pH 10.2) and normalized to time and protein content measured by bicinchoninic acid assay (Thermo Fisher Scientific, Waltham, MA).
Enzyme-linked immunosorbent assays were used to determine the levels of osteogenic factors in the conditioned media. Osteocalcin (OCN; Thermo Fisher), osteoprotegerin (OPG; R&D Systems, Inc.), osteopontin (OPN; R&D Systems, Inc.), vascular endothelial growth factor A (VEGF-A; R&D Systems, Inc.), BMP2 (R&D Systems, Inc.), bone morphogenetic protein 4 (BMP4; R&D Systems, Inc.), interleukin 6 (IL6; R&D Systems, Inc.), and interleukin 10 (IL10; R&D Systems, Inc.) were quantified according to the manufacturer’s protocol.

Osteoclast assays were performed as described (31 (link), 32 (link)). Bone marrow cells (BMCs) were plated in triplicate in 96-well flat-bottom plates at a density of 3 × 10⁴ cells/well with 10 ng/ml MCSF (R&D Systems, Minneapolis, MN). After 2 days, adherent bone marrow-derived macrophages (BMMs) were cultured in OC media supplemented with 10 ng/ml MCSF and either 5 ng/ml murine RANKL (PeproTech, Rocky Hill, NJ), 50ng/ml murine TNFα (PeproTech), 50ng/ml murine IL-6 (R&D Systems) or combined TNFα/IL-6. After 3 days, fresh cytokines were added. In some experiments, osteoprotegerin (OPG) (R&D Systems), anti-mouse IL-6 Receptor antibody (cMR16-1; Genentech, San Francisco, CA) (33 (link)) or control mouse IgG2a antibody (BioXCell, West Lebanon, NH) were added. Where indicated, cell proliferation was monitored using the alamarBlue assay according to the manufacturer’s instructions (Thermo Fisher Scientific). After 5 days, cells were fixed and stained for tartrate-resistant acid phosphatase (TRAP). Osteoclasts were quantified by counting TRAP-positive cells with 3 or more nuclei. All images were taken using a Nikon TMS-F inverted microscope at a final magnification of 100X.