Tms f inverted microscope

To measure resorptive function, osteoclasts were grown on either commercially available human bone particles (OsteoAssay Wells, Lonza, Basel, Switzerland) or hydroxyapatite coated wells (OsteoLogic slides, BD Biosciences, Franklin Lakes, NJ). Osteoclasts were cultured as above for 14 days in the presence of cytokines, which were replenished every 2–3 days. For the OsteoAssay, resorption activity was measured using an enzyme-lined immunoassay (ELISA) to detect carboxy-terminal collagen crosslinks (CTX) (Immunodiagostic Systems, Boldin, UK) in culture supernatants. For OsteoLogic plates, cells were removed and wells were stained with 5% (w/v) silver nitrate (Von Kossa reagent) and 1% pyrogallol. Images were taken using a Nikon TMS-F inverted microscope at a final magnification of 100X.

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.

Transwell chambers (8-μm) were used for both migration and invasion. For invasion, Matrigel-coated transwells were used (BD Biosciences). After irradiation (as described above), cells were trypsinized and brought into single-cell suspension. Next, 7500 cells were resuspended in serum-free medium and placed in the well of each chamber. Serum containing growth medium was used as a chemoattractant. After 24 hours of incubation, each well was fixed, rinsed, and stained with Crystal Violet. After serial washes and drying, the number of migrating or invading cells per high-power field were counted using a Nikon TMS-F inverted microscope at ×100 magnification. Using the surviving fraction of cells, as determined by the concomitant clonogenic survival assay described above, the percentage of surviving cells with migratory and/or invasive potential was calculated.