Murine Osteoblast-Osteoclast Co-culture System

To determine the involvement of Sema4D signaling in tenofovir mediated effects, we constructed a murine primary osteoblast-osteoclast precursor co-culture system based on previously described protocols [34 (link)]. Briefly, co-culture is performed as follows. Adherent BMCs from 6–8-wk-old female C57BL/6 or A2AKO mice (n = 5 each) were seeded at a density of 1 × 10⁵ cell/cm² density with osteogenic medium in the bottom chamber of transwell plates (Corning, New York, NY, USA), and atotal of 200,000 non-adherent cells were collected and seeded in α-MEM with 30 ng/mLM-CSF for two days in the top chamber of transwell plates. At day 3 (day 0 of differentiation), 30 ng/mLRANKL was added to cultures in the presence/absence of Sema4D antibody 10 ng/mL alone or in combination with tenofovir or dipyridamole 1 µM each. Cells and supernatant were collected at different time points for western blot analysis (n = 5 each).