Rm csf

Macrophages were prepared from bone marrow mononuclear cells as previously described (26 (link)). For each experiment mononuclear cells were obtained by flushing bone marrow from femurs, tibiae, and humeri of 3–5 WT or Chrm3^−/− mice with HyClone alpha MEM medium (Thermo-Scientific; Chicago, IL) pre-equilibrated at 37°C. Cells were cultured overnight in alpha MEM medium containing 10% FBS and 1% penicillin/streptomycin in a humidified incubator at 37°C with 5% CO₂. Non-adherent cells were collected by centrifugation after lysis of red blood cells using red blood cell lysis buffer (Sigma-Aldrich; St. Louis, MO) and mononuclear cells were counted and plated. Mature macrophages were generated by differentiating isolated mononuclear cells with 20 ng/mL rM-CSF (R&D Systems; Minneapolis, MN) for 7 days. WT or Chrm3^−/− macrophages were then treated with IFN-γ, IL-4, bethanechol, atropine, or a combination of these agents for 24 h to determine their ability to attain a CAM or AAM phenotype.

All animal experiments were performed in accordance with the guidelines of the Florida Atlantic University Institutional Animal Care and Use Committee (IACUC), protocol number A18–04. Osteoclastogenesis was performed as previously described.^{[32 (link)]} Briefly, whole bone marrow was flushed from both tibia and femur in hind limbs of either male or female mice. The marrow was plated at 0.5 × 10⁶ cells/mL in αMEM supplemented with 25 ng/mL recombinant macrophage-colony stimulating factor (rM-CSF) (R&D Systems, Minneapolis, MN). After 72 h, non-adherent cell population was plated at 1 × 10⁵ cells/250 μL/well in a 48 well plate in fresh media containing 25 ng/mL rM-CSF and 100 ng/mL RANKL. Inhibitors were added in triplicates at 10 μM final concentration at the time of plating. Media was refreshed every 2–3 d for 5–7 d. Upon osteoclast formation, cells were fixed by incubating in 4% paraformaldehyde for 10 min at room temperature. Samples were subsequently stained with TRAcP stain (Sigma-Aldrich, St. Louis, MO) per manufacturer instructions. Multinucleated osteoclasts were counted over 5 images from each triplicate sample.