Macrophage Differentiation and Phenotype

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.