Murine macrophage colony stimulating factor

Mouse BMDMs were derived from bone marrow cells of C57BL/6 mice as previously described (26 (link)). Briefly, following erythrocyte lysis, bone marrow cells were cultured in DMEM containing 10% FBS and 50 ng/ml murine macrophage colony-stimulating factor (R&D Systems) for 5 days. The differentiated cells were then split and plated for following experiments. Peritoneal macrophages were elicited in 8-week-old mice by intraperitoneal injection of 1 ml 4% Brewer thioglycollate (Sigma-Aldrich). Peritoneal cells were harvested 4 days later by lavage and plated for 2h, followed by extensive wash to remove non-adherent cells. The adherent cells were used as peritoneal macrophages. The animal protocol was approved by the University of Alabama at Birmingham Institutional Animal Care and Use Committee.

NHBE cells were purchased from Lonza (Walkersville, MD, USA), and cultured in collagen-coated plates in LHC-9 medium (Life Technologies, Grand Island, NY, USA). Human THP-1 cells (American Type Culture Collection, Manassas, VA, USA) were cultured for 3 days with 200 nM phorbol 12-myristate 13-acetate to induce macrophage differentiation (16 (link)). Human alveolar macrophages were isolated by centrifugation of bronchoalveolar lavage fluid from healthy adult volunteers using a protocol approved by the University of Arizona College of Medicine Institutional Review Board. Bone marrow cells were harvested from the femurs and tibias of C57BL6/J mice, and peritoneal macrophages were isolated from thioglycolate-treated C57BL6/J mice as described (17 ) using a protocol approved by the University of Arizona Collage of Medicine Institutional Animal Care and Use Committee. Bone marrow cells were cultured for 7 days with 10 ng/ml of murine macrophage-colony stimulating factor (R&D Systems) to induce macrophage differentiation. THP-1 cells and murine macrophages were seeded at 2.5 × 10⁶ cells/well in 6-well plates, and human alveolar macrophages were seeded at 5.0 × 10⁵ cells/well in 24-well plates, and cultured in RPMI-1640 containing 2.0 mM glutamine, 1.0 mM sodium pyruvate, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% FBS at 37°C in a humidified incubator containing 5% CO₂ in air.