Isolation and Culture of Macrophages

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.