Differentiation and Activation of Primary Macrophages

Primary macrophages were derived as previously described (3 (link)). Briefly, marrow was flushed from the femurs and tibias of 2- to 4-month-old 129SvEvTac mice (Taconic Laboratories, Hudson, NY). Cells were resuspended in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich, St. Louis, MO) supplemented with fetal bovine serum (10%), l-glutamine (2 mM), sodium pyruvate (1 mM), β-mercaptoethanol (50 µM), HEPES (10 mM), and penicillin-streptomycin (50 IU/ml penicillin and 50 µg/ml streptomycin). Cells were overlaid onto an equal volume of Histopaque-1083 (Sigma-Aldrich, St. Louis, MO) and centrifuged at 500 × g for 15 min. Monocytes at the interface were harvested and incubated for 6 days at 37°C in 5% CO₂ in supplemented DMEM that also contained 30% macrophage colony-stimulating factor (M-CSF) (conditioned medium from 3T3-MCSF cells) to promote differentiation of adherent monocytes into macrophages (54 (link)). BMDM and RAW264.7 Nramp1^G169 cells were activated with 20 ng/ml S. enterica serovar Typhimurium LPS from Sigma-Aldrich (where the dose is equivalent to a multiplicity of infection [MOI] of ~5,000 [20 pM] based on an E. coli dry weight of 2.8e−13 g/cell [55 ], 4.09 nmol LPS/mg dry weight of S. Typhimurium [56 (link)], and the formula weight of S. Typhimurium LPS [Sigma-Aldrich]) and 20 U/ml IFN-γ (PeproTech, Rocky Hill, NJ) or with only 20 U/ml IFN-γ for 18 h.