Inflammatory Macrophages

Inflammatory macrophages were obtained 4 days after the i.p. injection of 3% thioglycolate broth. Peritoneal resident macrophages or inflammatory (recruited) macrophages were collected by peritoneal lavage. Inflammatory macrophages were cultured in DMEM (Invitrogen Life Technologies), supplemented with 2 mM glutamine, 5 ×10⁵ M 2-ME, 10 μg/mL gentamicin, 1 mM sodium pyruvate, and 0.1 mM MEM non-essential amino acids (culture medium) plus 10% FBS. Cells were processed and analyzed prior or after culture by flow cytometry and functional assays. Cultures were treated with the following reagents: 0.2–0.5 ng/mL of recombinant IFN-γ (R&D Systems, EUA), 20 ng/mL of recombinant RANKL (R&D Systems, EUA), 200 ng/mL of LPS from Salmonella enterica serovar Typhimurium (Sigma), 10 μM of Bay 11-7082 (Santa Cruz Biotechnology, Dallas, USA) or DMSO (Sigma), 1 mM of N6-(1-imioetil) lysine (L-NIL) from Sigma, 100 μM of deferoxamine (DFO, Sigma) or N-acetyl-L-cysteine (NAC, Sigma). Cultures were maintained up to 3 days at 37°C with 7% of CO₂.