Generation of Bone Marrow-Derived Macrophages

Bone marrow (BM) cells were cultured 7 days in IMDM or RPMI 1640 supplemented with 100 IU/ml penicillin, 100 μg/ml streptomycin (Invitrogen, San Diego, CA), 10% heat inactivated fetal bovine serum (Biochrom GmbH, Berlin, DE) and 50 U/ml macrophage colony-stimulating factor (ImmunoTools, Friesoythe, Germany) or 30% L929 cell supernatant to generate BM-derived macrophages (BMDMs) (43 (link), 44 (link)). Cells were seeded in half-area 96-well plates (2.5 × 10⁴ cells/well), 96-well plates (2 × 10⁵ cells/well) and 6-well plates (3 × 10⁶ cells/well) without growth factors. Neutrophils were isolated from the bone marrow using the Neutrophil isolation kit (Miltenyi, Bergisch Gladbach, Germany) and plated in 96-well plates (10⁵ cells/well). Salmonella minnesota ultra pure lipopolysaccharide (LPS) was from List Biologicals Laboratories (Campbell, CA), Pam₃CSK₄ from EMC microcollections GmBH (Tübingen, Germany), and CpG ODN 1826 (CpG) and poly(I:C) from Invivogen (San Diego, CA). Monosodium urate (MSU) crystals were prepared as described (45 (link)). Listeria monocytogenes 10403 s was grown in brain heart infusion broth (BD Biosciences, Erembodegem, Belgium). Bacteria were washed with 0.9% NaCl and adjusted at 10¹⁰ cfu/ml. When required, bacteria were heat-inactivated for 2 h at 70°C.