Bone marrow derived macrophages (BMDMs) were differentiated from the bone marrow of 6–8 week old female Balb/c mice (ARC, Perth, Australia), as previously described . All procedures were in accordance with the guidelines of the UTS Animal Care and Ethics Committee (Protocol number: 2012–080). Briefly, bone marrow was flushed from freshly isolated femurs and tibias with RPMI 1640 (Life Technologies). Cells were collected by centrifugation (300×g, 5 min), and resuspended at a density of 2 × 106 cells/ml in BMDM media (RPM1 1640, supplemented with 10% v/v heat inactivated, certified low endotoxin, foetal calf serum (Life Technologies, catalogue number 10082147), beta-mercaptoethanol (715 nM), penicillin/streptomycin (1% v/v, Life Technologies) and macrophage colony-stimulating factor (M-CSF, 50 ng/ml, eBioscience). Cells (2 × 107 cells in 10 ml media) were then plated into sterile 90 mm petri dishes (Techno Plas, catalogue number S90001G). Cells were differentiated for 6 days, with the addition of 10 ml media at Day 3. At Day 6, media and non-adherent cells were removed, remaining non-adherent cells were rinsed away with 10 ml sterile saline (Baxter Healthcare), and then 10 ml fresh BMDM media was added. Cells were detached by gently scraping into media and collected by centrifugation (300×g, 5 min). The purity of the BMDM population was determined at day 6 by staining for CD11b by flow cytometry. The purity of the BMDM population was always greater than 98% CD11b+ (data not shown).