Isolation and culture of bone marrow derived macrophages (BMDM)

Bone marrow was harvested from C57BL/6 and Balb/c mice and differentiated into macrophages over 6 days in RPMI medium supplemented with 10% FCS, penicillin/streptomycin (100 U/ml), _L-Glutamine (2 mM), 2-mercaptoethanol (2-ME; 50 μM) and macrophage colony-stimulating factor (M-CSF;50 ng/ml; eBiosciences). For experimentation, cells were counted by trypan blue exclusion, seeded at a density of 1.25 x 10⁵ cells/well, and left to adhere overnight. Cells were stimulated in fresh RPMI medium with 10% FCS, penicillin/streptomycin (100 U/ml), and _L-Glutamine (2 mM) for 24 hrs. Cell-free supernatants were collected for measurement of cytokines (stored at -20°C until required). For dose-dependency response studies, Balb/c bone marrow derived macrophages (5.0 x 10⁵) were incubated for 30 min with full-length Sm16 (34–117) (5–50 μg/ml) and after washing in PBS were then stimulated with LPS (10 ng/ml) overnight. Ethical approval for these studies was granted by the University of Technology Sydney (UTS) Animal Care and Ethics Committee (Approval Number: 2017–1232) and experiments were conducted in accordance with the approved guidelines to be compliant with The Australian Code for the Care and Use of Animals for Scientific Purposes.