Isolation and Culture of Macrophages from Mice

BMDMs and alveolar macrophages were isolated and cultured under sterile techniques as described in detail by McMahan et al23 (link) and Manicone et al.26 (link) Briefly, for BMDMs, marrow from femurs and tibias of wild-type and Mmp10^−/− mice was recovered by brief centrifugation. Red blood cells were removed by adding lysis buffer (eBio-science, San Diego CA, USA). The remaining cells were suspended in Mac medium (RPMI 1640 with 10% fetal bovine serum and 20% medium conditioned by L929 cells as a source of CSF-1) and plated at 1.5×10⁶ cells/10 cm plate. Mac medium was replaced on days 3 and 6. Between days 7 and 10, plates were washed with PBS to remove nonadherent or dead cells. The macrophages were then removed from the plate using 5 mM ETDA in PBS, and cell counts and viability analysis were determined. Alveolar macrophages were isolated from BAL by centrifugation, pooled in pairs, and treated with 100 µg/mL of MWCNTs in low serum medium (RPMI 1640 + 2% FBS) for 2 or 24 h. Polymyxin B sulfate salt (10 µg/mL; Sigma-Aldrich, St Louis, MO, USA) was included in experimental treatments to control for potential lipopolysaccharide contamination.