Isolation and Characterization of Lung Macrophages

To validate the performance of CPM we sorted the population of macrophages from the lungs of various CC mice (Supplementary Table 1). To address this, the lungs were dissociated into single cell suspensions using Miltenyi Biotec lung dissociation kit (130-095-927), according to manufacturer’s instructions. Isolated lung cells were then enriched for CD45⁺ cells by a positive selection (CD45 microbeads, Miltenyi Biotec, 130-052-301), incubated with blocking solution (5% normal mouse serum, 5% normal rat serum, and 1% anti-Mouse CD16/CD32) for 30 min on ice, and stained with fluorochrome-conjugated antibody for CD11b (M1/70), CD64 (X54-5/7.1), I-A/I-E (M5/114.15.2), Ly6G (1A8), Ly6C (HK1.4), and CD45 (30-F11, Miltenyi Biotec). All antibodies were from Biolegend, unless otherwise mentioned (clone number in parentheses). Data was acquired with a SH800 flow cytometer (Sony Biotechnology) and analyzed with FlowJo v.10 Software. Mononulear phagocyte cells were gated as CD11b⁺CD45⁺Ly6G^-I-A/I-E⁺, as previously described22 (link), and the expression levels of Ly6C and CD64 were analyzed.