Isolation and Analysis of Murine Lung Immune Cells

Single-cell suspensions of mouse lung tissue were prepared as previously described (Tsukui et al. 2020 (link)). Briefly, lung tissues were perfused with PBS through the right ventricle and then cut into pieces for digestion in Liberase TL (Sigma) and DNase I (Sigma) solution at 37 °C for 30 min. Then the cells were passed through a 70 μm cell strainer, washed, and suspended in precooled PBS with 1% fetal bovine serum. For lymphocyte isolation, density gradient centrifugation was performed using Percoll (Cytiva, USA). Before staining with antibodies, the cells were blocked with an anti-CD16/32 antibody (clone 93, BioLegend, USA). Viability was assessed using the Zombie NIR™ Fixable Viability kit (BioLegend, USA). All of the following fluorescence-conjugated antibodies were purchased from BioLegend and eBioscience (USA): anti-CD45(30-F11), anti-TCR-β (H57-597), anti-CD4 (RM4-5), anti-CD8 (53-6.7), anti-CD69 (H1.2F3), anti-CXCR6 (3A051D1), anti-CD11b (M1/70), anti-Ly6G(1A8), anti-F4/80 (BM8), anti-iNOS (CXNFT), and anti-ARG1 (A1exF5). The iNKT cells were stained with fluorescence-conjugated mCD1d/PBS-57 tetramers provided by the National Institutes of Health (NIH) tetramer facility. Data were obtained from FACSVerse (BD Biosciences) and analyzed with FlowJo software (Tree Star).