Isolation and Characterization of Lung Immune Cells

Whole-lung single-cell suspensions were prepared as described previously (45 (link)). Briefly, lungs were minced and digested in buffer containing 2.2 mg/ml collagenase type IV (Worthington), 100 μg/ml DNase I (Zymo Research), and 5% fetal bovine serum (FBS) at 37°C rotating for 45 min. Digested samples were then passed through an 18-gauge needle, resuspended in red blood cell lysis buffer, diluted with PBS, passed through a 100-μm filter, and counted. For flow cytometry analysis, cells were stained with a viability dye (eFluor 780; eBioscience), anti-CD45 (Pacific Orange; Invitrogen), anti-CD11b (PECy5 [BioLegend] for cellularity, PerCPCy5.5 [BioLegend] for FLARE), anti-CD11c (phycoerythrin [PE], BioLegend), anti-Ly6G (fluorescein isothiocyanate [FITC], BioLegend), anti-CD64 (BV421; BioLegend), and anti-SiglecF (BV421; BD Bioscience). Samples were analyzed on a MACSQuant VYB flow cytometer (cellularity) or Beckman Coulter Cytoflex S (FLARE). Macrophages were identified as CD45⁺/Ly6G⁻/CD11b⁺/CD64⁺, neutrophils were identified as CD45⁺/SiglecF⁻/Ly6G⁺/CD11b⁺, and eosinophils were identified as CD45⁺/SiglecF⁺/CD11c^low. Analysis was performed with FlowJo version 9.9.6.