Lung tissue was digested using collagenase XI (0.7 mg/mL; Sigma-Aldrich, C7657), type IV DNase (30 μg/mL; Sigma-Aldrich, D5025), and Dispase (0.25 mg/mL) and filtered through a 70 μm filter to obtain a single-cell suspension. Cells were labeled with conjugated primary antibodies and analyzed using a 3-laser BD Fortessa analytical flow cytometer (BD Biosciences) and FlowJo software (BD Biosciences). Conjugated primary antibodies used for flow cytometry were as follows: CD45-BV510 (BioLegend, clone 30-F11), CD11b-PE-Cy7 (BioLegend, clone M1/70), Ly6G-APC-Cy7 (BioLegend, clone 1A8), CD64-APC (BioLegend, clone X54-5/7.1), F4/80-PE-Cy5 (BioLegend, clone BM8), Siglec F-PE (BD Pharmingen, clone E50-2440), Ly6C-PerCP-Cy5.5 (BD Pharmingen, clone AL-21), and I-A/I-E MHC-II-FITC (BD Pharmingen, clone 2G9). DAPI (Sigma-Aldrich) labeling was used to assess cell viability.

To isolate saccular lung neutrophils, P5 lung tissue was first digested and processed to a single-cell suspension as above. Cells were then labeled with conjugated primary antibodies and subjected to FACS on a FACSAria ll cell sorter (BD Biosciences) with FACSDiVa v6.1 software (BD Biosciences) to isolate CD45+ CD11b+ Ly-6G+ neutrophils that were collected for in vitro experiments.

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