Lung Cell Suspension Preparation

Lung cell suspensions were prepared as described before^{31 (link)}. Briefly, after perfusion with heparin in PBS, lungs were minced, digested with DNAse/collagenase, lysed for red blood cells, and pressed through a 0.7 μm filter to generate a single cell suspension. Cells were stained with appropriate fluorochrome-labeled specific antibodies or isotype control antibodies. Intracellular cytokine staining was performed using the BD Cytofix/Cytoperm kit (BD Biosciences). Mouse antibodies used include anti-CD11b (clone M1/70; Tonbo Biosciences), anti-CD11c (clone HL3; BD Biosciences), anti-Gr-1 (clone RB6–8C5, eBioscience), anti-CD3 (clone 500A2; BD Biosciences), anti-CD4 (clone RM4–5; BD Biosciences), anti-CD44 (clone IM7; eBioscience), and anti-IFN-γ (XMG1.2; BD Biosciences). Cells were processed with the Becton Dickinson (BD) Fortessa flow cytometer using FACS Diva software, or the BD FACSJazz flow cytometer using FACS Sortware software (BD). Flow cytometry experiments were analyzed using FlowJo (Tree Star Inc). As before^{34 (link)}, neutrophils were defined as CD11b⁺CD11c^-Gr-1^hi cells, monocytes were defined as CD11b⁺CD11c^-Gr-1^med cells, and recruited macrophages were defined as CD11b⁺CD11c^-Gr-1^low cells. Total numbers of cells within each gate were back calculated based on cell counts/individual lung sample.