Quantifying Airway Inflammatory Cells

After measurements of AHR, the trachea was cannulated and the bronchial alveolar lavage (BAL) fluid was collected as described before^{39 (link)}. Briefly, we tracheostomized and intubated the mice and then washed the airways three times with 1 mL of ice-cold PBS each time, followed by centrifuging at 400 × g for 7 min and harvesting the cells. Data were analyzed with FlowJo software (TreeStar, Ashland, Ore). The absolute cell numbers in BAL fluid were calculated by means of flow cytometry by staining the cells with phycoerythrin (PE)–anti-Siglec-F (E50-2440; BD Biosciences, San Jose, Calif; 1/1000), fluorescein isothiocyanate(FITC)–anti-CD19 (6D5; 1/400), peridinin-chlorophyll-protein complex (PerCP)/Cy5.5–anti-CD3ε (17A2; 1/200), allophycocyanin (APC)–anti–Gr-1 (RB6-8C5; 1/1000), PE/Cy7–anti-CD45 (30-F11; 1/500), APC/Cy7–anti-CD11c (N418; BioLegend, San Diego, Calif; 1/400), and eFluor450–anti-CD11b (M1/70; eBioscience, San Diego, Calif; 1/500) in the presence of anti-mouse FC-block (2.4G2; BioXcell, West Lebanon, NH; 1/200). We used CountBright Absolute Count Beads (Thermo Fisher Scientific, Waltham, Mass), according to the manufacturer’s instructions. At least 10⁵ CD45⁺ cells were acquired on a BD FACSCanto II (BD Biosciences) using the BD FACSDiva software v8.0.1.