Intravascular Leukocyte Discrimination Protocol

Intravascular staining for the discrimination of vascular and lung tissue leukocytes was conducted as previously described [26 (link)]. Anti-CD45.2 PE (clone 104, catalog number 12-0454-81; eBioscience, San Diego, California) was diluted to 25 mg/mL in sterile PBS; 100 μL of the solution was intravenously injected via the mouse tail 3 minutes before sacrificing the mice. The stained CD45.2⁺ cells are blood-borne in the mouse lungs and can be discriminated from cells localized in the lung tissue. Cellular staining and flow cytometry analyses for T helper (Th) cells and neutrophils were conducted as previously described [27 (link)]. In brief, cells were stained for surface markers with anti-CD4 fluorescein isothiocyanate (FITC; clone: GK1.5; eBioscience) for Th cells, and with anti-CD11b FITC (clone: M1/70; Biolegend, San Diego, California) and anti-Gr-1 APC (clone: RB6-8C5; Biolegend) for neutrophils. For intracellular staining, fixed cells were permeabilized and stained with anti–IL-17 FITC (catalog number 11-7177-81; eBioscience) and anti–interferon gamma (IFN-γ) APC (clone: XMG1.2; eBioscience) for 60 minutes in the dark at 4°C. Cells were washed with 1 mL of permeabilization buffer and resuspended in staining buffer for analysis. Samples were analyzed using a FACSCalibur or FACSCanto flow cytometer (BD Biosciences) and the FlowJo software (Tree Star, Ashland, Oregon).