Pac orange live dead fixable dead staining dye

Lungs were perfused with PBS containing EDTA (0.5 mM), minced, and digested in collagenase IV (5 mg/ml) and DNase I (23 (link)). Cells were filtered and washed with RBC lysis buffer (BD Biosciences, Franklin Lakes NJ) and kept on ice in media containing 10% serum. Dead cells were stained with Pac-Orange Live/Dead fixable dead staining dye (Invitrogen). Lung cells were then stained with fluorescent-labeled antibodies against various leukocyte surface markers (CD45, CD11b, CD11c, F4/80, CD103, MHCII, CD86, Clec9a, CD3ε, TCRβ, CD4, CD69, IFN-γ). Appropriate isotype-matched controls were used in all experiments. Antibodies were purchased from EBiosciences (San Diego, CA) or Biolegend (San Diego, CA). Cells were fixed and analyzed on a Canto2 (Becton-Dickinson, San Jose, CA) or FACSAria II (BD Biosciences) flow cytometer. Results were analyzed using FlowJo software (Tree Star, Ashland, OR). For analysis of intracellular IL-12 or IFN-γ, fresh aliquots of digested lung tissue were stimulated for 5 hours at 37°C, with Cell-stimulation Cocktail Buffer (40.5 μmol/L phorbol 12-myristate 13-acetate [PMA], 670 μmol/L ionomycin, 5.3 mmol/L brefeldin A, and 1 mmol/L monensin; eBioscience), fixed, permeabilized with Cell Permeabilization Buffer (eBioscience) and incubated with anti-mouse IL-12 clone C17.8 (eBioscience) or anti-mouse IFN-γ clone XMG1.2 (Biolegend).

Lungs from sham- and RV-treated immature wild-type BALB/c or TSLPR KO mice were perfused with PBS containing EDTA, minced and digested in collagenase IV. Cells were filtered and washed with RBC lysis buffer, and dead cells were stained with Pac-Orange Live/Dead fixable dead staining dye (Invitrogen). To identify ILC2s, cells were then stained with fluorescent-tagged antibodies for lineage markers (CD3ε, TCRβ, B220/CD45R, Ter-119, Gr-1/Ly-6G/Ly-6C, CD11b, CD11c, F4/80 and FcεRIα, all from Biolegend), anti-CD25 (Biolegend), and anti-CD127 (eBioscience), as described (21 (link)). Cells were fixed, subjected to flow cytometry and analyzed on a LSR Fortessa (BD Biosciences, San Jose, CA). Data were collected using FACSDiva software (BD Biosciences) and analyzed using FlowJo software (Tree Star, Ashland, OR).