7 aad viability solution

The left lungs of OVA–induced mice were processed by mincing and then passed through cell strainers. After Percoll density gradient centrifugation, the total number of cells per lung was determined. The single cell suspension was blocked with the blocking buffer (Biolegend, 101320) and stained with various antibodies for flow cytometry analysis (netrophils, CD11b⁺ Ly-6G⁺; eosinophils, CD11b⁺ SiglecF⁺). For intracelluar cytokine staining, cells were stimulated with phorbol myristate acetate (PMA) (100 ng/mL) and Ionomycin (1 µg/mL) in culture media with monensin (eBioscience, 00-4505-51). After 4 h, cells were washed, blocked with blocking buffer, and stained with the cell surface markers FITC-anti-mouse CD4 (RM4-5, BioLegend, 100510) and Pacific blue-anti-mouse CD8 (53-6.7, eBioscience, 48-0081-82) for 20 min, washed and fixed in 1% paraformaldehyde for 10 min, permeabilized in permeabilization buffer (eBioscience, 00-8333-56) for 5 min, and stained with specific cytokine antibodies APC-anti-IL-4 (11B11, eBioscience, 17-7041-82), PE-anti-IL-17 (ebio17B7, eBioscience, 12-7177-81) and Percp-anti-IFN-γ (G1.2, eBioscience, 45-7311-82) for 20 min. Dead cells were excluded by staining with the 7-AAD viability solution (BioLegend, 420402). Data were acquired on a BD FACS Calibur flow cytometer and analyzed by the FlowJo software.