Pe mouse igg1 isotype control

Lungs were minced and incubated in RPMI-1640 (Gibco, Grand Island, NY, USA) with 2 mg ml⁻¹ collagenase D for 30 min in 37 °C. After digestion, the lung cells were dispersed by gentle pipetting, followed by filtration through a 75 μm cell strainer. Then, 20 ng ml⁻¹ of phorbol 12-myristate 13-acetate and 1 μg ml⁻¹ of ionomycin (Sigma-Aldrich) were added to the cells. The cells were incubated at 37 °C in 5% CO₂ for 1 hour and then supplemented with 10 μg ml⁻¹ Brefeldin A (Sigma-Aldrich, Shanghai, CN) and incubated for another 4 hours. The cells were resuspended in 100 μl of staining buffer containing FITC-labeled mouse anti-rat CD3 and PerCP mouse anti-rat CD8a (BD Pharmingen) antibodies, and they were incubated for 30 minutes at 4 °C, fixed and permeabilized by addition of 500 μl of fixation/permeabilization solution (BD Pharmingen), vortexed and then incubated at RT in the dark for another 20 minutes. Subsequently, the cells were resuspended in 100 μl of Perm/Wash buffer containing PE mouse anti-rat IL-4 and Alexa Fluor® 647 mouse anti-rat IFN-γ antibodies and stained for 30 minutes at 4 °C. Mouse IgG1 PE isotype control (BD Pharmingen) and mouse IgG1 Alexa Fluor® 647 isotype control (BD Pharmingen) were used as negative controls. All samples were washed, resuspended in 2% paraformaldehyde and analyzed by flow cytometry (Accuri C6, BD Accuri).