Bronchoalveolar lavage fluid (BALF) analysis

After the mice were euthanized, collection of BALF was performed immediately with isotonic sterile PBS lavage for four times (0.5 ml each time). From each mouse, 2 ml BALF was collected separately. The cellular influx in the BALF was examined by using a cell counting plate. After centrifugation at 3,000 × g for 8 min at 37°C. After removing the supernatant, the precipitation cells were evenly spread on a slide and stained with 0.5 ml Wright-Giemsa reagent, which contains 0.5 g Wright pigment, 0.5 g giemsa pigment and 500 ml methanol for 1 min at 37°C. Cells were identified based on morphological features and counted in randomly selected areas of the slide using light microscopy. The levels of IL-4, IL-5, IL-13, IL-25 and IL-33 were measured in the supernatant of BALF using ELISA kits.