Cytospin device

After blood collection, a tracheotomy was performed on each mouse and the left lung was infused with PBS (4 °C, 0.7 mL) with the right bronchus tied via an endotracheal tube and aspirated after a short while. The aspiration step was repeated to collect a total 1.4 mL volume of bronchoalveolar lavage fluid (BALF). After centrifugal separation of the BALF at 800× g for 5 min, the upper part was gathered and stored under −80 °C until Th2 cytokine analysis. The pellet of the remaining BALF cell was resuspended in 500 μL of PBS (4 °C) and the total cell number was calculated. Then, 5 × 10³ BALF cells were incubated with 20 µM of 2′,7′-dichlorofluorescin diacetate (Sigma-Aldrich, St. Louis, MO, USA) for 20 min at 37 °C. The quantitation of ROS activity was conducted using a plate reader of fluorescence (485 nm excitation and 530 nm emission wavelengths: PerkinElmer, Waltham, MA, USA). Then, 200 ul of the remaining resuspended BALF were attached to slides utilizing a cytospin device (Hanil Science Industrial, Seoul, Korea) for determining the differential cell counts. The slides were dried and the Diff-Quik stain was performed using Diff-Quik^® reagent (Sysmex Co., Kobe, Japan). The levels of Th2 cytokines were obtained with each detection kit (R&D System, Minneapolis, MN, USA) and a plate reader of ELISA (450 nm wavelengths: Bio-Rad Laboratories, Hercules, CA, USA).

Mice were sacrificed with an overdose of pentobarbital 48 h after the final challenge. BALF was collected by washing lungs with ice-cold PBS (total volume of 1.8 mL) via tracheal cannulation. BALF was centrifuged and cell pellets were resuspended in 0.5 mL PBS, and cells in 100 μL of each solution spun onto a slide using a cytospin device (Hanil Science Industrial, Seoul, Korea). After the slides were dried, cells were stained using Diff-Quik Staining reagent (B4132-1A; Dade Behring, Deerfield, IL), according to the manufacturer’s instructions.