Diff quik reagent

BALF was collected by cannulating the trachea and lavaging the lung twice with 1 ml of sterile 0.9% NaCl containing 50 units/ml heparin. Approximately 1.8 ml of BALF was routinely recovered from each mouse and the total cell number was counted using a hemocytometer. After centrifugation with a Cytospin®4 (Thermo Fisher Scientific, Waltham, MA), cells were stained with Diff-Quik reagents (Sysmex, Kobe, Japan) and the ratios of macrophages, neutrophils and lymphocytes to total cell number was determined.

Invasion assays were performed using a BD Matrigel^® Invasion Chamber 24-well plate (BD Biosciences, San Jose, CA, USA). T-REx HeLa stable cell lines pre-cultured in medium containing 1 µg/ml doxycycline (DOX) for 24 h were seeded at a density of ~ 1 × 10⁵ cells/well in the upper chamber of the transwell with SFM containing 1 µg/ml DOX. The lower chamber was filled with a standard culture medium containing 1 µg/ml DOX. Invading cells were stained with Diff-Quik reagents (Sysmex, Kobe, Japan) and then counted. An MMP-12 inhibitor, MMP408, was purchased from Merck KGaA (Darmstadt, Germany).

On day 25, mice were anesthetized with alfaxalone (Jurox, Rutherford, Australia), and blood samples were collected from the cauda vena cava. The blood samples were centrifuged for 20 min at 200 g to separate the serum. The total immunoglobulin E (IgE) and OVA-specific IgE were measured by an enzyme linked immunosorbent assay (ELISA) (BioLegend Inc., San Diego, CA, USA). To collect the bronchoalveolar lavage fluid (BALF), we performed tracheostomy in the mice and inserted endotracheal tubes. PBS (0.7 mL) was injected into the lung and removed via the tube, and the process was repeated once. The collected BALF was centrifuged (200 g, 4 °C, 10 min). The supernatants were collected in new tubes for measuring pro-inflammatory cytokines interleukin (IL)-5 and IL-13 by using ELISA. The remaining pellet was dissolved in 200 μL of PBS, followed by centrifugation through Cytospin (Hanil Electric, Wonju, Korea) to attach inflammatory cells to the slides. Slides were stained by a Diff-Quik reagent (Sysmex, Kobe, Japan) for counting the different cells in the BALF.

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).