2.7. Bronchoalveolar lavage fluid (BALF) and inflammatory cells
BALF was collected by washing the airway lumens of the lungs three times with 1 mL ice cold PBS instilled into a tracheal polyethylene catheter. The fluid was centrifuged at 2,000 rpm for 5 min at 4°C. The supernatant was collected to determine histamine contents released from airway mast cells. Collected cell pellets were used to count the number of total cells and inflammatory cells, including macrophages, eosinophils, neutrophils, and lymphocytes. Total cells were counted using a hemocytometer, and differential inflammatory cells were counted using a cytospin at 500 rpm for 5 min followed by Diff-Quick (Sysmex Corporation, Kobe, Japan) or Wright-Giemsa staining (Sigma-Aldrich) according to the manufacturer’s protocol. Types of inflammatory cells were determined with a differential cell counter (Hwashin Technology, Seoul, Korea) using standard morphologic criteria (Saltini et al., 1984).