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).