Automated hematology analyzer xn 2000 5

The left (intratracheal instillation study and the single inhalation study) or right (repeated inhalation study) bronchus was tied off with a thread, and the opposite lung lobes were lavaged with 4–8 ml of saline. The total cell number in the BALF was counted using an Automated Hematology Analyzer (XN-2000 V; Sysmex Corp., Kobe, Japan). Cell populations on glass slides were prepared using Cytospin 4 (Thermo Fisher Scientific, Inc., Waltham, MA). After May-Grunwald-Giemsa staining, the differential white blood cell count was made by visual observation. To measure LDH activity, the BALF was centrifuged at 1960 rpm (800×g) for 10 min at 4 °C, and the supernatant was examined using an automatic analyzer (Hitachi 7080 or 7180, Hitachi High-Tech Corp., Tokyo, Japan).

For the 13-week inhalation study, the right bronchus was tied with a thread, and the left lung was lavaged. 4–7 ml of saline was injected into the lung through the trachea, in and out twice, and collected as BALF. In the macrophage or neutrophil depletion experiment, 6.2 ml of saline was injected into all lungs through the trachea, in and out twice, and collected as BALF. The total cell numbers in the BALF were counted using an automatic cell analyzer (Sysmex Corp., Automated Hematology Analyzer, XN-2000 V). Thin-layer cell populations on glass slides were prepared using Cytospin 4 (Thermo Fisher Scientific, Inc., Waltham, MA). After May-Grunwald-Giemsa staining, differential white blood cell count was conducted by visual observation. BALF was centrifuged at 1960 rpm (800×g) for 10 min at 4 °C, and the activity of LDH, ALP and γ-GTP and the phospholipid level in the supernatant was measured using an automatic analyzer (Hitachi 7080, Hitachi, High-Tech Corp., Tokyo, Japan).