Cells in the BALF were suspended in 1 ml PBS and mixed with 0.4% trypan blue stain in a 1:1 ratio. After mixing, 10 µl buffer was applied to the chamber slide, and the slide was inserted into an automatic cell counter. The total cell count was performed. The cell precipitation was resuspended to prepare a smear for staining. Inflammatory cells including eosinophils, neutrophils, lymphocytes and macrophages in the BALF were counted using Wright-Giemsa-staining. Briefly, slides were stained by fixing for 2 min with a one-step methanol-based Wright-Giemsa stain. Following that, the slides were stained in Diff-Quik I solution for 5–10 sec and taken out immediately. The slides were then stained in Diff-Quik II solution for 10–20 sec and taken out immediately at room temperature, according to the instructions of the Diff-Quik whole blood stain kit (Baxter Scientific). A total of 200–300 cells from each sample were then counted from a randomly chosen field using an automatic cell counter. The percentage of a leukocyte subset was multiplied by the total number of leukocytes to give the absolute number of the specific leukocyte subset.

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