Quantification of Neutrophils in BAL

At the end of the experiments, the absolute number of PMNs in BAL fluid was counted as described previously [27 (link)]. In brief, the collected BAL fluid was centrifuged and cell pellets were suspended in PBS. Cell suspension (300 μl) was cytocentrifuged onto a glass slide with a cytocentrifuge (Shandon, Southern Sewickley, PA, USA). Slides were stained with Diff-Quick dye (Dade Behring, Newark, DE, USA) and analyzed by light microscopy (magnification, 20× and 40×). The percentage of PMNs was calculated after counting at least 300 cells in five or more randomly selected fields in each slide.