Cellularity was determined by flow cytometry using reagents obtained from BD Biosciences (San Jose, CA) unless otherwise specified. The resuspended BALF and MLN cells were divided in two for differential and intracellular cytokine staining. One half of the BALF cells were stained for 30 minutes using the following antibodies at 1∶100 rat anti-mouse Ly-6G, rat anti-mouse Siglec-F, pan-leukocyte rat anti-mouse CD45, and Armenian hamster anti-mouse CD11c (eBioscience Inc. San Diego, CA). MLN cells were also stained to identify B cell populations using B220/CD45R antibodies. After staining, cells were washed and fixed with BD Cytofix. Cells were then washed and resuspended in FACS buffer.
Cell populations were evaluated on a BD LSRII (BD Biosciences, San Jose, CA). Neutrophils were defined as CD45hiLy-6Ghi, eosinophils as Ly-6GlowSiglecFhiCD11clow, and alveolar macrophages as Ly-6GlowSiglecFhiCD11chi as previously reported [14] (link), [19] (link). Total cell numbers were quantified using acridine orange nuclear staining and an automated cell counter (Cellometer AutoX4, Nexcelom Bioscience, Lawrence, MA). Total numbers of each cell population were obtained by multiplying the frequency of specific population by the total number of BALF cells recovered for each animal.
Cell populations were evaluated on a BD LSRII (BD Biosciences, San Jose, CA). Neutrophils were defined as CD45hiLy-6Ghi, eosinophils as Ly-6GlowSiglecFhiCD11clow, and alveolar macrophages as Ly-6GlowSiglecFhiCD11chi as previously reported [14] (link), [19] (link). Total cell numbers were quantified using acridine orange nuclear staining and an automated cell counter (Cellometer AutoX4, Nexcelom Bioscience, Lawrence, MA). Total numbers of each cell population were obtained by multiplying the frequency of specific population by the total number of BALF cells recovered for each animal.