Figure 2

Fluorescence activated cell sorting of cells in BAL fluid following LPS inhalation. Cells were identified in BAL fluid based upon their position on flow cytometry dot plots of size i.e. forward scatter (FSC-A) versus granularity i.e. side scatter (SSC-A) (A). PMLC were selected as HLA-DR + (B), and were subdivided into iPMLC and rPMLC subpopulations based upon their CD14 and CD16 expression (CD14++CD16- and CD14++CD16+, respectively; C). Representative samples of AM, iPMLC and rPMLC isolated from BAL fluid using FACS with >95% purity, and overlapped on SSC-A versus FSC-A dot plots are seen in (D). Like PMLC, it was possible to identify distinct populations of lymphocytes, neutrophils and alveolar macrophages in BAL fluid following LPS inhalation based upon their size and granularity (A). Lymphocytes were further selected for CD3 expression (E), neutrophils were classed as CD16+ HLA-DR- (F) and AM were selected as large CD16+ HLA- DR + cells (G).