Flow Cytometric Analysis of Lung Immune Cells

Lung single cell suspensions for flow cytometric analysis were prepared as previously described^{13 (link)}. Live cells were stained with fluorescently labelled antibodies against Siglec F, FcεRI, CD64, Ly-6G, CD11c, IFNGR1 and viability dye eFluor^™ 780 (ThermoFisher Scientific, reference: 65–0865–14). Antibodies used in this study are listed in Supplementary Table 2. After washing with FACS buffer (PBS containing 2% calf serum and 1 mM EDTA), cells were stained with streptavidin conjugated with PE-CF594 (1/300) for 15 mins at 4°C in the dark. Cells were subsequently fixed and permeabilized with 200 μl 1x Fixation/Permeabilisation Buffer (eBioscience, 00-5521-00) as per the manufacturer’s instructions, and then stained with a polyclonal FITC-anti-Legionella antibody (ViroStat, cat # 6053). Total numbers for each cell type were enumerated from the lungs by adding 2 × 10⁴ APC-labelled beads (BD Calibrite, reference: 340487) into each sample prior to flow cytometry analysis. Dead cells were detected and excluded based on eFluor 780 fluorescence. Data were analysed with FlowJo software.