Multicolor Flow Cytometry Profiling of Lung Immune Cells

Single cell suspensions extracted from the lungs were stained with a live/dead-Infrared marker (Thermo Fisher) and incubated with monoclonal antibody CD16/CD32 Becton Dickinson Biosciences (BD) to block Fc receptors, before adding a cocktail of directly conjugated mAbs directed against the following surface markers; CD11b-BV711 (M1/70, 563168), Ly6G-PerCp-Cy5.5 (1A8, 560602), CD45-BB515 (30-F11, 564590), SiglecF-PE-CF594 (E50-2440, 562757), CD103-BV421 (M290, 562771), CD11c-BV510 (HL3, 562949), CD3e-BV786 (145-2C11, 564379), CD19-BV605 (1D3, 563148), CD335-BV605 (29A1.4, 560469), and Ly6C-APC (AL-21, 560595) all purchased from BD and FceR1-PE-Cy7 (MAR-1, 25-5898-82), MHC-II-AF700 (M5/114.15.2, 56-5321-82) from Thermo Fisher (ebioscience). Data acquisition was performed with the BD LSR Fortessa (BD) and Diva software, and the analysis was performed with FlowJO X software (TreeStar, Ashland, OR). In initial control experiments, the right lobes were evaluated for the frequency of contaminating peripheral blood mononuclear cells after cardiac PBS infusion by staining with an anti-CD115 mAb (T38-320, 565249, BD), which is uniformly expressed on all circulating blood monocytes (13 (link)) but not on lung monocytes (14 (link)). The frequency of CD115⁺ cells was below 0.01% among the lung cells, arguing that we had little if any blood leukocyte contamination (Figure S1B).