Facs lsr 2

For FACS analysis as well as FACS sorting of single-cell suspensions, cells were first incubated with CD16/CD32-specific antibody (2.4G2; BD) for 10 min to block non-specific Fc-receptor interactions. A live death staining was performed using zombie aqua (eBioscience) according to the manufacturer’s instructions. To differentiate between DC subsets, cells were stained for 15 min at 4°C with combinations of antibodies specifically binding to CD11b (M1/70.15; Invitrogen), CD11c (HL3; BD), Siglec-H (eBio440c; eBioscience), CD69 (H1.2F3; BD), Clec9a (42D2; eBioscience), CD172 (SIRPα) (P84; Biolegend) and CD24 (M1/69; Biolegend). The cells were subsequently washed with 1 mL FACS buffer (PBS, 1% FCS) and then re-suspended in FACS buffer supplemented with 3% paraformaldehyde (PFA). Samples were measured using a FACS LSR II, and data were analyzed with FlowJo 7.6.5 software (TreeStar). Dead cells as well as cell duplets were excluded prior to subsequent analysis. DC subsets were sorted using the FACS Aria or FACS XDP and sorting efficiency ranged between 71.9–86.2% for the pDC, 81.4–94.4% for the CD11b-like DC and 44.54–83.2% for the CD8α-like DC.

To examine the absolute number of AM and other innate immune cells, BAL cells were collected as above and perfused lungs were isolated and minced through 70 μm cell strainer using MEM. The cells were stained with FITC-conjugated anti-CD11c (HL3), PE-conjugated anti-Siglec-F (E50-2440), PerCP-conjugated anti-Ly6C (AL-21), PE-Cy7-conjugated anti-Ly6G (1A8), APC-conjugated anti-CD11b (M1-70) or APC-conjugated anti-F4/80 (BM8), and APC-Cy7-conjugated anti-CD45 (30-F11) (all from BD Biosciences except anti-F4/80 from BioLegend). The cells were acquired using FACS LSR II and flow cytometric data were analyzed by using FlowJo software (Tree Star, San Carlos, CA, United States). We defined CD45⁺Ly6C^-Ly6G^-CD11c⁺Sigleg-F⁺F4/80⁺ as AMs, CD45⁺CD11b⁺Ly6C⁺Ly6G^- for inflammatory monocytes, and CD45⁺CD11b⁺Ly6G⁺Ly6C^- for neutrophils.