Siglecf e50 2440

Peritoneal cavity cells (PECs) were recovered in a total of 5 mL of ice-cold PBS. Splenic macrophage isolation were performed according to previous studies (33 (link)).Visceral fat was dissected and single cell dissociation and staining performed as previously described (34 (link)). For flow cytometry, cells were blocked with 0.6 µg Rat IgG and 0.6 µg α-CD16/32 (2.4G2) 5 min, stained for 25 min with antibodies to CD11b (M1/70), MHCII (M5/114.15.2), CD11c (N418), CD4(RM4-5), Ly6C(HK 1.4), Ly6G(1A8), CD19(1D3) and CD8(53-6.7) (all from BioLegend, San Diego, CA); SiglecF (E50-2440) (BD Bioscience, San Jose, CA); F4/80 (BM8) (eBioscience, Santa Clara, CA). Cells were analyzed on a Novocyte (ACEA Biosciences, San Diego, CA) or LSRII instrument (BD Bioscience, San Jose, CA) followed by data analysis using FlowJo v10 (Tree Star Inc.; Ashland, OR). t-SNE analyses were performed with FlowJo v10, involving concatenation of samples (5000 cells per biological replicate) from all groups before running the t-SNE analyses to generate plots consistent between groups. This was followed by analysis of separated groups for expression of the desired markers. The parameters used to run the t-SNE analyses are in Supplementary Table 1. Arg, Rα or TdTomato were excluded as parameters given that their expression was being analyzed, and they were negative in the some of the transgenic mouse groups.

Whole lungs were removed and chopped with razor blades, incubated with type IV collagenase (Worthington) at 37 °C for 40 min, then homogenized through a 70-µm cell strainer (Falcon). Remaining red blood cells were lysed using 1× red blood cell lysis buffer (BD Biosciences). Cells were stained with Fixable Viability Dye eFluor^® 455 (eBioscience). Anti-mouse immunophenotyping antibodies were diluted in FACS buffer (3% FBS, 2 mM EDTA, 1× PBS) to 5 µg per mL along with Fc block (anti-mouse CD16/CD32; 5 µg per mL, BD), and cells were stained for 30 min on ice in three groups (AMφ: CD45 (30-F11; BD), CD11c (HL3; BD), CD11b (M1/70; BD), and Siglec-F (E50-2440; BD); monocyte and neutrophil: Ly-6C (AL-21; BD), Ly-6G (1A8; BD), and CD11b (M1/70; BD); dendritic cell: CD45 (30-F11; BD), CD11c (HL3; BD), CD11b (M1/70; BD), MHC II (M5/114.15.2; BD), and CD103 (M290; BD); NK cells: CD3 (17A2; BD), NK-1.1 (PK136; BD), and CD49b (DX5; BD)). After the staining, cells were washed twice with FACS buffer and then fixed in 2% para-formaldehyde in FACS buffer for 15 min. Cell numbers were counted using AccuCount Fluorescent Particles (Spherotech). All data were collected on an LSR II flow cytometer (BD) and analyzed using FlowJo software.

Spleen cells were incubated with anti-mouse CD16/CD32 mAb (2.4G2; BD Biosciences, San Jose, CA) in FACS buffer (HBSS containing 2% FCS) for FcR blocking for 15 min on ice, and then incubated with BV421-conjugated anti-mouse CD3 mAb (145-2C11; Biolegend, San Diego, CA), APC-conjugated anti-mouse CD8 mAb (53-6.7; Biolegend), PE-conjugated anti-mouse CD4 (GK1.5 Biolegend) or Siglec F (E50-2440; BD Biosciences) mAb, PE-Cy7-conjugated anti-mouse CD19 mAb (6D5; Biolegend) or Gr1 (RB6-8C5; Affymetrix eBioscience, San Diego, CA) mAb for 20 min on ice. The expression profile of each cell surface marker on 7-aminoactinomycin D (7-AAD; Sigma-Aldrich Co. LLC, St. Louis, MO) -negative cells was analyzed on a MACSQuant Analyser (Miltenyi Biotec, Bergisch Gladbach, Germany) with MACSQuantify Software (Miltenyi Biotec) and FlowJo software (Tree Star Inc., Ashland, OR).