Anti siglecf pe

Bone marrow and spleen were mashed between two glass slides. Single-cell suspensions were filtered through a 70 µm cell strainer (BD Falcon) and washed twice with PBS/0.5% BSA. To block unspecific binding, cells were incubated with anti-CD16/CD32 (clone 2.4G2; made in-house) for 5 minutes on ice. Subsequently, cells were incubated with FITC labeled anti-B220 (made in-house), CD138PE (BD) and/or anti Siglec-F-PE (BD) for 20 minutes on ice. After washing, analysis was performed by flow cytometry (BD Biosciences LSRII).

Cells obtained from BAL or mediastinal lymph nodes of mice challenged with OVA or sterile saline in vivo were analyzed by flow cytometry (FACSAria II; BD Biosciences PharMingen or FACSCalibur; BD Biosciences PharMingen). We labeled the cells using monoclonal antibodies anti-CD3 (FITC or PECF-594), anti-CD4 (APC or FITC), anti-CD8 (PECy5), anti-SiglecF (PE) (BD Biosciences PharMingen), anti-Ly6G (FITC), anti-CD11c (PE) (Thermo Fisher Scientific, Swedesboro, NJ, USA) or polyclonal antibody anti-GcgR (PE) (Bioss Antibodies, Woburn, MA, USA). To evaluated GcgR expression on Total CD3⁺ cells, TCD4⁺ cells, eosinophils, neutrophils, and dendritic cells in BAL, we incubated these cells with primary polyclonal rabbit anti-GcgR (Santa Cruz Biotechnology) antibody for 30 min, washed with PBS, and then we incubated the cells with polyclonal anti-rabbit-Alexa 635 antibody (BD Biosciences PharMingen) for 30 min. In control nonspecific binding, we used an isotype-matched antibodies or the primary polyclonal rabbit antibody anti-GcgR (Santa Cruz Biotechnology) was omitted. The results were analyzed using Summit software version 4.3 (Beckman Coulter.). Gating strategies for identification of populations are shown in Figs S2, S3, S7 and S8.

hCD1Tg mice were immunized IT with either BCN or MA-BCN (2.5 μg of MA/mouse). 6 weeks later, lungs were isolated and single-cell suspension was stained with anti-SiglecF-PE (E50-2440, BD Bioscience). SiglecF-positive cells were enriched using an anti-PE MultiSort Kit (Miltenyi Biotec). 1 × 10⁵ flow through and AM-enriched fractions were co-cultured with 1x10⁵ hCD1Tg BMDCs and 3 × 10⁵ DN1 T cells for 48 hr and cells were stained for the expression of activation markers.

Colons were cut into ~1 cm² pieces, washed in 1X Hank’s Balanced Salt Solution (HBSS) containing 2% heat-inactivated fetal bovine serum (FBS), disrupted by vortexing, further treated with collagenase IV (20 mg/ml) and DNase I (10 mg/mL) in RPMI medium and passed through a 70 μM cell strainer to collect lamina propria cells. Cells were treated with blocking antibodies to CD16/32 (1:100), and stained for viability using Zombie Viability Dye V500 (1:400). Surface staining was done on ice for 20 min using anti-CD45 APC-eFluor780 (1:400, clone 30F11, eBioscience), anti-CD11b APC (1:400, clone M1/70, InVitrogen), anti-Gr1 PerCp-Cy5.5 (1:300, clone RB6-8C5, Biolegend), anti-Siglec F PE (1:400, clone E50-2440, BD), anti-CD3 FITC (1:200, clone 145-2C11, Biolegend), anti-CD49b FITC (1:200, clone DX5, eBioscience), anti-B220 FITC (1:300, clone RA3-6B2, eBioscience), anti-CD4 PerCp-Cy5.5 (1:400, clone RM4-5, eBioscience), and anti-CD8a AlexaFluor 700 (1:200, clone 53–6.7, Biolegend),. Intestinal epithelial cells were collected as described in^{22 (link)}. Cells were stained with anti-EpCAM PE-Cy7 (1:400, clone G8.8, eBioscience) and anti-FITC CD44 (1:200, clone IM7, eBioscience). Cells were analyzed on the Fortessa (BD Biosciences) and data analysis was performed on FlowJo (Tree Star).

Single-cell suspensions of mouse cells were stained using the following antibodies: anti-CD45-APC, anti-CD11b-PerCP-Cy5.5 (obtained from eBioscience, San Diego, CA), anti-Siglec-F-PE (BD Bioscience, San Jose, CA), DAPI (Sigma, St. Louis, MO). Colonic eosinophils were identified as: CD45⁺/CD11b⁺/Siglec-F⁺/SSC^high.