Lung dissociation kit

Mice lungs were collected in DMEM and washed to remove any blood. The tissues were then dissociated using the Miltenyi Lung dissociation kit, following manufacturer’s instructions with slight modifications^{47 (link),48 (link)}. Briefly, after coarse digestion of lungs, 0.1 mg/mL soybean trypsin inhibitor (Sigma) 60 U/mL DNAse I and 1 mg/mL type XI collagenase was added and tissues were digested at 37 °C for 30 min. Then, lungs were finely homogenized in Miltenyi Gentle MACs, followed by addition of 0.1 M EDTA in FBS to inactivate the enzymes. Cell suspension was then passed through 70 μm filter, washed with PBS by centrifugation at 800×g for 10 min. RBCs were lysed using ACK lysis buffer (Alfa Aesar) and washed with DPBS. Cells were then counted and 1–2 million cells were stained with live/dead marker (Life Technologies) before being incubated with rabbit anti-V5 (1:250, Abcam ab9116) antibody followed by AlexaFluor 488 conjugated donkey anti-rabbit secondary antibody (2 μg/ml, Invitrogen A-21206). Cells were then fixed with BD Cytofix/Cytoperm (BD Biosciences) and acquired on a BD LSRFortessa flow cytometer. Compensation was performed using beads (BD Biosciences) labeled with fluorophore conjugated antibodies. Live cells were gated on total cells and analyzed for V5 positive cells. Data was analyzed using Flow Jo software (Treestar, Inc.).

To analyze leukocytes from lung, lungs were removed from mice and were dissociated according to the protocol of lung dissociation kit from Miltenyi (130-095-927, Bergisch Gladbach, germany). Staining for flow cytometry was performed as described in splenocyte staining. Data were analyzed with BD FACS DIVA Software and FlowJo.

BAL and lung cells were treated with RBC lysing buffer (Sigma). Lungs were dissociated using a Lung Dissociation Kit (Miltenyi Biotec) and Gentle MACS (Miltenyi Biotec). LIVE/DEAD cells were stained with Fixable Aqua Dead Cell Staining Kit (Thermo Fisher Scientific), and after Fc block with the 2.4G2 mAb (eBioscience), cells were stained with the following Abs: DR3-PE (4C12; BioLegend), SiglecF (E50-2440; BD Biosciences), CD11b (M1/70; BD Biosciences), CD11c (HL3; BD Biosciences), Ly6G (1A8; BioLegend), ST2-PE (101001PE, MD), CD90.2 (30-H12; BioLegend). For lineage markers for ILC2 staining, the following Abs were used: CD3-FITC (145-2C11; eBioscience), CD4-FITC (GK1.5; eBioscience), CD8- FITC (5H-10-1; BioLegend), CD19-FITC (1D3; BioLegend), NK1.1-FITC (PK136; BioLegend), CD11b-FITC (M1/70; BioLegend), CD11c-FITC (HL3; BD Biosciences), and Gr1-FITC (RB6-8C5; BioLegend). Flow cytometry analysis was performed on a Fortessa (BD Biosciences), and data were analyzed using FlowJo Software (version 10; FlowJo, Ashland, OR). Live CD45+ lung immune cells were separated into T cells (CD3+, CD90.2+), macrophages (CD11b+, CD11c+ SiglecF+), DCs (CD11c+ MHC class II+), neutrophils (GR1+, CD11b+ SigF-), eosinophils (Ly6C+, SigF+, CD11c-) and ILC2 (Lin^- Thy1.2⁺ ST2⁺Sca1⁺).

The MLNs were harvested and ground on a 50 μm strainer to obtain a single-cell suspension. Ileal PPs were harvested, incubated for 20 min at 37 °C in Hank’s balanced salt solution (HBSS) buffer with 5 mM EDTA and 5% FBS, and then digested for 45 min in RPMI 1640 medium with collagenase D (Roche, 0.5 mg/mL)/DNase I (Sigma, 1 mg/mL) and 10% FBS. For isolated LP cells, small intestine pieces removed with PPs were incubated for 60 min at 37 °C in HBSS buffer with 10 mM EDTA and 5% FBS and then digested with the BD Horizon^TM Dri Tumor & Tissue Dissociation Reagent (TTDR, BD Bioscience, USA) according to the manufacturer’s protocol. Dead cells were removed using a dead cell removal kit (Miltenyi Biotec, Germany). To prepare the single cells from the lung tissue, perfusion was performed by injecting ice-cold PBS into the right ventricle of the heart. The lung tissue was then collected, minced, and digested using an enzyme mix from a Lung Dissociation kit (Miltenyi Biotec) according to the manufacturer’s protocol. T cells were enriched using the Pan T cell Isolation Kit II (Miltenyi Biotec).