Dnaase

To induce IL-33-mediated ILC2 expansion, 1 µg of recombinant mouse IL-33 (R&D Systems, Minneapolis, MN) was oropharyngeally administered to each of the IL2rγ^−/−, IL4Rα^−/−, and mye-IL4Rα^−/− mice on days 1, 3, and 5. On day 6, mice were euthanized to harvest lung lobes in order to prepare single cell lung digest. Briefly, lungs were perfused by flushing 5 ml of PBS through the right ventricle of the heart. Perfused lungs were transferred to C-tubes (Miltenyi Biotec, Gladbach, Germany) containing 2.5 ml HBSS containing 500U/ml collagenase Type I (Worthington, Lakewood, NJ) and 200U/ml DNAase (Sigma Aldrich, St. Louis, MO). Lung tissues were digested using a GentleMACS dissociator (Miltenyi Biotec, Gladbach, Germany) followed by incubation on a rocker at 37 °C. Cell suspensions were filtered through 70 μm nylon cell strainer followed by centrifugation at 300×g for 10 min. Cells were resuspended in 10 ml of DMEM/F12 medium. Single -cell suspensions were stained with various fluorochrome-conjugated anti-mouse monoclonal antibodies for cellular phenotyping as follows: Lin (CD3e, B220, CD11b, TER-119, Gr-1, CD11c, FceR1α, CD8a, CD4), CD45, CD90.2, IL-33R, and CD278. Information for antibodies is included in Supplemental Table 1. Data were acquired using a CytoFLEX flow cytometer (Beckman Coulter Life Sciences, Indianapolis, IN) and analyzed using CytExpert software.

For ex vivo studies, tumors were dissected, mechanically minced, and digested using a mixture of 1 mg/ml collagenase type IV (Sigma-Aldrich) and 20 µg/ml DNAase (Sigma-Aldrich). After 30 min of digestion at 37 °C, the cells were passed through a 70 μm filter, washed and then passed through a 40 µm filter. Single cell suspensions were then stained with antibody cocktails for various TIL subsets. In some experiments, isolated cells were restimulated with phorbol 12-myristate 13-acetate (PMA) (50 ng/ml) and ionomycin (1 μg/ml; Sigma-Aldrich) in the presence of GolgiPlug (BD Biosciences; 1:1000) and GolgiStop (BD Biosciences; 1:1500) for 4 h prior to flow cytometry analysis. Samples were analyzed by FACS with Fixable Yellow used to discriminate viable and dead cells. Lymphocytes were distinguished by CD3⁺/TCRβ⁺, CD45⁺ cells. BD fluorosphere counting beads were added to cocktails before running samples. A total of 20,860 beads were added per sample.

Cortical tissues were isolated from postnatal day 1 rats (Sprague–Dawley rats from Charles River) and digested in Dulbecco's modified eagle medium (DMEM, Thermo Fischer Scientific, UK) containing 0.1% Trypsin and 0.05% DNAase (Sigma–Aldrich, UK) for 20 min in an incubator. The tissues were dissociated to single cell suspension by trituration through 1 mL and 200 µL Gilson pipette tips and the suspension was centrifuged at 600 rpm for 5 min. The supernatant was replaced by Neurobasal medium conaining 2% B27 and 0.25% Glutamax (all from Thermo Fischer Scientific, UK) and the cell pellet was gently resuspended. Finally, cells were plated on the MEAs containing the culture medium (NbActiv 4, BrainBits LLC, USA) at a cell density of 700–900 cells per mm². The cultures were maintained by replacing half of the medium every 3 days.

After asphyxiation by carbon dioxide in rats, 20 mg of mucosa was weighed and added to 5 ml of DMEM solution (Biowest, France) containing 5 mg of collagenase type II (Sigma-Aldrich, USA) and 50 µg of DNAase (Sigma-Aldrich, USA) and digested at 37 °C for 1.5 h. The resulting suspension was filtered and centrifuged at 450 g for 5 min. Nasal mucosal cells were obtained by filtration and centrifugation at 450 g for 5 min and resuspended in appropriate PBS. The nasal mucosal cell suspension was divided into several portions, some of which were incubated with IL4-PE (Biolegend, USA), IL5-APC (Biolegend, USA), IL17-AF488 (Biolegend, USA), and IFNγ-PE (Biolegend, USA) at 4 °C for 60 min in the darkness. Another portion of mucosa was incubated with CD4-BV650 (Biolegend, USA) and IL4-PE (Biolegend, USA) or IFNγ-PE (Biolegend, USA) for 60 min at 4 °C in the dark. After rinsing with PBS and resuspension, mucosal samples were transferred to a flow cytomete (FACS Celesta, USA) and analyzed with FlowJo software (version 10.6.2, Treestar, USA).