Flow cytometry

For FACS analysis, dead single-cell preparations of the small intestine or colon lamina propria were excluded using a live/dead cell viability assay in the Brilliant Violet 510. A lineage cocktail containing R-phycoerythrin-conjugated monoclonal antibodies against CD3, CD5, and CD19 was used, except where stated, and ILC were identified as Lin⁻ and CD45⁺ (PE-Cy7). Surface staining was performed with antibodies diluted in FACS buffer at 4°C for 20 min in the dark after blocking the Fc receptors with purified anti-CD16/CD32 (BioLegend). When needed, cells were fixed and intracellularly stained using the Foxp3 Staining Buffer Set (eBioscience), according to the manufacturer’s instructions. For functional experiments, MNK3 or primary cells were cultured in 96-well plates with complete media and stimulated with cytokines and/or Golgi Plug (BD Biosciences) for 3 hours at 37°C. Following incubation, Live⁺ Lin^– (CD3^–CD5^–CD19^–) CD45^intCD90.2^highILC3s were stained for viability, surface molecules, fixed in 2% paraformaldehyde (PFA), and intracellularly stained using the BD Biosciences Fixation/Permeabilization Solution Kit (Table S3). Samples were acquired using BD FACS-Symphony™ with BD FACSDiva™ Software (BD Biosciences). All FACS data were analyzed using FlowJo v.9.5.2 software (Tree Star).