In vitro Expansion and Manipulation of ILC2s

Sorted ILC2s were cultured (37°C, 10% CO₂) at 3,000–10,000 cells per well in 200 µl high-glucose DMEM supplemented with 10% heat-inactivated FBS, penicillin/streptomycin/l-glutamine, 2-mercaptoethanol, recombinant mouse cytokines, IL-33, TSLP, and IL-7 at 10 ng/ml (R&D Systems). At harvest, cell-free culture supernatants were collected, and cells were analyzed by flow cytometry or collected in TRIzol LS (Thermo Fisher Scientific) for RNA extraction. On day 8 of culture, ILC2s were transfected with miRNA mimics and siRNAs (Dharmacon). miRIDIAN miRNA mimics were used at 500 nM per transfection. siGENOME SmartPools and ON-TARGETplus Individual siRNAs were used at 500 nM. Cells were transfected using the Neon Transfection system (Invitrogen) as previously described (Steiner et al., 2011 (link)). Cells and culture supernatants were harvested on day 10 for further analysis.