Il 22 2g12a41

For functional analyses, cells were stimulated with human (h) IL-1β (50 ng/ml) and IL-23 (50 ng/ml) with or without IL-2 (1,000 U/ml) overnight with subsequent addition of Brefeldin A solution (1,000×, BioLegend, 1,000-fold dilution) after 1 h of stimulation. The cells were stained extracellularly for detection of ILC3 and NK cells as well as intracellularly for analysis of IL-22 (2G12A41, BioLegend) production using the intracellular staining kit (BioLegend). In vitro generated ILC3 were flow cytometrically sorted (see RNA sequencing and analyses for gating) and 50,000 cells were subsequently stimulated for 5 days. Supernatant was collected at day 5. LEGENDplex Human Th Panel and the LEGENDplex Human Hematopoietic Stem Cell Panel from LEGENDplex (Cat: 740722 and 740611, BioLegend, San Diego, California) were used and experiments were done according to the manufacturer’s instructions.

For functional analyses, cells were stimulated with human (h) IL-1β (50 ng/ml) and IL-23 (50 ng/ml) with or without IL-2 (1,000 U/ml) overnight with subsequent addition of Brefeldin A solution (1,000×, BioLegend, 1,000-fold dilution) after 1 h of stimulation. The cells were stained extracellularly for detection of ILC3 and NK cells as well as intracellularly for analysis of IL-22 (2G12A41, BioLegend) production using the intracellular staining kit (BioLegend). In vitro generated ILC3 were flow cytometrically sorted (see RNA sequencing and analyses for gating) and 50,000 cells were subsequently stimulated for 5 days. Supernatant was collected at day 5. LEGENDplex Human Th Panel and the LEGENDplex Human Hematopoietic Stem Cell Panel from LEGENDplex (Cat: 740722 and 740611, BioLegend, San Diego, California) were used and experiments were done according to the manufacturer’s instructions.