Il 12 il 18

Flow cytometry was used to analyze α4β7⁺ ILC progenitors, CD117⁺ and CD127⁺ ILC precursors as well as mature ILCs. The gating strategy for ILCs was as shown (fig. S1). To evaluate the intracellular IL-13, IL-22 and IFN-γ expressions in ILCs, cells were stimulated with 10 ng/ml PMA (Sigma) + 1 μg/ml ionomycin (Sigma) or 10 ng/ml of IL-12+IL-18 (for ILC1, R&D Systems), IL-25+IL-33 (for ILC2, Shenandoah) and IL-1β (Stemcell)+IL-23 (R&D Systems) (for ILC3) overnight and in the presence of 2 μg/mL brefeldin A (BD Biosciences) for the last 4 h. For intracellular staining, cells were first stained for surface markers and fixed, followed by permeabilization and staining for intracellular proteins. For CD107a degranulation assay, sorted NK cells were incubated with K562 cells at effector:target of 5:1, and the detail of the assay is previously described (57 (link)). All flow cytometry data were acquired in LSR II (BD Biosciences) and analyzed using FlowJo (BD Biosciences) or Kaluza (Beckman Coulter) analysis software. As negative controls, fluorochrome conjugated isotype-matched antibodies from the respective companies were utilized. Viability of cells was analyzed using flow cytometry with the fixable viability dye eFluor™ 780 (eBioscience). The detail of the antibodies used for surface or intracellular proteins staining is as mentioned in table S5.