Surface expression of TSLPR, IL-17BR, and ST2 reveals different ILC2 subpopulations. ILC2s were stimulated with ECs or EC combinations, and surface expression of EC receptors was evaluated by flow cytometry. (A) Percentage of IMDM-cultured ILC2s that show double-positive expression for IL-17BR and TSLPR. (B) Percentage of IMDM-cultured ST2⁺ ILC2s. (C) Contour plots showing the percentage of cells in each gate for IL-17BR (x-axis) and TSLPR (y-axis), comparing IMDM and SFEM II. (D) viSNE plots, as analyzed in Cytobank, showing the population clustering for each receptor. Red indicates the highest expression and blue the lowest expression. (E) Photographs depicting ILC2s when stimulated with double or triple cytokine combinations, or the latter combination with the addition of IL-2 (phase contrast microscopy, original magnification ×100). Data are shown as mean ± SEM from 2 independent experiments, with n = 2 or 3 donors each. Significance was calculated using 1-way ANOVA followed by correction for multiple comparisons, where *P < .05, **P < .01, ***P < .001, and ****P < .0001. Statistics above the bars represent comparison with media baseline; comparison between samples is represented by the connecting line. ns, not significant.