Gating and sorting strategies for analysis and purification of ILCs from different organs. (A) Backgating of ILC populations on the CD45⁺ live lymphocyte gate in naïve C57BL/6. (B) MFI for CD127 expression on the different ILC populations in kidney, lung, and SILP. Data are representative of at least two independent experiments with similar results with n = 3–5 mice per group. (C) Sorting strategy for kidney, lung, and SILP ILC2s from naïve C57BL/6 mice without using genetically modified fluorescent reporter mice for RNAseq analyses (see Fig. 1). While the Lin⁻CD127⁺CD25⁺Sca-1⁺ gate reliably identified GATA-3⁺ ILC2s in the kidney and lung, SILP ILC2s were better identified by the Lin⁻CD127⁺CD25⁺KLRG1⁺ gate. (D) Gating on all GATA-3⁺ ILC2s showed that the gating strategy depicted in C represents >80% of the total ILC2 population in naïve C57BL/6 mice. (E) Sorting strategy for IL-33–expanded ILC2s from CD45.1 BALB/c mice (see Fig. 2, A and B, for experimental setup and sorting purity). (F) Sorting strategy for IL-33–expanded effector ILC2s from C57BL/6 Red5 mice (see Fig. 3 for sorting purity). (G) Sorting strategy for IL-33–expanded ILC2s from BALB/c IL-13fm donor mice and BALB/c Rag2^−/−Il2rg^−/− mice recipient mice (see Fig. 4 A for experimental setup and Fig. S3, A and B, for sorting purity). Numbers in flow cytometry plots indicate the percentage of events in the repective gates. Symbols in B represent individual data points and bars indicate mean ± SEM. Statistical analysis was performed using one-way ANOVA with Tukey’s post-hoc test (*P < 0.05, **P < 0.01, ***P < 0.001).