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. 2024 May 29;18(3):101366. doi: 10.1016/j.jcmgh.2024.101366

Figure 4.

Figure 4

Isolation of activated ILC2s and 3D co-culture with Meta1 gastroids. (A) H&E and immunofluorescence staining of stomach tissues from untreated and L635-treated mice. Yellow arrows point to ILC2 cells double positive for GATA3 and TdTomato. Quantitation of GATA3 positive cells (ILC2s) and GATA3 and TdTomato double-positive cells (activated ILC2s). Scale bar: 100 μm. Mean ± SD (n = 6–8 from 3 different experiments). ∗∗P < .01. (B) Schematic illustration of activated ILC2s isolation from L635-treated mice using FACS. (C) FACS plots showing tdTomato positive cells (ILC2s) in untreated and L635-treated mice. Cell number quantitation shows significant increase of tdTomato positive cells (activated ILC2s) in stomachs from treated mice. Mean ± SD (N = 4). ∗P < .05. (D) Representative bright field images of Matrigel domes containing Meta1 gastroids only or co-cultured with ILC2s. Immunofluorescence imaging shows proximity of activated ILC2s (IL-13-tdTomato positive) to the Meta1 gastroid (GFP-positive membrane). Quantitation of gastroid diameters demonstrates significantly higher sizes in Meta1 gastroids co-cultured with ILC2s compared with gastroids only after 5 days of plating. Scale bar: 500 μm. Mean ± SD (N = 4 independent experiments). ∗P < .05. (E) Immunoblotting shows significant up-regulation of phosphorylated STAT6 in Meta1 gastroid co-cultured with ILC2s. Mean ± SD (N = 4 independent experiments). ∗∗∗∗P < .0001.