IFN-γ–induced PC degranulation. (A) RT-PCR expression analysis of Cd3e (T cells) and Cd45 (leukocytes) in whole mouse small intestinal RNA or in RNA from freshly isolated crypts and cultured organoids. Hprt expression was used for normalization. Representative results of 2 independent experiments. (B) Morphology of small intestinal organoids 2 d after addition of mouse recombinant TNF, IL-1b, IL-6, IL-22 (all at 20 ng/ml) or IFN-γ (5 ng/ml) to the culture medium. PCs were observed in all cultures (blue arrowheads) except after IFN-γ treatment. Representative images from three independent experiments. (C) Small intestinal organoids were stimulated with IFN-γ (2 d; 5 ng/ml) followed by lysozyme immunostaining (red) and UEA-1 lectin staining (green). Blue arrow shows accumulation of UEA-1⁺ mucus in the crypt lumen. 3D projected confocal images show nuclei (gray) stained with TO-PRO3. Representative images from two independent experiments. (D) Organoids were stimulated for 2 d with indicated concentrations of IFN-γ and epithelial lysozyme content was analyzed by Western blot; results are representative for two independent experiments. (E) Quantification of Western blot data in D by densitometric analysis. Mean normalized lysozyme signal ± SD of triplicate wells is shown; **, P < 0.01; Student’s t test. (F) Histological analysis of organoids 2 d after addition of 5 ng/ml IFN-γ by PAS and alkaline phosphatase (AP) staining. Black arrows show loss of mucus-filled goblet cells. Images are representative from two independent experiments. Bars, 50 µm.