Figure 5.

Assessment of apoptosis by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay and 4′,6-diamino-2-phenylindole (DAPI) nuclear staining. Results for the guinea pig primary gastric epithelial cells (A) or fibroblasts (B), untransfected or transfected with siRNA, in medium only or exposed to H. pylori glycin acid extract) (GE)presented as the percentage of apoptotic cells stained in the TUNEL assay and counted using the Image J software version 1.48v (National Institute of Health, United States) (i). (ii) Representative images of cells stained in the TUNEL assay (red nuclei) and photographed in the fluorescence microscope (Axio Scope A1, Zeiss, Germany) at wavelengths: 550 nm (excitation) and 590 nm (emission), at magnification x20. (iii) The percentage of cells stained with DAPI, indicating the chromatin condensation. (iv) Representative images of cells stained with DAPI (blue nuclei) and photographed in the fluorescence microscope (Axio Scope A1, Zeiss, Germany) at wavelengths: 345 (excitation) and 455 nm (emission), at magnification ×40. (v) Representative images of untransfected cells cultured in medium only (medium) or transfected with siRNA IL-33 and cultured in medium only (medium/siRNA IL-33) photographed in the phase-contrast microscope (YS100 Nikon, Tokyo, Japan), at magnification ×20. The results of four independent experiments performed in triplicates for each experimental variant are presented. Statistical analysis was performed using the nonparametric U Mann-Whitney test with significance for *, ** p < 0.05, * unstimulated (only medium) vs. GE stimulated, untransfected or tranfected cells, ** untransfected vs. transfected cells, in medium only or in medium with GE.