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. 2010 Aug 9;78(10):4157–4165. doi: 10.1128/IAI.00524-10

FIG. 2.

FIG. 2.

Localization of replicating H. pylori in infected AGS cells. (a) Immunofluorescent staining of H. pylori in AGS cells. AGS cells were infected with HP238 at an MOI of 50 for 1 h. The infected cells were collected, fixed, and stained with anti-H. pylori antibody (red) and DAPI (blue) at various time points postinfection. The samples were observed with confocal or phase-contrast microscopy. The cell boundary is shown by dashed line. The plasma membrane-associated H. pylori bacteria are indicated by arrows. (b) Internalization of H. pylori on EEA1+ endosomes. AGS cells were infected with HP238 at an MOI of 20 for 1 h. The infected cells were collected, fixed, and stained with anti-H. pylori antibody (red) and anti-EEA1 antibody (green). The samples were observed with confocal microscopy. Arrows indicate the colocalization of H. pylori with the EEA1 early endosome marker. Arrowheads indicate no colocalization. (c) H. pylori on LAMP1+ lysosomes. As stated for panel b, the infected cells were stained with anti-H. pylori antibody (red) and anti-LAMP1 antibody (green). Arrows indicate the colocalization of H. pylori with the LAMP1 lysosome marker. Arrowheads indicate no colocalization. (d) Flow cytometric staining of H. pylori in AGS cells. AGS cells were infected with HP238 at an MOI of 25 for 1 h. Gentamicin (Gm; 150 μg/ml) was added to kill the extracellular bacteria. The infected cells were then cultured in gentamicin-containing medium (25 μg/ml) or gentamicin-free medium beginning at 5.5 h. At various time points, the infected cells were collected and stained with anti-H. pylori antibody without fixation. The mean fluorescence intensity of plasma membrane-associated-H. pylori in an arbitrary unit is shown to indicate the increased amount of anti-H. pylori staining. (e) Release of H. pylori into the culture supernatant. As stated for panel d, the supernatant from the infected AGS cells was collected for the recovery of the viable H. pylori (CFU). *, P < 0.05. Each experiment was repeated at least three times with the same results. Values are the means ± standard deviations from one representative experiment.