A Model of Intracellular Persistence of Pseudomonas aeruginosa in Airway Epithelial Cells

Figure 3

Detection of intracellular P.a. by confocal microscopy. (a) Z-projection images (AF488, AF647, and merged channels) to distinguish extracellular from intracellular bacteria in AEC at 4 h p.i or uninfected negative controls. (b) Z-projection and orthogonal views (merged channels) to localize bacteria at different time points. Green and red arrows denote the plane of cut for the and plane projections, respectively. Extracellular P.a. (gray arrowhead) was double-stained by the total (AF488 green) and extracellular (AF647 red) stains, while intracellular P.a. (white arrowhead) was single stained with the total (green) stain. The actin cytoskeleton was stained with phalloidin (grey) and the AEC nuclei with DAPI (blue). (c) The percentage of AEC harboring intracellular P.a. signals among all AEC. (d) The percentage of AEC harboring a given number of intracellular P.a. per AEC at different time points (among all AEC with intracellular P.a.). For all experiments, BEAS-2B cells were infected with PAO1 at MOI 1, and confocal microscopy images were analyzed manually. For (a) and (b), representative images are shown, with . Results in (c) and (d) shown are from pooled data (≥ 1000 cells or ≥ 16 fields of view per conditions) from 2 independent experiments.

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