FIG. 1.

Schematic diagram (A) and immunofluorescence microscopy pictures (B) illustrating the in vitro traversal model. Human corneal epithelial cells were grown air lifted on Transwell tissue culture inserts (3-μm pore size) under high-calcium conditions (1.15 mM) to induce a polarized multilayered epithelium. To measure bacterial traversal, bacteria were added only to the apical compartment, and bacteria traversing to the basal compartment were enumerated at various times postinoculation. Fluorescence microscopy using rhodamine-conjugated phalloidin (red) to label the actin cytoskeleton and DAPI (blue) to label cell nuclei showed that the cells formed a multilayered epithelium to which P. aeruginosa (FITC labeled using antibody to PAO1 [green]) can adhere and traverse (B). Magnification, ∼×1,000. Bar, 10 μm.