Figure 1. Schematic drawing of trans-epithelial migration assay with chemical inhibition targeting epithelium or neutrophils.

Epithelial cells are matured on the underside of a Transwell filter with 3μm pore size. Chemical inhibitors (represented by darkened background) are then applied to the epithelium for two hours prior to infection, or the neutrophils for 30–60 minutes prior to migration. Transwells are washed and infected with PAO1 or mock infection. Neutrophils, unwashed, are then applied to the basolateral surface and allowed to undergo trans-epithelial migration. The transwell with the darkened epithelium represents migration across epithelium pre-treated with chemical inhibitor and washed prior to infection. The transwell with the neutrophils in a darkened suspension indicate neutrophils pre-treated with chemical inhibitor then applied, unwashed, to the epithelium to measure trans-epithelial migration.