Confocal Imaging of Pseudomonas Infection in A549 Cells

For confocal imaging, 2 ×10⁵ A549 cells were seeded in 24-well plates containing 11 mm round glass coverslips and phosphate-free DMEM medium with 5% (v/v) FBS one day prior to infection. Infections with green fluorescence-labelled P. aeruginosa cells containing a σ^VreI-dependent red fluorescence transcriptional fusion were performed at a MOI of 10. At 0 and 10 hpi, the cells were fixated with 4% (v/v) paraformaldehyde in PBS. Samples were washed with PBS and coverslips were mounted on glass slides containing Fluoroshield mounting medium with DAPI (Sigma-Aldrich) to retain fluorescence and stain the A549 cells DNA. Confocal images were generated with a Nikon A1R confocal scanning laser microscope. NIS-Elements and ImageJ software were used to process the confocal images. Total corrected cellular fluorescence (TCCF) was calculated using the following equation: TCCF = integrated density − (area of selected cell × mean fluorescence of background readings), as described before^{56 (link),57 (link)}.