Detection of Bacterial Adherence to Host Cells Using Fluorescence Imaging

Take a multi-well plate containing adherent epithelial cell monolayer susceptible to bacterial adherence.

Add varying numbers of fluorescent protein-tagged Pseudomonas aeruginosa, a pathogenic bacterium, to achieve the desired bacteria-to-epithelial cell ratio.

Centrifuge to bring the bacteria closer to the epithelial cells, facilitating interactions.

Type IV pili, proteinaceous fibers on the bacteria, play a pivotal role by binding to specific glycolipids on the epithelial cells, mediating bacterial adherence to the host cells.

Post-incubation, wash with a buffer to remove non-adherent bacteria. Fix the cells to preserve the cell structure.

Add DAPI, a fluorescent stain that binds strongly to A-T-rich DNA regions, staining the nucleus. Cover the cells with buffer to prevent cell drying.

Acquire images of the GFP-labeled bacteria attached to epithelial cells with DAPI-stained nucleus. Enumerate the adherent bacteria on a single epithelial cell.

Increased adherent bacteria per host cell in a dose-dependent manner, enables quantification of host-pathogen interactions.

For bacterial adherence assay, wash the previously cultured A549 cell monolayers three times by adding 100 microliter