Encyclopedia of Experiments: Immunology
A subscription to JoVE is required to view this content. Sign in or start your free trial.
Transcript
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.
