An Assay to Study the Role of Vitronectin in Bacterial Adherence to Host Epithelial Cells
Transcrição
Take a multi-well plate containing adherent epithelial cell monolayers. Replace media with serum-free media, preventing serum protein interference. Post-incubation, remove media. Wash the cells with buffer. Add chilled serum-free media containing vitronectin. Incubate at lower temperatures.
The N-terminal RGD-domain of vitronectin binds to the epithelial cell surface integrin receptors. Lower temperatures inhibit cellular internalization of the vitronectin-integrin complex.
Following integrin receptor saturation, remove media. Wash with buffer to remove excess vitronectin, which could impact the bacterial vitronectin-dependent integrin binding.
Add Haemophilus influenzae type f, or Hif, a gram-negative bacterial pathogen, suspension. Incubate.
Protein H on the Hif surface binds to the vitronectin's C-terminus, facilitating Hif adherence to the epithelial cell and subsequent internalization.
Remove media and wash with buffer to remove unbound Hif. Use a cell detachment enzyme to harvest the cells. Add serum-containing media to stop the enzymatic reaction. Transfer the cell suspension to a glass tube containing glass beads. Vortex to lyse the cells, releasing internalized and surface-attached bacteria.
Dilute the lysate with serum-free media and seed on a chocolate agar plate. Incubate for Hif to form colonies. Count the colonies.
A higher number of colonies indicates enhanced Hif adherence to the epithelial cells in the presence of vitronectin.
