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An Assay to Study the Role of Vitronectin in Bacterial Adherence to Host Epithelial Cells

An Assay to Study the Role of Vitronectin in Bacterial Adherence to Host Epithelial Cells

Transkript

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.

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