Micro-Parallel Plate Flow Chamber Model for Studying Bacterial Adhesion to Human Endothelial Cells

Take a gelatin-coated coverslip and seed it with human primary endothelial cells. Incubate to allow cell adherence and proliferation.

Place the coverslip at the base of a micro-parallel plate flow chamber.

Attach the upper parts of the chamber.

Connect the inlet to a syringe filled with a calcium ionophore solution.

Perfuse the chamber with the solution.

The ionophore binds to calcium ions and transports them across the membrane, increasing intracellular calcium levels.

This triggers the release of a vesicle containing the von Willebrand factor (VWF), a glycoprotein.

VWF forms elongated strings on the cell surface under flow conditions.

Now, perfuse fluorescently labeled bacteria through the inlet to apply a fluid flow that mimics shear stress in blood vessels.

The bacteria bind to VWF via surface adhesins.

Wash with a buffer to remove unbound bacteria.

Using fluorescence microscopy, image and quantify bacterial adhesion to the host cells.

For experiments with endothelial cells, coat plastic slips with one milliliter of a 1% gelatin solution in PBS and incubate them for 30 minutes at 37 degrees Celsius.

Then, seed human umbilical vein endothelial cells on the gelatin-coated plastic slips and gr