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 grow them to 70 to 80% confluency. Mount the endothelial cell-coated plastic slip in the bottom part of the flow chamber. Then, connect the inlet and outlet tubing to the upper part of the flow chamber and connect the outlet tubing to a waste container.

Gently place the upper part of the flow chamber on the bottom part and assemble the flow chamber while avoiding the formation of air bubbles. Inject one milliliter of medium through the chamber to make sure that the chamber is not leaking and to remove excess coating solution.

Next, set up the infusion pump for a one milliliter syringe so that it flows at 75 microliters per minute. Working in a dark room, fill a one milliliter syringe with one milliliter of fluorescently labeled bacteria and place it into the infusion pump. Connect the outlet of the syringe to the inlet of the flow chamber.

Taking care to avoid air bubbles, start the infusion pump for 10 minutes at 75 microliters per minute. After 10 minutes, remove the first syringe and connect a one milliliter syringe containing PBS or DMEM to the inlet tube. Start the infusion, pump, and rinse for 10 minutes to wash away any unbound bacteria.

Once rinsed, leave the infusion pump on and take at least 15 black and white images using an exposure time of 1.5 seconds in random locations, spread over the coated surface of the flow chamber.