Evaluating Pneumococcal Attachment to Primary Human Endothelial Cells Under Constant Media Flow

Place a gelatin-coated channel slide, connected to a microfluidic pump, onto a microscope stage.

The slide contains primary human endothelial cells cultured under flowing media, with strings of von Willebrand factor or VWF, a glycoprotein, on the cell surface.

These strings are generated by stimulating the cells with histamine.

Controlled media flow, applied through the microfluidic pump, promotes VWF secretion and uncoiling. This exposes specific binding domains on the VWF strings.

Stop the media flow, add a green fluorophore-conjugated VWF-specific antibody, and resume the flow to label the strings with the antibody.

Pause the media flow and introduce red fluorescent protein-expressing pneumococci bacteria.

Restart the flow to distribute the bacteria, enabling attachment to the exposed domains through surface adhesins.

Using fluorescence microscopy, visualize real-time bacterial binding to the strings.

Stop the media flow and acquire Z-stack images to quantify bacterial attachment to the endothelial cells.

For microscopic visualization, place the fluidic unit into the 37 degrees Celsius heating chamber. Place the channel slide on the stage of a prewarmed microscope.

Control the cell morpho