This article describes a method to visualize bacterial binding to von Willebrand factor (VWF) strings on human endothelial cells using fluorescence microscopy. The technique involves stimulating cells with histamine to promote VWF secretion and then introducing fluorescently labeled antibodies and bacteria to study their interactions.
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 morphology and the integrity of the HUVEC layer prior to injection of histamine and bacteria to the flow circulation. Maintain the flow setting. Induce the release of VWF from endothelial viable Palade bodies by injecting 136 microliters of a 100 millimolar histamine stock solution through an injection port into the ECGMS medium circulating in the perfusion tubing.
For immunofluorescence detection of multimerized VWF strings, inject 20 micrograms of a VWF specific FITC conjugated antibody in a volume of 200 microliters of PBS into the circulating 13.6 milliliters of ECGMS medium. To quantify Pneumococcal attachment to the VWF strings generated on HUVEC cell surfaces, inject 1.35 times 10 to the eighth CFU per milliliter RFP expressing Pneumococci in a maximum volume of one milliliter into the ECGMS medium using the injection port. Select a 63 times oil immersion objective for microscope magnification and adjust the fluorescence filter settings in the microscope software to the RFP channel with a 540 nanometer detection filter for detection of RFP expressing Pneumococci.
For quantification of bacterial attachment to the VWF strings, create snapshots of Z stacks of at least 30 representative field views each containing approximately 10 morphologically intact HUVEC and count the amount of Pneumococci.
繁體中文
