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

JoVE Encyclopedia of Experiments

Microbiologie

0 weergaven • 3:20 min • September 26th, 2025

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

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.

08:30

Onderzoeken van von Willebrand Factor pathofysiologie met behulp van een Flow kamer Model van von Willebrand Factor-bloedplaatjes String vorming

Gerelateerde video's

0 Bekeken

07:50

Een In Vitro Model van een Parallel-plaat perfusie-systeem te bestuderen van de naleving van de bacteriële om te enten van weefsels

Gerelateerde video's

0 Bekeken

07:21

Geautomatiseerde, high-throughput detectie van bacteriële hechting aan gastheercellen

Gerelateerde video's

0 Bekeken

12:43

Parallelle plaat flow kamer en Continuous Flow Circuit te endotheliale progenitorcellen Evalueer onder laminaire stroom Afschuifspanning

Gerelateerde video's

0 Bekeken

03:10

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

Gerelateerde video's

0 Bekeken

11:22

Kwantitatief In vitro Assay om neutrofieladhesie Maatregel om Activated primaire humane microvasculaire endotheelcellen onder statische omstandigheden

Gerelateerde video's

0 Bekeken

11:38

Een Flow adhesieanalyse om te studeren leukocyten rekrutering van humane lever Sinusvormige endotheel onder omstandigheden van Afschuifspanning

Gerelateerde video's

0 Bekeken

12:42

Human Neutrophil Flow Kamer adhesietest

Gerelateerde video's

0 Bekeken

11:26

Real-time beeldvorming van endotheelcellen-cel Junctions Tijdens Neutrophil Transmigration onder fysiologische Flow

Gerelateerde video's

0 Bekeken

10:24

Kwantificering van endotheelcellen Hechtvermogen In Vitro

Gerelateerde video's

0 Bekeken

Laatst bijgewerkt: 1 augustus 2026