Assessing Internalized Porphyromonas gingivalis in Host Endothelial Cells

JoVE Encyclopedia of Experiments

Immunologie

0 weergaven • 2:37 min • July 8th, 2025

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During Porphyromonas gingivalis infection, the fimbriae on the anaerobic bacteria bind to specific receptors on host cells.

This binding initiates intracellular signaling cascades, resulting in actin rearrangement and bacterial internalization within phagosomes.

To assess the survival of internalized Porphyromonas gingivalis within endothelial cells in vitro, take a multi-well plate containing endothelial cells adhered to coverslips.

Add Porphyromonas gingivalis suspension. Incubate to facilitate bacterial internalization.

Remove media and wash using an anaerobic buffer to remove non-internalized bacteria.

Add media containing antibiotics, impermeable to the endothelial cells, to eliminate the surface-attached bacteria.

Incubate with a mild surfactant to lyse the endothelial cells, releasing the bacteria without causing their lysis.

Dilute the collected lysate with media. Prepare serial dilutions of the lysate.

Plate the dilutions on blood agar plates. Incubate under anaerobic conditions for Porphyromonas gingivalis to grow and form visible colonies.

Count the colonies to assess the ability of Porphyromonas gingivalis to invade and survive within endothelial cells.

Following infection of HUVEC cells, wash the cells three times with anaerobic PBS. Next, add 2 milliliters of VEGF medium supplemented with pre-optimized concentrations of antibiotics to the HUVEC cells. Incubate the cells for one hour. Aspirate the medium, and add 2 milliliters of 1% saponin solution in BHI to each well. Incubate for 15 minutes.

Next, scrape the lysed cells, and collect the lysate in a centrifuge tube. Dilute the lysate 1 to 1 with BHI medium, and then prepare serial dilutions starting at 1 to 100. Plate 200 microliters of the suitable concentrations on blood agar plates. Allow the bacteria to grow for seven days in an anaerobic chamber, and then count the colonies.

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Laatst bijgewerkt: 15 augustus 2026