eoe sub articles

EoE Sub Articles

1. Survival of internalized bacteria.
<ol>
	<li>Prepare P. gingivalis anaerobic bacteria until they reach mid-log growth (OD660 0.5-0.7).</li>
	<li>Centrifuge bacteria at 5,000 x g for 10 min.</li>
	<li>Place pelleted P. gingivalis back in the chamber, and discard the supernatant. Wash with PBS, and pellet bacteria again before resuspending in VEGF media. Prepare suspensions for all bacterial strains to be tested at OD660 of 0.7 which corresponds to the ...

assessing internalized porphyromonas gingivalis in host endothelial cells

Source: Wunsch, C. M., et al. <a href="https://app.jove.com/v/53408">Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells.</a> J. Vis. Exp. (2015)
In this video, we demonstrate an assay to assess the survival of Porphyromonas gingivalis inside human endothelial cells. The measure of survival and growth of internalized Porphyromonas gingivalis is evaluated by the number of colonies on the blood agar plate.

Assessing Internalized Porphyromonas gingivalis in Host Endothelial Cells

1. Survival of internalized bacteria.

	Prepare P. gingivalis anaerobic bacteria until they reach mid-log growth (OD660 0.5-0.7).
	Centrifuge bacteria at ...

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.