Visualization of Internalized Anaerobic Bacteria within Host Endothelial Cells

Published: November 30, 2023

Abstract

Source: Wunsch, C. M. et. al., Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells. J. Vis. Exp. (2015)

In this video, we demonstrate a procedure to visualize internalized Porphyromonas gingivalis, an anaerobic pathogenic bacterium, within human endothelial cells using confocal microscopy. The bacteria are stained with a fluorescent dye while the host endothelial cell actin cytoskeleton is labeled with a different fluorescent dye. The internalized Porphyromonas gingivalis inside endothelial cells are visualized using confocal microscopy.

Protocol

1. Internalization of Bacteria into Host Cells (Fluorescent Microscopy) Note: P. gingivalis is labeled with 2',7'-Bis-(2-Carboxyethyl)-5-(and-6)-Carboxyfluorescein, Acetoxymethyl Ester (BCECF-AM). BCECF-AM is a non-fluorescent membrane-permeable dye; its conversion to fluorescein BCECF via the action of intracellular esterases can indicate cell viability. P. gingivalis is labeled with the BCECF-AM dye and then used to infect eukaryotic …

開示

The authors have nothing to disclose.

Materials

Vinyl Anaerobic Chamber-Type B Coy Laboratory Products Model 2000 incubator
TSA II Trypticase Soy Agar w/5% Sheep Blood BBL 221261
Human Umbilical Vein Endothelial Cells 10-donor Pool LifeLine Technology FC-0044
VascuLife VEGF Medium Complete Kit LifeLine Technology LL-0003
TrypKit LifeLine LL-0013
BCECF-AM LifeTechnologies B1150
TRITC Phalloidin Sigma-Aldrich P1951
18 mm Circular Coverslips Electron Microscopy Sciences 72222-01
VectaShield Mounting Medium with DAPI Vector Laboratories H-1200

Play Video

記事を引用
Visualization of Internalized Anaerobic Bacteria within Host Endothelial Cells. J. Vis. Exp. (Pending Publication), e21796, doi: (2023).

View Video