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 …
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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 |
