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.
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 cells. Following infection, cells are fixed and labeled with DAPI and TRITC-phalloidin. The DAPI stain used to stain the eukaryotic cell nucleus will also label bacterial cell nucleus, which provides a countermeasure to identify non-viable bacteria that cannot metabolically cleave BCECF-AM. Host cells are highlighted with TRITC-phalloidin, a red actin dye.
The authors have nothing to disclose.
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 |