A Bacterial Infection Assay for Studying Anaerobic Bacteria-Host Cell Interactions
Transkript
For culture and experimentation with Porphyromonas gingivalis, a vinyl anaerobic chamber is maintained with an artificial atmosphere containing 80% nitrogen, 10% hydrogen, and 10% carbon dioxide.
Place 30 blood agar plates, 500 milliliters of PBS, and milliliters of vascular endothelial growth factor, or VEGF medium, inside the anaerobic chamber 24 hours in advance to equilibrate the reagents.
Seed 4 times 10 to the 5 human umbilical vein endothelial cells, or HUVECs, per well in six-well plates in VEGF medium and leave them in the cell culture incubator overnight for attachment. Prepare Porphyromonas gingivalis cultures in brain-heart infusion, or BHI medium, as described in the accompanying text, and allow the cultures to reach log phase while the optical density of the culture at 660 nanometers ranges between 0.5 to 0.7.
Transfer the bacteria to a 15-milliliter tube and wrap a piece of parafilm around the cap to prevent aerobic exposure. Then, centrifuge the bacteria at 5,000 times g for 10 minutes. Inside the anaerobic chamber, wash the pelleted bacteria with anaerobic PBS, and spin down again at 5,000 times g for 10 minutes. Resuspend the bacteria in VEGF medium.
To infect the HUVECs with Porphyromonas gingivalis, transfer the six-well plates from the incubator to the anaerobic chamber. Aspirate the medium from the test wells, and wash three times with anaerobic PBS.
Determine the cell number from one representative well by trypan blue exclusion assay. Then, add 2 milliliters per well of anaerobic VEGF medium and place the plates at 37 degrees Celsius in the anaerobic incubator for 20 minutes to equilibrate.
Determine the optical density of the bacteria and resuspend them in an adequate volume of VEGF medium to infect cells at a multiplicity of infection of 100 to 1 bacteria-to-cells ratio. Add the bacterial suspension to the cells and incubate for 30 minutes at 37 degrees Celsius for infection. For cell lysis, inside the anaerobic chamber, prepare a 1% weight per volume saponin solution in BHI medium, and filter through a 0.2-micron filter.
To study bacterial interactions with host cells, remove the six-well plate from the incubator and aspirate the infection medium. Wash three times with anaerobic PBS. Then, add 2 milliliters of saponin solution to each well and incubate for 15 minutes to allow cell lysis.
Next, scrape the cells from the bottom of the well, and dilute the cell lysate 1 to 1 with BHI medium. Then, make serial dilutions starting at 1 to 100. Plate 200 microliters of a suitable dilution on blood agar plates. Wrap the plates with parafilm, and incubate them in a 37 degrees Celsius anaerobic incubator for seven days to allow colony formation. Then, using a light box, count the colony-forming units, or CFUs on each plate.
