Assessing Internalized Porphyromonas gingivalis in Host Endothelial Cells

1. Survival of internalized bacteria.

1. Prepare P. gingivalis anaerobic bacteria until they reach mid-log growth (OD₆₆₀ 0.5-0.7).
2. Centrifuge bacteria at 5,000 x g for 10 min.
3. 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 mid-log phase (~7 x 10⁸ cells/ml). The bacteria are now ready for infection.
4. Transfer 6-well plates containing HUVECs from the tissue culture incubator into the anaerobic chamber. Remove media and wash three times with anaerobic PBS. Add 2 ml of anaerobic VEGF media to each well and place the plates at 37 °C in the anaerobic incubator for 20 min to equilibrate the temperature for infection.
5. Infect host cells with bacteria at a multiplicity of infection (MOI bacteria: host) of 100:1.
   Note: HUVEC cell number is determined by performing a trypan exclusion test on a single well before infection. Bacterial cell number is determined via optical density (e.g., OD of 0.5 = 5 x 10⁸ cells/ml). Bacterial concentration is adjusted to proper MOI based on HUVEC's concentration.
6. Place 6-well plates with infected HUVECs into an anaerobic incubator and allow bacteria to interact with host cells for 30 min.
7. Prepare saponin in BHI (1.0% w/v) inside the anaerobic chamber and filter through a 0.2 µm filter.
8. Remove plates from the incubator. Wash three times with anaerobic PBS and add 2 ml VEGF media with supplemented antibiotic (300 µg/ml of gentamicin and 400 µg/ml of metronidazole).
9. Incubate for 1 hr. Be sure to test the antibiotics so they are 100% effective at killing the desired bacterial strain and make sure that they do not penetrate host cells.
10. Aspirate media, add 2 ml of filtered 1.0% saponin. Incubate for 15 min to allow host cell lysis.
11. Scrape the bottom of each well with a cell scraper. Collect the cell mixture from each well and make a 1:1 dilution in BHI.
12. Prepare serial dilutions of the sample (1:100, 1:1,000).
13. Plate 200 µl of desired dilution on blood agar plates. Wrap plates in parafilm and place them in an anaerobic incubator.
14. After seven days of incubation at 37 °C remove plates and count CFUs.