Visualization of Internalized Anaerobic Bacteria within Host Endothelial Cells

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.

1. Autoclave coverslips. Aseptically add coverslips to 12-well plates before seeding endothelial cells at 5 x 10⁴ cells/well. (Prepared day before experiment)
2. Have endothelial cells prepared on 18 mm (#1.5 thickness) circular coverslips in 12-well plates as described above.
3. Prepare anaerobic bacteria grown to mid-log phase (OD₆₆₀ = 0.5-0.7).
4. Wash bacteria 2x with anaerobic PBS by centrifuging at 5,000 x g and suspending the pellet in PBS at 5-7 x 10⁸ cells/ml.
5. Add 20 µl of 0.2 mM BCECF-AM to 2 ml of bacterial suspension (5-7 x 10⁸ cells/ml) to a final concentration of BCECF-AM of 2 µM.
6. Incubate at 37 °C for 30 min in the dark.
7. Transfer plates with endothelial cells seeded on 18 mm (#1.5 thickness) circular coverslips from tissue culture incubator into the anaerobic chamber. Wash with PBS and exchange with anaerobic VEGF media.
   Note: Verify that HUVECs are healthy under a light microscope. HUVECS should be ~80% confluent, morphology should be comparable to the manufacturers.
8. Centrifuge labeled bacteria at 5,000 x g for 10 min to remove residual BCECF-AM dye. Suspend in 2 ml anaerobic VEGF media.
9. Infect host cells with labeled bacteria at MOI of 100:1 (bacteria: host).
10. Incubate in an anaerobic chamber at 37 °C for 30 min.
11. After infection wash cells with PBS three times and fix in freshly prepared 4.0% paraformaldehyde for 10 min.
    Note: After fixing cells, experiments can be conducted outside of the anaerobic chamber.
12. Wash coverslips with PBS three times.
13. Add 1 ml of 0.2% Triton X-100 for 10 min.
14. Wash coverslips with PBS three times.
15. Add 50 µl of TRITC phalloidin (50 µg/ml) to coverslips for 45 min.
16. Wash coverslips three times, remove from the 12-well plate and place on a slide with a soft-set mounting medium containing DAPI. Seal the sides with nail polish.
    Note: Slides may be stored for a couple months in the dark. Avoid light exposure to prevent photo-bleaching.
17. View slides using a confocal microscope.
    1. Here, use a 34 channel spectral system (32-channel array detector and two side PMT detectors, plus a transmitted light detector) configured around an AxioObserver (inverted) stand with a motorized XY stage. The system has five lasers: blue diode (405 nm), multi-line Argon (458, 488, 514 nm), green diode (561 nm), red HeNe (633 nm) and a 440 nm pulsed laser. Equip a Fluorescence Lifetime Imaging system with 2 hybrid GaAsP detectors (for FRET-FLIM).
    2. Detect the fluorescence from DAPI and TRITC in one channel using a dual-band filter with excitation wavelengths of 340-380 nm and 540-560 nm, and an emission filter of 435-485 nm and 570-590 nm respectively. Detect the fluorescence from BCECF-AM using a filter with excitation wavelength of 440-500 nm and an emission filter of 510-590 nm.
       Note: Controls for BCECF-AM should be done on every bacterial strain being studied to ensure proper labeling of viable bacteria. First validate that nonviable bacteria are DAPI-positive and BCECF-negative. Second, ensure that live bacteria can metabolize BCECF-AM into fluorescein BCECF. Variable concentrations of the bacteria or BCECF-AM dye may need to be tested for optimal labeling.