All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.
- Intracellular replication assay
NOTE: All volumes are consistent with an assay using two 6-well plates.
- Subculture overnight Shigella cultures via 1:50 dilution into fresh media.
- Vortex, then add 100 µL of each overnight culture to 5 mL of fresh TSB (Tryptic soy broth) or TSB + BS (Bile salts) in a suitably sized culture tube.
NOTE: Limit culture volume to <20% of culture flask or tube volume to ensure proper aeration. - Incubate at 37 °C with shaking at 250 rpm until cells reach an OD600 (Optical density) of 0.7 (mid-log phase of Shigella growth); about 2-2.5 h.
NOTE: During the subculture, aliquot 50 mL of DMEM + 50 mg/mL gentamicin and a sufficient volume of PBS (Phosphate-buffered saline) for all washing steps and place in a 37 °C water bath. Allow media to reach 37 °C before use.
- Transfer 2 x 108 CFUs (Colony forming units) subcultured Shigella to individual 2 mL microcentrifuge tubes.
NOTE: 2 x 108 CFUs corresponds to approximately 1 mL of bacterial cells at an OD600 of 0.7. Use OD600 readings to approximate CFU/mL according to the calibration of each individual spectrophotometer. - Wash Shigella samples 1x with PBS.
- Pellet cells by centrifugation at 17,000 x g for 2 min at room temperature. Aspirate the supernatant, then add 1 mL of warm PBS and resuspend the pellet well, gently pipetting the sample up and down until the mixture is fully homogeneous (8-10x).
- Repeat the washing step one additional time.
- Pellet cells by centrifugation at 17,000 x g for 2 min at room temperature, aspirate the supernatant, and resuspend the pellets in 2 mL of warm DMEM.
NOTE: The final concentration of resuspended bacteria will be 1 x 108 CFU/mL.
- Vortex, then add 1 mL (1 x 108 CFUs) of resuspended Shigella plus 1 mL of DMEM (Dulbecco’s Modified Eagle Medium) to each well of prepared HT-29 colonic epithelial monolayers in 6-well plates.
NOTE: Infections are normally performed at a multiplicity of infection (MOI; ratio of bacterial to epithelial cells) of 100. To test different MOIs, dilute resuspended Shigella in DMEM to the desired concentration, then add 1 mL of diluted bacteria to HT-29 monolayers. For example, to test an MOI of 10, dilute bacteria 1:10 by adding 150 µL of 1 x 108 CFU/mL bacteria to 1.35 mL of DMEM, then apply 1 mL (1 x 107 CFUs) to HT-29 cells. - To promote bacterial contact with the HT-29 cells, centrifuge the 6-well plates at 2,000 x g for 10 min at room temperature or 37 °C if the temperature setting can be adjusted.
NOTE: Centrifugation promotes bacterial contact with the HT-29 cells, which bypasses the need for adherence factors and allows the bacteria to quickly invade the cells. - Incubate 6-well plates at 37 °C with 5% CO2 for 45 min.
- During the incubation, determine the bacterial infection titer.
- Prepare 10-fold serial dilutions of resuspended Shigella cells into PBS.
- Plate 100 µL of the 1 x 10-5 and 1 x 10-6 dilutions onto TSB + Congo red plates and incubate overnight at 37 °C.
NOTE: Plating 100 µL from the 1 x 10-5 and 1 x 10-6 dilutions corresponds to a final dilution factor of 1 x 10-6 and 1 x 10-7, respectively.
- Thoroughly wash infected HT-29 cells 3x with 1 mL of PBS.
- Aspirate media from each well.
NOTE: When aspirating media from 6-well plates, guide the tip of the aspirator along the bottom side of the wells, trying to avoid contact with the HT-29 cells. - Add 1 mL of warm PBS to each well and wash gently.
NOTE: To gently wash 6-well monolayers with PBS, move the plate up and down and side to side on the benchtop. Washing plates in a circular motion and/or removing the plate from the benchtop surface can cause the mechanical removal of cells from plastic. - Repeat the washing steps two additional times.
- Remove PBS by aspiration, then add 2 mL of warm DMEM supplemented with 50 µg/mL gentamicin to each well and incubate for 30 min at 37 °C with 5% CO2.
- Thoroughly wash infected HT-29 cells 3x with 1 mL of PBS.
- Repeat washing step 1.8.
- Remove PBS by aspiration, then add 2 mL of warm DMEM with 50 µg/mL gentamicin to each well of the 6-well plates and incubate at 37 °C with 5% CO2 for the desired length of time to allow for intracellular replication (up to 24 h).
- Thoroughly wash cells 2x with 1 mL of PBS.
- Repeat washing step 1.8.
- Remove PBS by aspiration and lyse HT-29 cells by adding 1 mL of PBS + 1% Triton X-100 to each well.
- Incubate 6-well plates at 37 °C for 5 min.
- Use a cell scraper or bent pipette tip to scrape the lysed cells from the bottom of the well and transfer the full 1 mL into a fresh 1.7 mL microcentrifuge tube.
- Determine the number of intracellular bacteria.
- Vortex each tube (from step 6.15) for at least 30 s to further displace Shigella from the lysed eukaryotic cells.
- Prepare 10-fold serial dilutions of lysates into PBS.
- Plate 100 µL of the 1 x 10-2, 1 x 10-3, and 1 x 10-4 dilutions onto TSB + Congo Red plates and incubate overnight at 37 °C.
NOTE: Plating 100 µL from the 1 x 10-2, 1 x 10-3, and 1 x 10-4 dilutions corresponds to a final dilution factor of 1 x 10-3, 1 x 10-4, and 1 x 10-5, respectively.
