Intracellular Replication Assay for Shigella in Human Colonic Epithelial Cells

Take a culture of Shigella, a pathogenic bacterium.

Add the bacterial suspension and culture medium to the differentiated human colonic epithelial monolayer in a culture plate.

Centrifuge to promote direct contact between bacteria and epithelial cells.

Incubate to allow bacterial internalization.

Wash with buffer to remove the extracellular bacteria.

Add antibiotic-supplemented culture medium. During incubation, antibiotics selectively eliminate the extracellular bacteria without affecting intracellular ones.

Remove the media. Wash the cells with buffer to remove the dead bacteria.

Add fresh antibiotic-supplemented culture medium to prevent cross-contamination.

Incubate to allow internalized bacteria to multiply.

Remove media and wash the cells with buffer.

Add detergent-containing buffer to lyse the epithelial cells containing bacteria.

Scrape the lysed cells and collect in a microcentrifuge tube.

Vortex to release the bacteria from the lysed cells.

The recovered bacteria are ready for downstream analysis.

To begin, take the overnight-grown Shigella cultures and vortex them. Add 100 microliters of the culture to 5 milliliters of fresh TSB in a culture tube. Incubate the cultures at 37 degrees Celsius while shaking at 250 RPM until an optical density of 0.7 is reached at 600 nanometers.

Transfer two times ten to the eight colony-forming units of subcultured Shigella to 2 milliliter microcentrifuge tubes. Pellet cells by centrifugation at 17,000 g for 2 minutes at room temperature. Aspirate the supernatant.

Add 1 milliliter of warm PBS and resuspend the pellet thoroughly. After centrifuging the cell once again, resuspend the pellet in 2 milliliters of warm DMEM and vortex the cell suspension. Then, add 1 milliliter of resuspended Shigella and 1 milliliter of DMEM to each well of HT-29 colonic epithelial monolayers in six-well plates.

To ensure bacterial contact with HT-29 cells, centrifuge the six-well plates at 2,000 g for 10 minutes at room temperature or 37 degrees Celsius. Incubate six-well plates at 37 degrees Celsius and 5% carbon dioxide for 45 minutes. After incubation, aspirate media from each well. Add 1 milliliter of warm PBS to each well and wash gently.

Then, add 2 milliliters of warm DMEM with 50 micrograms per milliliter of gentamicin and incubate for 30 minutes at 37 degrees Celsius with 5% carbon dioxide. Wash infected HT-29 cells three times with 1 milliliter of PBS. Add 2 milliliters of warm DMEM with gentamycin to each well and incubate for up to 24 hours for intracellular replication at 37 degrees Celsius with 5% carbon dioxide.

At the end of the incubation, wash cells two times with 1 milliliter of PBS. Add 1 milliliter of PBS with 1% Triton X-100 to each well to lyse HT-29 cells. Then, using a cell scraper or bent pipette tip, scrape the lysed cells from the well bottoms and transfer the mixture into a 1.7 milliliter microcentrifuge tube.

Vortex each tube for at least 30 seconds to further displace Shigella from the lysed eukaryotic cells. Prepare tenfold serial dilutions of the lysates in PBS. Plate 100 microliters of the serial dilutions onto Congo red plates with TSB and incubate overnight at 37 degrees Celsius.