High-Throughput Imaging of Intracellular Brucella abortus Infection in Host Epithelial Cells

Begin with epithelial cells in which expression of the interferon adaptor protein, a key mediator of innate immunity, has been reduced, making the cells more susceptible to Brucella infection.

Replace the medium with an infection medium containing green fluorescent protein–expressing Brucella abortus, a facultative intracellular pathogen, at the desired concentration.

Centrifuge the plate to promote contact between bacteria and host cells, then incubate to allow bacterial internalization.

Wash the cells with serum-containing medium, and treat them with an antibiotic to eliminate extracellular bacteria.

Incubate further to allow Brucella to multiply intracellularly.

Wash the cells with buffer and fix them with paraformaldehyde to preserve intracellular structures.

Rinse off the excess fixative. Then, add detergent-supplemented buffer to permeabilize the cells.

Wash away the residual detergent, then add DAPI to stain the nuclei, and perform a final wash.

Using an automated microscope, acquire high-throughput images of Brucella infection in the host epithelial cells.

Using an automated plate washer, exchange the transfection medium with 50 microliters of infection medium. Inside the hood, transfer the plate into the centrifuge cage and centrifuge the 384-well plate at 400 times g and four degrees Celsius for 20 minutes.

Then, use parafilm to seal the plate and incubate it on a prewarmed aluminum plate at 37 degrees Celsius and 5% CO₂ for four hours. After the incubation with the automated plate washer, use DMEM with 10% FCS containing 100 micrograms per milliliter of Gentamicin or GM to wash the cells to inactivate extracellular bacteria. Then, use parafilm to seal the plate and place it back on the prewarmed aluminum plate in the incubator for another 40 hours for the endpoint assay.

To fix the cells, first use PBS to wash the wells. Then, exchange the PBS with 50 microliters of 3.7% PFA in 0.2 molar HEPES at pH 7.4 and incubate the cells at room temperature for 20 minutes.

After the incubation, use 50 microliters of PBS to exchange the fixation medium. To stain the samples with the automated plate washer, begin by using 50 microliters of 0.1% Triton X-100 in PBS for 10 minutes to permeabilize the cells.

After using PBS to wash the cells three times, add 50 microliters of PBS with one microgram per milliliter of DAPI and incubate at room temperature for 30 minutes. Then, use PBS to wash the cells three times before protecting them from light. Transfer the plate to the automated microscope.