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, tran