An Assay to Quantify Cytosolic and Vacuolar Salmonella in Infected Primary Macrophages

Take a culture of bone marrow-derived macrophages. Remove the media, and add Salmonella bacteria, expressing a fluorescent cytoplasmic protein.

Centrifuge to facilitate bacteria-macrophage contact, and incubate.

The bacteria bind to macrophage receptors, facilitating internalization inside a phagosome.

The phagosome fuses with endosome-derived vesicles, and simultaneously, the bacteria secrete effectors, remodeling the phagosome into a Salmonella-containing vacuole or SCV.

A subset of SCVs ruptures, releasing internalized bacteria into the cytoplasm.

Add an antibiotic to eliminate non-internalized bacteria. Aspirate the media containing dead bacteria.

Introduce a buffer containing the detergent digitonin, which binds to cholesterol.

Digitonin binding to the cholesterol-rich plasma membrane results in pore-formation, while the cholesterol-deficient SCV membrane remains intact — a phenomenon termed differential permeabilization.

Add fluorescently labeled antibodies that enter permeabilized cells to bind cytosolic bacteria.

Use a detergent to lyse the cells and SCVs, releasing all internalized bacteria.

Using flow cytometry, quantify dual-stained cytosolic bacteria and single-stained vacuolar bacteria.