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