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

Source: Meunier, E., et al. Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization. J. Vis. Exp. (2015).

This video demonstrates an assay to quantify cytosolic and vacuolar Salmonella typhimurium inside bone marrow-derived macrophages using differential permeabilization. The macrophages are infected with a fluorescent protein-expressing Salmonella, which is internalized inside a specialized vacuolar structure called Salmonella-containing vacuole or SCV. A subset of bacteria escape the SCV and enter the cytosol. The cholesterol-rich plasma membrane of macrophages is permeabilized using digitonin, leaving the SCV intact. Upon labeling the cytosolic bacteria with a fluorescently tagged antibody, the percentage of vacuolar and cytosolic bacteria is quantified using fluorescence-activated cell sorting (FACS).