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

In This Article

Abstract

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).

Protocol

1. Digitonin Assay with mCherry-expressing Salmonella (FACS-based Analysis)

Preparing bacteria
Note: Salmonella wild-type SL1344 (Streptomycin resistant) expressing mCherry from a plasmid (pFPV encoding mCherry under the rpsM promoter) is used in this assay. Bacterial phagocytosis and proliferation were not altered by the constitutive expression of mCherry (data not shown).
1. Inoculate the strain 1 day before the infection in 3 ml of LB medium supplemented with Streptomycin (90 µg/ml) and Ampicillin (100 µg/ml, mCherry expressing vector) in a shaker at 37 °C O/N.

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Results

Bacterial compartmentalization analysis using flow cytometry; process diagram showing FACS setup.
Figure 1. Schematic representation of protocol 1. Infected cells containing mCherry-positive Salmonella either in intact vacuoles or the cytosol are differentially permeabilized with digitonin. Cytosolic Salmonella are stained with anti-<.......