Related Experiment Video
Updated: Apr 5, 2026
Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization
Published on: July 28, 2015
Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization
1Focal Area Infection Biology, Biozentrum, University of Basel.
Abstract:
Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like Shigella flexneri and Francisella novicida need to induce the rupture of the phagosome. In contrast, Salmonella typhimurium replicates in a vacuolar compartment, known as Salmonella-containing vacuole (SCV). However certain mutants of Salmonella fail to maintain SCV integrity and are thus released into the cytosol. The percentage of cytosolic vs. vacuolar bacteria on the level of single bacteria can be measured by differential permeabilization, also known as phagosome-protection assay. The approach makes use of the property of detergent digitonin to selectively bind cholesterol. Since the plasma membrane contains more cholesterol than other cellular membranes, digitonin can be used to selectively permeabilize the plasma membrane while leaving intracellular membranes intact. In brief, following infection with the pathogen expressing a fluorescent marker protein (e.g. mCherry among others), the plasma membrane of host cells is permeabilized with a short incubation in digitonin containing buffer. Cells are then washed and incubated with a primary antibody (coupled to a fluorophore of choice) directed against the bacterium of choice (e.g. anti-Salmonella-FITC) and washed again. If unmarked bacteria are used, an additional step can be done, in which all membranes are permeabilized and all bacteria stained with a corresponding antibody. Following the staining, the percentage of vacuolar and cytosolic bacteria can be quantified by FACS or microscopy by counting single or double-positive events. Here we provide experimental details for use of this technique with the bacterium Salmonella typhimurium. The advantage of this assay is that, in contrast to other assay, it provides a quantification on the level of single bacteria, and if analyzed by microscopy provides the exact number of cytosolic and vacuolar bacteria in a given cell.
Insights
This study details a phagosome-protection assay to quantify intracellular bacteria, differentiating between those in vacuoles and the cytosol. The method uses digitonin to selectively permeabilize host cell membranes for accurate bacterial localization.
Area of Science:
- Microbiology
- Cell Biology
- Immunology
Background:
- Intracellular bacterial pathogens exhibit diverse replication strategies, residing either within pathogen-containing vacuoles (PCVs) or the host cell cytosol.
- Pathogens like Shigella flexneri and Francisella novicida must breach the phagosome to access the cytosol, while Salmonella typhimurium typically replicates within a Salmonella-containing vacuole (SCV).
- Certain Salmonella mutants may compromise SCV integrity, leading to cytosolic release, necessitating methods to distinguish these populations.
Purpose of the Study:
- To present a detailed methodology for the phagosome-protection assay, a technique enabling single-bacterium resolution of intracellular bacterial localization.
- To provide experimental specifics for applying this assay to Salmonella typhimurium infections.
- To highlight the assay's advantage in offering precise quantification of cytosolic versus vacuolar bacteria.
Main Methods:
- The assay employs digitonin, a cholesterol-binding detergent, to selectively permeabilize the host cell plasma membrane while preserving intracellular membranes.
- Following selective permeabilization and antibody staining against the bacteria, flow cytometry (FACS) or microscopy quantifies cytosolic and vacuolar bacterial populations.
- The protocol includes steps for both fluorescently tagged and untagged bacteria, ensuring versatility.
Main Results:
- The phagosome-protection assay allows for the discrimination of intracellular bacteria based on their location (cytosolic vs. vacuolar) at a single-bacterium level.
- Microscopy analysis provides exact counts of bacteria within each compartment per cell.
- The method is validated for Salmonella typhimurium, offering a quantitative approach to studying bacterial intracellular dynamics.
Conclusions:
- The phagosome-protection assay is a powerful tool for precisely quantifying the proportion of cytosolic and vacuolar intracellular bacteria.
- This technique offers significant advantages over other methods by providing single-bacterium resolution and detailed cellular localization data.
- Accurate determination of bacterial location is crucial for understanding host-pathogen interactions and bacterial pathogenesis.
More Related Videos
10:01Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites
Published on: March 16, 2018
11:10High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages
Published on: August 11, 2014