Vacuolar Rupture Assay
Vacuolar Rupture Assay - Related Videos
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Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens
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Cited by 22 •
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We describe a method for tracking the endomembrane rupture elicited by the intracellular bacteria Shigella flexneri and Mycobacterium tuberculosis upon host cell invasion. Our assay makes use of CCF4, a host cytoplasmic FRET probe in live or fixed cells. This reporter is degraded by an enzyme activity present on the bacterial surface.
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An Assay to Quantify Cytosolic and Vacuolar Salmonella in Infected Primary Macrophages
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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,...
09:25
Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization
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Cited by 19 •
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We describe the quantification of cytosolic and vacuolar Salmonella typhimurium in bone-marrow derived macrophages using differential digitonin permeabilization.
13:55
Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions
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Cited by 40 •
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Vacuolar and cytosolic pH can be measured in live yeast (S. cerevisiae) cells using ratiometric fluorescent dyes localized to specific cellular compartments. We describe procedures for measuring vacuolar pH with BCECF-AM, which localizes to the vacuole in yeast, and cytosolic pH with a cytosolic ratiometric pH-sensitive GFP (yeast pHluorin).
05:16
A Photodynamic Approach to Study Function of Intracellular Vesicle Rupture
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AlPcS2a-mediated chromophore-assisted laser inactivation (CALI) is a powerful tool for studying spatiotemporal damage of intracellular vesicles (IVs) in live cells.
FAQs
Damage changes the accessibility of the vacuole’s luminal glycans. Once the membrane loses integrity, these glycans become exposed to the cytosol, allowing fluorescently tagged galectin-3, galectin-8, or related galectins to bind them. Each binding event can contribute to a visible fluorescent punctum, converting membrane damage into a measurable cellular signal.
The number or presence of galectin-associated puncta provides an indicator of vacuolar damage or loss of membrane integrity. A stronger punctate signal can therefore support comparisons between experimental conditions, such as different pathogen strains or host-cell states. The readout focuses on membrane disruption rather than directly measuring every stage of infection.
These galectins act as fluorescent reporters for exposure of vacuolar luminal glycans to the cytosol. Their binding creates puncta that can be detected and quantified in infected cells. Using tagged galectins connects a molecular consequence of membrane damage, glycan exposure, with an observable signal suitable for assessing intracellular infection conditions.
The assay can reveal effects caused by pathogen strain, host-cell condition, immune defenses, or microbial virulence factors. Differences in the resulting puncta signal may indicate that these variables influence vacuolar stability or pathogen behavior inside cells. Comparing matched conditions helps investigators examine how host and microbial properties shape infection progression and intracellular survival.
A typical workflow uses infected cells containing pathogen-associated vacuoles, introduces or expresses fluorescently tagged galectin reporters, and examines the cells for galectin-binding puncta. Researchers then quantify the signal and compare it across strains or cellular conditions. This sequence links infection, reporter localization, and quantitative assessment of vacuolar damage within the same cell-based experiment.
Researchers can apply it when they need to determine whether pathogen strains or host-cell conditions differ in their effects on vacuolar integrity. The resulting puncta measurements provide a comparative readout of damage, helping evaluate how microbial virulence factors and immune defenses influence the intracellular environment. Such comparisons can clarify relationships between membrane disruption and infection progression.
In this context, the assay connects a structural event, loss of vacuolar membrane integrity, with biological outcomes relevant to infection. Its measurements can support investigation of pathogen escape, innate immune sensing, and intracellular survival. By quantifying damage in infected cells, researchers can examine how host defenses and microbial virulence shape the course of intracellular infection.