Salmonella Phagosome Isolation
Salmonella Phagosome Isolation - Related Videos
10:33
Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages
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Cited by 5 •
2017
We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.
03:37
Isolation of Phagosomes Containing Magnetic Bead-Tagged Bacteria from Infected Macrophages
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2026
Source: Gutiérrez, S., et al., Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages. J. Vis. Exp. (2017)This video demonstrates the magnetic isolation of phagosomes containing biotin–streptavidin bead–coated bacteria from infected macrophages. The protocol includes buffer treatments, cell disruption, magnetic separation, and washing to yield purified phagosomes for analysis.
14:39
Measuring Phagosome pH by Ratiometric Fluorescence Microscopy
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Cited by 26 •
2015
Phagosomal pH influences phagosome maturation, oxidant production, phagosomal killing as well as antigen presentation. Here we describe a ratiometric method for measuring time-course and endpoint pH changes in individual phagosomes in living phagocytes using fluorescence microscopy.
06:27
An Assay to Quantify Cytosolic and Vacuolar Salmonella in Infected Primary Macrophages
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2025
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,...
12:42
Imaging the Neutrophil Phagosome and Cytoplasm Using a Ratiometric pH Indicator
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Cited by 13 •
2017
This manuscript describes a simple method to measure the phagosomal pH and area as well as the cytoplasmic pH of human and mouse neutrophils using the ratiometric indicator seminaphthorhodafluor (SNARF)-1, or S-1. This is achieved using live-cell confocal fluorescence microscopy and image analysis.
FAQs
The central technical challenge is maintaining the integrity of Salmonella-containing host membranes during cell disruption. Fractionation conditions must preserve the bacteria-containing vacuoles long enough for them to separate from soluble cytoplasmic material and unrelated membranes. If these compartments are damaged or mixed extensively, measurements of membrane composition, maturation, or trafficking may no longer represent the infected-cell state.
Density-based centrifugation enriches compartments according to their physical properties, allowing bacteria-containing vacuoles to be separated from surrounding cellular material. Enrichment alone does not establish compartment identity, so researchers assess fractions with compartmental markers. This combined physical and marker-based approach helps distinguish the targeted vacuoles from other membrane populations and supports interpretation of their composition and maturation.
Marker profiles can reveal how the Salmonella-containing compartment relates to vesicle maturation and intracellular trafficking. Examining these signals alongside the isolated material helps researchers determine whether the compartment has characteristics associated with changing host membrane pathways. That information is important because bacterial survival may depend on how the vacuole interacts with, or alters, normal cellular transport and immune processes.
A representative workflow begins with infected host cells, followed by disruption under conditions intended to preserve bacteria-containing vacuoles. The resulting cellular material is subjected to physical separation, including density-based centrifugation, to enrich the desired fractions. Researchers then assess the recovered fractions using compartmental markers. These linked steps connect sample preparation with verification of what was isolated.
Interpretability depends on both physical enrichment and biological validation. A useful fraction should emerge from the separation process as an enriched membrane population and show compartmental marker patterns consistent with the targeted vacuole. Evaluating these features together reduces the risk of attributing properties of surrounding cellular material to the Salmonella-containing compartment itself.
In Immunology and Infection research, isolated fractions provide material for examining phagosomal membrane composition, vesicle maturation, and intracellular trafficking. They also help clarify how Salmonella survives within host cells and alters antimicrobial defenses. By connecting compartment properties with immune-pathway interactions, the approach can generate mechanistic insight into intracellular infection and identify processes relevant to potential therapeutic targets.