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"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy (TIRFM)
Published on: August 26, 2016
In vitro phagosome-endosome fusion
Isabelle Vergne1, Vojo Deretic
1Department of Molecular Genetics and Microbiology, University of New Mexico School of Medicine, Albuquerque, NM, USA.
Methods in Molecular Biology (Clifton, N.J.)
|April 22, 2008
Summary
This study presents a rapid, sensitive assay to measure phagosome-endosome fusion in leukocytes. The method uses biotin-streptavidin complex formation and fluorescence detection to quantify fusion events, aiding pathogen effector identification.
Area of Science:
- Cell Biology
- Immunology
- Microbiology
Background:
- Phagolysosome biogenesis is crucial for leukocyte pathogen clearance.
- This process involves sequential membrane fusion between phagosomes and endosomes.
- Studying this pathway is vital for understanding host-pathogen interactions.
Purpose of the Study:
- To describe a novel, sensitive, and rapid in vitro assay for measuring phagosome-endosome fusion.
- To enable the study of phagolysosome biogenesis and identify pathogen-derived inhibitors.
- To quantify fusion events with both early and late endosomal compartments.
Main Methods:
- The assay utilizes biotinylated-peroxidase loaded endosomes and streptavidin-conjugated magnetic beads containing phagosomes.
- Fusion is detected via the formation of a stable biotin-streptavidin complex.
- Quantification is achieved through a fluorescence-based method measuring peroxidase activity.
Main Results:
- The described method provides a sensitive and rapid measurement of phagosome-endosome fusion.
- It allows for the assessment of fusion with distinct endosomal compartments.
- The assay is suitable for studying pathogen effectors that interfere with phagolysosome biogenesis.
Conclusions:
- This assay is a valuable tool for investigating phagolysosome biogenesis.
- It facilitates the identification of novel pathogen virulence factors.
- The method offers a robust approach for quantitative analysis of membrane fusion events in cellular immunity.
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