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Updated: Jan 1, 2026
Quantification of Efferocytosis by Single-cell Fluorescence Microscopy
Published on: August 18, 2018
Real-time imaging of multivesicular body-plasma membrane fusion to quantify exosome release from single cells
Maarten P Bebelman1,2, Philippe Bun3,4, Stephan Huveneers5
1Amsterdam UMC, Vrije Universiteit Amsterdam, Department of Pathology, Cancer Center Amsterdam, de Boelelaan 1117, Amsterdam, The Netherlands.
Abstract:
Exosomes are small extracellular vesicles with a diameter of 40-150 nm, and are implicated in cellular homeostasis and cell-cell communication. They can be secreted in bulk in response to cell-extrinsic and cell-intrinsic signals that cause multivesicular body (MVB) fusion with the plasma membrane (PM). However, research on the regulation of exosome release is hampered by the failure of current methods to capture the dynamics of exosome release. Here we describe how live imaging with tetraspanin-based pH-sensitive fluorescent reporters can quantify the MVB-PM fusion rate of single cells. Our approach enables identification of exogenous stimuli, signaling pathways, and fusion complexes, and can map subcellular sites of fusion events. In addition, dual-color imaging can be used to assess simultaneous release of different cargo by MVB exocytosis. This protocol describes the complete imaging experiment, consisting of transient expression of tetraspanin reporters (2 d), live-cell (dual-color) total internal reflection fluorescence microscopy (30-60 min per condition), and semiautomatic image analysis by using a newly developed ImageJ macro (±30 min per condition).
Insights
Researchers developed a new live imaging technique to track the release of exosomes, which are crucial for cell communication. This method quanties multivesicular body (MVB) fusion with the plasma membrane (PM) in real-time.
Area of Science:
- Cell Biology
- Molecular Biology
- Biophysics
Background:
- Exosomes, small extracellular vesicles, mediate cellular homeostasis and intercellular communication.
- Exosome release involves multivesicular body (MVB) fusion with the plasma membrane (PM), a process regulated by various signals.
- Current methods lack the dynamic resolution to study exosome release regulation.
Purpose of the Study:
- To develop a novel live imaging method for quantifying exosome release dynamics.
- To enable the identification of stimuli, pathways, and molecular complexes regulating MVB-PM fusion.
- To map subcellular sites of exosome release events.
Main Methods:
- Utilized tetraspanin-based pH-sensitive fluorescent reporters for live imaging.
- Employed dual-color total internal reflection fluorescence microscopy (TIRFm) to observe MVB-PM fusion.
- Developed a semiautomatic ImageJ macro for quantitative image analysis.
Main Results:
- Successfully quantified the MVB-PM fusion rate at the single-cell level.
- Demonstrated the ability to identify regulatory factors and map fusion sites.
- Showcased dual-color imaging for assessing simultaneous release of different exosome cargo.
Conclusions:
- The developed live imaging protocol provides a powerful tool for studying exosome biogenesis and release.
- This method overcomes limitations of previous techniques, offering dynamic insights into MVB exocytosis.
- Facilitates deeper understanding of cell-cell communication mechanisms mediated by exosomes.
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