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Real-time imaging of multivesicular body–plasma membrane fusion to quantify exosome release from single cells


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).

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Fig. 1: Model for the visualization of MVB–PM fusion with CD63-pHluorin.
Fig. 2: Simultaneous dual-color TIRF microscopy of TSPAN-pHluorin with TSPAN-pHuji reporters.
Fig. 3: Visualization of individual fusion events.
Fig. 4: Setting the TIRF angle for a CD63-pHluorin-expressing HeLa cell.
Fig. 5: Analysis of MVB–PM fusion activity with the AMvBE macro.

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Data availability

The data that support this study are available from the corresponding author upon reasonable request.

Code availability

The AMvBE macro, a Readme file, and example data can be found in Supplementary Software 1. The software in this protocol has been peer reviewed.


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We acknowledge the NeurImag facility of the Institute of Psychiatry and Neuroscience of Paris where the AMvBE macro has been developed. This Protocol was funded by a Dutch Organizations for Scientific Research–Amsterdam Institute for Molecules, Medicines, and Systems STAR Graduate Program grant (022.005.031) to M.P.B., a Dutch Cancer Fund (KWF-5510) and a Cancer Center Amsterdam–VU University Medical Center grant to D.M.P., and a European Molecular Biology Organization grant (EMBO ALTF 1383-2014) and a Fondation ARC pour la Recherché sur le Cancer fellowship (PJA 20161204808) to F.J.V.

Author information

Authors and Affiliations



M.P.B. and F.J.V. performed the experiments, prepared the figures, and wrote the paper. G.v.N., P.B., S.H., and D.M.P. provided critical feedback and helped to shape the paper. P.B. developed the AMvBE macro. S.H. helped to set up the dual-color TIRF experiments. D.M.P. conceived the study. F.J.V. and D.M.P. supervised the study.

Corresponding authors

Correspondence to D. Michiel Pegtel or Frederik J. Verweij.

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Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Protocols thanks David Perrais and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Key references using this protocol

Verweij, F. J. et al. J Cell Biol, 217 (3) 1129–1142 (2018):

Supplementary information

Supplementary Software 1

AMvBE macro, Readme file and example data

Reporting Summary

Supplementary Video 1

TIRF microscopy of a CD63-pHluorin expressing HeLa cell at 8× speed. Scale bar, 20 μm

Supplementary Video 2

Dual-color TIRF microscopy of a CD63-pHluorin (green) and CD81-pHuji (red) expressing HeLa cell at 8× speed. Scale bar, 20 μm

Supplementary Video 3

Dual-color TIRF microscopy of a CD63-pHluorin (green) and CD63-mRFP (red) expressing HeLa cell at 8× speed. Scale bar, 20 μm. White arrows highlight MVBs containing CD63-pHluorin and CD63-mRFP that are visible in red before fusion

Supplementary Video 4

Dual-color TIRF microscopy of a CD63-C-term-pHluorin (green) and CD63- pHuji (red) expressing HeLa cell at 8× speed. Scale bar, 20 μm. Original source: ref. 17

Supplementary Video 5

TIRF microscopy of HeLa cells stably expressing CD63-pHluorin 6 weeks post-transduction at 8× speed. Scale bar, 20 μm

Supplementary Video 6

TIRF microscopy of a CD63-pHluorin expressing HUVEC cell at 8× speed. Scale bar, 20 μm

Supplementary Video 7

TIRF microscopy of a CD63-pHluorin expressing HEK293T cell at 8× speed. Scale bar, 20 μm

Supplementary Video 8

Example of the summary TIFF file as created by the AMvBE macro upon analysis of an MVB-PM fusion event (highlighted by a green circle) or a neutral CD63-pHluorin-positive vesicle moving in the TIRF plane (highlighted by a red circle). Scale bar, 5 μm

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Bebelman, M.P., Bun, P., Huveneers, S. et al. Real-time imaging of multivesicular body–plasma membrane fusion to quantify exosome release from single cells. Nat Protoc 15, 102–121 (2020).

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