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Author Notes:

Corresponding author at: Division of Cardiology, Emory University School of Medicine, Atlanta, GA, United States. Email: wdgray@emory.edu.

We would like to thank the following: the Emory Blood Bank for providing packed red blood cells; the Emory/Georgia Tech Center for Health Discovery and Well Being for human plasma samples; and the Emory+Children's Pediatric Research Center Flow Cytometry Core.

The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

MethodsX thanks the reviewers of this article for taking the time to provide valuable feedback.


Research Funding:

This work was supported by the National Heart Lung and Blood Institute of the National Institutes of Health as a Program of Excellence in Nanotechnology (HHSN268201000043C, R01HL109559, R01HL124879), and a VA Merit Award (I01 BX000704).

Research reported in this publication was supported by the National Heart, Lung, and Blood Institute of the National Institutes of Health under Award Number T32HL007745.


  • Extracellular vesicles
  • Calcein AM
  • Flow cytometry

An accurate, precise method for general labeling of extracellular vesicles


Journal Title:



Volume 2


, Pages 360-367

Type of Work:

Article | Final Publisher PDF


Extracellular, membrane vesicles (microvesicles, exosomes) are secreted by cells and may serve as mediators of intercellular communication. Methods for detecting them by flow cytometry have included the use of agents that fluorescently stain vesicle membrane, or fluorescent antibodies that target specific cell-of-origin antigens. However, these methods may falsely detect cell debris or require prior cell-of-origin knowledge. Here, we demonstrate the suitability of calcein AM for detection of intact extracellular vesicles (EVs) by flow cytometry. Calcein AM is non-fluorescent until it passively enters EVs, after which it is activated and becomes fluorescent and EV-impermeant. Permeabilized/lysed EVs label positive with antibodies and lipophilic membrane stain, whereas no labeling was observed with calcein. In contrast to methods that use antibodies or membrane stains, calcein AM allows for the differentiation between intact EVs and debris. Calcein AM can be used for detection of intact EVs from numerous cell types.

Copyright information:

© 2015 Published by Elsevier B.V.

This is an Open Access work distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/).

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