An accurate, precise method for general labeling of extracellular vesicles

• Type of Material: Article
• Authors: Charles Searles Jr, Emory University; Adam Mitchell, Emory University; Warren Gray, Emory University
• Language: English
• Date: 2015-01-01
• Publisher: Elsevier
• Publication Version: Final Published Version
• Copyright Statement: © 2015 Published by Elsevier B.V.
• License: Creative Commons Attribution 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.1016/j.mex.2015.08.002
• Title of Journal or Parent Work: MethodsX
• ISSN: 2215-0161
• Volume: 2
• Start Page: 360
• End Page: 367
• Grant/Funding Information: 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.
• Keywords: Extracellular vesicles; Flow cytometry; Calcein AM
• Research Categories: Health Sciences, General; Health Sciences, Medicine and Surgery

Abstract

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.