Tech Tip: Fluorescent Detection of Exosomes by Flow Cytometry

In this Tech Tip, we share the expertise we have acquired for optimal fluorescent staining and detection of EVs.

eBook: Exploring Extracellular Vesicles: Biomarkers and Beyond

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ExoBrite™ True EV Membrane Stains are lipophilic dyes designed as the best choice for pan-EV labeling

ExoBrite™ EV Stains Comparison Guide

ExoBrite™ EV Surface Stains

The Best Choice for Pan-EV Staining

ExoBrite™ True EV Membrane Stains are novel membrane dyes designed specifically for EV detection. While other membrane dyes like PKH, DiO, and DiI, are commonly used for labeling EVs, these dyes often have poor solubility and thus form aggregates that can be confused with EVs.

ExoBrite™ True EV Membrane Stains were designed specifically to address the issues of membrane dye aggregation and are the best choice for general pan-EV staining.  They demonstrate higher (near-complete) coverage of EVs over competitor membrane dyes in fluorescence nanoparticle tracking analysis (fNTA). They also offer clear differentiation of EVs from non-specific particles during flow detection, which is not usually possible with other membrane dyes.

Bright & Sensitive EV Stains with Broad Compatibility

ExoBrite™ Annexin EV Staining Kits are uniquely formulated conjugates of Annexin V designed with broad compatibility for EVs derived from different sources. Similar to the ExoBrite™ CTB EV Stains, these Annexin V-based conjugates were developed for bright and sensitive staining of isolated EVs by flow cytometry with little to no background aggregation. ExoBrite™ Annexin EV Staining Kits also offer the broadest coverage for EVs. We tested EVs derived from 9 cell lines and ExoBrite™ Annexin EV Stains showed strong staining for all of them.

Advantages of ExoBrite™ Annexin EV Stains:

• Optimally formulated Annexin V conjugates for staining purified EVs
• Broad compatibility, stained EVs isolated from all 9 sources tested
• Bright signal and low background staining for flow cytometry
• Compatible with antibody co-staining
• Available in 4 colors for flexible experimental design

Sensitive Stains for Purified or Bead-Bound EVs with Broad Compatibility

ExoBrite™ WGA EV Stains are fluorescent conjugates of wheat germ agglutinin (WGA) optimized for bright and sensitive staining of EVs. Similar to ExoBrite™ Annexin EV Stains, these WGA-based conjugates offer broad compatibility with EVs. The stains were validated for EVs derived from all 9 cell lines tested. In addition, ExoBrite™ WGA EV Stains are less prone to aggregation than hydrophobic membrane dyes and may be used to stain purified and bead-bound EVs.

Advantages of ExoBrite™ WGA EV Stains:

• Optimally formulated WGA conjugates for staining purified or bead-bound EVs
• Broad compatibility, stained EVs isolated from all 9 sources tested
• Bright signal and low background staining for flow cytometry
• Compatible with antibody co-staining
• Available in 4 colors for flexible experimental design

Bright EV Stains with Excellent Signal-to-Noise

ExoBrite™ CTB EV Staining Kits were designed to overcome some of the challenges of EV detection. ExoBrite™ stains bind to molecules in the EV membrane providing bright, specific staining of isolated EVs by flow cytometry. A key advantage of ExoBrite™ stains is that, unlike many other dyes, they show little to no background aggregates of a similar size as EVs. Also, unlike most membrane dyes, ExoBrite™ stains do not bind non-specifically to polystyrene beads, meaning that they can be used to stain bead-bound EVs.

Advantages of ExoBrite™ CTB EV Stains:

• Optimally formulated CTB conjugates for staining EVs
• Designed for detection by flow cytometry
• Bright fluorescence and low background for excellent signal-to-noise
• Compatible with antibody co-staining
• Stain purified or bead-bound EVs
• Available in 4 colors for flexible experimental design

The most common proteins used as EV markers are CD9, CD63, and CD81, members of the tetraspanin family. While antibodies targeting these proteins are available by commercial suppliers, few are validated or perform well for detection of EVs or exosomes.  The antibodies and dye labels of ExoBrite™ Flow Antibody Conjugates were carefully selected and validated for robust detection of isolated EVs. In addition, the antibodies are provided in a proprietary buffer formulation for reduced antibody aggregation and brighter EV staining for optimal accuracy and signal-to-noise.

ExoBrite™ Isotype Control Flow Antibody, which have been found to not react with any target in human cells and have the same isotype as the tetraspanin antibodies, are also available as a negative control.

Convenient & Optimized Capture of EVs

Streamline your EV and exosome isolation workflows with ExoBrite™ Streptavidin Magnetic Beads, developed to offer lower background and higher sensitivity for EV capture than similar products. These streptavidin-coated and magnetic polystyrene beads (4.5 um diameter) may be used to isolate EVs from cell culture medium or other biological fluids without an overnight precipitation step.

See beyond the diffraction limit with
ExoBrite™ STORM CTB EV Staining Kits

Characterizing exosomes and EVs by imaging remains a challenge due to their small size and the resolution limit of light microscopy. Super-resolution microscopy techniques such as direct stochastic optical reconstruction microscopy (dSTORM) push beyond the diffraction limit of traditional light microscopy, allowing single-molecule resolution of subcellular structures such as EVs.

ExoBrite™ STORM CTB EV Stains were developed for STORM imaging of EVs and are available in four CF® Dyes validated for STORM.  ExoBrite™ 560/585 has been validated for dSTORM on the ONI Nanoimager S Mark II, allowing the study of fine morphological details and co-staining with EV biomarkers such as tetraspanin proteins CD9, CD63, and CD81.

Advantages of ExoBrite™ STORM CTB EV Stains:

• Dive deeper into EV morphology and structure
• Optimally formulated CTB conjugates for imaging EVs by STORM
• Available in 4 CF® Dyes validated for STORM imaging
• Allows antibody co-staining and localization studies with EV biomarkers

ExoBrite™ Western Antibodies were validated to offer bright signal and low background of EV markers CD9, CD63, and CD81 in EV extracts by near-IR fluorescent western blot. The antibodies are available with two near-infrared fluorescent dyes, ExoBrite™ 680/700 and ExoBrite™ 770/800, which offer greater signal-to-noise than dyes with visible light emission for western blotting.

An ExoBrite™ Calnexin Western Antibody detects a protein of the endoplasmic reticulum that is not found in EVs. It is offered as a negative control to assess the purity of isolated EV extracts.

Efficient total RNA extraction from purified EVs

The ExoBrite™ EV Total RNA Isolation Kit was designed to address the challenges of EV RNA extraction by offering an optimized and easy-to-use kit for total RNA isolation, including mRNA and miRNAs, from purified EVs. The isolated EV RNA can then be used for downstream analysis such as qPCR or RNAseq.

Advantages of ExoBrite™ EV Total RNA Isolation Kit:

• Optimized for total RNA extraction from purified EVs
• Recover ~10-20 ng of RNA from 1×10¹⁰ SEC-enriched EVs
• Compatible with downstream applications such as qPCR or RNAseq
• Simple column-based purification
• No phenol/chloroform or ethanol precipitation steps