Extracellular vesicles (EVs) are small, membrane-bound particles secreted from cells and thought to function as cellular messengers, carrying cargo from one cell to another. There are several subtypes of EVs that vary in function, cargo, and size, which can range from ~30-1000 nm in diameter. The smallest type of EV is the exosome, which is ~30-150 nm in size. EVs originate within a cellular compartment called the multivesicular body (MVB), which is itself derived from invagination of endosomes. The vesicles are released when the MVB fuses with the plasma membrane and releases its cargo.

In biomedical research, EVs and their cargo are used as diagnostic biomarkers for cancer and other diseases. EVs can be isolated from blood or other biological fluids using techniques such as ultracentrifugation, PEG precipitation, and immuno-capture beads.

EV membranes harbor transmembrane proteins originating from the plasma membrane of the cell of origin, which often includes proteins of the tetraspanin family (such as CD9, CD63, and CD81). Inside, EVs contain cytoplasmic components such as proteins and RNA. EV components can be analyzed using methods like RNAseq, western blotting, and flow cytometry. For characterization by flow cytometry, researchers can use dyes that stain EV components like membranes and nucleic acids, and antibodies that bind to tetraspanins or other proteins of interest.

Read our blog article to to learn more about EVs and their growing potential in both medicine and research.

The small size of EVs makes them challenging to handle and analyze. In flow cytometry, EVs can be difficult to distinguish from cell debris and other small particles, with their size at or below the limits of some flow cytometers’ sensitivity. Staining with certain dyes or antibodies can help to distinguish EVs from other particles. However, it is important to use stains that won’t form aggregates, since these small particles could themselves be confused for stained EVs. Such aggregates have been a frustration for EV and exosome researchers.

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.

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. ExoBrite™ Flow Antibody Conjugates were designed and validated for flow cytometry to offer bright signal and low background of EV markers in purified and bead-bound EVs.

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.