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

Exosome 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, exosomes contain cytoplasmic components such as proteins and RNA. Exosome components can be analyzed using methods like RNAseq, western blotting, and flow cytometry. For characterization by flow cytometry, researchers can use dyes that stain exosome 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 exosomes and their growing potential in both medicine and research.

The small size of exosomes makes them challenging to handle and analyze. In flow cytometry, exosomes 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 exosomes 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 exosomes. Such aggregates have been a frustration for 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 exosomes.

Exosome staining you can trust

ExoBrite™ EV Membrane Staining Kits were designed to overcome some of the challenges of exosome detection. ExoBrite™ stains bind to molecules in the exosome 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 exosomes or 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 exosomes.

ExoBrite™ EV Membrane Stains have been validated in flow cytometry for their ability to stain exosomes and EVs derived from several different cultured cell lines, and isolated by several different methods, including size exclusion chromatography (SEC), polyethylene glycol (PEG) precipitation, and magnetic bead immunoprecipitation (IP).

ExoBrite™ EV Membrane Stain advantages:

• Designed for exosome detection by flow cytometry
• Bright fluorescence and low background for excellent signal-to-noise
• Compatible with antibody co-staining (unlike CellBrite® Fix or MemBrite® Fix)
• Stain purified or bead-bound exosomes
• Available in 4 colors for flexible experimental design

One advantage of ExoBrite™ EV Membrane Stains is the ability to co-stain with antibodies, for exosome protein profiling studies. Co-staining can be performed concurrently or sequentially, and can be performed on purified or bead-bound exosomes. ExoBrite™ EV Membrane Stains are general exosome stains that should label all exosomes in a sample,* allowing antibodies to be used to quantify how many exosomes express the protein of interest. For example, in the exosomes that we tested (MCF-7-derived), the tetraspanin CD9 is highly and ubiquitously expressed.** We have found that when we performed co-staining with ExoBrite™ 560/585 EV Membrane Stain and CD9(H19a)-CF®488A, they show near-complete overlap of their positive populations (see Figure, right).

Tips for optimal ExoBrite™/antibody co-staining of purified exosomes:

• Stain purified exosomes in 1 mL with 10X ExoBrite™ (ExoBrite™ staining is somewhat reduced during simultaneous co-staining so 1X is not recommended)
• Try starting with at least 0.1 ug/mL antibody

*In the exosomes that we have tested, ExoBrite™ EV Membrane Stains label all exosomes. There may be exosome types with different staining levels.

**Expression levels of tetraspanin proteins (CD9, CD61, & CD81) and other proteins differ greatly between cell types and exosome sources.

See beyond the diffraction limit with ExoBrite™ EV stains

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™ EV Membrane Stains have been validated for dSTORM by the ONI Nanoimager, allowing the study of fine morphological details and co-staining with EV biomarkers such as tetraspanin proteins CD9, CD63, and CD81.

• ExoBrite™ 560/585 and 650/665 EV Membrane Stains are validated for dSTORM imaging of EVs
• Understand more about vesicle morphology and structure
• Allows antibody co-staining and localization studies with EV biomarkers

The most common proteins used as exosome 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 exosome markers in purified and bead-bound exosomes.

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.

ExoBrite™ Western Antibodies were validated to offer bright signal and low background of exosome 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 exosomes. It is offered as a negative control to assess the purity of isolated exosome extracts.