Support & Resources

+1

EXOSOME & EV LABELING

Fluorescent staining kits for bright, clean staining of EVs and exosomes
ExoBrite™ EV Membrane Stains have been validated for super-resolution dSTORM imaging of isolated EVs
Validated antibodies for detection of exosome markers in purified or bead-bound exosomes by flow cytometry
Validated antibodies for detection of exosome markers in EV extracts by western blot

What Are Extracellular Vesicles?

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.

 

Exosome analysis: Small targets, big challenge

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.

The method used to enrich or purify EVs prior to analysis is also an important factor. Samples enriched with a simple PEG precipitation step often contain a large amount of non-EV lipid particles, which may also bind to membrane dyes. These impurities can be reduced by using other EV purification methods, such as size exclusion chromatography (SEC) or immunoprecipitation. Magnetic beads bound to anti-tetraspanin antibodies can give pure exosome clusters. However, these beads may bind non-specifically with a lot of hydrophobic dyes, such as membrane dyes.

We have screened a large collection of our antibodies and dyes to look for those that both stain exosomes well, and show minimal to no aggregation (ie, signal in the absence of exosomes). Continue reading below to explore our validated EV antibodies and dyes.

EXOBRITE™ EV MEMBRANE STAINING KITS

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
SEC-purified MCF-7-derived exosomes were stained with ExoBrite™ EV Membrane Stains.

Less background and better coverage over other exosome/EV stains

ExoBrite™ EV Membrane Stains have less background and more complete exosome staining than other classic dye or competitor dyes. Exosomes were purified from MCF-7 cell supernatant using size exclusion chromatography (SEC). The purified exosomes were stained in PBS with the indicated dyes (top row). The exosome population was gated, with the number showing the percentage of particles falling within the exosome gate. Each dye was also added to filtered PBS (bottom row), to look for dye aggregation and non-specific background. Red arrows indicate these dye aggregates. Lipophilic dyes like DiO show a high number of particles of similar size to exosomes, making them unsuited for exosome staining. CellMask™ also shows an unacceptable amount of dye aggregation falling within the exosome gate. The ExoFlow-ONE™ and ExoGlow™ dyes form aggregates that can mostly be gated away from the true exosomes, but they also show less-complete coverage of exosomes than ExoBrite™ membrane stains (Click to enlarge).
ProductEx/EmDetection channelsSizeCatalog Number
ExoBrite™ 410/450
EV Membrane Staining Kit
416/452 nmPacific Blue™100 Labelings30111-T
500 Labelings30111
ExoBrite™ 490/515
EV Membrane Staining Kit
490/516 nmFITC100 Labelings30112-T
500 Labelings30112
ExoBrite™ 560/585
EV Membrane Staining Kit
562/584 nmPE, Cy®3100 Labelings30113-T
500 Labelings30113
ExoBrite™ 640/660
EV Membrane Staining Kit
642/663 nmAPC100 Labelings30114-T
500 Labelings30114

Purified exosomes stained with ExoBrite™ EV membrane stains

SEC-purified MCF-7-derived exosomes were stained with ExoBrite™ 490/515 EV Membrane Stain (right). Specific staining was seen, compared with the same stain in buffer (left). Exosomes were detected on a CytoFLEX LX flow cytometer in the FITC channel.
SEC-purified MCF-7-derived exosomes were stained with ExoBrite™ 560/585 EV Membrane Stain (right). Specific staining was seen, compared with the same stain in buffer (left). Exosomes were detected on a CytoFLEX LX flow cytometer in the PE channel.

Bead-bound EVs stained with ExoBrite™ EV membrane stains

Flow cytometry of bead-bound exosomes derived from MCF-7 cells. Unstained exosomes (gray) or stained with 10X ExoBrite™ 490/515 (green). Exosomes were detected on a CytoFLEX LX flow cytometer in the FITC channel.
Flow cytometry of bead-bound exosomes derived from MCF-7 cells. Unstained exosomes (gray) or stained with 10X ExoBrite™ 560/585 (orange). Exosomes were detected on a CytoFLEX LX flow cytometer in the R-PE channel.
Flow cytometry of bead-bound exosomes derived from MCF-7 cells. Unstained exosomes (gray) or co-stained with 10X ExoBrite™ 490/515 and CD9(H19a)-CF®568 (pink). Exosomes were detected on a CytoFLEX LX flow cytometer in the FITC and R-PE channels.
Flow cytometry of bead-bound exosomes derived from MCF-7 cells. Unstained exosomes (gray) or co-stained with 10X ExoBrite™ 560/585 and CD81-CF®488A (pink). Exosomes were detected on a CytoFLEX LX flow cytometer in the FITC and R-PE channels.

ExoBrite™ EV membrane stain/antibody co-staining

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.

SEC-purified MCF-7-derived exosomes were stained simultaneously with 1 mL 10X ExoBrite™ 560/585 EV Membrane Stain and 0.1 ug/mL CD9(H19a)-CF®488A. When gated on ExoBrite™ 560/585-positive particles, ~95% were also positive for CD9 (top row). Likewise, when gated on CD9-positive particles, ~ 95% were also positive for ExoBrite™ 560/585 (bottom row)
CellMask is a trademark of Thermo Fisher Scientific; ExoFlow-ONE and ExoGlow are trademarks of System Biosciences.

SUPER-RESOLUTION IMAGING OF EXOSOMES

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
Super-resolution dSTORM images of human colorectal cancer cell line derived EVs stained for tetraspanins and with ExoBrite™ stains at 1x concentration. EVs stained with ExoBrite™ 560/585 (blue) and CF®647-labeled CD9, CD63, and CD81 antibody cocktail (purple)(left). EVs stained with ExoBrite™ 650/665 (purple), CF®568-labeled CD63 antibody (blue), and CF®488A-labeled CD9 antibody (yellow)(right). Samples were prepared using the ONI EV Profiler assay and acquired using on an ONI Nanoimager. Data was processed using a beta-release of the online ONI localization microscopy data analysis platform named CODI.

ExoBrite™ Flow Antibody Conjugates

Validated antibodies for detection of exosome markers by flow cytometry

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™ Flow Antibody advantages:

  • Developed for detection of exosome markers CD9, CD63, and CD81 by flow cytometry
  • Validated for purified or bead-bound exosomes
  • Bright signal and low background
  • ExoBrite™ Isotype Control Flow Antibody available
  • Colors available for Pacific Blue™, FITC, and PE channels

Purified exosomes stained with ExoBrite™ Flow Antibodies

SEC-purified MCF-7-derived exosomes were stained with ExoBrite™ CD9 560/585 Flow Antibody (left). Specific staining was seen, compared with the same antibody in buffer (right). Exosomes were detected on a CytoFLEX LX flow cytometer in the PE channel.
SEC-purified MCF-7-derived exosomes were stained with ExoBrite™ CD63 490/515 Flow Antibody (left). Specific staining was seen, compared with the same antibody in buffer (right). Exosomes were detected on a CytoFLEX LX flow cytometer in the FITC channel.
SEC-purified MCF-7-derived exosomes were stained with ExoBrite™ CD81 490/515 Flow Antibody (left). Specific staining was seen, compared with the same antibody in buffer (right). Exosomes were detected on a CytoFLEX LX flow cytometer in the FITC channel.

ProductConjugatesDetection ChannelsSizesCatalog Number
ExoBrite™ CD9 Flow AntibodyExoBrite™ 410/450
ExoBrite™ 490/515
ExoBrite™ 560/585
R-PE
Pacific Blue ™
FITC
PE
25 tests
100 tests
P003-410… P003-RPE
ExoBrite™ CD63 Flow AntibodyP004-410… P004-RPE
ExoBrite™ CD81 Flow AntibodyP005-410… P005-RPE
ExoBrite™ IgG1 Isotype Control Flow AntibodyP008-410… P008-RPE

ExoBrite™ Western Antibody Conjugates

Validated antibodies for detection of exosome markers by western blotting

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.

ExoBrite™ Western Antibody advantages:

  • Developed for detection of exosome markers CD9, CD63, and CD81 by fluorescent western blot
  • Validated for use with EV extracts
  • Bright signal and low background
  • Available in 2 near-infrared colors
  • Negative control ExoBrite™ Calnexin Western Antibody available

Western detection of human CD9 in MCF-7 cell and exosome lysate using ExoBrite™ 680/700 CD9 Western Antibody, showing enrichment of CD9 in the exosome prep.
Western detection of human CD63 in MCF-7 cell and exosome lysate using ExoBrite™ 680/700 CD63 Western Antibody, showing enrichment of CD63 in the exosome prep.
Western detection of human CD81 in MCF-7 cell and exosome lysate using ExoBrite™ 680/700 CD81 Western Antibody, showing enrichment of CD81 in the exosome prep.
Western detection of human calnexin in MCF-7 cell and exosome lysate using ExoBrite™ 770/800 calnexin Western Antibody, showing the lack of calnexin in the exosome prep.

ProductConjugatesSizesCatalog Number
ExoBrite™ CD9 Western AntibodyExoBrite™ 680/700
ExoBrite™ 770/800
25 tests
100 tests
P003-680, P003-770
ExoBrite™ CD63 Western AntibodyP004-680, P004-770
ExoBrite™ CD81 Western AntibodyP006-680, P006-770
ExoBrite™ Calnexin Western AntibodyExoBrite™ 770/800P007-770

Back to top