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EXOSOME & EV LABELING

EV and exosome staining basics and challenges
Antibodies that have been validated for staining of exosomes
Dyes to stain exosome and EV membranes for flow cytometry

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.

 

Exosome Analysis: A Small 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 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.

VALIDATED EXOSOME ANTIBODIES

The most common proteins used as exosome markers are CD9, CD63, and CD81, members of the tetraspanin family. Tetraspanins are plasma membrane proteins with many proposed functions, including activation and sorting of other membrane proteins. They are also thought to play a role in the targeting of proteins to Multivesicular Bodies (MVBs) and exosomes. These tetraspanins are broadly expressed on many cell types and can therefore be detected on many types of exosomes, but their expression levels do vary depending on the cell type of origin.

Other proteins that are often detected in EVs include Heat Shock proteins (ie, HSP70), membrane trafficking proteins, MVB-biogenesis proteins (ie, Alix, TSG101), and cell adhesion molecules (ie, Ep-CAM, ICAM-1). Ep-CAM is mainly expressed in epithelial cells, including the cell line MCF-7.

Biotium offers several different antibody clones against exosome marker proteins, each available in a wide variety of conjugate choices such as bright CF® Dyes and biotin. The antibodies listed below have been validated specifically for exosome detection by flow cytometry, using enriched MCF-7-derived EVs bound to CD63-specific magnetic beads (Thermo Fisher Scientific).

Exosome antibodies validated in flow

TargetHostCloneNotes:
CD9 (human)MouseCD9/1619Recommended clone: bright staining
MouseCD9/2343Recommended clone: bright staining
CD63 (human, mouse)MouseMX-49.129.5Recommended clone: bright staining
CD63 (human)MouseLAMP3/968Recommended clone: bright staining
CD81 (human, mouse, rat)Mouse1.3.3.22Recommended clone: bright staining
MouserC81/3442Recommended clone: bright staining
RabbitC81/2885RRecommended clone: good staining
Ep-CAM (human)MouseVU-1D9Ep-CAM expression on MCF7 derived exosomes is low but detectable
MouserVU-1D9
MouseEGP40/826+EGP40/837
+EGP40/1110+EGP40/1120
MouseHEA125
All antibodies were validated using PEG-enriched MCF-7 cell derived exosomes, immobilized on CD63-specific magnetic beads (Thermo Fisher Scientific)
Flow cytometry of bead-bound exosomes derived from MCF-7 cells. Unstained exosomes (gray) or exosomes stained with CF568-labeled CD9 monoclonal antibody (CD9/1619) (orange)
Flow cytometry of bead-bound exosomes derived from MCF-7 cells. Unstained exosomes (gray) or exosomes stained with CF568-labeled CD9 monoclonal antibody (CD9/2343) (orange)
Flow cytometry of bead-bound exosomes derived from MCF-7 cells. Unstained exosomes (gray) or stained with CF568-labeled CD63 monoclonal antibody (MX-49.129.5) (orange)
Flow cytometry of bead-bound exosomes derived from MCF-7 cells. Unstained exosomes (gray) or stained with CF555-labeled CD63 monoclonal antibody (LAMP3/968) (orange)

Flow cytometry of bead-bound exosomes derived from MCF-7 cells. Unstained exosomes (gray) or exosomes stained with CF568-labeled CD81 monoclonal antibody (1.3.3.22) (orange)
Flow cytometry of bead-bound exosomes derived from MCF-7 cells. Unstained exosomes (gray) or exosomes stained with CF568-labeled CD81 monoclonal antibody (rC81/3442) (orange)
Flow cytometry of bead-bound exosomes derived from MCF-7 cells. Unstained exosomes (gray) or exosomes stained with CF568-labeled CD81 monoclonal antibody (C81/2885R) (orange)
Flow cytometry of bead-bound exosomes derived from MCF-7 cells. Unstained exosomes (gray) or stained with CF568-labeled Ep-CAM monoclonal antibody (HEA125) (orange)

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MEMBRANE STAINS FOR EXOSOMES

EVs and exosomes are vesicles enclosed by a single lipid bilayer. These membranes can be stained using many of the  same dyes used to stain cellular membranes. However, not all membrane dyes are equally well-suited for exosome staining. Biotium has curated a selection of membrane dyes in a variety of colors that show bright staining of exosomes, with minimal to no aggregation.

All of the validated membrane and surface stains below were tested on PEG-enriched EVs derived from HeLa cells. Cholera Toxin B conjugates were also validated using enriched MCF-7-derived EVs bound to CD63-specific magnetic beads (Thermo Fisher Scientific). CellBrite® Steady dyes bind non-specifically to the beads.

CellBrite® Steady Membrane Stains have been validated for EV detection by flow cytometry

The CellBrite® Steady Dyes are unique fluorescent membrane probes. Designed for use in long-term cell imaging studies, we have found them to show excellent labeling of EVs for detection by flow cytometry, showing bright staining with minimal aggregation. CellBrite® Steady Dyes are available in several colors. We have seen the best EV staining with CellBrite® Steady 405, 488, 550, and 650.

Enriched exosomes were stained with 10X CellBrite® Steady 488 (right). Specific staining was seen, compared with CellBrite® Steady 488 in buffer (left). Exosomes were detected on a Cytoflex LX flow cytometer in the FITC channel.
Enriched exosomes were stained with 0.5X CellBrite® Steady 550 (right). Specific staining was seen, compared with CellBrite® Steady 550 in buffer (left). Exosomes were detected on a Cytoflex LX flow cytometer in the PE channel.
Enriched exosomes were stained with 2X CellBrite® Steady 650 (right). Specific staining was seen, compared with CellBrite® Steady 650 in buffer (left). Exosomes were detected on a Cytoflex LX flow cytometer in the APC channel.

CellBrite® Fix Membrane Stains have been published for labeling EVs

CellBrite® Fix Membrane Stains are fluorogenic membrane dyes that accumulate in the cell membrane, where they become fluorescent. The dyes have an amine-reactive group for covalent attachment to membrane proteins.

Highlighted Citation: CellBrite® Fix Membrane Stains were used to label EVs containing viral particles by Santiana et. al in a study investigating the role of vesicle enclosure in viral transmission. Results show CellBrite® Fix 488 was suitable for labeling isolated EVs for super-resolution microscopy as well as confocal microscopy to compare relative infection rates of cultured murine macrophage-like cells treated with EVs containing noroviruses.

View the publication here.

Structured illumination microscopy performed on Wa-containing vesicles co-stained with CellBrite® Fix 488 fluorescent membrane dye and antibodies against rotavirus nucleocapsids. Arrows point to the dual-labeled vesicles. Scale bar, 400 nm. Credit: Santiana et al. doi:10.1016/j.chom.2018.07.006

Membrane Dyes for EV Detection by Flow

Membrane DyeValidated in flow with:Staining LevelNotes
CellBrite® Steady• PEG-enriched HeLa-derived EVsStrong staining• Fluorescent membrane probes
• 4 colors validated for exosome detection (405, 488, 550, 650)
• Non-covalent labeling
• May also bind to non-EV membranous particles
• Dyes bind non-specifically to magnetic beads
MemBrite® Fix 405/430• PEG-enriched HeLa-derived EVsStrong staining• Fluorescent membrane probe for the Pacific Blue channel
• Reactive dye for covalent labeling
• May not be suitable for antibody co-staining
Cholera Toxin B• PEG-enriched HeLa-derived EVs
• CD63 bead-bound MCF-7-derived EVs
Strong staining• Fluorescent dye that binds lipid rafts
• Non-covalent labeling
• Only CF®488A conjugate binds to small exosomes
• All dye conjugates bind to larger EVs
• May also bind to non-EV membranous particles
CellBrite® Fix• PEG-enriched HeLa-derived EVsWeak staining• Fluorogenic membrane probes
• Available in 3 colors (488, 555, 640)
• Reactive dye for covalent labeling
• May not be suitable for antibody co-staining