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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: 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.

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 (i.e., HSP70), membrane trafficking proteins, MVB-biogenesis proteins (ie, Alix, TSG101), and cell adhesion molecules (i.e., 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

AntibodyCloneValidated in flow with:Staining level:Notes:
CD9 (human)CD9/1619• Bead-bound MCF-7-derived EVs1
• SEC-purified MCF-7-derived exosomes2
Strong stainingRecommended clone: bright staining
CD9 (human)CD9/2343• Bead-bound MCF-7-derived EVs1
• SEC-purified MCF-7-derived exosomes2
Strong stainingRecommended clone: bright staining
CD63 (human, mouse)MX-49.129.5• Bead-bound MCF-7-derived EVs1See NotesLabels bead-bound EVS but not purified exosomes
CD63 (human)LAMP3/968• Bead-bound MCF-7-derived EVs1See NotesLabels bead-bound EVS but not purified exosomes
CD81 (human, mouse, rat)1.3.3.22• Bead-bound MCF-7-derived EVs1
• SEC-purified MCF-7-derived exosomes2
Strong stainingRecommended clone: bright staining
CD81 (human, mouse, rat)rC81/3442• Bead-bound MCF-7-derived EVs1
• SEC-purified MCF-7-derived exosomes2
Strong stainingRecommended clone: bright staining
rabbit CD81 (human, mouse, rat)C81/2885R• Bead-bound MCF-7-derived EVs1Moderate stainingLower signal than other CD81 clones
Ep-CAM (human)VU-1D9• Bead-bound MCF-7-derived EVs1See NotesEp-CAM expression on MCF7 derived exosomes is low but detectable
Ep-CAM (human)rVU-1D9• Bead-bound MCF-7-derived EVs1See Notes
Ep-CAM (human)EGP40/826+EGP40/837
+EGP40/1110+EGP40/1120
• Bead-bound MCF-7-derived EVs1See Notes
Ep-CAM (human)HEA125• Bead-bound MCF-7-derived EVs1See Notes
1Bead-bound EVs were prepared using PEG-enriched MCF-7 cell derived EVs, immobilized on CD63-specific magnetic beads (Thermo Fisher Scientific)
2SEC-purified MCF-7 cell derived exosomes were prepared using qEV size exclusion columns (SECs) (IZON)

 

Bead-bound EVs stained with tetraspanin antibodies conjugated to CF® dyes

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.

Membrane dyes for EV detection by flow cytometry

Membrane DyeValidated in flow with:Staining LevelNotes
CellBrite® Steady• PEG-enriched HeLa-derived EVs
• SEC-purified MCF-7-derived exosomes1
Strong staining• Fluorescent membrane probes
• All 5 colors validated for exosome detection
• Non-covalent labeling
• Dyes bind non-specifically to magnetic beads
MemBrite® Fix 405/430• PEG-enriched HeLa-derived EVs
• SEC-purified MCF-7-derived exosomes1
Strong staining• Fluorescent membrane probe for the Pacific Blue channel
• Reactive dye for covalent labeling
• May not be suitable for antibody co-staining
CellBrite® Fix 488 & 555• SEC-purified MCF-7-derived exosomes1Moderate staining• Fluorogenic membrane probes for the FITC and PE/Cy3 channels
• Reactive dye for covalent labeling
• May not be suitable for antibody co-staining
• Used in publication as exosome stain3
Di-8-ANEPPS• SEC-purified MCF-7-derived exosomes1Moderate staining• Red fluorescent membrane probe
• Non-covalent labeling
• Less soluble than other dyes
• Used in publications as exosome stain4,5
1SEC-purified MCF-7 cell derived exosomes were prepared using qEV size exclusion columns (SECs) (IZON)
2Bead-bound EVs were prepared using PEG-enriched MCF-7 cell derived EVs, immobilized on CD63-specific magnetic beads (Thermo Fisher Scientific)
3Cell Host & Microbe (2018) Aug 8;24(2):208-220.e8 doi: 10.1016/j.chom.2018.07.006
4Cytometry A. (2016) Feb;89(2):196-206 doi: 10.1002/cyto.a.22787
5Anal. Chem. (2021) 93, 14, 5897–5905 doi.org/10.1021/acs.analchem.1c00253

CellBrite® Steady Membrane Stains validated for exosome & EV detection

The CellBrite® Steady Dyes are unique fluorescent membrane probes. Originally designed for use in long-term cell imaging studies, we have found them to show good staining of exosomes and EVs for detection by flow cytometry, showing bright specific signal. We have observed that CellBrite® Steady dyes bind non-specifically to immuno-capture beads, so we do not recommend these dyes for staining bead-bound exosomes.

CellBrite® Steady Dyes are available in five colors: 405, 488, 550, 650, and 685. We have verified staining of SEC-purified MCF-7-derived exosomes with all of the colors. In PEG-enriched EVs derived from HeLa cells, we have validated all of the colors except 685.

SEC-purified MCF-7-derived exosomes were stained with 0.02X CellBrite® Steady 405 (right). Specific staining was seen, compared with CellBrite® Steady 405 in buffer (left). EVs were detected on a CytoFLEX LX flow cytometer in the Pacific Blue channel.
PEG-enriched HeLa-derived EVs were stained with 2X CellBrite® Steady 650 (right). Specific staining was seen, compared with CellBrite® Steady 650 in buffer (left). EVs were detected on a CytoFLEX LX flow cytometer in the APC channel.


MemBrite® Fix 405/430 validated for exosome & EV detection

MemBrite® Fix Cell Surface Stains covalently label the surface of live cells. We have found that MemBrite® Fix 405/430 also shows good staining of enriched EVs and purified exosomes. The staining is covalent and so will withstand fixation and other downstream processes, without loss of signal or dye transfer.

MemBrite® Fix 405/430 shows strong staining of PEG-enriched EVs derived from HeLa cells, as well as SEC-purified exosomes derived from MCF-7 cells. Staining was performed with 1X MemBrite® Fix 405/430 without the pre-staining solution. It is likely that MemBrite® Fix dyes will bind non-specifically to immuno-capture beads, so we do not recommend these dyes for staining bead-bound exosomes. We have found that MemBrite® Fix 488/515 and MemBrite® Fix 640/660 do not show good EV or exosome staining.

SEC-purified MCF-7-derived exosomess were stained with 1X MemBrite® Fix 405/430 (right). Specific staining was seen, compared with MemBrite® Fix 405/430 in buffer (left). EVs were detected on a CytoFLEX LX flow cytometer in the Pacific Blue channel.
PEG-enriched HeLa-derived EVs were stained with 1X MemBrite® Fix 405/430 (right). Specific staining was seen, compared with MemBrite® Fix 405/430 in buffer (left). EVs were detected on a CytoFLEX LX flow cytometer in the Pacific Blue channel.


CellBrite® Fix 488 Membrane Stain published for EV labeling

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.

CellBrite® Fix 488 & 555 dyes show clear staining of SEC-purified exosomes derived from MCF-7 cells, though there is also some non-specific background in buffer alone. Purification or washing of the exosomes after staining is recommended. CellBrite® Fix 640 does not stain exosomes.

It is likely that CellBrite® Fix dyes will bind non-specifically to immuno-capture beads, so we do not recommend these dyes for staining bead-bound exosomes.

SEC-purified MCF-7-derived exosomes were stained with 0.5X CellBrite® Fix 555 (right). Specific staining was seen, compared with CellBrite® Fix 555 in buffer (left). Exosomes were detected on a CytoFLEX LX flow cytometer in the PE channel.

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.

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

 

View the publication here.

 

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