ExoBrite™ EV Surface Stain Sampler Kit, Green

Kit includes each of ExoBrite™ 490/515 EV Surface Stains (CTB, WGA, and Annexin V) for assessing which stain offers the best coverage for EV samples of interest.

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ExoBrite™ EV Surface Stain Sampler Kit, Green

ExoBrite™ EV Surface Stain Sampler Kit, Green - Image 2

SEC purified MCF-7-derived EVs stained with ExoBrite™ 490/515 WGA EV Stain (left) compared to the same stain in buffer (right). EVs were detected on a CytoFLEX LX flow cytometer in the FITC channel.

ExoBrite™ EV Surface Stain Sampler Kit, Green - Image 3

SEC purified HeLa-derived EVs stained with ExoBrite™ 490/515 Annexin EV Stain (left) compared to the same stain in buffer (right). EVs were detected on a CytoFLEX LX flow cytometer in the FITC channel.

ExoBrite™ EV Surface Stain Sampler Kit, Green - Image 4

SEC purified MCF-7-derived EVs stained with ExoBrite™ 490/515 CTB EV Stain (left) compared to the same stain in buffer (right). EVs were detected on a CytoFLEX LX flow cytometer in the FITC channel.

ExoBrite™ EV Surface Stain Sampler Kit, Green - Image 5

ExoBrite™ 490/515 CTB EV Stain was used to stain either SEC-purified, Jurkat-derived EVs, or artificial liposomes. Robust staining was seen with the EVs, but the liposomes were not stained. The liposomes in this experiment were prepared from Presome® ACD-1, and were verified to be of similar size and concentration to the EVs using the lipophilic dye di-8-ANNEPS (data not shown). Analysis was performed on a CytoFLEX flow cytometer with SSC detected from the 405 nm laser, and ExoBrite™ detected in the FITC channel. Presome® is a registered trademark of NIPPON FINE CHEMICAL CO., LTD.

ExoBrite™ EV Surface Stain Sampler Kit, Green - Image 6

ExoBrite™ 490/515 WGA EV Stain was used to stain either SEC-purified, Jurkat-derived EVs, or artificial liposomes. Robust staining was seen with the EVs, but the liposomes were not stained. The liposomes in this experiment were prepared from Presome® ACD-1, and were verified to be of similar size and concentration to the EVs using the lipophilic dye di-8-ANNEPS (data not shown). Analysis was performed on a CytoFLEX flow cytometer with SSC detected from the 405 nm laser, and ExoBrite™ detected in the FITC channel. Presome® is a registered trademark of NIPPON FINE CHEMICAL CO., LTD.

ExoBrite™ EV Surface Stain Sampler Kit, Green - Image 7

ExoBrite™ 490/515 Annexin EV Stain was used to stain either SEC-purified, Jurkat-derived EVs, or artificial liposomes. Robust staining was seen with the EVs, but the liposomes were not stained. The liposomes in this experiment were prepared from Presome® ACD-1, and were verified to be of similar size and concentration to the EVs using the lipophilic dye di-8-ANNEPS (data not shown). Analysis was performed on a CytoFLEX flow cytometer with SSC detected from the 405 nm laser, and ExoBrite™ detected in the FITC channel. Presome® is a registered trademark of NIPPON FINE CHEMICAL CO., LTD.

ExoBrite™ EV Surface Stain Sampler Kit, Green - Image 8

ExoBrite™ CTB EV Stains have less background and more complete staining of extracellular vesicles (EVs) than other classic and competitor dyes. EVs were purified from MCF-7 cell supernatant using size exclusion chromatography (SEC). The purified EVs were stained in PBS with the indicated dyes (top row). The stained EV 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 EVs, making them unsuited for small particle staining. CellMask™ also shows an unacceptable amount of dye aggregation falling within the EV gate. The ExoFlow-ONE™ and ExoGlow™ dyes form aggregates that can mostly be gated away from the EVs, but they also show less-complete coverage of EVs than ExoBrite™ CTB stains.

HeLa EVs were stained with ExoBrite™ 490/515 WGA Stain with or without ExoBrite™ EV Stain Enhancer. The Enhancer reduces dye aggregates and increases the number of detected stained EVs, for improved signal-to-noise.

Jurkat EVs were stained with ExoBrite™ 490/515 Annexin Stain with or without ExoBrite™ EV Stain Enhancer. The Enhancer reduces dye aggregates and increases the number of detected stained EVs, for improved signal-to-noise.

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Product Description

The ExoBrite™ EV Surface Stain Sampler Kit, Green was developed to offer each of Biotium’s ExoBrite™ EV Surface Stains (CTB, WGA, and Annexin V) for assessing which stain offers the best coverage for the EV samples of interest.

Features

Optimally formulated ExoBrite™ 490/515 Annexin, CTB, and WGA conjugates for staining purified EVs
Broad compatibility with EVs isolated from different sources
Designed for detection by flow cytometry
Bright signal and low background
Compatible with antibody co-staining

Kit Components

ExoBrite™ 490/515 Annexin EV Stain
ExoBrite™ 490/515 WGA EV Stain
ExoBrite™ 490/515 CTB EV Stain
ExoBrite™ Reconstitution Solution
ExoBrite™ 1X PBS Solution
50X Annexin Binding Buffer

Bright & Specific EV Staining with Minimal Aggregation

ExoBrite™ EV Surface Stains are conjugates of probes for labeling EV membrane surface targets using Biotium’s unique fluorescent dyes for superior brightness and specificity. The stains were designed to overcome some of the challenges of EV detection, particularly in flow cytometry. For example, some lipophilic membrane dyes used to stain EVs can form aggregates of a similar size as exosomes or EVs, thus confounding analysis. ExoBrite™ EV stains have been formulated for bright and specific staining of EV surface targets with minimal aggregation in flow cytometry. In addition, ExoBrite™ EV Stains do not bind non-specifically to polystyrene beads, and therefore unlike hydrophobic membrane dyes, they can be used to stain bead-bound EVs.

A Convenient Sampler Kit for Comparing Samples

A major issue for EV detection includes varying signal and coverage using tetraspanin antibody staining of EVs isolated from different cell types or biological fluids. The ExoBrite™ EV Surface Stain Sampler Kit, Green includes each of Biotium’s ExoBrite™ EV Surface Stains (CTB, WGA, and Annexin V) to allow users to assess which stain(s) offer the best coverage for their EV samples. ExoBrite™ CTB EV Stains are conjugates of cholera toxin subunit B (CTB), which binds to GM1 gangliosides that are found on the surface of mammalian lipid rafts and some EV populations. ExoBrite™ WGA EV Stains are uniquely formulated conjugates of wheat germ agglutinin (WGA), a carbohydrate-binding lectin with high affinity for N-acetylglucosamine moieties of glycoproteins frequently exposed on EV membranes. ExoBrite™ Annexin V EV Stains is a calcium-dependent phospholipid-binding protein with high affinity for phosphatidyleserine (PS), which is exposed on the surface of apoptotic cells and also used as a marker for EV from a variety of sources.

The ExoBrite™ EV Stain Enhancer may be used to improve the signal-to-noise when staining with ExoBrite™ WGA and Annexin EV Stains. The enhancer has shown to be beneficial for staining EVs with Annexin V, WGA, and other lectins. It is easy to use, simply add it directly to your staining reaction, and does not interfere with antibody staining.

EVs are often labeled with fluorescent antibodies targeting one or more of the tetraspanin proteins CD9, CD63, and CD81. ExoBrite™ EV Stains can be combined with antibody staining, for multi-parameter analysis. Biotium offers a selection of fluorescent ExoBrite™ Flow Antibodies against CD9, CD63, and CD81 that are optimized for detection of free or bead-bound EVs by flow cytometry.

Notes:

ExoBrite™ EV Stains have been found to label EVs derived from several tested cell lines (see Validated EV Sources below), but may not stain EVs from every source.
In our testing, we have found that ExoBrite™ 490/515 dye may bind to streptavidin coated surfaces or beads if free biotin binding sites are not blocked. We recommend performing a biotin blocking step after binding your biotinylated capture antibody to streptavidin beads or surfaces when using ExoBrite™ 490/515 conjugates. Alternatively, consider using a different ExoBrite™ dye for staining EVs captured on streptavidin beads or surfaces.

ExoBrite™ EV Stains Comparison Guide

ExoBrite™ EV Surface Stain	Pros	Cons
ExoBrite™ True EV Membrane Stains	• Near-complete staining of EVs in a sample • Broad compatibility with different EV sources • Validated for flow and fNTA	• Can't be used to stain bead-bound EVs • May have more aggregation than CTB & Annexin
ExoBrite™ Annexin EV Staining Kits	• Broad compatibility with different EV sources • Validated for flow and fNTA • Low background aggregates	• May not stain every EV in a sample • Doesn't work well on bead-bound EVs
ExoBrite™ WGA EV Staining Kits	• Broad compatibility with different EV sources • Can be used with bead-bound EVs	• May not stain every EV in a sample • Doesn't work well for fNTA
ExoBrite™ CTB EV Staining Kits	• Validated for flow and fNTA • Extremely low background • Can be used with bead-bound EVs	• May not stain every EV in a sample • Does not stain EVs from every source
ExoBrite™ Antibodies	• Highly specific for human tetraspanins CD9, CD63, CD81, and other EV markers • Validated for EV flow • Broad compatibility for different EV sources • Can be used with bead-bound EVs • Can be used for WB	• Depends on the expression level of the target protein on the EVs
ExoBrite™ EV Stain Enhancer	• Improves signal-to-noise by reducing or eliminating aggregates of certain EV stains • Validated with several different lectins and Annexin V • Does not interfere with antibody staining of EVs • Easy to use, just add directly to the staining reaction	• Not recommended for use with lipophilic EV stains

Validated EV Sources for ExoBrite™ EV Surface Stains

EV Source	ExoBrite™ True EV Membrane Stains	ExoBrite™ CTB Stains	ExoBrite™ WGA Stains	ExoBrite™ Annexin Stains
A549 cells	Yes	Yes	Yes	Yes
CHO cells	Yes	No	Yes	Yes
hASC (human adipose stem cells)	ND	No¹	ND	ND
HEK293 cells	Yes	Yes¹	Yes	ND
HeLa cells	Yes	No	Yes	Yes
HUVEC (human umbilical vein endothelial cells)	ND	No¹	ND	ND
J774 cells	Yes	Yes	Yes	Yes
Jurkat cells	Yes	Yes	Yes	Yes
MCF-7 cells	Yes	Yes	Yes	Yes
Plasma	ND	No	ND	Yes
Raji cells	ND	Yes	Yes	Yes
RAW 264.7 cells	Yes	ND	ND	ND
Serum	ND	No	ND	Yes
Skeletal myoblasts	ND	Yes¹	ND	ND
THP-1 cells	Yes	ND	ND	ND
U2OS cells	Yes	No	Yes	Yes
U937 cells	Yes	No	Yes	Yes
NIH3T3 cells	Yes	ND	ND	ND
HepG2 cells	ND	ND	Yes	ND
Yeast (S. cerevisiae)	Yes	No	Yes	Yes

¹Customer-reported data
Value of “Yes” or “No” indicates coverage of EVs based on Biotium’s internal data or customer-reported data. Value of “ND” indicates no data.

Biotium also offers other validated ExoBrite™ reagents for flow cytometry, western blotting, or super-resolution imaging.

Learn about Biotium's new ExoBrite™ True EV Membrane Stains. These genuine lipophilic membrane dyes are designed for superior pan-EV labeling over other membrane dyes including PKH, DiO, DiI, and DiD. Biotium also offers ExoBrite™ Antibody Conjugates for optimal detection of CD9, CD63, and CD81 EV markers by flow cytometry and western blotting. For super-resolution imaging by STORM, learn about our ExoBrite™ STORM CTB EV Staining Kits available in four CF® Dyes validated for STORM.

Product Attributes

Dye

ExoBrite™ 490/515

Colors

Green

Documents, Protocols, SDS and COA

Protocols

PI-30127

SDS

SDS 30127_ExoBrite™ EV Surface Stain Sampler Kit, Green_US_en

SDS 30127_ExoBrite™ EV Surface Stain Sampler Kit, Green_CA_en

SDS 30127_ExoBrite™ EV Surface Stain Sampler Kit, Green_BE_en

COA

Request the Certificate of Analysis

FAQs

Exosome & EV Staining

ExoBrite™ Antibodies for western validation of immunomodulatory MSC EVs

Extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) are emerging as powerful, cell-free immunomodulatory therapies for inflammatory diseases such as COVID-19. However, because the mechanism is poorly understood, optimizing EV-based therapies remains challenging.

In a 2025 Springer Nature study, Infante et al. investigated how COVID-19 patient serum reshapes the transcriptome and paracrine activity of Wharton’s jelly–derived MSC stem cells (WJ-MSCs). WJ-MCSs exposed to serum from hospitalized COVID patients showed downregulation of NEAT1 and MALAT1, two pro-inflammatory two long noncoding RNAs (lncRNAs). Furthermore, the researchers found that EVs derived from the treated cells had enhanced immunosuppressive activity when administered to T-cells.

The researchers isolated EVs from WJ-MSC cells after NEAT1 and/or MALAT1 knockdown, and tested whether there was an effect on T-cell proliferation. A Western blot of EVs derived from control and lncRNA-knockdown MSCs were probed with ExoBrite™ 680/700 CD81 Western Antibody. ExoBrite™ 770/800 Calnexin Western Antibody was also used as an endoplasmic reticulum marker to assess cellular contamination.

EV enriched samples in control, NEAT1 knockdown, MALAT1 knockdown, and NEAT1/MALAT1-double knockdown were confirmed by bright CD81 detection and the absence of Calnexin. They found that the MALAT1 knockdown EVs were found to have an inhibitory effect on T-cell proliferation. These results illustrate the importance of EV characterization using tools like Biotium’s ExoBrite™ antibodies in translational EV research.

Isolation and characterization of EVs from various lncRNA knock-down WJ-MSCs. Western blot analysis using ExoBrite™ 680/700 CD81 and ExoBrite™ 770/800 Calnexin in EV and MSC lysates. Asterisk (*) indicates reduced conditions used in the MSCs lysate. Modified from Infante et. al. Reproduced under CC BY 4.0.

Learn more about Biotium’s many stains and antibodies for EV research, including ExoBrite™ CD9/CD63/CD81 Antibody Cocktails for flexible and bright multiplexing detection by flow cytometry. Biotium also offers ExoBrite™ stains for pan-EV labeling, optimized fluorescent conjugates of CTB, WGA, and Annexin V for EV detection, ExoBrite™ antibodies for STORM imaging, and more.

Full Citation:

Infante, A., Cabodevilla, L., Gener, B. et al. Modulation of NEAT1 and MALAT1 expression in WJ-MSCs by Covid-19 serum: a foundation for EVs-mediated therapy. Respir Res 26, 313 (2025). https://doi.org/10.1186/s12931-025-03394-4

Can other membrane stains besides ExoBrite™ True EV Membrane Stains be used to stain extracellular vesicles (EVs)?

While early studies of EVs attempted to use first-generation membrane dyes like DiI or PKH to stain EVs, more recently this class of dyes has been found to be largely unsuitable for EV staining due to their high degree of aggregation. Dye aggregation not only generates nonspecific particles that are indistinguishable from EVs in flow cytometry, but also results in poor EV labeling efficiency. Biotium developed the ExoBrite™ True EV Membrane Stains in response to our customers difficulties with using traditional membrane dyes to stain EVs. See our Literature Digest for more information.

Which of Biotium's antibodies can be used to stain EVs?

We strongly recommend our ExoBrite™ Flow Antibody Conjugates for staining both purified or bead-bound EVs. The antibodies are validated and optimized to offer bright signal and low background. They are available against human or mouse CD9, CD63, and CD81 tetraspanin proteins.

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