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ExoBrite™ Annexin EV Staining Kits

Fluorescent Annexin V conjugates that are optimized for bright and low background staining of extracellular vesicles for flow cytometry.

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ExoBrite™ Annexin EV Staining Kits
ExoBrite™ Annexin EV Staining Kits - Image 2SEC-purified HeLa-derived EVs were stained with ExoBrite™ Annexin EV Stains. 410/450 was detected in the Pacific Blue channel, 490/515 was detected in the FITC channel, 560/585 was detected in the PE channel, and 650/665 was detected in the APC channel.
ExoBrite™ Annexin EV Staining Kits - Image 3ExoBrite™ 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™ Annexin EV Staining Kits - Image 4SEC purified HeLa-derived EVs stained with ExoBrite™ 410/450 Annexin EV Stain (left) compared to the same stain in buffer (right). EVs were detected on a CytoFLEX LX flow cytometer in the Pacific Blue channel.
ExoBrite™ Annexin EV Staining Kits - Image 5SEC 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™ Annexin EV Staining Kits - Image 6SEC purified HeLa-derived EVs stained with ExoBrite™ 560/585 Annexin EV Stain (left) compared to the same stain in buffer (right). EVs were detected on a CytoFLEX LX flow cytometer in the PE channel.
ExoBrite™ Annexin EV Staining Kits - Image 7SEC purified HeLa-derived EVs stained with ExoBrite™ 650/665 Annexin EV Stain (left) compared to the same stain in buffer (right). EVs were detected on a CytoFLEX LX flow cytometer in the APC channel.
ExoBrite™ EV Stain Enhancer (100X)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.
ExoBrite™ Annexin EV Staining Kits - Image 9SEC-purified HeLa-derived EVs were stained first with ExoBrite™ 560/585 CD9 in 100 uL, followed by 1X ExoBrite™ 650/665 Annexin EV Stain. EVs were detected on a CytoFLEX LX flow cytometer in the PE and APC channels. When we gated on ExoBrite™ 650/665 Annexin-positive particles, ~50% were also positive for CD9.
ExoBrite™ Annexin EV Staining Kits - Image 10SEC-purified HeLa-derived EVs were stained first with ExoBrite™ 560/585 CD63 in 100 uL, followed by 1X ExoBrite™ 650/665 Annexin EV Stain. EVs were detected on a CytoFLEX LX flow cytometer in the PE and APC channels. When we gated on ExoBrite™ 650/665 Annexin-positive particles, ~16% were also positive for CD63.

Product Description

ExoBrite™ Annexin EV Staining Kits were designed to overcome some of the challenges of EV detection, particularly in flow cytometry. ExoBrite™ Annexin EV Stains bind to molecules in the EV membrane for bright, specific staining, with little to no background.

Features

  • Optimally formulated Annexin V conjugates for staining purified EVs
  • Broad compatibility with EVs isolated from all sources tested
  • Designed for detection by flow cytometry
  • Bright signal and low background
  • Compatible with antibody co-staining
  • Available in 4 colors

Kit Components

  • ExoBrite™ Annexin EV Stain
  • 50X Annexin Binding Buffer

Validated EV Sources

  • MCF-7, J774, U2OS, Jurkat,
    HeLa, Raji, CHO, U937, A549

ExoBrite™ Annexin EV Stains are uniquely formulated conjugates of Annexin V, a 35-36 kDA calcium-dependent phospholipid-binding protein with high affinity for phosphatidyleserine (PS). Annexin V conjugates have been used to detect EVs due to the presence of PS on most EV membranes.

ExoBrite™ Annexin EV Stains were designed to overcome some of the challenges of EV detection, particularly in flow cytometry. For example, lipophilic membrane dyes commonly used to stain EVs can form aggregates of a similar size as exosomes or EVs, thus confounding analysis. Conversely, ExoBrite™ Annexin EV Stains are specially formulated to minimize aggregation in flow cytometry, allowing EVs to be identified with bright staining with minimal background. In addition, ExoBrite™ Annexin EV Stains were designed to offer broad coverage of EVs isolated from different sources. We tested EVs derived from 9 cell lines and ExoBrite™ Annexin EV Stains showed strong staining for all of them.

The ExoBrite™ EV Stain Enhancer may be used to improve the signal-to-noise when staining with ExoBrite™ 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™ Annexin staining can be combined with antibody staining, for multi-parameter analysis.

Notes:

  1. ExoBrite™ Annexin EV Stains have been found to label EVs derived from all cell lines tested (see Validated EV Sources below), but may not stain EVs from every source.
  2. 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™ Annexin EV Staining Kits

ProductEx/EmDetection channelsSizeCatalog Number
ExoBrite™ 410/450
Annexin EV Staining Kit		416/452 nmPacific Blue™100 Labelings30119-T
500 Labelings30119
ExoBrite™ 490/515
Annexin EV Staining Kit		490/516 nmFITC100 Labelings30120-T
500 Labelings30120
ExoBrite™ 560/585
Annexin EV Staining Kit		562/584 nmPE100 Labelings30121-T
500 Labelings30121
ExoBrite™ 650/665
Annexin EV Staining Kit		652/668 nmAPC100 Labelings30122-T
500 Labelings30122

ExoBrite™ EV Stains Comparison Guide

ExoBrite™ EV Surface StainProsCons
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 SourceExoBrite™ True EV Membrane StainsExoBrite™ CTB StainsExoBrite™ WGA StainsExoBrite™ Annexin Stains
A549 cellsYesYesYesYes
CHO cellsYesNoYesYes
hASC (human adipose stem cells)NDNo1NDND
HEK293 cellsYesYes1YesND
HeLa cellsYesNoYesYes
HUVEC (human umbilical vein endothelial cells)NDNo1NDND
J774 cellsYesYesYesYes
Jurkat cellsYesYesYesYes
MCF-7 cellsYesYesYesYes
PlasmaNDNoNDYes
Raji cellsNDYesYesYes
RAW 264.7 cellsYesNDNDND
SerumNDNoNDYes
Skeletal myoblastsNDYes1NDND
THP-1 cellsYesNDNDND
U2OS cellsYesNoYesYes
U937 cellsYesNoYesYes
NIH3T3 cellsYesNDNDND
HepG2 cellsNDNDYesND
Yeast (S. cerevisiae)YesNoYesYes
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™ CTB EV Stains (cholera toxin B conjugates) and ExoBrite™ WGA EV Stains (wheat germ agglutinin) optimized for bright and sensitive staining of EVs. The ExoBrite™ EV Surface Stain Sampler Kit contains 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. 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.

Note: We do not recommend using ExoBrite™ 410/450 Annexin EV Stain or ExoBrite™ 490/515 Annexin EV Stain to stain bead-bound EVs. For bead-bound EVs we recommend using ExoBrite™ 560/585 Annexin EV Stain, ExoBrite™ 650/665 Annexin EV Stain, as well as ExoBrite™ CTB EV Stains.

Product Attributes

Size
100 labelings, 500 labelings
Colors
Blue, Green, Orange-red, Far-red

Documents, Protocols, SDS and COA

FAQs

Exosome & EV Staining

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

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.

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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