Check Out Our helpful Tech Tips on EV Isolation and Detection
Tech Tip: Exosome Isolation and Staining Protocols
In this Tech Tip, Biotium scientists provide detailed protocols for isolating EVs from cell supernatants using PEG precipitation or size exclusion chromatography (SEC).
Robust Stains Designed to Detect EVs, Not Dye Aggregates
Membrane dyes, while common tools for labeling EVs, can pose significant challenges when used for EV staining. For example, membrane dyes such as PKH, DiO, and DiI, have poor solubility and thus form aggregates that can be confused with EVs. To overcome these challenges, Biotium developed ExoBrite™ True EV Membrane Stains and ExoBrite™ EV Surface Stains (Annexin V, WGA, and CTB) to offer higher coverage and signal-to-noise over membrane dyes commonly used for EV staining. View our comparison table and validated EV sources below to select the best stain for your next experiment.
ExoBrite™ CTB, WGA, and Annexin V Conjugates offer bright staining of EVs with broad compatibility and low background
![](https://biotium.com/wp-content/uploads/2024/03/ExoBrite-CTB-Annexin-WGA-separate-e1714693445818.png)
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 | • May not stain every EV in a sample • Does not stain EVs from every source |
ExoBrite™ Antibodies | • Highly specific for human tetraspanins CD9, CD63, or CD81 • 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 Surface Stains
Product | Conjugate | Detection channels | Size | Catalog Number |
---|---|---|---|---|
ExoBrite™ True EV Membrane Stains | N/A | FITC, PE | 100 Labelings, 500 Labelings | 30129, 30130 |
ExoBrite™ Annexin EV Staining Kits | Annexin V | Pacific Blue™, FITC, PE, Cy®3, APC | 100 Labelings, 500 Labelings | 30119-30122 |
ExoBrite™ WGA EV Staining Kits | Wheat Germ Agglutinin (WGA) | 30123-30126 | ||
ExoBrite™ CTB EV Staining Kits | Cholera Toxin Subunit B (CTB) | 30111-30114 | ||
ExoBrite™ EV Surface Stain Sampler Kit, Green | Annexin V, Wheat Germ Agglutinin (WGA), Cholera Toxin Subunit B (CTB) | FITC | 100 Labelings | 30127 |
ExoBrite™ STORM CTB EV Staining Kits | Cholera Toxin Subunit B (CTB) | FITC, Texas Red®, Cy®5, Cy®5.5 | 100 Labelings, 500 Labelings | 30115-30118 |
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 |
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 | Yes | ND | ND | ND |
Serum | ND | No | ND | Yes |
Skeletal myoblasts | ND | Yes1 | ND | ND |
THP-1 | Yes | ND | ND | ND |
U2OS cells | Yes | No | Yes | Yes |
U937 cells | Yes | No | Yes | Yes |
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.
EXOBRITE™ TRUE EV MEMBRANE STAINS
The Best Choice for Pan-EV Staining
ExoBrite™ True EV Membrane Stains are novel membrane dyes designed specifically for EV detection. While other membrane dyes like PKH, DiO, and DiI, are commonly used for labeling EVs, these dyes often have poor solubility and thus form aggregates that can be confused with EVs.
ExoBrite™ True EV Membrane Stains were designed specifically to address the issues of membrane dye aggregation and are the best choice for general pan-EV staining. They demonstrate higher (near-complete) coverage of EVs over competitor membrane dyes in fluorescence nanoparticle tracking analysis (fNTA). They also offer clear differentiation of EVs from non-specific particles during flow detection, which is not usually possible with other membrane dyes.
Advantages of ExoBrite™ True EV Membrane Stains
- Lipophilic membrane dyes designed for pan-EV labeling
- Superior alternative to common membrane dyes like PKH, DiO, DiI, DiD
- Bright signal with near-complete coverage of EVs in a sample
- Broad compatibility with EVs isolated from different sources
- Available in two colors for detection by flow in FITC or PE channels
- Compatible with antibody co-staining
Discover Superior EV Coverage and Signal-to-Noise Over PKH, DiO, and Other Membrane Dyes
![](https://biotium.com/wp-content/uploads/2024/04/ExoBrite-515_540-True-Membrane-PKH-DiO-scaled.jpg)
![](https://biotium.com/wp-content/uploads/2024/04/ExoBrite-515_540-True-Membrane-PKH-DiO-zeta.jpg)
ExoBrite™ True EV Membrane Stains
Product | Ex/Em | Detection Channels | Size | Catalog Number |
---|---|---|---|---|
ExoBrite™ 400/460 True EV Membrane Stain | 402/460 nm | Pacific Blue® | 100 Labelings | 30136-T |
500 Labelings | 30136 | |||
ExoBrite™ 515/540 True EV Membrane Stain | 515/542 nm | FITC | 100 Labelings | 30129-T |
500 Labelings | 30129 | |||
ExoBrite™ 555/575 True EV Membrane Stain | 556/576 nm | PE | 100 Labelings | 30130-T |
500 Labelings | 30130 | |||
ExoBrite™ 645/675 True EV Membrane Stain | 644/671 nm | APC | 100 Labelings | 30137-T |
500 Labelings | 30137 |
EXOBRITE™ ANNEXIN EV STAINING KITS
Bright & Sensitive EV Stains with Broad Compatibility
ExoBrite™ Annexin EV Staining Kits are uniquely formulated conjugates of Annexin V designed with broad compatibility for EVs derived from different sources. Similar to the ExoBrite™ CTB EV Stains, these Annexin V-based conjugates were developed for bright and sensitive staining of isolated EVs by flow cytometry with little to no background aggregation. ExoBrite™ Annexin EV Staining Kits also offer the broadest coverage for EVs. We tested EVs derived from 9 cell lines and ExoBrite™ Annexin EV Stains showed strong staining for all of them.
Advantages of ExoBrite™ Annexin EV Stains:
- Optimally formulated Annexin V conjugates for staining purified EVs
- Broad compatibility, stained EVs isolated from all 9 sources tested
- Bright signal and low background staining for flow cytometry
- Compatible with antibody co-staining
- Available in 4 colors for flexible experimental design
![](https://biotium.com/wp-content/uploads/2023/09/ExoBrite-490-Annexin-liposome-figure.png)
![](https://biotium.com/wp-content/uploads/2023/09/ExoBrite-655-Annexin-560-CD9-costain.png)
ExoBrite™ Annexin EV Staining Kits
Product | Ex/Em | Detection channels | Size | Catalog Number |
---|---|---|---|---|
ExoBrite™ 410/450 Annexin EV Staining Kit | 416/452 nm | Pacific Blue™ | 100 Labelings | 30119-T |
500 Labelings | 30119 | |||
ExoBrite™ 490/515 Annexin EV Staining Kit | 490/516 nm | FITC | 100 Labelings | 30120-T |
500 Labelings | 30120 | |||
ExoBrite™ 560/585 Annexin EV Staining Kit | 562/584 nm | PE | 100 Labelings | 30121-T |
500 Labelings | 30121 | |||
ExoBrite™ 655/670 Annexin EV Staining Kit | 652/668 nm | APC | 100 Labelings | 30122-T |
500 Labelings | 30122 |
EXOBRITE™ WGA EV STAINING KITS
Sensitive Stains for Purified or Bead-Bound EVs with Broad Compatibility
ExoBrite™ WGA EV Stains are fluorescent conjugates of wheat germ agglutinin (WGA) optimized for bright and sensitive staining of EVs. Similar to ExoBrite™ Annexin EV Stains, these WGA-based conjugates offer broad compatibility with EVs. The stains were validated for EVs derived from all 9 cell lines tested. In addition, ExoBrite™ WGA EV Stains are less prone to aggregation than hydrophobic membrane dyes and may be used to stain purified and bead-bound EVs.
Advantages of ExoBrite™ WGA EV Stains:
- Optimally formulated WGA conjugates for staining purified or bead-bound EVs
- Broad compatibility, stained EVs isolated from all 9 sources tested
- Bright signal and low background staining for flow cytometry
- Compatible with antibody co-staining
- Available in 4 colors for flexible experimental design
![](https://biotium.com/wp-content/uploads/2023/10/ExoBrite-410-490-560-640-WGA-flow-qEV.png)
![](https://biotium.com/wp-content/uploads/2023/10/ExoBrite-490-WGA-liposome-figure.png)
![](https://biotium.com/wp-content/uploads/2023/10/ExoBrite-640-WGA-490-CD81-costain.png)
ExoBrite™ WGA EV Staining Kits
Product | Ex/Em | Detection channels | Size | Catalog Number |
---|---|---|---|---|
ExoBrite™ 410/450 WGA EV Staining Kit | 416/452 nm | Pacific Blue™ | 100 Labelings | 30123-T |
500 Labelings | 30123 | |||
ExoBrite™ 490/515 WGA EV Staining Kit | 490/516 nm | FITC | 100 Labelings | 30124-T |
500 Labelings | 30124 | |||
ExoBrite™ 560/585 WGA EV Staining Kit | 562/584 nm | PE | 100 Labelings | 30125-T |
500 Labelings | 30125 | |||
ExoBrite™ 640/660 WGA EV Staining Kit | 642/663 nm | APC | 100 Labelings | 30126-T |
500 Labelings | 30126 |
EXOBRITE™ CTB EV STAINING KITS
Bright EV Stains with Excellent Signal-to-Noise
ExoBrite™ CTB EV Staining Kits were designed to overcome some of the challenges of EV detection. ExoBrite™ stains bind to molecules in the EV 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 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 EVs.
Advantages of ExoBrite™ CTB EV Stains:
- Optimally formulated CTB conjugates for staining EVs
- Designed for detection by flow cytometry
- Bright fluorescence and low background for excellent signal-to-noise
- Compatible with antibody co-staining
- Stain purified or bead-bound EVs
- Available in 4 colors for flexible experimental design
![](https://biotium.com/wp-content/uploads/2021/08/ExoBrite-410-490-560-640-SEC-exosomes-944x1024.jpg)
![](https://biotium.com/wp-content/uploads/2021/08/ExoBrite-490-CTB-liposome-figure.png)
![](https://biotium.com/wp-content/uploads/2021/08/ExoBrite-560-CTB-490-CD9-costain.png)
ExoBrite™ CTB EV Staining Kits
Product | Ex/Em | Detection channels | Size | Catalog Number |
---|---|---|---|---|
ExoBrite™ 410/450 CTB EV Staining Kit | 416/452 nm | Pacific Blue™ | 100 Labelings | 30111-T |
500 Labelings | 30111 | |||
ExoBrite™ 490/515 CTB EV Staining Kit | 490/516 nm | FITC | 100 Labelings | 30112-T |
500 Labelings | 30112 | |||
ExoBrite™ 560/585 CTB EV Staining Kit | 562/584 nm | PE, Cy®3 | 100 Labelings | 30113-T |
500 Labelings | 30113 | |||
ExoBrite™ 640/660 CTB EV Staining Kit | 642/663 nm | APC | 100 Labelings | 30114-T |
500 Labelings | 30114 |
CellMask is a trademark of Thermo Fisher Scientific; ExoFlow-ONE and ExoGlow are trademarks of System Biosciences.
ExoBrite™ Flow Antibody Conjugates
Validated antibodies for detection of EV markers by flow cytometry
The most common proteins used as EV 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 EV markers in purified and bead-bound EVs.
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.
Advantages of ExoBrite™ Flow Antibodies:
- Developed for detection of EV markers CD9, CD63, and CD81 by flow cytometry
- Validated for purified or bead-bound EVs
- Bright signal and low background
- ExoBrite™ Isotype Control Flow Antibody available
- Colors available for Pacific Blue™, FITC, and PE channels
Purified EVs stained with ExoBrite™ Flow Antibodies
Product | Conjugates | Detection Channels | Sizes | Catalog Number |
---|---|---|---|---|
ExoBrite™ CD9 Flow Antibody | ExoBrite™ 410/450 ExoBrite™ 490/515 ExoBrite™ 560/585 ExoBrite™ 650/665 ExoBrite™ R-PE | Pacific Blue ™ FITC PE APC | 25 tests 100 tests | P003-410… P003-RPE |
ExoBrite™ CD9 (Mouse) Flow Antibody | P018-410… P018-650 | |||
ExoBrite™ CD63 Flow Antibody | ExoBrite™ 410/450 ExoBrite™ 490/515 ExoBrite™ 560/585 ExoBrite™ R-PE | Pacific Blue ™ FITC PE | 25 tests 100 tests | P004-410… P004-RPE |
ExoBrite™ CD63 (Mouse) Flow Antibody | ExoBrite™ 410/450 ExoBrite™ 490/515 ExoBrite™ 560/585 | P022-410… P022-560 | ||
ExoBrite™ CD81 Flow Antibody | ExoBrite™ 410/450 ExoBrite™ 490/515 ExoBrite™ 560/585 ExoBrite™ R-PE | Pacific Blue ™ FITC PE | 25 tests 100 tests | P005-410… P005-RPE |
ExoBrite™ CD81 (Mouse/Rat) Flow Antibody | ExoBrite™ 410/450 ExoBrite™ 490/515 ExoBrite™ 560/585 | P019-410… P019-560 | ||
ExoBrite™ IgG1 Isotype Control Flow Antibody | ExoBrite™ 410/450 ExoBrite™ 490/515 ExoBrite™ 560/585 ExoBrite™ 650/665 ExoBrite™ R-PE | Pacific Blue ™ FITC PE APC | 25 tests 100 tests | P008-410… P008-RPE |
ExoBrite™ Streptavidin Magnetic Beads
Convenient & Optimized Capture of EVs
Streamline your EV and exosome isolation workflows with ExoBrite™ Streptavidin Magnetic Beads, developed to offer lower background and higher sensitivity for EV capture than similar products. These streptavidin-coated and magnetic polystyrene beads (4.5 um diameter) may be used to isolate EVs from cell culture medium or other biological fluids without an overnight precipitation step.
Advantages of ExoBrite™ Streptavidin Magnetic Beads:
- Combine with a biotinylated antibody (not included) for isolation of EVs
- Isolate EVs from biofluids without an overnight precipitation step
- Lower background and higher sensitivity than competitors
- Convenient magnetic-based separation
- Detect downstream with microscopy, flow cytometry, or western blot
![](https://biotium.com/wp-content/uploads/2023/11/28000-CD9-biotin-CTB560.jpg)
![](https://biotium.com/wp-content/uploads/2023/11/28000-CD63-biotin-CTB560.jpg)
![](https://biotium.com/wp-content/uploads/2023/11/28000-CD81-biotin-CD9-560.jpg)
![](https://biotium.com/wp-content/uploads/2023/11/28000-CD81-biotin-CTB560.jpg)
SUPER-RESOLUTION IMAGING
See beyond the diffraction limit with
ExoBrite™ STORM CTB EV Staining Kits
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™ STORM CTB EV Stains were developed for STORM imaging of EVs and are available in four CF® Dyes validated for STORM. ExoBrite™ 560/585 has been validated for dSTORM on the ONI Nanoimager S Mark II, allowing the study of fine morphological details and co-staining with EV biomarkers such as tetraspanin proteins CD9, CD63, and CD81.
Advantages of ExoBrite™ STORM CTB EV Stains:
- Dive deeper into EV morphology and structure
- Optimally formulated CTB conjugates for imaging EVs by STORM
- Available in 4 CF® Dyes validated for STORM imaging
- Allows antibody co-staining and localization studies with EV biomarkers
![](https://biotium.com/wp-content/uploads/2023/03/Exobrite-Fig-4.4.23-800x596-1.png)
Product | Ex/Em (nm) | Laser Line(s) (nm) | Detection Channel | Size | Catalog Number |
---|---|---|---|---|---|
ExoBrite™ STORM CF®505 CTB EV Staining Kit | 505/519 | 488 | FITC | 100 Labelings | 30115-T |
500 Labelings | 30115 | ||||
ExoBrite™ STORM CF®583R CTB EV Staining Kit | 583/609 | 555 or 561 | Rhodamine or Texas Red® | 100 Labelings | 30116-T |
500 Labelings | 30116 | ||||
ExoBrite™ STORM CF®647 CTB EV Staining Kit | 652/668 | 633-640 | Cy®5 | 100 Labelings | 30117-T |
500 Labelings | 30117 | ||||
ExoBrite™ STORM CF®680 CTB EV Staining Kit | 681/698 | 633-640 | Cy®5.5 | 100 Labelings | 30118-T |
500 Labelings | 30118 |
ExoBrite™ Western Antibody Conjugates
Validated antibodies for detection of EV markers by western blotting
ExoBrite™ Western Antibodies were validated to offer bright signal and low background of EV 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 EVs. It is offered as a negative control to assess the purity of isolated EV extracts.
Advantages of ExoBrite™ Western Antibodies:
- Developed for detection of EV 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
Product | Conjugates | Sizes | Catalog Number |
---|---|---|---|
ExoBrite™ CD9 Western Antibody | ExoBrite™ 680/700 ExoBrite™ 770/800 | 25 tests 100 tests | P003-680, P003-770 |
ExoBrite™ CD63 Western Antibody | P004-680, P004-770 | ||
ExoBrite™ CD81 Western Antibody | P006-680, P006-770 | ||
ExoBrite™ Calnexin Western Antibody | ExoBrite™ 770/800 | P007-770 |
TOTAL RNA EXTRACTION
Efficient total RNA extraction from purified EVs
The ExoBrite™ EV Total RNA Isolation Kit was designed to address the challenges of EV RNA extraction by offering an optimized and easy-to-use kit for total RNA isolation, including mRNA and miRNAs, from purified EVs. The isolated EV RNA can then be used for downstream analysis such as qPCR or RNAseq.
Advantages of ExoBrite™ EV Total RNA Isolation Kit:
- Optimized for total RNA extraction from purified EVs
- Recover ~10-20 ng of RNA from 1×1010 SEC-enriched EVs
- Compatible with downstream applications such as qPCR or RNAseq
- Simple column-based purification
- No phenol/chloroform or ethanol precipitation steps