ExoBrite™ EV Surface Stain Sampler Kit, Green
Cat# 30127
Size : 1kit
Brand : Biotium
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.
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.
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.
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™ 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™ 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™ 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™ 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.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 | Yes |
| 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 | Yes | No | ND | Yes |
| Raji cells | ND | Yes | Yes | Yes |
| RAW 264.7 cells | Yes | Yes | Yes | Yes |
| Serum | Yes | 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 | Yes | Yes | Yes |
| HepG2 cells | Yes | No | Yes | Yes |
| Yeast (S. cerevisiae) | 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.
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.
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