ExoBrite™ 650/665 Annexin EV Staining Kit, 100 labelings
Katalog-Nummer 30122-T
Size : 1kit
Marke : Biotium
ExoBrite™ Annexin EV Staining Kits
Fluorescent Annexin V conjugates that are optimized for bright and low background staining of extracellular vesicles for flow cytometry.
SEC-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™ 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.
SEC 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.
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 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.
SEC 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.
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.
SEC-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.
SEC-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:
- 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.
- 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
| 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™ 650/665 Annexin EV Staining Kit | 652/668 nm | APC | 100 Labelings | 30122-T |
| 500 Labelings | 30122 |
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™ 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.
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