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ExoBrite™ STORM CTB EV Staining Kits

Fluorescent cholera toxin subunit B (CTB) conjugates that are optimized for direct stochastic optical reconstruction microscopy (dSTORM) imaging of EVs and exosomes.

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100 labelings
500 labelings
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Product Description

ExoBrite™ STORM CTB EV Stains incorporate a selection of CF® Dyes validated for stochastic optical reconstruction microscopy (dSTORM) and were developed for STORM imaging of extracellular vesicles (EVs).

Features

  • Optimally formulated CTB conjugates for staining EVs
  • Developed for super-resolution imaging by STORM
  • Bright signal and low background
  • Stain purified or bead-bound exosomes
  • Compatible with antibody co-staining
  • Available in 4 colors

Kit Components

  • ExoBrite™ STORM CTB EV Stain
  • ExoBrite™ Reconstitution Solution
  • 1 vial of stain makes 100 uL of 500X staining solution

Note: The name of this product has been revised from ExoBrite™ STORM EV Membrane Staining Kits.

Extracellular vesicles (EVs), including exosomes, are lipid-bound vesicles that are released from cells. EVs display specific surface proteins and can carry nucleic acids and other cargo, allowing them to transfer biological information between cells in different parts of the body. Therefore, EVs are increasingly studied for their potential use in drug delivery and medical diagnostic applications. 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 the proteins found on EVs.

ExoBrite™ STORM CTB EV Stains are optimally formulated fluorescent conjugates of cholera toxin subunit B (CTB), which binds to GM1 gangliosides that are commonly found on the surface of mammalian lipid rafts and EVs. The stains were developed for STORM imaging of EVs and incorporate a selection of our STORM-validated CF® Dyes. Unlike other lipophilic dyes traditionally used to stain EV membranes, ExoBrite™ STORM CTB EV Stains were formulated to show little to no background from aggregation in buffer only controls, allowing EVs to be accurately identified.

The ExoBrite™ STORM dyes CF®505, CF®583R, CF®647, and CF®680 have all been validated for use in dSTORM. In addition, ExoBrite™560/585 CTB EV Stain has also been validated for dSTORM on the ONI Nanoimager S Mark II together with fluorescent tetraspanin antibodies, allowing single-EV characterization studies.

Super-resolution dSTORM images of human colorectal cancer cell line derived EVs. EVs were stained with anti-tetraspanin antibodies (an anti-CD9/CD63/CD81 cocktail, all conjugated to CF®647, shown in magenta) together with 1X ExoBrite™ 560/585 (in cyan). Samples were prepared using ONI’s EV Profiler Kit and acquired using the Nanoimager S Mark II from ONI (Oxford Nanoimaging, UK). Data was processed using a beta-release version of CODI, ONI’s cloud-based data analysis platform. Scale bars are 500 nm (zoomed out, left panel) and 50 nm for single-EV panels (right). Image courtesy of ONI.

ExoBrite™ STORM CTB EV Staining Kits

ProductEx/Em (nm)Laser Line(s) (nm)Detection ChannelSizeCatalog Number
ExoBrite™ STORM CF®505 CTB EV Staining Kit505/519488FITC100 Labelings30115-T
500 Labelings30115
ExoBrite™ STORM CF®583R CTB EV Staining Kit583/609555 or 561Rhodamine or
Texas Red®
100 Labelings30116-T
500 Labelings30116
ExoBrite™ STORM CF®647 CTB EV Staining Kit652/668633-640Cy®5100 Labelings30117-T
500 Labelings30117
ExoBrite™ STORM CF®680 CTB EV Staining Kit681/698633-640Cy®5.5100 Labelings30118-T
500 Labelings30118

Validated EV Sources for ExoBrite™ CTB EV Stains

EV SourceBiotium DataCustomer Reported
A549 cellsHigh
CHO cellsLow
hASC (human adipose stem cells)Low
HEK293T cellsHigh
HeLa cellsLow
HUVEC (human umbilical vein endothelial cells)Low
J774 cellsHigh
Jurkat cellsHigh
MCF-7 cellsHigh
PlasmaLow
Raji cellsHigh
Skeletal myoblastsHigh
U2OS cellsLow
U937 cellsLow

Biotium also offers other ExoBrite™ EV Stains and ExoBrite™ Antibody Conjugates

ExoBrite™ staining can be combined with antibody staining (for example, antibodies against tetraspanin proteins CD9, CD63, & CD81), for multi-parameter analysis. Biotium offers a selection of validated ExoBrite™ antibodies optimized for detection of EVs by flow cytometry or western blot. Biotium also offers ExoBrite™ CTB EV Stains validated for detection of purified or bead-bound EVs by flow cytometry.

Product Attributes

Size
100 labelings, 500 labelings
Colors
Green, Orange-red, Far-red
Dye
ExoBrite™ STORM CF®505, ExoBrite™ STORM CF®583R, ExoBrite™ STORM CF®647, ExoBrite™ STORM CF®680

Documents, Protocols, SDS and COA

FAQs

Product shipping, storage, shelf life, & solubility

Bioscience kits
The guaranteed shelf life from date of receipt for bioscience kits is listed on the product information sheet. Some kits have an expiration date printed on the kit box label, this is the guaranteed shelf life date calculated from the day that the product shipped from our facility. Kits often are functional for significantly longer than the guaranteed shelf life. If you have an older kit in storage that you wish to use, we recommend performing a small scale positive control experiment to confirm that the kit still works for your application before processing a large number of samples or precious samples.

Antibodies and other conjugates
The guaranteed shelf life from date of receipt for antibodies and conjugates is listed on the product information sheet. Antibodies and other conjugates often are functional for significantly longer than the guaranteed shelf life. If you have an older conjugate in storage that you wish to use, we recommend performing a small scale positive control experiment to confirm that the product still works for your application before processing a large number of samples or precious samples.

For lyophilized antibodies, we recommend reconstituting the antibody with glycerol and antimicrobial preservative like sodium azide for the longest shelf life (note that sodium azide is not compatible with HRP-conjugates).

Chemicals, dyes, and gel stains
Biotium guarantees the stability of chemicals, dyes, and gel stains for at least a year from the date you receive the product. However, the majority of these products are highly stable for many years, as long as they are stored as recommended. Storage conditions can be found on the product information sheet or product safety and data sheet, material safety data sheet, and on the product label. Fluorescent compounds should be protected from light for long term storage.

If you have a Biotium compound that has been in storage for longer than one year that you wish to use, we recommend performing a small scale positive control experiment to confirm that the compound still works for your application before processing a large number of samples or precious samples.

Expiration date based on date of manufacture (DOM)
If your institution requires you to document expiration date based on date of manufacture for reagents, please contact techsupport@biotium.com for assistance.

Chemical products with special stability considerations:

Esters

Ester compounds include the following:

  • Succinimidyl esters (SE, also known as NHS esters), such as our amine-reactive dyes
  • Acetoxymethyl esters (AM esters), such as our membrane-permeable ion indicator dyes
  • Diacetate-modified dyes, like ViaFluor™ 405, CFDA, and CFDA-SE cell viability/cell proliferation dyes

Ester dyes are stable in solid form as long as they are protected from light and moisture. Esters are not stable in aqueous solution. Concentrated stock solutions should be prepared in anhydrous DMSO (see Biotium catalog no. 90082). Stock solutions in anhydrous DMSO can be stored desiccated at -20°C for one month or longer. Esters should be diluted in aqueous solution immediately before use. Succinimidyl esters (SE) should be dissolved in a solution that is free of amine-containing compounds like Tris, glycine, or protein, which will react with the SE functional group. AM esters and diacetate compounds should be dissolved in a solution that is free of serum, because serum could contain esterases that would hydrolyze the compound.

A note on CF® Dye succinimidyl ester stability

Succinimidyl esters (SE) are generally susceptible to hydrolysis, which can result in lower labeling efficiency. Many commercially available fluorescent dyes used for life science research are heavily sulfonated dyes which makes them particularly hygroscopic, worsening the hydrolysis problem. In addition, for several commercially available SE reactive dyes, the SE group is derived from an aromatic carboxylic acid, while the SE group in all of Biotium’s CF® Dyes is prepared from an aliphatic carboxylic acid. This structural difference reduces the susceptibility of CF® Dye SE reactive groups to hydrolysis, resulting in relatively stable reactive dyes with consistently higher labeling efficiency compared to other SE derivatives of other fluorescent dyes.

Maleimides, MTS and thiosulfate dyes
Like the succinimidyl ester dyes, these dyes are also susceptible to hydrolysis, although generally to a much lower degree. Thus, for long term storage, anhydrous DMSO is recommended for making stock solutions.

Other reactive dyes
Amines, aminooxy (also known as oxylamine), hydrazide, azide, alkyne, BCN, and tyramide reactive dyes, as well as dye free acids, are generally stable in aqueous solution when stored at -20°C for 6-12 months or longer, as long as no compounds are present that may react with the dye’s functional group. See the product information sheets for specific reactive dyes more information.

Coelenterazines and D-luciferin

Coelenterazines are stable in solid form when stored as recommended; they are not stable in aqueous solution. Concentrated coelenterazine stock solutions (typically 1-100 mg/mL) should be prepared in ethanol or methanol; do not use DMSO or DMF to dissolve coelenterazines, because these solvents will oxidize the compounds. Ethanol or methanol stocks of coelenterazine can be stored at -20°C or below for six months or longer; alcohol stocks may evaporate during storage, so use tightly sealing screw cap vials and wrap the vials with Parafilm for long term storage. Propylene glycol also can be used as a solvent to minimize evaporation. If the solvent evaporates, the coelenterazine will still be present in the vial, so note the volume in the vial prior to storage so that you can adjust the solvent volume to correct for evaporation if needed. Prepare working solutions in aqueous buffers immediately before use. Coelenterazines are stable for up to five hours in aqueous solution.

Aquaphile™ coelenterazines are water soluble formulations of coelenterazines. They are stable in solid form when stored as recommended. Aquaphile™ coelenterazines should be dissolved in aqueous solution immediately before use. They are stable for up to five hours in aqueous solution.

Note that coelenterazines are predominantly yellow solids, but may contain dark red or brown flecks. This does not affect product stability or performance. If your coelenterazine is uniformly brown, then it is oxidized and needs to be replaced.

D-luciferin is stable in solid form and as a concentrated stock solution when stored as recommended; it is not stable at dilute working concentrations in aqueous solution. Prepare concentrated D-luciferin stock solutions (typically 1-100 mg/mL) in water, and store in aliquots at -20°C or below for six months or longer. Prepare working solutions immediately before use.

For dyes or reagents that are supplied lyophilized (as solids), it is hard to compare quantities based on appearance of the dye in the tube, because during the lyophilization process the dye can dry down in different ways, either spread out all over the tube, clumped together, or coating the sides or bottom of the tube. Centrifugation of the tube may not help in collecting the dye solid to the bottom of the tube as this generally works for solutions. However, lyophilized solids are packaged based on highly accurate absorbance measurement of the reagent solution prior to drying, so the vial will contain the correct amount of dye.

Biotium ships all antibodies (primary, secondary and conjugates) at room temperature. We guarantee their quality and performance under these conditions based upon our stability testing. Antibodies were subjected to accelerated stability testing by storing them at various temperatures (4°C, room temperature, or 37°C) for 1 week to mimic simulated shipping conditions and tested in immunostaining experiments. All antibodies showed the expected brightness and specificity, even after storage at sub-optimal temperatures for a week or longer. You can also download our Product Storage Statement here.

In line with our goal to be more environmentally friendly by reducing the use of excess packaging, and lowering shipping costs for our customers, products that have passed our stability testing are shipped at room temperature.

Once you have received the antibody vial, please follow the long-term storage instructions on the product information (PI) sheet.

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