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CellBrite Fix Membrane Stains

CellBrite™ Fix Membrane Stains are a new class of fluorogenic membrane dyes that covalently stain the plasma membrane in live cells. They are unique among membrane stains in that they can withstand both fixation and detergent permeabilization.

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Absorption and emission of CellBrite Fix 555 dye in water.Absorption and emission of CellBrite Fix 640 dye in water.Comparison of traditional carbocyanine dye and CellBrite Fix before and after fixation in HeLa cells. CellBrite Fix 555 gave more even surface staining and was better retained after fixation/permeabilization compared to DiI.HeLa cell membranes were stained with CellBrite Fix 555 (red) for 15 minutes at 37°C, then fixed with MeOH. Mitochondria were stained with CF®640R Anti-Mitochondrial Marker clone 111-3 (cyan) and nuclei were stained with NucSpot® 470 (green).HeLa cells labeled with CellBrite Fix for 30 minutes at room temperature, then fixed with paraformaldehyde and permeabilized with Triton X-100.HeLa cells labeled with CellBrite Fix 640 for 30 minutes at room temperature, then fixed with methanol.
Catalog #

30089, 30089-T, 30088, - 30088-TView allHide

Dye

CellBrite Fix 555, CellBrite Fix 640

Size

Trial size (1 vial), Set of 5 vials

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Product Description

CellBrite™ Fix Membrane Stains Features

  • The only membrane dyes that withstand fixation and permeabilization
  • Bright, uniform cell surface staining in 15 minutes
  • Available with visible red and far-red fluorescence
  • Each vial makes 20 uL of 1000X dye

CellBrite™ Fix dyes are a series of proprietary fluorophores developed by Biotium to rapidly stain the outer plasma membranes of live cells. Other membrane dyes like DiO, DiI, Vybrant® membrane dyes, CellMask™, CellVue®, or PKH dyes can be fixed with formaldehyde. But they are not compatible with detergent permeabilization or methanol fixation, because these treatments extract lipophilic dyes from membranes. In contrast, CellBrite™ Fix Membrane Stains are unique in that their surface staining can withstand permeabilization and methanol fixation, allowing plasma membrane staining to be combined with intracellular staining with antibodies. Unlike lectins such as WGA, which bind specific targets that may vary between cell types, CellBrite™ Fix dyes are general membrane stains.

CellBrite™ Fix dyes are amine-reactive and are designed to accumulate at the cell membrane, where they become covalently attached to membrane proteins. As a result, surface staining is well-retained after permeabilization or methanol fixation, with only a slight increase in intracellular fluorescence compared to formaldehyde fixation alone. CellBrite™ Fix dyes are only weakly fluorescent in aqueous media but become intensely fluorescent upon membrane staining. This fluorogenic property of the dyes makes the staining very specific with low background. Due to their better water solubility, CellBrite™ Fix dyes yield much more uniform staining compared to lipophilic carbocyanine dyes like DiO and DiI. The dyes are non-cytotoxic and do not readily transfer between cells. They also can be used to stain yeast and bacteria (gram-positive or gram-negative).

Because CellBrite™ Fix staining is covalent, the fluorophores remain on the outer leaflet of the cell membrane, unlike the traditional membrane dyes, such as DiO and DiI, which are known to undergo flip-flop transmembrane movement during staining. Thus, CellBrite™ Fix dyes may be uniquely suited for membrane potential detection when coupled with a quencher dye whose partition into the membrane is membrane-potential dependent, similar to the DiO/DPA system used for this purpose (see J Neurosci 29(29), 9197(2009)). We welcome any collaborator who may be interested in working with Biotium to validate CellBrite™ Fix dyes for this application to contact us at techsupport@biotium.com.

CellBrite™ Fix Membrane Stains are provided in lyophilized format. A vial of anhydrous DMSO is included for dye reconstitution. After reconstitution, each vial yields 20 uL of 1000X dye (download the product protocol for more information). The dyes are available with visible red and far-red fluorescence. More colors are in development.

DyeAbs/EmSizeCatalog number
CellBrite™ Fix 555542/571 nmTrial size (1 vial)30088-T
Set of 5 vials30088
CellBrite™ Fix 640638/667 nmTrial size (1 vial)30089-T
Set of 5 vials30089

Vybrant® and CellMask™ are trademarks of Thermo Fisher Scientific. CellVue® is a registered trademark of Millipore Sigma.

 

Support & Faq

CellBrite™ Cytoplasmic Membrane Stains

CellBrite™ and CellBrite™ Fix dyes are designed to label cell membranes. Original CellBrite™ dyes contain lipophilic carbocyanine dyes (DiB, Neuro-DiO, DiI, or DiD). These dyes accumulate in membrane lipids where they become fluorescent. Like other membrane dyes like VyBrant® DiO, VyBrant® DiI, ViBrant® DiR, CellMask™, CellVue® Claret, or PKH dyes, original CellBrite™ dyes can be fixed with formaldehyde, but are poorly retained after permeabilization with detergents or fixation with alcohol. CellBrite™ NIR dyes are CellBrite™ dyes with near-infrared fluorescence compatible with small animal NIR imaging systems.

CellBrite™ Fix dyes are a novel class of dye for labeling the cell surface. They are fluorogenic dyes that rapidly accumulate in the plasma membrane, where they react covalently with the cell surface. As a result, surface staining is well-retained after permeabilization or methanol fixation, with only a slight increase in intracellular fluorescence compared to formaldehyde fixation alone. CellBrite™ Fix dyes have better water solubility than classic lipophilic dyes, and as a result they yield much more uniform staining compared to lipophilic carbocyanine dyes like DiO and DiI. CellBrite™ Fix dyes are non-toxic and do not readily transfer between cells. They also can be used to stain yeast and bacteria (gram-positive or gram-negative).

Vybrant® and CellMask™ are trademarks of Thermo Fisher Scientific. CellVue® is a registered trademark of Millipore Sigma.

Category: CellBrite™ Cytoplasmic Membrane Stains

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Original CellBrite™ Cytoplasmic Membrane Stains are lipophilic carbocyanine dyes. These dyes undergo an increase in fluorescence when they insert into lipid bilayers. Lipophilic carbocyanine dyes stably label the plasma membrane and other intracellular membranes of cells. They also can be used to stain fixed cells or artificial lipid bilayers.

CellBrite™ Fix Membrane Stains are fluorogenic membrane dyes that react covalently with proteins on the cell surface. They have better water solubility than original CellBrite™ dyes and as a result generally give more even staining of the cell surface compared to original CellBrite™ dyes. CellBrite™ Fix are well retained after fixation and permeabiliation or methanol fixation. However, CellBrite™ Fix dyes cannot be used to stain the plasma membrane of cells after they are already fixed (the dyes stain intracellular membranes of fixed cells).

For all membrane stains, if cells are returned to culture after staining, membrane internalization will occur over time, resulting in predominantly intracellular staining.

Category: CellBrite™ Cytoplasmic Membrane Stains

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Lipophilic carbocyanine dyes like our original CellBrite™ Cytoplasmic Membrane Stains have been used to stain neuronal cells in culture for several weeks, and in vivo for up to a year. The dyes do not appreciably affect cell viability, and do not readily transfer between cells with intact membranes, allowing cell migration and tracking studies in mixed populations. Over time, the dyes will be internalized and become intracellular as membranes turn over. Long term stability of labeling may vary between cell types, depending on rates of membrane turnover or cell division.

CellBrite™ Fix Membrane Stains label the cell surface covalently. Like original CellBrite™ dyes, they are non-toxic and do not readily transfer between cells, but will be internalized and become intracellular as membranes turn over.

Category: CellBrite™ Cytoplasmic Membrane Stains

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Product shelf life, solubility, shipping and storage, and stability

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 are generally susceptible to hydrolysis, which can result in lower labeling efficiency. Heavily sulfonated dyes, such as the Alexa Fluor® dyes, DyLight® dyes and IRDyes® are particularly hygroscopic, worsening the hydrolysis problem. For example, the percent of active Alexa Fluor® 488 succinimidyl ester (SE) could be well below 50% by the time of application (according to the manufacturer’s product datasheet). In a number of Alexa Fluor® SE reactive dyes, the SE group is derived from an aromatic carboxylic acid, while in all of Biotium’s CF™ dyes the SE group 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.

Category: Mix-n-Stain™ Antibody Labeling Kits, DNA Quantitation Kits, NucView™ Caspase 3 Enzyme Substrates, Luciferase Assays, CF™ Dyes, Product shelf life, solubility, shipping and storage, and stability

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Many of our solid compounds are packaged by lyophilization, in which case they usually do not appear as fluffy powders, but form a film or coating on the sides of the vial. Simply add the appropriate volume of the recommended solvent to the vial to make the desired concentration stock solution, and swirl or gently vortex to mix. Make sure the solvent comes in contact with the inside walls of the vial to fully recover the product.

Category: Product shelf life, solubility, shipping and storage, and stability

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Most of our products are stable at room temperature for many days, but we recommend storage at 4°C or -20°C to prolong shelf life. In the case of many of our aqueous dye solutions, the compounds are very stable at room temperature, but we recommend cold storage to prevent the growth of mold or other microbes over time. Therefore, to save on shipping costs, products with recommended storage at 4°C or -20°C may ship at ambient temperature without affecting product performance. When you receive the product, place it under the recommended storage conditions.

Category: Mix-n-Stain™ Antibody Labeling Kits, DNA Quantitation Kits, NucView™ Caspase 3 Enzyme Substrates, Luciferase Assays, CF™ Dyes, Product shelf life, solubility, shipping and storage, and stability

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Shipping/Shelf-life

Shipment Method: Shipping and handling methods will be assessed and calculated at time of shipment based upon item(s) storage temperature conditions. Expedited shipment may be requested at time of checkout. Please note that products with recommended storage at 4°C or -20°C may ship at ambient temperature. This will not affect product performance. When you receive the product, place it under the recommended storage conditions.