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CellBrite® Steady Membrane Staining Kits

Cell surface staining kits with fluorescent membrane dye plus enhancer for imaging live cell surface for several hours to days.

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100 labelings
500 labelings
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Product Description

CellBrite® Steady Membrane Staining Kits allow fluorescence imaging of cell surface for up to several days in culture. The CellBrite® Steady Dyes are unique fluorescent membrane probes that distribute between the cell surface and intracellular compartments, so cells retain cell surface staining over time. With the use of CellBrite® Steady Enhancer, intracellular staining can be reduced or eliminated for imaging of cell outlines or boundaries.

  • Retained on cell surface, unlike other membrane dyes that are rapidly internalized
  • Image live cell surface membranes for 24 hours or longer
  • Optional Enhancer masks intracellular signal for selective imaging of cell surface
  • Rapid, even staining in complete cell culture medium
  • Dye colors from blue to near-IR, with STORM-compatible options

Unlike other membrane/cell surface stains that are rapidly lost from the cell surface by endocytosis after labeling, CellBrite® Steady Dyes equilibrate between intracellular compartments and the plasma membrane. Cells retain surface staining in addition to intracellular staining over the course of hours to days in culture. CellBrite® Steady Enhancer is an optional reagent included in the kits that can be used to mask intracellular fluorescence of CellBrite® Steady Dyes, for more selective visualization of cell boundaries.

Kit Components

Kit sizes are based on 200 uL labeling volume, actual number of labelings may vary based on culture chamber size/staining volume.

  • Trial size kit (100 labelings):
    • CellBrite® Steady Dye, 1000X in DMSO, 20 uL
    • CellBrite® Steady Enhancer, 1000X in DMSO, 20 uL
  • Full size kit (500 labelings):
    • CellBrite® Steady Dye, 1000X in DMSO, 100 uL
    • CellBrite® Steady Enhancer, 1000X in DMSO, 100 uL

Selecting a CellBrite® Steady Dye

CellBrite® Steady Dyes are available in colors from blue to near-infrared. CellBrite® Steady 550, 650, and 685 fluorophores are compatible with super-resolution imaging by STORM. Masking of intracellular fluorescence by CellBrite® Steady Enhancer tends to show more complete masking of intracellular fluorescence for far-red CellBrite® Steady 650 and CellBrite® Steady 685 dyes.

Tips for Success

Washing after staining is optional for imaging by confocal microscopy, but is required for imaging by epifluorescence. For dyes with fluorescence in the visible range (405, 488, and 550), staining may or may not be visible through the microscope eyepieces without washing, so you may need to use brightfield or another marker to focus on cells initially for no-wash confocal imaging.

CellBrite® Steady Dyes have low toxicity and can be continuously incubated in cell culture medium. Enhancer may be toxic to some cell types, especially at higher concentrations. Enhancer may be incubated together with membrane dyes or added after staining to minimize any potential effect of Enhancer on cells. See the Product Protocol for details.

Find the Right Stain for Your Application

CellBrite® Steady Dyes must be used for staining live cells. They cannot be used to stain cells that are already fixed (the dyes primarily label intracellular membranes in fixed cells). Our original CellBrite® Cytoplasmic Membrane Dyes can be used to stain cells after fixation. To find the right stain for your application, see our Membrane & Cell Surface Stains Comparison, or download our Membrane & Surface Stains Brochure.

Membrane staining with CellBrite® Steady Dyes is retained immediately after fixation with formaldehyde, but does not tolerate methanol or detergent. If cells are stored after formaldehyde fixation, over time the dyes will redistribute to stain cytoplasmic structures. Enhancer does not tolerate fixation; if Enhancer-treated cells are fixed after staining, masking will be lost and intracellular fluorescence will return. For fixable cell surface staining that tolerates permeabilization for immunofluorescence staining, we recommend our CellBrite® Fix Membrane Stains or MemBrite® Fix Cell Surface Staining Kits.

CellBrite® Steady Dyes readily transfer between cells, and are not recommended for cell tracking, co-culture, or transplantation studies. Our stable, non-toxic ViaFluor® SE Cell Proliferation Kits can be used to covalently label cells for long-term tracking by microscopy or flow cytometry. See our Tech Tip: Using ViaFluor® SE Stains for Cell Tracing and Co-Culture to learn more.

CellBrite® Steady Dyes have been validated for labeling of purified extracellular vesicles (EVs) and exosomes. However, our tests have shown CellBrite® Steady to bind non-specifically to bead-bound exosomes. For optimal labeling of both purified and bead-bound exosomes we recommend our ExoBrite™ True EV Membrane Stains. Learn more about our ExoBrite™ stains and validated antibodies for Exosome & EV Labeling.

Watch our video where Technical Applications Scientist II, Jacqueline Steenhuis PhD answers your top questions about Biotium’s various membrane stains for fluorescence microscopy.

For additional support or product recommendations, contact us at techsupport@biotium.com.

 

CellBrite® Steady Membrane Staining Kits

ProductEx/EmSizeCatalog Number
CellBrite® Steady 405 Membrane Staining Kit406/428 nm100 Labelings30105-T
500 Labelings30105
CellBrite® Steady 488 Membrane Staining Kit505/529 nm100 Labelings30106-T
500 Labelings30106
CellBrite® Steady 550 Membrane Staining Kit562/579 nm100 Labelings30107-T
500 Labelings30107
CellBrite® Steady 594 Membrane Staining Kit580/615 nm100 Labelings30142-T
500 Labelings30142
CellBrite® Steady 650 Membrane Staining Kit656/676 nm100 Labelings30108-T
500 Labelings30108
CellBrite® Steady 685 Membrane Staining Kit686/708 nm100 Labelings30109-T
500 Labelings30109
CellBrite® Steady 750 Membrane Staining Kit755/789 nm100 Labelings30141-T
500 Labelings30141

Product Attributes

Dye
CellBrite® Steady 594, CellBrite® Steady 750, CellBrite® Steady 405, CellBrite® Steady 488, CellBrite® Steady 550, CellBrite® Steady 650, CellBrite® Steady 685
Size
100 labelings, 500 labelings
Probe cellular localization
Membrane/cell surface
For live or fixed cells
For live/intact cells
Assay type/options
Long term staining (24-72h)
Colors
Blue, Green, Red, Far-red, Near-infrared

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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