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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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CellBrite® Steady Membrane Staining Kits
CellBrite® Steady Membrane Staining Kits - Image 2Live MCF7 cells stained with CellBrite® Steady dyes for 30 minutes and imaged by confocal microscopy without a wash step.
CellBrite® Steady Membrane Staining Kits - Image 3Live HeLa cells co-stained with CellBrite™ Steady 488 (green) with Enhancer and NucSpot® Live 650 (magenta) for 45 minutes. Cells were imaged by confocal microscopy without a wash step.
CellBrite® Steady Membrane Staining Kits - Image 4Comparison of live cell staining with CellBrite™ Steady Dyes with or without Enhancer. Live HeLa cells were stained for the indicated times. Over 24 hours, the dyes equilibrate between the plasma membrane and intracellular compartments. Co-incubating or post-treating with Enhancer masks intracellular staining, allowing selective imaging of cell boundaries. Cells were imaged by confocal microscopy without a wash step.
CellBrite® Steady Membrane Staining Kits - Image 5Live HeLa cells stained with the lipophilic carbocyanine dyes Neuro-DiO, DiI, or DiD or CellBrite® Steady Membrane Dyes and Enhancer. Lipophilic carbocyanine dyes are rapidly internalized into intracellular compartments, so surface staining must be imaged within short time frames. CellBrite® Steady Dyes stain cell membranes evenly, and surface staining can be clearly imaged for 24 hours with the use of Enhancer.
CellBrite® Steady Membrane Staining Kits - Image 6Long-term staining with CellBrite™ Steady Dyes and Enhancer. MCF7 cells stained with 1X CellBrite™ Steady 405 or CellBrite™ Steady 488 dye, co-incubated with 0.2X Enhancer for up to six days. Cells were imaged by confocal microscopy without a wash step.
CellBrite® Steady Membrane Staining Kits - Image 7Live MCF7 cells stained with 1X CellBrite™ Steady 650 or CellBrite™ Steady 685 dye, co-incubated with 0.2X Enhancer for up to three days. Cells were imaged by confocal microscopy without a wash step.
CellBrite® Steady Membrane Staining Kits - Image 8HeLa cells co-stained with CellBrite® Steady 488 (green) and MitoView™ 633 mitochondrial stain (magenta).
CellBrite® Steady Membrane Staining Kits - Image 9Enriched EVs were stained with 0.5 uM CellBrite® Steady 405 (right). Specific staining was seen, compared with CellBrite® Steady 405 in buffer (left). EVs were detected on a Cytoflex LX flow cytometer in the Pacific Blue channel.
CellBrite® Steady Membrane Staining Kits - Image 10Enriched EVs were stained with 0.5 uM CellBrite Steady® 488 (right). Specific staining was seen, compared with CellBrite® Steady 488 in buffer (left). EVs were detected on a Cytoflex LX flow cytometer in the FITC channel.
CellBrite® Steady Membrane Staining Kits - Image 11Enriched EVs were stained with 0.5 uM CellBrite® Steady 550 (right). Specific staining was seen, compared with CellBrite® Steady 550 in buffer (left). EVs were detected on a Cytoflex LX flow cytometer in the PE channel.
CellBrite® Steady Membrane Staining Kits - Image 12Enriched EVs were stained with 0.5 uM CellBrite® Steady 650 (right). Specific staining was seen, compared with CellBrite® Steady 650 in buffer (left). EVs were detected on a Cytoflex LX flow cytometer in the APC channel.
CellBrite® Steady Membrane Staining Kits - Image 13Excitation and emission spectra of CellBrite™ Steady Dyes in methanol.
CellBrite® Steady Membrane Staining Kits - Image 14SEC-purified MCF-7-derived exosomes were stained with 0.02X CellBrite® Steady 405 (right). Specific staining was seen, compared with CellBrite® Steady 405 in buffer (left). EVs were detected on a Cytoflex LX flow cytometer in the Pacific Blue channel.

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

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

Citations

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FAQs

CellBrite® & MemBrite® Membrane & Cell Surface Stains

While CellBrite® Cytoplasmic Membrane Dyes can stain formaldehyde-fixed cells, they generally do not give good results in cryosections, possibly because the cell membrane integrity is disrupted, exposing other membrane structures to the dyes. Some customers have reported success using these dyes with vibratome sections.

CellBrite® Cytoplasmic Membrane Dyes are not suitable for membrane staining in FFPE samples as membrane lipids are extracted during the dewaxing and rehydration process. Similarly, acetone or methanol fixation of cryosections will extract lipids, leading to poor staining.

CellBrite® Fix, MemBrite® Fix, and CellBrite® Steady are recommended for use on live cells only. In fixed cells or sections they will label intracellular structures.

In some tissue types, lectins such as CF® Dye WGA Conjugates, CF® Dye Concanavalin A Conjugates, or CF® Dye PNA Conjugates may be useful for staining cell boundaries in FFPE or frozen sections. However, the staining pattern of lectins is highly dependent on cell and tissue type, so we recommend consulting the literature before trying these stains for your tissue of interest.

Alternatively, immunostaining using cell surface-specific antibodies could be done.

So far we have not found a universal plasma membrane stain for tissue sections. This is an application of interest to us and our customers, so we are working to find new solutions.

 

CellBrite® Cytoplasmic Membrane Dyes are too prone to aggregation to efficiently stain EVs. Some of the CellBrite® Fix, MemBrite® Fix, and CellBrite® Steady dye options have been reported for this application, however we do not recommend them. For optimal staining of exosome membranes we recommend our ExoBrite™ True EV Membrane Stains, which are novel lipophilic membrane dyes specifically designed and optimized for efficient staining of EV membranes with minimal dye aggregation. See our Extracellular Vesicle Research page for more information about our complete line of EV stains and antibodies.

Lipophilic carbocyanine dyes like CellBrite® and CellBrite® NIR Cytoplasmic Membrane Dyes can be used to stain formaldehyde-fixed cells. Fixation with methanol or other solvents is not recommended. Permeabilization with detergents after staining will extract the dyes and alter the staining pattern. However, we’ve seen good results when formaldehyde-fixed cells are permeabilized before staining with CellBrite® dyes (See Tech Tip: Combining Lipophilic Membrane Dyes with Immunofluorescence).

CellBrite® Fix, MemBrite® Fix, and CellBrite® Steady cannot be used to stain the plasma membrane of fixed samples; these dyes will primarily stain intracellular structures in cells that are already fixed.

To find the right dye for your workflow, see our Comparison of Membrane & Cell Surface Stains, or download our Membrane & Surface Stains Brochure.

Microbiology: Dyes for bacteria and yeast

To date, we have not identified a fluorescent cellular stain that will detect bacteria but not mammalian cells with high specificity, or vice versa. While some mammalian cell stains show weak staining of bacteria, they usually do show some signal, and will frequently stain dead bacteria more intensely than live bacteria.

We offer a selection of antibodies for specific bacterial antigens, which potentially have applications for differential staining of bacteria vs. mammalian cells, but we have not validated them in co-culture models.

Also see our Viability PCR Technology Page to learn about how PMA dye can be used for highly specific detection of microbial cell viability in complex samples.

CellBrite® and MemBrite® Stains were originally developed for staining mammalian cells in culture, but some of the stains also have been validated for other organisms and applications. For dyes to stain yeast or bacteria membranes, see Cellular Stains in Different Organisms. For information on staining other organisms or cell types, please see our Tech Tip: Researching Applications for Membrane Dyes.

The CellBrite® Cytoplasmic Membrane Dyes do not stain bacteria. The reactive CellBrite® Fix dyes stain both gram-positive and gram-negative bacteria, while the MemBrite® Fix dyes stain only gram-positive bacteria. However we have not tested these dyes for cell division tracking in bacteria.

There is literature describing the use of CFSE to track bacterial cell division,  the ViaFluor® SE cell proliferation dyes are likely to work in a similar manner, but we have not tested this.

See our Cellular Stains Table for a comprehensive list of cellular stains with their ability to stain various cell types.

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