FAQs

CellBrite® Cytoplasmic Membrane Stains are lipophilic dyes for simple, non-toxic, stable labeling of membranes in live or fixed cells. Cells can be fixed with formaldehyde before or after CellBrite® staining. But the staining has poor tolerance for permeabilization or methanol fixation, so CellBrite® staining is not easily combined with intracellular immunofluorescence (IF) staining. The dyes also do not stain bacteria or yeast. CellBrite® NIR dyes are CellBrite® dyes with near-infrared fluorescence compatible with small animal NIR imaging systems.

CellBrite® Fix and MemBrite® Fix stains were developed to overcome some of these shortcomings. They are novel covalent stains that can be fixed and permeabilized for IF staining. CellBrite® Fix Membrane Stains are fluorogenic reactive membrane dyes that rapidly accumulate at the plasma membrane. When they incorporate into lipids, they become fluorescent, and at the same time react covalently with membrane proteins for stable labeling. Staining takes only 15 minutes in a single step with no wash. CellBrite® Fix stains mammalian cells, yeast, and bacteria.

MemBrite® Fix Cell Surface Stains do not bind lipids, but label cell surface proteins. MemBrite® Fix requires a two-step staining protocol with washing, but offers a more extensive choice of dye colors than CellBrite® Fix. MemBrite® Fix also can be used to stain yeast. But unlike original CellBrite® dyes and lectins, CellBrite® Fix and MemBrite® Fix cannot be used on cells that are already fixed.

To select a dye that’s right for your application, see our Membrane and Cell Surface Stains Comparison, or download our Membrane & Surface Stains Brochure.

CellBrite® Cytoplasmic Membrane Dyes do not efficiently stain EVs. Some of the CellBrite® Fix, MemBrite® Fix, and CellBrite® Steady dye options can be used for this application. However, 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

Biotium’s CellBrite® Cytoplasmic Membrane Dyes are dye delivery solutions that can be added directly to normal culture media to uniformly label suspended or adherent cells in culture. The PKH dyes are structurally related dyes for cell membrane labeling. But unlike CellBrite®, labeling with PKH dyes requires multiple steps and subjects cells to an iso-osmotic mannitol loading medium that can negatively affect cell membrane integrity and viability.

CellBrite® Fix dyes also feature rapid and simple labeling in isotonic buffer. Cells can be fixed and permeabilized after labeling with CellBrite® Fix, unlike original CellBrite® dyes or PKH dyes, which don’t tolerate detergent.

DiI (DiIC18(3); 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine) is a widely used lipophilic carbocyanine dye that labels cell membranes by inserting its two long (C18) hydrocarbon chains into the lipid bilayer. It has been extensively used for the antero- and retro-grade labeling of neurons.

Neuro-DiI (also available as a ready-to-use staining solution, CellBrite® Green) was developed by Biotium as an alternative to DiI. It has two additional t-butyl groups (one on each side of the dye) attached to the dye chromophore which introduce structural flexibility in the dye making it much more soluble in both organic solvents and in cell membranes. Improved dye solubility in the membranes enables rapid dispersion of the dye molecules, away from each other much more rapidly than regular DiI which has a relatively flat structure that promotes dye aggregation, and slows down dispersion of dye molecules. Neuro-DiI therefore results in more uniform, rapid and stable staining as compared to DiI.

FAST™ DiI uses a different strategy to solubilize the dye. Replacing the saturated C18 lipophilic chain of regular DiI with unsaturated linoleyl chains gives the dye molecules a bent structure, making them less likely to aggregate and thus disperse faster in the membrane.

Both Neuro-DiI and FAST™ DiI would diffuse faster than regular DiI in membranes, but a comparison of the relative diffusion rate between the two dyes has not been made. Neuro-DiI may have a potential advantage over FAST™ DiI. The t-butyl groups of Neuro-DiI render it more hydrophobic than FAST™ DiI, likely resulting in more stable membrane staining and make dye transfer between cells less likely.

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® 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 artificial lipid bilayers. Immediately after staining cultured cells, the dyes primarily localize to the plasma membrane. If cells are cultured over time after staining, the labeled membranes are internalized and staining gradually becomes mostly intracellular.

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 (see note below). 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. Staining with the covalent stains CellBrite® Fix and MemBrite® Fix lasts up to 48 hours in tissue culture cells (see note below).

Note: Over time, all cell surface stains will be internalized and become intracellular as membranes turn over by endocytosis. The rate of internalization may vary by cell type, rate of membrane turnover, and rate of cell division. In immortalized cells in culture, most of the surface staining becomes internalized over the course of about 24 hours for CellBrite®, CellBrite® Fix, and MemBrite® Fix stains.

For an alternative stable and fixable stain for long term cell tracking or tracking cells in mixed cultures, see our ViaFluor® SE Cell Proliferation Dyes. These dyes covalently label intracellular proteins throughout the cell and are non-toxic and fixable.

Cells can be fixed with formaldehyde after labeling with the original CellBrite® or CellBrite® NIR cytoplasmic membrane dyes. Permeabilization of cells with detergents or solvents, or mounting medium containing glycerol may adversely affect staining. Permeabilization with digitonin (10 ug/mL to 1 mg/mL) has been reported to be compatible with lipophilic carbocyanine dye staining. 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).

The CF® Dye WGA Conjugates can also be used to label the cell surface or plasma membrane in fixed cells/tissues or FFPE sections. On tissue sections however, the lectins would label glycoproteins on all cell membranes, external (plasma membrane) as well as internal (organelle membranes). WGA staining can also be tissue- and cell-type dependent i.e. it is based on the expression pattern of glycoproteins on the cell membranes.

CellBrite® Fix Membrane Stains and MemBrite® Fix Cell Surface Stains belong to a new class of membrane dyes designed to covalently label the cell surface. They can withstand fixation and permeabilization, or fixation with alcohol after labeling of live cells. CellBrite® Fix and MemBrite® Fix cannot be used to label the plasma membranes of fixed cells or tissues (the dyes label the cytoplasm in fixed cells).

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

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.

The loading buffer in the CellBrite® Blue kit is required for solubilizing the DiB dye solution. The other CellBrite® dyes are more soluble and do not require this.

It’s common for DiB Loading Buffer (30024B) to solidify into a gel during storage. This does not affect the product, but the buffer must be in liquid form before use. Heat the solidified gel to 50-60°C for 5-10 minutes and vortex periodically until it has formed a clear liquid. DiB Loading Buffer is viscous, so pipet it slowly to ensure the correct volume is added.

You can heat the DiB Cell Labeling Solution to 37°C for 10 minutes or longer, and pipette the solution up and down or vortex to mix completely until is is completely dissolved.

CellBrite® dyes are ready-to-use solutions of the lipophilic carbocyanine dyes DiI, DiO etc. It is challenging to get uniform labeling with CellBrite® dyes due to their hydrophobicity, CellBrite® Red (#30023), which is DiD,  is particularly difficult as the hydrophobic dye tends to aggregate and give uneven staining. Warming the dye vial to ~50°C to dissolve any dye aggregates before staining could be tried. Also rather than adding the dye directly to the medium, we recommend diluting in it medium (1:200 dilution) first, and then adding the medium with the dye to the cells. Also, be aware that these dyes will be internalized if cells are cultured after staining.

Alternatively, for live cell staining, CellBrite® Steady or the reactive dyes CellBrite® Fix or MemBrite® Fix would be more suitable as they result in more even staining.

CellBrite® dyes are not compatible with glycerol-based mounting media as this results in altered staining and high background. Organic mounding media are also not suitable. We recommend imaging in directly in PBS (or other aqueous buffer). Coverslips should be mounted using PBS and sealed with a suitable coverslip sealant such as CoverGrip™ or nail polish. Stained samples can be stored in PBS at 4°C for several weeks or longer.

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.

The lipophilic CellBrite® dyes would not be suitable for solvent-cleared samples as membrane lipids would be extracted during the solvent treatment step. The reactive membrane dyes, CellBrite® fix and MemBrite® too would not work as these are live cell stains, and in dead/ fixed samples, they would stain all accessible targets resulting in whole cell staining.

However, if staining of live cells is possible, the reactive CellBrite® fix and MemBrite® fix membrane dyes may be used before fixation and solvent-clearing, but these dyes may not penetrate multilayer cells or tissues effectively.

Staining using CF® Dye WGA Conjugates may be an option, but staining can be cell- and tissue-type dependent i.e. it is based on the expression pattern of glycoproteins on the cell membranes. You may verify is WGA staining is an option for your cell or tissue type based on published literature. It is also important to note that if tissue sections are used, the lectins would stain glycoproteins on all cell membranes, external (plasma membrane) as well as internal (organelle membranes).

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

To find the right dye for your workflow, check out the links below:

Tech Tip: Cell Surface Stains for Live & Fixed Cells

Comparison of Membrane & Cell Surface Stains

Or download our Membrane & Surface Stains Brochure.

Lipophilic carbocyanine dyes like CellBrite® Cytoplasmic Membrane Dyes may stain tissue slices or organotypic cultures, but due to their lipophilicity, they probably will not penetrate through multiple cell layers in a thick tissue slice. The dyes have been used for cell tracing in organoids and labeling hydrogel cultures. However, for this application, usually the cells are labeled before they are placed in organoid culture or embedded in hydrogel.

This class of dyes have been widely used for neuronal tracing in organisms, tissue slices, Drosophila embryos, and chicken embryos by microinjection or spot-labeling with crystals of solid dye. Neuronal tracing also can be done in formaldehyde-fixed tissues or embryos.

See our Tech Tip: Researching Applications for Membrane Dyes for more information and selected references.

Matrigel is a registered trademark of Corning Inc.

Lectins such as CF® Dye WGA Conjugates, CF® Dye Concanavalin A Conjugates, or CF® Dye PNA Conjugates can be used to label the cell surface or plasma membrane in FFPE or frozen sections. However lectins would label glycoproteins on all cell membranes, external (plasma membrane) as well as internal (organelle membranes). Lectin staining also is tissue- and cell-type dependent, based on the expression pattern of glycoproteins on the cell membranes. Therefore they generally do not selectively label all cell outlines or boundaries in a tissue section.

Other cell surface membrane stains such as the lipophilic CellBrite® or CellBrite® NIR 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® Cytoplasmic Membrane Dyes may stain formaldehyde-fixed cryosections. However, because the cell membrane integrity is disrupted in frozen sections, CellBrite® dyes probably will not selectively stain the plasma membrane in tissue sections and will likely stain intracellular structures as well.

The reactive, fixable membrane stains, CellBrite® Fix and MemBrite® Fix are for live cell staining only. In fixed sections, they would indiscriminately react with target proteins throughout the cell, staining the entire cell.

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

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

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.

Yes, CellBrite® Cytoplasmic Membrane Dye staining can be done in buffer instead of culture medium. If you are staining adherent cells, we recommend using a buffer like HBSS with calcium/magnesium, which helps maintain cell adherence and morphology.

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. If you are staining a non-mammalian cell type or organism that grows in water or other medium, please see our Tech Tip: Researching Applications for Membrane Dyes. For dyes to stain yeast or bacteria membranes, see Cellular Stains in Different Organisms.

Yes, CellBrite® Cytoplasmic Membrane Dye staining can be done at room temperature or lower, instead of 37°C, this may reduce dye internalization during staining. Be aware that staining at 4°C may result in dye precipitation or uneven staining.

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. If you are staining a non-mammalian cell type or organism that grows at a temperature below 37°C, please see our Tech Tip: Researching Applications for Membrane Dyes. For dyes to stain yeast or bacteria membranes, see Cellular Stains in Different Organisms.