Membrane staining is useful in defining cell boundaries in multicolor imaging studies, but finding dyes compatible with the various viability, fixation, and experimental staining conditions dictated by a particular workflow or application can be challenging. In order to solve this issue, Biotium has created a versatile selection of highly fluorescent and photostable stains to visualize cell boundaries under a wide variety of conditions.
To select the cell surface stain that is right for you, read about the different types of stains in Table 1 and consult the selection table at the end of the page
Jump to a section:
- The best choice for staining fixed cells: CytoLiner™ Fixed Cell Membrane Stains
- The unrivaled option for long-term live-cell imaging: CellBrite® Steady Membrane Stains
- Need to stain and then fix your cells? Choose CellBrite® Fix or MemBrite® Fix Membrane Stains
- The original CellBrite® Cytoplasmic Membrane Dyes
- Lectin conjugates for robust cell surface staining
- Looking for something more specialized?
Table 1. Cell Surface Stain Overview
Table 2. Find the right stain for your application or workflow: To compare and choose the right stain for your application, see the table below.
| Sample/Workflow | CytoLiner™ Fixed Cell Membrane Stains | CellBrite® Steady Membrane Stains | CellBrite® Fix Membrane Stains | MemBrite® Fix Membrane Stains | CellBrite® Cytoplasmic Membrane Dyes | CF® Dye Lectin Conjugates |
|---|---|---|---|---|---|---|
| Stain live | No1 | Yes | Yes | Yes | Yes | Yes |
| Stain for real time live cell imaging | No | Yes | No | No | Yes3 | No3,7 |
| Stain live, then fix with PFA | No1 | No | Yes | Yes | Yes | Yes |
| Stain live, then fix with methanol | No | No | Yes | Yes | No | Yes |
| Stain live, fix with PFA, & then permeabilize | No | No | Yes | Yes | No | Yes |
| Fix (PFA), then stain | Yes | No | No | No | Yes | Yes |
| Fix (methanol), then stain | No | No | No | No | No | Yes |
| Stain bacterial cells | ND* | No | Yes | Yes2 | No | Yes4 |
| Stain yeast | ND* | No | Yes | Yes | No | Yes5,6 |
| Dye transfer between cells | Yes | Yes | Minimal | Minimal | Minimal | Possible |
| Non-toxic, stable | Yes | Yes | Yes | Yes | Yes | Possibly toxic7 |
| Compatible with Poly-lysine or collagen-coated culture ware | Yes | Yes | No | No | No | Yes |
1. CytoLiner™ Fixed Cell Membrane Stains may exhibit high background if used to stain live cells before fixation
2. Gram-positive only
3. Staining is well-retained by live cells/tissues but internalized rapidly and diluted by cell division in cultured cells
4. CF® Dye WGA stain gram-positive only
5. CF® Dye ConA only
6. CF® Dye WGA stains bud scars only
7. Lectins may be toxic or stimulatory to live cells, depending on cell type.
1. The best choice for staining fixed cells: CytoLiner™ Fixed Cell Membrane Stains
CytoLiner™ Fixed Cell Membrane Stains are a new generation of membrane stains developed to address the limitations of traditional lipophilic dyes in fixed cells; they offer robust signal and unparalleled reliability for plasma membrane staining of formaldehyde-fixed cells for imaging applications.
While traditional lipophilic membrane stains, like DiO and DiI, often produce highly variable staining in formaldehyde-fixed cells due to their poor water solubility, CytoLiner™ stains reliably stain the plasma membrane in formaldehyde-fixed cells. These stains are not compatible with cells fixed using solvents like methanol, ethanol, or acetone or with paraffin-embedded samples (such as FFPE tissues) because these treatments will remove the lipids required for CytoLiner™ staining.
CytoLiner™ stains may be combined with downstream staining with antibodies or other probes after mild permeabilization for intracellular immunofluorescence co-staining protocols. The stains are also compatible with poly-L-lysine coated cultureware and Transwell® membranes and are available in a wide range of five colors from blue to near-IR for flexible panel design.
2. The unrivaled option for long-term live-cell imaging: CellBrite® Steady Membrane Stains
Designed for long-term live cell staining, cells labeled with CellBrite® Steady Membrane Stains retain both surface and intracellular staining over time and can be imaged for 48 hours or longer in live cells. This contrasts with the CellBrite® Cytoplasmic Membrane Stains or other lipophilic membrane dyes that are eliminated from the cell surface by endocytosis.
Intracellular staining with CellBrite® Steady Membrane Stains can become more prominent with longer incubation times. CellBrite® Steady Enhancer, an optional reagent provided in the kit, can be applied to reduce intracellular staining and improve selective imaging of cell outlines and boundaries over several hours or days.
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, the dyes will redistribute to stain cytoplasmic structures relatively quickly. CellBrite® Steady Enhancer cannot be used in a workflow that includes cell fixation.
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 the cell cytoplasm 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.
3. Need to stain and then fix your cells? Choose CellBrite® Fix or MemBrite® Fix Membrane Stains
CellBrite® Fix and MemBrite® Fix are covalent stains compatible with downstream fixation and permeabilization for multicolor IF staining. While staining is non-toxic and homogenous, these dyes cannot be used on pre-fixed cells and the intracellular labeling of cells with compromised cell membranes is intense. If live cells are cultured after staining, the dyes will be internalized over time by endocytosis, so we recommend fixing shortly after labeling. For cells already fixed with formaldehyde, we recommend Cytoliner™ Fixed Cell Membrane Stains. CellBrite® Fix and MemBrite® Fix Stains work by covalently labeling cell surface proteins and, therefore, also react with other proteins like those used in cell culture surface coatings. Please see Table 2 for details on the different workflows of our stains.
CellBrite® Fix Membrane Stains require only a single step, allowing researchers to stain their cells rapidly in only 15 minutes. They are reactive membrane dyes that associate with the membrane and then covalently bind to membrane proteins so that the cells can be fixed and permeabilized without loss of fluorescence. CellBrite® Fix can be used to stain mammalian cells, yeast, and bacteria.
MemBrite® Fix Cell Surface Stains require a two-step procedure, but this stain is offered in a more extensive choice of dye colors, including STORM-compatible MemBrite® Fix-ST dyes. MemBrite® Fix does not have an affinity for the membrane but will react with extracellular membrane protein domains. MemBrite® Fix also can be used to stain gram-positive bacteria and yeast.
4. The original CellBrite® Cytoplasmic Membrane Dyes
CellBrite® Cytoplasmic Membrane Dyes are ready-to-use dye delivery solutions for lipophilic carbocyanine dyes (DiO, DiI, DiD, and DiR) that label membranes in live or fixed cells. The dyes are available with colors ranging from blue to near-IR and can be added directly to standard culture media of suspension or adherent cell cultures. Staining is non-toxic and stable, with very little dye transfer between cells, making them suitable for cell labeling, tracking mixed cell populations, and cell fusion studies. The CellBrite® NIR dyes and near-infrared DiR are suitable for small animal imaging. These dyes do not stain bacteria or yeast.
While labeling with lipophilic carbocyanine dyes is very stable, the cellular localization of staining changes over time in live cells. Immediately after staining, the dyes primarily label the plasma membrane but become internalized by endocytosis over the course of hours to a day, so staining becomes localized to intracellular vesicles and lysosomes.
Cells can be fixed with formaldehyde (PFA) before or after CellBrite® staining. However, the staining will have poor tolerance for permeabilization or methanol fixation and cannot be used on FFPE sections, as membrane lipids are extracted during the dewaxing and rehydration process. Therefore, it is not compatible with some immunofluorescence (IF) protocols. Good results can be obtained when cells are PFA-fixed and permeabilized with detergent prior to staining; see our Tech Tip: Combining Lipophilic Membrane Dyes with Immunofluorescence.
For staining formaldehyde-fixed cells, we strongly recommend using CytoLiner™ Fixed Cell Membrane Stains, which offer more robust and consistent results over the CellBrite® Cytoplasmic Membrane Dyes. Biotium also offers a selection of stand-alone lipophilic carbocyanine dyes such as Neuro-DiO, Neuro-DiI, and dilinoleyl dyes which have structural features designed to make the probes diffuse faster in cell membranes. Our ViaFluor® SE Dyes, which stain the cytoplasm, may be a more suitable option for long-term imaging of cell morphology. See our Tech Tip: Using ViaFluor® SE Dyes for Cell Tracing and Co-Culture.
5. Lectin conjugates for robust cell surface staining
Conjugated to our bright and photostable CF® Dyes, lectins label glycoproteins on the surface of live or fixed cells. Lectin staining is compatible with PFA, solvent fixation, and permeabilization. In pre-fixed and permeabilized cells, lectins stain both the cell surface and organelles in the secretory pathway. Lectins, such as the CF® Dye WGA Conjugates or CF® Dye Concanavalin A Conjugates, can be used to label the cell surface or plasma membrane in FFPE or frozen sections. However, this staining is tissue-specific and may also be sample-specific, as lectin conjugates can stain intracellular structures if the cell membrane is compromised. Immunostaining using cell surface-specific antibodies is also an excellent option for FFPE tissue.
WGA and Con A lectin conjugates are widely used for cell surface staining in mammalian cells and are also useful for bacterial Gram stains and yeast stains. While WGA and Con A broadly stain commonly cultured cell types, other lectins may be better suited for staining different cell or tissue types. All of Biotium’s lectin conjugates can be viewed on our website.
Because lectin staining may be cell-type or tissue-dependent, we recommend considering our CellBrite®, MemBrite®, or Cytoliner™ stains as you search for the most suitable stain.
6. Looking for something more specialized?
Biotium offers a variety of stains for different organelles and cellular structures. For researchers interested in staining extracellular vesicles (EVs), we developed the ExoBrite™ stains and antibodies, which offer higher coverage and signal-to-noise over membrane dyes commonly used for EV staining. Check out our Extracellular Vesicle Research page for more information. For microbiologists, we have a wide range of fluorescent dyes and assay kits to stain bacteria, yeast, and fungi, including dyes to assess cell viability and bacterial gram stain type. An overview of these products can be viewed on our Microbiology Stains & Kits technology page. We also offer a selection of voltage sensitive membrane potential dyes and nerve terminal dyes for tracking synaptic vesicle trafficking.