Researching Applications for Membrane Dyes

Membrane Stains for Different Cell Types, Organisms, and Sample Preparations

For staining mammalian cells in 2D culture, please see our Membrane & Cell Surface Stains Comparison Guide and our Membrane & Surface Stains Brochure to select the right dye for your experiment.

If you wish to use a membrane dye for a different cell type, organism, culture system, or specimen preparation, we recommend performing a literature search for dyes used in your organism or application. Below we’ve provided some background information to help get you started. The references provided are not intended to be comprehensive, but to provide examples as a starting point for your own literature search and experimental design.

Classes of Membrane Dyes

Long-Chain Lipophilic Carbocyanine Dyes

The class of dyes that includes CellBrite® and CellBrite® NIR Cytoplasmic Membrane Dyes is the long-chain lipophilic carbocyanine dyes. These dyes have been widely used for decades for many applications in a wide variety of organisms. It may be useful to search for literature on the widely used orange fluorescent dye DiIC18(3), commonly called DiI (pronounced “Dye-Eye” and spelled with the capital letter I). Another historically widely used dye is green fluorescent DiO (DiOC18(3)).

Cationic Styryl Dyes (Nerve Terminal Dyes)

Another class of dyes used for membrane labeling is the cationic styryl dyes, commonly known as FM® dyes, offered by Biotium under the names SynaptoGreen™ and SynaptoRed™. These are amphipathic dyes that accumulate in the plasma membrane by virtue of their positive charge and lipophilic chains. Because they are less hydrophobic than long chain carbocyanine dyes, they can be washed off of membranes. This makes the dyes useful for tracking vesicle uptake and release, and therefore they are often called nerve terminal dyes. These dyes also have been used to stain the plasma membrane of various organisms that do not stain efficiently with lipophilic carbocyanine dyes. Learn more about Nerve Terminal Dyes.

Neutral Lipid Dyes

Small, neutral lipophilic dyes are used to stain lipid droplets. This class of dyes includes Nile Red, BODIPY, Oil Red O, and Biotium’s LipidSpot™ Lipid Droplet Stains. They also have been used to stain plasma membrane of some organisms.

Biotium’s Unique Membrane & Cell Surface Stains

Our CellBrite™ Fix Membrane Stains and MemBrite™ Fix Cell Surface Staining Kits covalently react with membrane proteins on cells, for staining that can be fixed and permeabilized for immunofluorescence staining. CellBrite® Steady Membrane Staining Kits are designed for long-term live cell imaging of the plasma membrane.

Tips for Searching Published Literature

If there are publications for the use of one dye for a particular application, then other dyes in the same class may be suitable too. But it is important to note that the different dyes in each class also have different physical properties that may make them more or less optimal for a specific experiment or organism.

Useful keywords to combine with your organism or application of interest for literature search:

• DiI (Note: last letter is capital I, not lower case L)
• DiO
• CellBrite
• FM1-43
• FM4-64
• SynaptoGreen
• SynaptoRed
• Nile Red
• BODIPY
• LipidSpot

Selected References for Different Sample Types

Exosomes/Extracellular Vesicles (EVs)

Lipophilic carbocyanine dyes do not efficiently stain EVs, but CellBrite® Fix, MemBrite® Fix, CellBrite® Steady, and other dyes have been used for this application.  See our Exosome & EV Labeling Technology Page for recommended exosome membrane stains.

Neurons in Culture, Slices, or Organs

Lipophilic carbocyanine dyes like CellBrite® Cytoplasmic Stains 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.

Stock solutions like the CellBrite® Dyes may be diluted in buffer for microinjection. We also supply the dyes in solid form. Neuro-DiO in Vegetable Oil, Neuro-DiI in Vegetable Oil, and DiI in Vegetable Oil are formulated specifically for microinjection.

CellBrite® Dyes can be used to label the surface of dissociated neurons in culture to visualize processes and dendritic spines. However, staining of dense cultures or slices by incubation with dye is not likely to result in clear staining of cell outlines due to the density of cellular processes. In addition, the dyes will not penetrate efficiently through multiple cell layers in a thick tissue slice. Therefore other labeling methods such as staining of neuronal cultures with dye crystals and microinjection of brain slices have been used.

• Trends Neurosci (1989) 12(9): 333-341. https://doi.org/10.1016/0166-2236(89)90040-4
  (Carbocyanine dyes for neuronal tracing)
• J Histochem Cytochem (1990) 38(5): 725-733. https://doi.org/10.1177/38.5.2185313
  (Neuronal tracing with DiI in fixed chicken embryos)
• PLoS One (2011) 6(10): e26549. doi: 10.1371/journal.pone.0026549
  (Staining of dendritic spines in cultured neurons using DiI crystals)
• J Neurosci (1994) 14(3): 1060-1078. https://www.jneurosci.org/content/14/3/1060.short
  (Microinjection of neuronal slices with DiI and DiO)

Organoids, Spheroids, or Matrigel®/Hydrogel Cultures

Lipophilic carbocyanine dyes, including CellBrite® Dyes, have been used to trace cells in spheroid and hydrogel cultures; usually cell labeling is done before spheroid formation or embedding in hydrogels.

• bioRxiv (2018) doi: https://doi.org/10.1101/454397
  (CellBrite® Cytoplasmic Membrane Dyes, cell labeling before spheroid formation)
• Applied Materials Today 23 (2021) 101013. https://doi.org/10.1016/j.apmt.2021.101013
  (CellBrite® Cytoplasmic Membrane Dyes, cell labeling before hydrogel culture)
• PLoS ONE (2020) 15(1), e0220019. https://doi.org/10.1371/journal.pone.0220019 (30023)
  (CellBrite® Cytoplasmic Membrane Dyes, cell labeling before Matrigel® culture)

Tissue Sections

Lipophilic carbocyanine dyes like CellBrite® Cytoplasmic Stains are not recommended for paraffin embedded sections, because paraffin tissue processing extracts most of the cellular lipids from the samples. Similarly, acetone or methanol fixation of cryosections will extract lipids, leading to poor staining.

Lipophilic dyes will 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.

CellBrite® 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. CellBrite® Dyes have been used for cell tracing in organoids, but for this application, usually the cells are labeled before they are placed in organoid culture.

Transwell® Filter Inserts

We do not recommend using CellBrite® Cytoplasmic Membrane Dyes for staining Transwell® inserts. Due to their hydrophobic nature, the dyes will bind the insert membrane and cause high background.  Customers have reported that MemBrite® Fix Stains do not cause high background on Transwell® supports, but that the staining protocol may require optimization to achieve uniform staining of different cell types on Transwells®.

Selected References for Non-Mammalian Organisms

Bacteria, Yeast, & Fungi

We have tested a variety of our cellular stains in bacteria and yeast. The CellBrite® Cytoplasmic Membrane Dyes do not efficiently stain these cell types, but we have found that the stains listed below can be used. See our Cellular Stains in Different Organisms selection table, and Microbiology Technology Page for more information.

Bacteria:

• SynaptoGreen™ C4 or SynaptoRed™ C2
• CellBrite® Fix for fixable staining
• MemBrite® Fix for fixable staining (gram-positive only)
• WGA CF® Dye Conjugates (fluorescent Gram stain)

Yeast:

• SynaptoGreen™ C4 or SynaptoRed™ C2
• CellBrite® Fix or MemBrite® Fix for fixable staining
• Calcofluor White
• Concanavalin A CF® Dye Conjugates
• WGA CF® Dye Conjugates (selectively stain bud scars)

Cationic styryl dyes like SynaptoGreen™ C4 and SynapoRed™ C2 also have been using for staining membranes of filamentous fungi.

• J  Vis Exp (2019) 153, e60613. doi:10.3791/60613
  (SynaptoGreen™ C4, SynaptoRed™ C2, filamentous fungi T. atroviride, N. crassa)

Dictyostelium & Amoebae

DiI has been used to label the plasmalemma of Amoeba proteus and Dictyostelium by incubation with dye in buffer.

• PLoS ONE 7(12): e50846. doi:10.1371/journal.pone.0050846
  (DiI in Amoeba Proteus)
• J Cell Biol (1996) 132(3) 335-344. https://rupress.org/jcb/article/132/3/335/15180
  (DiI in Dictyostelium)

Drosophila

CellBrite® Fix has been used to stain dissected Drosophila fat body.

• BioRxiv (2020) https://doi.org/10.1101/2020.09.24.311670
  (CellBrite® Fix 488, Drosophilia fat body)

Nematodes

DiI has been used to label cuticular structures and neurons in C. elegans by incubation with dye in buffer.

• J Vis Exp (2012) 59: 3362. doi: 10.3791/3362
  (DiI labeling of C. elegans cuticle)
• Science (2008) 322(5902): 744-747. doi: 10.1126/science.1163074
  (DiI labeling of C. elegans sensory neurons)

Plankton, Microalgae, & Marine Larvae

The lipophilic dyes DiO, Nile Red, and BODIPY have been used to stain freshwater microalgae by incubation with dye in buffer. CellBrite® Fix 488 has been used to stain marine algae. DiI (the dye in CellBrite® Orange) and DiD (the dye in CellBrite® Red) have been used to stain aquatic larvae of a variety of marine organisms by incubation with dye in seawater. Note: Lipophilic carbocyanine dyes may be prone to precipitate in high salt solutions like seawater.

• Cytometry Part (2012) A81A: 618-626. DOI: 10.1002/cyto.a.22066
  (DiO, Nile Red, and BODIPY in microalgae)
• Limnology Oceanography (2020) 9999, 1-14. https://doi.org/10.1002/lno.11585
  (CellBrite® Fix 488, Florenciella algae)
• J Biol Methods (2020) 7(2): e132. doi: 10.14440/jbm.2020.320
  (DiI, DiD in aquatic larvae)

Plants

Staining of plant cell membranes may require disruption of cell walls. Lipophilic carbocyanine dyes like DiOC6(3) have been used to stain onion bulb epidermal cells, as wells as tobacco and Torenia cells by incubation with dye in water. A variety of lipophilic dyes including FM®4-64 and carbocyanine dyes have been used to stain epidermal strips and protoplasts of Arabidopsis by incubation with dyes in water. FM®1-43 and FM®4-64 have been used to stain pollen tubes of tobacco and Arabidopsis.

• Protoplasma (2007) 231: 99-111. DOI 10.1007/s00709-006-0227-6
  (DiOC6(3) in onion bulb epidermal cells)
• Cell Biol International (1999) 23(10): 653-661. https://doi.org/10.1006/cbir.1999.0431
  (DiOC6(3) in tobacco and Torenia plant cells)
• Plant Methods (2012) 8: 28. https://doi.org/10.1186/1746-4811-8-28
  (FM®4-64, carbocyanine dyes, sphingolipid conjugates, Arabidopsis tissue and protoplasts)
• Plant Cell (2008) 20(11): 3050-3064. doi: 10.1105/tpc.108.058826
  (FM®1-43 and FM®4-64 labeling of tobacco and Arabidopsis pollen tubes)

Sea Urchin Oocytes & Embryos

DiI has been used to stain sea urchin embryos by incubation with dye in seawater,  and by microinjection to label endoplasmic reticulum in sea urchin eggs. FM®1-43 has been reported to stain sea urchin embryos, oocytes, and eggs by incubation with dye in seawater. Note: Lipophilic carbocyanine dyes (like DiI and CellBrite® Dyes) may be more likely to precipitate in high salt solutions like seawater compared to the SynaptoGreen™/SynaptoRed™ (FM®) dyes.

• Dev Biol (1998) 196: 95-106. https://doi.org/10.1006/dbio.1998.8857
  (DiI labeling of sea urchin embryos in seawater)
• J Cell Biol (1991) 114(5): 929-940. https://doi.org/10.1083/jcb.114.5.929
  (Microinjection of DiI to label ER in sea urchin oocytes)
• FASEB J (2000) 14(11): 1559-1566. https://doi.org/10.1096/fj.99-0812com
  (FM®1-43 labeling of sea urchin embryos in seawater)
• Dev Biol (2003) 2(15) 353-370. https://doi.org/10.1016/S0012-1606(03)00301-4
  (FM®1-43 labeling of sea urchin oocytes and eggs in seawater)

Sponges

The DiI derivative CM-DiI has been used for labeling and cell lineage tracing in A. queenslandica sponge larvae by incubation with dye in seawater.

• BMC Biology (2014) 12:26. https://doi.org/10.1186/1741-7007-12-26
  (CM-DiI labeling of A. queenslandica sponge larvae)

Viruses

FM®1-43 has been used to stain the lipid envelope of EhV-86 virus for microscopic imaging.

• J Gen Virol (2009) 90: 2306-2316. https://doi.org/10.1099/vir.0.011635-0
  (FM®1-43, EhV-86 virus)

Xenopus Oocytes

Xenopus oocytes (and those of other aquatic and amphibious organisms) are surrounded by a protective glycoprotein envelope, called the vitelline envelope. This may prevent staining of the oocyte plasma membrane by some lipophilic dyes unless it is removed or enzymatically permeabilized.

Lipophilic carbocyanine dyes (like the CellBrite® Dyes) have not been widely reported in the literature to stain Xenopus oocyte cell surface membrane, so these dyes may not be optimal for this application. DiI has been used for microinjection into Xenopus oocytes to stain ER membranes.

SynaptoGreen™ C4 (FM®1-43) has been reported to stain Xenopus oocyte plasma membranes by incubation with dye in buffer after removal of the vitelline envelope.

• Mol Biol Cell (2004) 15(10): 4347-4760. https://doi.org/10.1091/mbc.e04-03-0265
  (Microinjection of DiI to label ER in Xenopus oocytes)
• Cell Physiol Biochem (2000) 10:99-107. https://doi.org/10.1159/000016339
  (FM®1-43 in Xenopus oocytes after removal of vitelline envelope)

Zebrafish

DiI has been used for neuronal tracing in live and fixed zebrafish tissues, and to trace xenografted cells, as in other organisms. DiI also has been used to selectively label zebrafish thrombocytes in vivo by injection.

• Blood Cells Molecules Diseases (2002) 28(3) 418-427. https://doi.org/10.1006/bcmd.2002.0527
  (In vivo injection of DiI to label of zebrafish thrombocytes)