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Cell and Neuronal Tracing
Cell tracers are non-toxic probes that can track the flow of capillaries and define neuronal cell connectivity and translocation to monitor movement and localization in single cells, whole cell populations, or intact organisms. These fluorescent tracers can be introduced through iontophoresis, microinjection, or pinocytosis. Additionally, transport can be retrograde, anterograde, or bidirectional. While most of these fluorescent probes are effective in experiments involving fresh tissue sections and cultured cells, there are special requirements for fixed tissues and in vivo imaging.
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Properties of cell tracers
| Cholera toxin subunit B | DiI and derivatives | Hydrazides and Biocytins | |
|---|---|---|---|
| Readout | Direct fluorescence imaging of cells, selective for ganglioside GM1 | Labels cell membranes and uniformly label neurons via lateral diffusion in the plasma membrane | Direct fluorescence imaging of cells |
| Tissue sections | Yes | Yes | Yes |
| In vivo neurons | No | Yes | Yes |
| Fixed tissue sections | No | Yes | No |
| Cultured cells | Yes | Yes | Yes |
| Cell loading | Microinjection, iontophoresis | Microinjection or iontophoresis; for tissue sections, crystal and paste placement options available | Microinjection or pinocytosis |
| Staining mode | Exogenous | Exogenous | Endogenous |
| Best for | Cell populations | Cell populations | Single cells |
| Fixable | Yes | No | Yes |
| Transport | Retrograde | Bidirectional | Bidirectional |
| Photobleaching resistance |  |  | |
| Gap junction transfer | No | No | Yes |
Selection guide for cell tracers
These conjugates are useful for tissue sections, and cultured cells. They are fixable and photostable retrograde tracers introduced via microinjection or iontophoresis.
| Carrier | Standard filter set(s) | Fluorophore | Ex/Em (nm) | Quantity | Cat. No. | |
|---|---|---|---|---|---|---|
| Cholera toxin subunit B | ||||||
| Cholera Toxin Subunit B, Alexa Fluor 488 Conjugate | Cholera toxin subunit B (Recombinant) | FITC | Alexa Fluor 488 | 495/519 | 100 µg | C34775 |
| Cholera Toxin Subunit B, Alexa Fluor 555 Conjugate | TRITC | Alexa Fluor 555 | 555/565 | 100 µg | C34776 | |
| Cholera Toxin Subunit B, Alexa Fluor 594 Conjugate | Texas Red | Alexa Fluor 594 | 590/617 | 100 µg | C34777 | |
| Cholera Toxin Subunit B, Alexa Fluor 647 Conjugate | Cy5 | Alexa Fluor 647 | 650/668 | 100 µg | C34778 | |
| Cholera Toxin Subunit B, Biotin-XX Conjugate | NA | Biotin-XX | NA | 100 µg | C34779 | |
| Cholera Toxin Subunit B, Horseradish Peroxidase Conjugate | NA | Horseradish peroxidase | NA | 100 µg | C34780 | |
| Lipid raft kits | ||||||
| Vybrant Alexa Fluor 488 Lipid Raft Labeling Kit | Cholera toxin subunit B (Recombinant) and anti-cholera toxin subunit B antibody (anti-CT-B) | FITC | Alexa Fluor 488 | 495/519 | 1 kit | V34403 |
| Vybrant Alexa Fluor 555 Lipid Raft Labeling Kit | TRITC | Alexa Fluor 555 | 555/565 | 1 kit | V34404 | |
| Vybrant Alexa Fluor 594 Lipid Raft Labeling Kit | Texas Red | Alexa Fluor 594 | 590/617 | 1 kit | V34405 | |
These cell tracers, also called lipophilic tracers, are useful for tissue sections, in vivo neurons, fixed tissue sections, and cultured cells. They are anterograde and retrograde tracers used for long term studies.
| Standard filter set (s) | Ex/Em (nm) | Format | Fixable | Quantity | Cat. No. | |
|---|---|---|---|---|---|---|
| DiI and derivatives | ||||||
| DiI Stain – DiIC18(3) | Rhodamine | 549/565 | solid | No | 100 mg | D282 |
| DiI Stain – DiIC18(3), crystalline | crystal | 25 mg | D3911 | |||
| DiD – DiIC18(5) | Cy5 | 644/663 | solid | 10 mg | D7757 | |
| DiR – DiIC18(7) | Cy5.5 | 750/780 | solid | 10 mg | D12731 | |
| CellTracker DiI | ||||||
| CellTracker CM-DiI Dye, solution | Rhodamine | 549/565 | solution | Yes | 20 x 50 µg | C7000 |
| CellTracker CM-DiI Dye | solid | 1 mg | C7001 | |||
| Neurotrace DiI | ||||||
| NeuroTrace DiI Tissue-Labeling Paste | Rhodamine | 549/565 | paste | No | 500 mg | N22880 |
| NeuroTrace CM-DiI Tissue-Labeling Paste | paste | Yes | 100 mg | N22883 | ||
| Vybrant DiI and derivatives | ||||||
| Vybrant DiO Cell-Labeling Solution | GFP | 484/501 | solution | No | 1 mL | V22886 |
| Vybrant DiI Cell-Labeling Solution | Rhodamine | 549/565 | solution | No | 1 mL | V22885 |
| Vybrant CM-DiI Cell-Labeling Solution | solution | Yes | 1 mL | V22888 | ||
| Vybrant DiD Cell-Labeling Solution | Cy5 | 644/663 | solution | No | 1 mL | V22887 |
| Vybrant Multicolor Cell-Labeling Kit | — | Various | solution | No | 1 mL each | V22889 |
These cell tracers, also called polar tracers, are useful for tissue sections, in vivo neurons, and cultured cells. They are anterograde and retrograde tracers used to trace neuronal projections and investigate gap junctions.
| Carrier | Standard filter set(s) | Fluorophore | Ex/Em (nm) | Quantity | Cat. No. | |
|---|---|---|---|---|---|---|
| Hydrazides | ||||||
| Alexa Fluor 350 Hydrazide | Hydrazide, sodium salt | DAPI | Alexa Fluor 350 | 346/442 | 5 mg | A10439 |
| Alexa Fluor 488 Hydrazide | FITC | Alexa Fluor 488 | 495/519 | A10436 | ||
| Alexa Fluor 568 Hydrazide | Rhodamine | Alexa Fluor 568 | 578/603 | A10437 | ||
| Alexa Fluor 594 Hydrazide | Texas Red | Alexa Fluor 594 | 590/617 | A10438 | ||
| Alexa Fluor 555 Hydrazide | Hydrazide, Tris(triethylammonium) salt | TRITC | Alexa Fluor 555 | 555/565 | 1 mg | A20501MP |
| Alexa Fluor 633 Hydrazide | Texas Red | Alexa Fluor 633 | 632/647 | A30634 | ||
| Alexa Fluor 647 Hydrazide | Cy5 | Alexa Fluor 647 | 650/668 | A20502 | ||
| Biocytin | ||||||
| Alexa Fluor 488 Biocytin | Biocytin, sodium salt | FITC | Alexa Fluor 488 | 495/519 | 250 mg | A12924 |
| Alexa Fluor 594 Biocytin | Texas Red | Alexa Fluor 594 | 590/617 | A12922 | ||
These conjugates are useful for tissue sections, and cultured cells. They are fixable and photostable retrograde tracers introduced via microinjection or iontophoresis.
| Carrier | Standard filter set(s) | Fluorophore | Ex/Em (nm) | Quantity | Cat. No. | |
|---|---|---|---|---|---|---|
| Cholera toxin subunit B | ||||||
| Cholera Toxin Subunit B, Alexa Fluor 488 Conjugate | Cholera toxin subunit B (Recombinant) | FITC | Alexa Fluor 488 | 495/519 | 100 µg | C34775 |
| Cholera Toxin Subunit B, Alexa Fluor 555 Conjugate | TRITC | Alexa Fluor 555 | 555/565 | 100 µg | C34776 | |
| Cholera Toxin Subunit B, Alexa Fluor 594 Conjugate | Texas Red | Alexa Fluor 594 | 590/617 | 100 µg | C34777 | |
| Cholera Toxin Subunit B, Alexa Fluor 647 Conjugate | Cy5 | Alexa Fluor 647 | 650/668 | 100 µg | C34778 | |
| Cholera Toxin Subunit B, Biotin-XX Conjugate | NA | Biotin-XX | NA | 100 µg | C34779 | |
| Cholera Toxin Subunit B, Horseradish Peroxidase Conjugate | NA | Horseradish peroxidase | NA | 100 µg | C34780 | |
| Lipid raft kits | ||||||
| Vybrant Alexa Fluor 488 Lipid Raft Labeling Kit | Cholera toxin subunit B (Recombinant) and anti-cholera toxin subunit B antibody (anti-CT-B) | FITC | Alexa Fluor 488 | 495/519 | 1 kit | V34403 |
| Vybrant Alexa Fluor 555 Lipid Raft Labeling Kit | TRITC | Alexa Fluor 555 | 555/565 | 1 kit | V34404 | |
| Vybrant Alexa Fluor 594 Lipid Raft Labeling Kit | Texas Red | Alexa Fluor 594 | 590/617 | 1 kit | V34405 | |
These cell tracers, also called lipophilic tracers, are useful for tissue sections, in vivo neurons, fixed tissue sections, and cultured cells. They are anterograde and retrograde tracers used for long term studies.
| Standard filter set (s) | Ex/Em (nm) | Format | Fixable | Quantity | Cat. No. | |
|---|---|---|---|---|---|---|
| DiI and derivatives | ||||||
| DiI Stain – DiIC18(3) | Rhodamine | 549/565 | solid | No | 100 mg | D282 |
| DiI Stain – DiIC18(3), crystalline | crystal | 25 mg | D3911 | |||
| DiD – DiIC18(5) | Cy5 | 644/663 | solid | 10 mg | D7757 | |
| DiR – DiIC18(7) | Cy5.5 | 750/780 | solid | 10 mg | D12731 | |
| CellTracker DiI | ||||||
| CellTracker CM-DiI Dye, solution | Rhodamine | 549/565 | solution | Yes | 20 x 50 µg | C7000 |
| CellTracker CM-DiI Dye | solid | 1 mg | C7001 | |||
| Neurotrace DiI | ||||||
| NeuroTrace DiI Tissue-Labeling Paste | Rhodamine | 549/565 | paste | No | 500 mg | N22880 |
| NeuroTrace CM-DiI Tissue-Labeling Paste | paste | Yes | 100 mg | N22883 | ||
| Vybrant DiI and derivatives | ||||||
| Vybrant DiO Cell-Labeling Solution | GFP | 484/501 | solution | No | 1 mL | V22886 |
| Vybrant DiI Cell-Labeling Solution | Rhodamine | 549/565 | solution | No | 1 mL | V22885 |
| Vybrant CM-DiI Cell-Labeling Solution | solution | Yes | 1 mL | V22888 | ||
| Vybrant DiD Cell-Labeling Solution | Cy5 | 644/663 | solution | No | 1 mL | V22887 |
| Vybrant Multicolor Cell-Labeling Kit | — | Various | solution | No | 1 mL each | V22889 |
These cell tracers, also called polar tracers, are useful for tissue sections, in vivo neurons, and cultured cells. They are anterograde and retrograde tracers used to trace neuronal projections and investigate gap junctions.
| Carrier | Standard filter set(s) | Fluorophore | Ex/Em (nm) | Quantity | Cat. No. | |
|---|---|---|---|---|---|---|
| Hydrazides | ||||||
| Alexa Fluor 350 Hydrazide | Hydrazide, sodium salt | DAPI | Alexa Fluor 350 | 346/442 | 5 mg | A10439 |
| Alexa Fluor 488 Hydrazide | FITC | Alexa Fluor 488 | 495/519 | A10436 | ||
| Alexa Fluor 568 Hydrazide | Rhodamine | Alexa Fluor 568 | 578/603 | A10437 | ||
| Alexa Fluor 594 Hydrazide | Texas Red | Alexa Fluor 594 | 590/617 | A10438 | ||
| Alexa Fluor 555 Hydrazide | Hydrazide, Tris(triethylammonium) salt | TRITC | Alexa Fluor 555 | 555/565 | 1 mg | A20501MP |
| Alexa Fluor 633 Hydrazide | Texas Red | Alexa Fluor 633 | 632/647 | A30634 | ||
| Alexa Fluor 647 Hydrazide | Cy5 | Alexa Fluor 647 | 650/668 | A20502 | ||
| Biocytin | ||||||
| Alexa Fluor 488 Biocytin | Biocytin, sodium salt | FITC | Alexa Fluor 488 | 495/519 | 250 mg | A12924 |
| Alexa Fluor 594 Biocytin | Texas Red | Alexa Fluor 594 | 590/617 | A12922 | ||
Cholera toxin subunit B
Cholera toxin originates from Vibrio cholerae and is comprised of subunits A and B. Subunit A is an ADP-ribosyltransferase that disrupts protein signaling and will lead to cellular dehydration. Alternatively, subunit B is nontoxic and is involved in the binding of ganglioside GM1 to label lipid rafts (Figure 1). In neurons, cholera toxin subunit B binds to glycosphingolipids in axonal membranes. Introduced via microinjection or iontophoresis, cholera toxin subunit B are fixable and photostable retrograde tracers.
While cholera toxin subunit B Alexa Fluor conjugates offer sensitive and selective binding and neuronal tracing, cholera toxin subunit B biotin-XX and horseradish peroxidase conjugates are available for use with tyramide signal amplification (TSA).
Learn more about cholera toxin b and other protein conjugates
Figure 1. J774 mouse macrophage cell stained with Alexa Fluor 555 dye-labeled cholera toxin subunit B. A J774 mouse macrophage cell sequentially stained with BODIPY FL ganglioside GM1 and then with Alexa Fluor 555 dye-labeled cholera toxin subunit B (also available as a component of Vybrant Alexa Fluor 555 Lipid Raft Labeling Kit). The cell was then treated with an anti-CT-B antibody (a component of Vybrant Alexa Fluor 555 Lipid Raft Labeling Kit) to induce crosslinking. Alexa Fluor 555 dye fluorescence (left panel, red) and BODIPY FL dye fluorescence (center panel, green) were imaged separately and overlaid to emphasize the coincident staining (right panel, yellow). Nuclei were stained with blue-fluorescent Hoechst 33258.
DiI and derivatives
DiI and its derivatives are lipophilic tracers, or carbocyanine dyes, that label cell membranes without visibly affecting cell viability. These dyes are weakly fluorescent in water but are highly fluorescent in membranes where they diffuse laterally in the plasma membrane. Lipophilic tracers are useful for live cell staining in tissue sections, in vivo neurons (Figure 2), and cultured cells. Additionally, they can be used in fixed tissue sections, where they can be used to trace neuronal projections (Figure 3). DiI and its derivatives can also be used for long-term studies.
These lipophilic tracers can be loaded via microinjection or iontophoresis and used in both anterograde and retrograde transport studies. They come as crystal and paste preparations which provides convenient options for loading tissues.
Figure 2. Tracer DiI as a diagnostic tool to evaluate retinal and tectal patterns in a chimeric five-day-old zebrafish brain. Axons in the dorsonasal retina of a zebrafish embryo labeled with DiI, and nuclei counterstained with SYTOX Green nucleic acid stain. This digital image, previously published on the cover of Development 125 (13) (1999), was created by Alexander Picker and Michael Brand, Neurobiology, University of Heidelberg. The image is used with the permission of The Company of Biologists, Ltd.
Figure 3. Confocal microscope image of retrogradely labeled neurons in the ventrobasal nucleus of the thalamus in newborn mouse. Paired dye deposits of DiI (DiI18(3); pseudocolored green) and DiD (DiIC18(5); pseudocolored red) were injected into this 500 µm-thick section, which was then fixed with paraformaldehyde and incubated for two weeks at 37°C to allow retrograde dye transport to proceed. Image contributed by Ariel Agmon, West Virginia University.
Hydrazides and Biocytins
Hydrazides and biocytins are fixable and photostable polar tracers. They have low molecular weights (<1,000 Da) and bright fluorescent signals. Hydrazides and biocytins can be used to investigate cell-cell and cell-liposome fusion. These cell-impermeant polar tracers can also be used to trace neuronal projections and investigate gap junctions (Figures 4, 5). Since they can cross gap junctions, biocytins and hydrazides are often used to investigate neuronal communication along with labeled dextrans, which are retained within the cell (Figure 6). Biocytins can also be detected using fluorescent streptavidin conjugates for signal amplification such as TSA and ELF technologies.
These polar tracers can be loaded using microinjection, pinocytosis, or other techniques that temporarily permeabilize the cell membrane. Additionally, electroporation can be used to transport these tracers into cells.
Figure 4. Rabbit retinal ganglion cells loaded with Alexa Fluor 488 hydrazide and Alexa Fluor 568 hydrazide. Three rabbit retinal ganglion cells injected with red-fluorescent Alexa Fluor 568 hydrazide (top cell), green-fluorescent Alexa Fluor 488 hydrazide (middle cell), or both the Alexa Fluor 488 and the Alexa Fluor 568 hydrazides (bottom cell), where yellow fluorescence indicates the co-localization of the two dyes. Image contributed by Michiel van Wyk, University of Queensland, Australia.
Figure 5. Neural cells loaded with Alexa Fluor 488 hydrazide and Alexa Fluor 568 hydrazide. Processes of a Leydig neuron iontophoretically filled with green-fluorescent Alexa Fluor 488 hydrazide and the cell body of a giant glial cell filled with red-fluorescent Alexa Fluor 568 hydrazide in a leech ganglion. Image contributed by F. Britz, C. Lohr, J. Schmidt and J.W. Deitmer, University of Kaiserslautern, Germany.
Figure 6. Co-stain of neuron with Dextran and Hydrazide. Confocal image stack of a 10,000 MW Calcium Green dextran-labeled climbing fiber in a sagittal cerebellar slice, showing incoming axon and terminal arborization (in yellow). The Purkinje cell innervated by this fiber was labeled with Alexa Fluor 568 hydrazide.
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