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CELLULAR STAINS

Cell Stains & Dyes for Live & Fixed Cells

Biotium offers a variety of stains for different organelles and cellular structures. Scroll down or select an organelle below to learn more.

Organelle MEMBRANE & CELL SURFACE NEURONAL STAINS CYTOPLASM CY T OSKELE T ON L YSOSOMES VESICLE TRAFFICKING NUCLEUS MITOCHONDRIA LIPID DROPLETS EXOSOMES AND EVS

Other Cellular Stain Resources

Explore Cellular Stains Comparison Guides to browse targets, applications, fixability, and stains for different organisms.

Cellular Stains Selection Guides

Find a membrane stain that fits your workflow with our Membrane and Cell Surface Stain Comparison Guide.

Membrane & Cell Surface Stains Comparison

 

Nucleus

NucSpot® Nuclear Stains
NucSpot® Nuclear Stains are cell membrane-impermeant fluorescent DNA stains available in a variety of colors from green to near-infrared (near-IR). NucSpot® Stains have minimal fluorescence until they bind to DNA and can be used for no-wash nuclear staining. Unlike other nucleic acid dyes such as propidium iodide (PI), TOTO®, TO-PRO®, and similar dyes that stain both the nucleus and cytoplasm, NucSpot® Nuclear Stains selectively stain the nucleus in fixed and permeabilized cells without the need for RNase treatment.

NucSpot® Nuclear Stains also can be used for selective staining of dead cells in unfixed cell cultures for analysis by flow cytometry or fluorescence imaging. Several of the dyes can be continuously incubated with cells for multi-day imaging. NucSpot® 470 and NucSpot® Far-Red can be used for DNA content analysis of cell cycle by flow cytometry in fixed and permeabilized cells without requiring RNase treatment, unlike propidium iodide.

NucSpot® Live 488 and NucSpot® Live 650 Nuclear Stains
NucSpot® Live Nuclear Stains specifically stain nuclei in live or fixed cells with no need for washing. NucSpot® Live 488 has green fluorescence (Ex/Em 500/515 nm), while NucSpot® Live 650 has far-red fluorescence (650/675 nm) for detection in the Cy®5 channel. Unlike Draq5™, NucSpot® Live 650 has low cytotoxicity and can be used for longer term imaging. NucSpot® Live 650 dye is also compatible with super-resolution imaging by SIM and STED.

RedDot™1 and RedDot™2 Far-Red Nuclear Stains
RedDot™1 and RedDot™2 are far-red nuclear counterstains for the Cy®5 channel. RedDot™1 is an alternative to DRAQ5™ that rapidly and specifically stains nuclei in live cells. It can be used for cell cycle analysis by flow cytometry. It also can be used for cell normalization for In Cell Western™. RedDot™1 shows cytotoxicity within a few hours of staining, so for long-term live cell imaging, we recommend NucSpot® Live 650.

RedDot™2 is membrane impermeant and can be used to selectively stain dead cells, or as a nuclear counterstain for fixed cells. RedDot™2 shows better nuclear specificity in fixed cells than DRAQ7™, which requires a blocking step for nuclear-specific counterstaining. RedDot™2 has also been validated for tissue clearing protocols such as CUBIC.

Live-or-Dye NucFix™ Red
Live-or-Dye NucFix™ Red is a unique, cell membrane impermeable dye that specifically stains the nuclei of dead cells. The dye is able to enter into dead cells that have compromised membrane integrity and covalently label the cell nucleus, allowing for clear differentiation of live and dead cells by either microscopy or flow cytometry. Unlike other commonly used nuclear stains such as propidium iodide or DRAQ7™, NucFix™ covalently attaches to DNA, allowing the cells to be fixed and permeabilized without loss of fluorescence or dye transfer between cells.

Classic Blue Nuclear Stains
DAPI and Hoechst are widely used blue fluorescent nuclear counterstains. They are minor groove-binding DNA dyes that are minimally fluorescent in solution, but have strong fluorescence enhancement upon binding DNA.

DAPI is less membrane permeant than Hoechst, and is typically used to stain fixed cells at concentrations around 1 ug/mL. Antifade mounting medium with DAPI, like Biotium’s EverBrite™ Mounting Media, can be used for one-step mounting and counterstaining. Staining live cells with DAPI requires higher concentration (~10 ug/mL). We offer DAPI dilactate, a more water soluble DAPI salt, which can be used at higher concentrations.

Hoechst dyes are membrane-permeant and can be used for live or fixed cell staining and cell cycle analysis. Hoechst 33342 and Hoechst 33528 both are quenched by BrdU labeled DNA, and have been used in cell division studies. The two dyes are spectrally similar. Hoechst 33342 is slighly more cell permeable than Hoechst 33258, but both dyes are commonly used as nuclear stains for live or fixed cells at 1 ug/mL.

While Hoechst and DAPI show less cytotoxicity than intercalating DNA dyes, they bind DNA in living cells and are potentially hazardous. Biotium offers Hoechst 33342, Hoechst 33258, and DAPI as 10 mg/mL solutions in water, for greater convenience and safety compared to weighing out the powdered dyes.

DAPI and Hoechst undergo photoconversion by UV excitation to form green fluorescent dyes, which can lead to artifacts in multi-color imaging. See our Tech Tip Avoiding Artifacts from UV Photoconversion of DAPI and Hoechst for more information.

Asymmetric Cyanine Dyes and Other Dead Cell Nuclear Stains
Biotium offers a selection of cyanine dyes for nucleic acids. Cyanine dyes have high affinity for DNA and RNA. Several of the membrane-impermeant dyes are useful dead cell stains. Oxazole Yellow (equivalent to YO-PRO®-1) is unique in that it selectively stains early apoptotic cells. Membrane-impermeant cyanine dyes also can be used as nuclear counterstains, but unlike RedDot™ or NucSpot® dyes, they stain both RNA and DNA, and so require RNase treatment for selective nuclear staining.

We also offer a variety of other cell-membrane impermeant nuclear stains for dead cell staining. See the table below for more information. For more application specific information, please see our nuclear stains comparison table.

 

Formaldehyde-fixed, Triton X-100-permeabilized HeLa cells stained with NucSpot® Live 488.
Live HeLa cells stained with NucSpot® Live 650 overnight at 37°C and imaged in the Cy® 5 channel.
NucView 405 Caspase-3 Substrate, 1 mM in DMSO
RedDot™ 1 (red) in live HeLa. Apoptotic cells are stained with NucView® 405 (blue) and CF®488A Annexin V (green).
RedDot™ 2 nuclear counterstain (magenta) with CF® dye secondary antibodies and CF®405M phalloidin.

Nuclear Stains

Catalog
No.

Color (Ex/Em*)

Cell
permeability

For live /
fixed cells

Fix after
staining?

Features

EverBrite™ Mounting Medium w/DAPI
EverBrite™ Hardset w/DAPI
Drop-n-Stain EverBrite™ w/DAPI
23002
23004
23009
Blue
(358/461 nm)
N/AMounting fixed samplesN/A• Hardset and wet-set mounting media
• Broad dye compatibility
• Available with DAPI for one-step mounting and staining
• Drop-n-Stain EverBrite™ comes in convenient dropper bottle
DAPI
DAPI, 10 mg/mL in H2O
DAPI, dilactate 10 mg in H2O
40011
40043
40009
Blue
(358/461 nm)
Membrane
permeant
Live cells
Fixed cells
Yes• Classic nuclear counterstain for fixed cells
• Can be used at higher concentrations to stain live cells
• Dilactate salt has improved water solubility
Hoechst 33258, 10 mg/mL in H2O
Hoechst 33258, pentahydrate
Hoechst 33342, 10 mg/mL in H2O
Hoechst 33342, trihydrochloride trihydrate
40044
40045
40046
40047
Blue
(358/461 nm)
Membrane
permeant
Live cells
Fixed cells
Yes• Classic nuclear counterstain for live cells
• Can also be used on fixed cells
Oxazole Gold (SYBR® Gold)40094Green
(496/539)
Membrane
permeant
Live cellsNo 2• Aka SYBR® Gold, an ultrasensitive DNA and RNA gel stain
• Can be used for live cell staining of nuclei and mitochondrial DNA
NucSpot® 470 Nuclear Stain40083Green
(460/546 nm)
Membrane
impermeant
Dead cells
Fixed cells
No 1• Nuclear-specific green counterstain for fixed cells
• Selectively stains dead cells in live cultures
• Excellent match for blue LED excitation sources
Thiazole Green (SYBR® Green I)40086Green
(498/522 nm)
Membrane
permeant
Live cells
Fixed cells
No 2• Aka SYBR® Green I, a well known DNA gel stain and qPCR dye
• Can be used as a green nuclear stain for all cells in live cultures
• Loses nuclear specificity after fixation
• Can be excited by 488 nm laser line
NucSpot® Live 488 Nuclear Stain40081Green
(500/515 nm)
Membrane
permeant
Live cells
Fixed cells
Yes• Low-toxicity nuclear stain
• Fix before or after labeling
• Live cell staining may require VRP (included)
Live-or-Dye NucFix™ Red32010Red
(520/610 nm)
Membrane
impermeant
Dead cellsYes• Reactive nuclear stain for dead cells
• Specifically stains dead cell nuclei
• Fix/permeabilize without dye transfer between cells
NucSpot® 555/570 Nuclear Stain41033Red
(559/566 nm)
Membrane
impermeant
Dead cells
Fixed cells
No 1• Nuclear-specific red counterstain for fixed cells
• Specifically stains dead cells in live cultures
NucSpot® 568/580 Nuclear Stain41036Red
(572/583 nm)
Membrane
impermeant
Dead cells
Fixed cells
No 1• Nuclear-specific red counterstain for fixed cells
• Specifically stains dead cells in live cultures
• Suitable for multi-day live cell imaging
NucSpot® 594/615 Nuclear Stain41037Red
(603/613 nm)
Membrane
impermeant
Dead cells
Fixed cells
No 1• Nuclear-specific red counterstain for fixed cells
• Specifically stains dead cells in live cultures
• Suitable for multi-day live cell imaging
NucSpot® 650/665 Nuclear Stain41034Far-red
(653/671 nm)
Membrane
impermeant
Dead cells
Fixed cells
No 1• Nuclear-specific far-red counterstain for fixed cells
• Specifically stains dead cells in live cultures
NucSpot® Live 650 Nuclear Stain40082Far-red
(650/675 nm)
Membrane
permeant
Live cells
Fixed cells
Yes• Low-toxicity nuclear stain for the Cy®5 channel
• Fix before or after labeling
• Live cell staining may require VRP (included)
• Compatible with SIM, STED, or STORM
RedDot™1 Far-Red Nuclear Stain40060Far-red
(662/694 nm)
Membrane
permeant
Live cellsNo 2• For short-term live cell staining (≤4 hours)
• Analyze DNA content/cell cycle by flow cytometry
• Useful for cell number normalization for In Cell Western®
• Can be excited at wavelengths between 488 and 647 nm
• Detect in Cy®5 or APC channel

RedDot™2 Far-Red Nuclear Stain40061Far-red
(665/695 nm)
Membrane
impermeant
Dead cells
Fixed cells
No 1• Far-red nuclear stain for dead or fixed cells
• Selectively stains dead cells
• Specific nuclear counterstain for fixed cells
• Can be excited at wavelengths between 488 and 647 nm
• Detect in Cy®5 or APC channel
NucSpot® 680/700 Nuclear Stain41035Near-IR
(683/707 nm)
Membrane
impermeant
Dead cells
Fixed cells
No 1• Nuclear-specific near-IR counterstain for fixed cells
• Specifically stains dead cells in live cultures
NucSpot® 750/780 Nuclear Stain41038Near-IR
(757/780 nm)
Membrane
impermeant
Dead cells
Fixed cells
No 1• Nuclear-specific near-IR counterstain for fixed cells
• Specifically stains dead cells in live cultures
• Suitable for multi-day live cell imaging
* With DNA
1 Dye can transfer from dead to live cells after fixation.
2 Loses nuclear specificity after fixation.
In Cell Western is a registered trademark of LI-COR® Bioscience. Cy Dye is a registered trademark of Cytiva.

 

Dead Cell Nucleic Acid Stains

Catalog No.

Color (Ex/Em*)

Features

Oxazole Blue, 1 mM in DMSO 40091Blue
(434/457 nm)
• Blue cell-impermeant dye
• Selectively stains early apoptotic cells
• Equivalent to PO-PRO™-1 Iodide
Oxazole Blue Homodimer, 1 mM in DMSO 40093Blue
(433/457 nm)
• Blue cell-impermeant dye
• Equivalent to POPO™-1 Iodide
NucSpot® 470, 1000X in DMSO 40083Green
(460/546 nm)
• Green cell-impermeant dye
• Nuclear-specific counterstain in fixed cells
• Useful for cell cycle analysis in fixed cells
• Excellent match for blue LED excitation sources
Oxazole Yellow, 1 mM in DMSO 40089Green
(491/506 nm)
• Green cell-impermeant dye
• Selectively stains early apoptotic cells
• Equivalent to YO-PRO®-1 Iodide
Oxazole Yellow Homodimer, 1 mM in DMSO 40090Green
(491/508 nm)
• Green cell-impermeant dye
• Equivalent to YOYO®-1 Iodide
TO Iodide, 1 mM in DMSO40088Green
(515/531 nm)
• Green cell-impermeant dye
• Equivalent to TO-PRO®-1 Iodide
Thiazole Orange Homodimer,
1 mM in DMSO
40079Green
(514/531 nm)
• High affinity dimeric cyanine dye
• Dead cell stain and electrophoresis dye
• Equivalent to TOTO®-1 Iodide
NucSpot® 555/570 Nuclear Stain41033Red
(559/566 nm)
• Red cell-impermeant dye for the Cy®3 or PE channels
• Nuclear-specific counterstain in fixed cells
NucSpot® 568/580 Nuclear Stain41036Red
(572/583 nm)
• Red cell-impermeant dye for the Cy®3 or PE channels
• Nuclear-specific counterstain in fixed cells
• Suitable for multi-day live cell imaging
NucSpot® 594/615 Nuclear Stain41037Red
(603/613 nm)
• Red cell-impermeant dye for the Texas Red® or PE-Texas Red® channels
• Nuclear-specific counterstain in fixed cells
• Suitable for multi-day live cell imaging
Propidium Iodide, 100 mg40016Red
(530/622 nm)
• Widely used dead cell stain
• Can be excited by 488 nm laser line for detection in the PE channel by flow cytometry
• Useful for cell cycle analysis in fixed cells (with RNase treatment)
Propidium Iodide, 1 mg/mL in Water40017
Propidium Iodide Buffer, 50 ug/mL40048
Ethidium Homodimer I, 1 mg 40010Red
(527/624 nm)
• High-affinity membrane-impermeant nucleic acid stain
• >30-fold fluorescence enhancement upon binding to DNA/RNA
• High-purity grade not available from other manufacturers
Ethidium Homodimer I, 2 mM in DMSO 40014
Ethidium Homodimer III, 1 mg 40050Red
(532/625 nm)
• Developed at Biotium as an alternative to Ethidium Homodimer I
• 45% brighter than EthDI when bound to DNA
Ethidium Homodimer III, 1 mM in DMSO 40051
Oxazole Red, 1 mM in DMSO40105Far-red (613/629 nm)• Far-red cell-impermeant dye for the PE-Cy®5, or APC channel
• Useful dead cell stain
• Equivalent to YO-PRO®-3
Oxazole Red Homodimer, 1 mM in DMSO40106Far-red (612/631 nm)• Far-red cell-impermeant dye for the PE-Cy®5, or APC channel
• Useful dead cell stain
• Equivalent to YOYO®-3
7-AAD, 1 mg 40037Far-red
(546/647 nm)
• Far-red dye for flow cytometry detection in the PE-Cy®5 channel
• Can be excited by the 488 nm or 532 nm laser line
• Useful for cell cycle analysis in fixed cells
7-AAD, 1 mg/mL solution 40084
NucSpot® Far-Red, 1000X in DMSO40085Far-red
(597/667 nm)
• Designed as improved replacement for 7-AAD
• For flow cytometry in the PE-Cy®5 or APC channel
• Useful for cell cycle analysis in fixed cells
• Less bleed into the PE-Texas Red® channel
RedDot™2 Far-Red Nuclear Stain40061Far-red
(665/695 nm)
• Far-red cell-impermeant dye for the Cy®5 channel
• Nuclear-specific counterstain in fixed cells
• Replaces Draq7™
Thiazole Red, 1 mM in DMSO40087Far-red
(642/657 nm)
• Far-red cell-impermeant dye for the Cy®5 channel
• Dead cell stain and electrophoresis dye
• Equivalent to TO-PRO®-3 Iodide
Thiazole Red Homodimer,
1 mM in DMSO
40080Far-red
(642/661 nm)
• High affinity dimeric cyanine dye for the Cy®5 channel
• Useful dead cell stain
• Equivalent to TOTO®-3 Iodide
NucSpot® 650/665 Nuclear Stain41034Far-red
(653/671 nm)
• Far-red cell-impermeant dye for the Cy®5 or APC channels
• Nuclear-specific counterstain in fixed cells
NucSpot® 680/700 Nuclear Stain41035Near-IR
(683/707 nm)
• Near-IR cell-impermeant dye for the Cy®5.5 channel
• Nuclear-specific counterstain in fixed cells
NucSpot® 750/780 Nuclear Stain41038Near-IR
(757/780 nm)
• Near-IR cell-impermeant dye for the Cy®7 or APC-Cy®7 channels
• Nuclear-specific counterstain in fixed cells
• Suitable for multi-day live cell imaging
* When bound to nucleic acids
SYBR, PO-PRO, POPO, Texas Red, TOTO, TO-PRO, YO-PRO, and YOYO are trademarks or registered trademarks of Thermo Fisher Scientific. Cy Dye is a registered trademark of Cytiva. Draq7 is a trademark of Biostatus Ltd.

 

Dyes for Cell Cycle Analysis

Catalog No.

Live or Fixed Cells?RNase treatment required?

Color (Ex/Em*)

Features

NucSpot® 470 Nuclear Stain, 1000X in DMSO 40083FixedNoGreen
(460/546 nm)
• Nuclear-specific green counterstain for fixed cells
• Selectively stains dead cells in live cultures
• Excellent match for blue LED excitation sources
Propidium Iodide, 100 mg40016FixedYesRed
(530/622 nm)
• Widely used dead cell stain
• Can be excited by 488 nm laser line for detection in the PE channel
Propidium Iodide, 1 mg/mL in Water40017
Propidium Iodide Buffer, 50 ug/mL40048
7-AAD, 1 mg 40037FixedNoFar-red
(546/647 nm)
• Far-red dye for detection in the PE-Cy®5 channel
• Can be excited by the 488 nm or 532 nm laser line
7-AAD, 1 mg/mL solution 40084
NucSpot® Far-Red, 1000X in DMSO40085FixedNoFar-red
(597/667 nm)
• Designed as improved replacement for 7-AAD
• For the PE-Cy®5 or APC channel
• Less bleed into the PE-Texas Red® channel
RedDot™1 Far-Red Nuclear Stain, 200X in Water 40060LiveNoFar-red
(662/694 nm)
• For short-term live cell staining (≤4 hours)
• Useful for cell number normalization for In Cell Western®
• Can be excited at wavelengths between 488 and 647 nm
• Detect in Cy®5 or APC channel
* When bound to nucleic acids
Cy Dye is a registered trademark of Cytiva. In Cell Western is a registered trademarks of LI-COR Inc.

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MEMBRANE & CELL SURFACE

CellBrite® Cytoplasmic Membrane Dyes
Lipophilic carbocyanine dyes are widely used for labeling neurons in tissues by retrograde labeling, and to label membranes in a wide variety of cell types. The dyes are weakly fluorescent in solution, but become highly fluorescent in lipid bilayers. Staining is very stable with low toxicity and very little dye transfer in between cells, making the dyes suitable for long-term cell labeling and tracking studies. Cell populations can be labeled with different fluorescent colors for identification after mixing. When live cells are cultured after staining, the dyes are internalized over time by endocytosis. Cells can be fixed either before or after staining, although permeabilization affects the staining pattern.

Unlike PKH dyes, a different class of membrane stains, CellBrite® dyes do not require a complicated hypoosmotic labeling protocol. They are ready-to-use dye solutions that can be added directly to normal culture media to label suspended or adherent cells. We offer a selection of CellBrite® dyes with fluorescence ranging from blue to near-infrared.

CellBrite® Fix and MemBrite® Fix: Surface Stains That Tolerate Fix & Perm
CellBrite® Fix and MemBrite® Fix dyes are new classes of dyes developed by Biotium to rapidly stain the outer plasma membranes of live cells. While other lipophilic membrane dyes can be fixed with formaldehyde, they have poor tolerance for detergent permeabilization or methanol fixation. In contrast, CellBrite® Fix and MemBrite® Fix stains are unique in that they can withstand permeabilization and methanol fixation, allowing surface staining to be combined with intracellular immunofluorescence.

CellBrite® Fix dyes are fluorogenic membrane dyes that accumulate in the cell membrane, where they become fluorescent. The dyes have an amine-reactive group for covalent attachment to membrane proteins. MemBrite® Fix are non-lipophilic dyes that react covalently with cell surface proteins by a different mechanism than CellBrite® Fix, and are available with a wider selection of colors.

Due to their high water solubility, CellBrite® Fix and MemBrite® Fix dyes yield much more uniform staining than lipophilic dyes like DiO and DiI. They are non-cytotoxic and do not readily transfer between cells, and also stain yeast and bacteria. Surface staining is well-retained after permeabilization or methanol fixation, with only slight a increase in intracellular fluorescence. Unlike lectins, which bind specific targets, CellBrite® Fix and MemBrite® Fix are general surface stains. However, CellBrite® Fix and MemBrite® Fix dyes cannot be used to stain cells that are already fixed.

CellBrite® Steady Membrane Staining Kits for Multi-Day Cell Surface Imaging
Our recently released CellBrite® Steady Membrane Staining Kits are unique fluorescent membrane probes designed for multi-day cell surface imaging. Unlike other membrane/cell surface stains that are rapidly lost from the cell surface by endocytosis, CellBrite® Steady Dyes equilibrate between intracellular compartments and the plasma membrane. This allows the cells to retain surface staining as well as intracellular staining for several days. These kits also include CellBrite® Steady Enhancer as an optional reagent that can be used to mask intracellular fluorescence of CellBrite® Steady Dyes, providing more selective visualization of cell boundaries. Dye colors are available from blue to near-IR, with STORM-compatible options.

Lectin Conjugates: CF® Dye Labeled WGA & Con A
Lectins are carbohydrate binding proteins that recognize specific sugar moieties on glycoproteins. They can be used to selectively stain the cell surface of live cells, and withstand fixation and permeabilization. When cells are fixed and permeabilized before staining, fluorescent lectins stain both cell surface and organelles in the secretory pathway. WGA and Con A lectins are widely used for cell surface staining. Unlike CellBrite® or MemBrite® stains, lectin staining may be cell type dependent. We offer WGA and Con A with a wide selection of bright and photostable CF® Dyes. We also offer PNA lectin conjugates, which have more specialized applications.

Surface Stains for Yeast and Bacteria
CellBrite® Fix stains yeast and bacteria cell surface. MemBrite® Fix and Con A conjugates also can be used for staining yeast. WGA conjugates can be used as yeast bud scar stains, while in bacteria they are useful fluorescent Gram stains. Also see our full selection of Microbiology Products.

Other Membrane and Lipid Probes
We offer a number of other probes for specialized membrane staining applications, such as labeled phospholipids, cholera toxin conjugates for lipid raft labeling, vesicle trafficking probes, nerve terminal dyes, and membrane potential dyes. Download our Cellular Stains Brochure to learn more.

Find the Right Stain for Your Application or Workflow
See our Membrane & Cell Surface Stain Comparison guides or download our brochure to compare products and find the right stain for your workflow.

Membrane & Cell Surface Stains Comparison

HeLa stained with CellBrite® Fix 555 (red), fixed and stained with CF®640R anti-Mitochondrial Marker clone 113-1 (cyan).
HeLa cells fixed with formaldehyde, then stained with CellBrite® Blue, washed and imaged.
Live HeLa cells stained with CF® 594 Con A (red) and Hoechst 33342 (blue) for 10 minutes at 37°C, then washed and imaged.
HeLa cells co-stained with CellBrite® Steady 488 (green) and MitoView™ 633 mitochondrial stain (magenta).
MemBrite® Fix labels cells more evenly than traditional membrane stains, and tolerates permeabilization. HeLa cells were labeled with DiI or MemBrite® Fix 568/580, then fixed/permeabilized with formaldehyde followed by Triton® X-100, or fixed with methanol. Unlike DiI, MemBrite® Fix labeling is covalent, so it does not redistribute or wash off after permeabilization.
MemBrite® Fix kits offer a wide selection of dye colors, with excellent tolerance for common fixatives like PFA and methanol. Cells were labeled with the indicated dye for 5 minutes at 37°C, then fixed with ice-cold methanol for 5 minutes at -20°C.

Stain Cell Surfaces in Whole Organisms

MemBrite® Fix stains have been used to label cell surfaces in whole organisms. In the following figures provided by Patrick Lemaire’s lab of CRBM (Centre de Recherche en Biologie cellulaire de Montepellier), MemBrite® Fix 543/560 was used to stain whole embryos of the tunicate Phallusia mammillata followed by downstream hybridization chain reaction fluorescent in situ hybridization (HCR-FISH) for detection of a RNA transcript. Cell boundaries were clearly labeled through the entirety of the Z-stack from the animal hemisphere to the vegetal hemisphere of the embryo.

Embryos of the tunicate Phallusia mammillata were stained using MemBrite® Fix 543/560 (red) at stage 12: mid gastrula, followed by downstream HCR-FISH. The embryos were mounted with DAPI (blue) and imaged using confocal microscopy. Left: plane through the animal hemisphere of the embryo. Center: the center of the embryo. Right: plane through the vegetal hemisphere of the embryo with the FISH-labeled RNA transcript (green). Scale bar is 100 um. Credit: Ingrid Mormin, research assistant, Patrick Lemaire’s lab, CRBM-CNRS UMR5237.
Embryo of the tunicate Phallusia mammillata stained with MemBrite® Fix 543/560 (red) at stage 12: mid gastrula and mounted with DAPI (blue). Images progress through the z-stack from the animal hemisphere to the vegetal hemisphere of the embryo. Credit: Ingrid Mormin, research assistant, Patrick Lemaire’s lab, CRBM-CNRS UMR5237.

Membrane & Cell Surface Stains

Features

CellBrite® Cytoplasmic Membrane Dyes• Easy to use dye solutions for stable, non-toxic membrane labeling
• Fix before or after labeling
• Blue, green, red, and far-red options
• Don’t tolerate permeabilization or methanol fixation
CellBrite® NIR Near-Infrared Cytoplasmic Membrane Dyes• CellBrite® dyes with near-IR emission ranging from 724-820 nm
• Compatible with NIR small animal imaging systems
• Don’t tolerate permeabilization or methanol fixation
CellBrite® Fix Membrane Stains• The only fluorogenic membrane dyes that can be fixed & permeabilized for IF
• Bright, uniform live cell surface staining in 15 minutes
• Available with green, red, and far-red fluorescence
• Stain yeast and bacteria
MemBrite® Fix Cell Surface Staining Kits• Covalent, rapid, and uniform live cell surface protein labeling
• Can be fixed & permeabilized for IF staining
• Stain yeast and gram-positive bacteria
• Choice of 8 colors from blue to near-IR
CellBrite® Steady Membrane Staining Kits• 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
CF® Dye WGA Conjugates• Binds to sialic acid and N-acetylglucosamine
• Stain cell surface glycoproteins in live or fixed cells
• Bacterial Gram stain and yeast bud scar stain
• 13 CF® dye colors plus HRP
CF® Dye Con A Conjugates• Binds α-mannopyranosyl and α-glucopyranosyl residues
• Stain cell surface glycoproteins in live or fixed cells
• Stains yeast cell wall
• 10 CF® dye colors from UV to NIR
CF® Dye Cholera Toxin Subunit B • Labels lipid rafts
• Withstands fixation and permeabilization
• Wide selection of CF® dye colors

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Mitochondria

MitoView™ Dyes
In metabolically active cells, mitochondria produce a membrane potential by maintaining a proton gradient across the inner and outer membranes. Loss of mitochondrial membrane potential is a hallmark for apoptosis. Biotium’s MitoView™ mitochondrial dyes are available in a variety of colors. MitoView™ 633 is a cationic membrane potential-sensitive dye for mitochondria in mammalian cells and yeast. The dye preferentially localizes to the matrix in mitochondria with intact membrane potential in live cells. Staining is lost when mitochondria become depolarized during cell death, allowing monitoring of cell viability.

We also offer MitoView™ Green, a membrane-potential independent dye that also accumulates within mitochondria and interacts with the mitochondrial matrix via hydrophobic interactions. It can be used to image mitochondria following mitochondrial depolarization, or after fixation, but fixed cell staining tends to be less specific compared to live cell staining. Blue fluorescent MitoView™ 405 and near-infrared MitoView™ 720 dyes localize to the cytoplasm after mitochondrial depolarization, but still retain fluorescence.

JC-1 and Other Classic Mitochondrial Dyes
Ratiometric dyes like JC-1 constitute another class of potential-dependent mitochondrial dyes. In healthy cells, JC-1 dye aggregates in mitochondria as a function of membrane potential, resulting in red fluorescence with brightness proportional to the membrane potential. Conversely, in apoptotic and necrotic cells with diminished mitochondrial membrane potential, JC-1 exists in a green fluorescent monomeric form in the cytosol, allowing cell viability to be assessed by measuring the ratio of red to green fluorescence by flow cytometry or fluorescence microplate reader.

Green fluorescent Rhodamine 123 is a potentiometric dye that accumulates in the mitochondria and is widely used for flow cytometry studies of mitochondrial membrane potential. Red fluorescent TMRM and TMRE are similar potentiometric dyes that are preferred for quantitative membrane potential measurements. Nonyl acridine orange (NAO) is a membrane potential-independent dye that is reported to bind the phospholipid cardiolipin in the inner mitochondrial membrane. We also offer a number of other classic mitochondrial dyes. See the Cellular Stains Brochure for more information.

MitoView™ Fix 640
MitoView™ Fix 640 is a far-red-mitochondrial stain suitable for long-term mitochondrial imaging. The cell membrane-permeant dye is non-toxic and offers mitochondrial staining that is well retained for at least 72 hours. In addition, MitoView™ Fix 640 has a reactive group that can covalently attach to proteins inside the mitochondria, allowing for stable staining that withstands fixation and permeabilization for downstream immunofluorescence workflows. In our testing, MitoView™ Fix 640 offers better specificity after PFA fixation when compared to the similar MitoTracker® Deep Red FM dye. For staining mitochondria in fixed cells or tissue sections, we recommend using one our Mitochondrial Marker Antibodies, available with a wide selection of bright and photostable CF® dyes and other conjugations.

Live HeLa cells stained with MitoTracker® Deep Red FM or MitoView™ Fix 640 in culture medium for 2 hours at 37°C and imaged without a wash step. Cells were then fixed with 4% PFA/PBS for 20 minutes at RT. MitoView™ Fix 640 staining retains better specificity after PFA fixation compare to MitoTracker® Deep Red FM.
MitoView™ 405.
MitoView™ Green.
MitoView™ 633
MitoView™ 720 in the Cy®5 channel.

Mitochondria DyesAbs/EmDetection channelMitochondrial LocalizationPotential-dependent?Fixable?Catalog no.Size
MitoView™ 405398/440 nmDAPIMatrixPartial1No70070-T50 ug
7007020×50 ug
MitoView™ Green490/523 nmFITC, GFPMatrixNoNo270054-T50 ug
7005420×50 ug
MitoView™ 633622/648 nm3Cy®5, APC3MatrixYesNo70055-T50 ug
7005520×50 ug
MitoView™ 650644/670 nmCy®5, APCMatrixPartial1No70075-50ug50 ug
7007520×50 ug
MitoView™ 720720/758 nm4Cy®5, Cy®74MatrixPartial1No70068-T50 ug
7006820×50 ug
MitoView™ Fix 640648/670 nmCy®5MatrixYes5Yes70082-50ug50 ug
7008210×50 ug
Nonyl Acridine Orange (NAO)495/522 nmFITC/GFPIMMNoYes67001250 mg
Aquaphile™ JC-1510/527 nm (monomer)
585/590 nm (aggregate)
Green/Red7MatrixRatiometric7No7007650 uL
JC-1 (iodide salt)700145 mg
JC-1 (chloride salt)700115 mg
Rhodamine 123505/534 nmFITC/GFPMatrixYesNo7001050 mg
TMRE549/574 nmCy®3MatrixYesNo7001625 mg
TMRE, 2 mM in DMSO700050.5 mL
TMRM549/574 nmCy®3MatrixYesNo7001725 mg
DASPEI461/589 nmRedMatrixYesNo70018100 mg
DiIC1(5)638/658 nmCy®5MatrixYesNo70015100 mg
1. Dyes with partial mitochondrial membrane potential dependence localize to the cytoplasm after mitochondrial depolarization, but still retain fluorescence.
2. MitoView™ Green is not fixable but can be used to stain cells after formaldehyde fixation.
3. MitoView™ 633 also has red fluorescence in the Cy®3 channel and is not recommended for use with other red dyes.
4. While optimal for Cy®7 settings, MitoView™ 720 is bright enough to be imaged in the Cy®5 channel.
5. MitoView™ Fix 640 staining is initially potential-dependent, but reacts covalently with mitochondrial proteins for staining that is retained after fixation and permeabilization.
6. Formaldehyde fixation only. Not compatible with solvent-based fixation or permeabilization.
7. JC-1 forms red fluorescent aggregates in polarized mitochondria, and green fluorescent monomers in cytoplasm.


Matrix: Mitochondrial matrix; IMM: Inner mitochondrial membrane
Cy Dye is a registered trademark of Cytiva.

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Lysosomes

LysoView™ Dyes
LysoView™ dyes are fluorescent stains for imaging lysosome localization and morphology in live cells. LysoView™ dyes belong to a family of lysosomotropic dyes that contain weakly basic amines that accumulate in acidic organelles in mammalian cells and yeast. Red-fluorescent LysoView™ 540 and far-red fluorescent LysoView™ 633 dye fluorescence is pH-sensitive, resulting in specific lysosomal staining without a wash step. We also offer LysoView™ 650, a far-red lysosome dye that is compatible with super-resolution imaging by SIM and STED.

“Light-On” LysoView™ 555
“Light-On” LysoView™ 555 is a UV-activatable lysosome stain. In cells, the dye initially shows low fluorescence, but brief exposure to UV excitation from a mercury arc lamp activates bright red fluorescence localizing to lysosomes. Lysosomal fluorescence fades over the course of several minutes after UV exposure, but can be re-activated in the same cells multiple times by exposure to UV light. Therefore the dye provides a novel tool for UV-activated, reversible fluorescence imaging of lysosomes.

HeLa cells stained with LysoView™ 540 (red) and Hochst 33342 (blue).
Live HeLa cells stained with LysoView™ 650 (magenta) and MitoView™ Green.
Lysosomes and vacuoles of live S. cerevisiae yeast stained with LysoView™ 633.
Lysosome StainsAbs/Em (nm)
(pH ≤ 5)
Detection
channel
FeaturesCatalog numberSize
LysoView™ 405, 1000X in DMSO318, 400/ 464DAPI, Pacific Blue™Blue fluorescent lysosome stain70066-T10 uL
7006650 uL
LysoView™ 488, 1000X in DMSO496/526GFP, FITCGreen fluorescent dye validated in SIM70067-T10 uL
7006750 uL
LysoView™ 540, 1000X in DMSO540/634TRITC, Cy®3, PEOrange, pH-sensitive fluorescence**70061-T10 uL
7006150 uL
LysoView™ 550, 1000X in DMSO542/567TRITC, Cy®3, PEBright & photostable orange dye70083-T10 uL
7008350 uL
LysoView™ 594, 1000X in DMSO585/634Texas Red®Bright & photostable red dye70084-T10 uL
7008450 uL
LysoView™ 633 (lyophilized solid)634/657Cy®5, APCFar-red, pH-sensitive fluorescence70058-T1 vial*
7005810 vials*
LysoView™ 640, 1000X in DMSO640/671Cy®5, APCBright & photostable far-red dye70085-T10 uL
7008550 uL
LysoView™ 650, 1000X in DMSO650/675Cy®5, APCPhotostable far-red dye compatible with SIM, STED70059-T10 uL
7005950 uL
LysoView™ 680, 1000X in DMSO674/711Cy®5.5Unique near-IR lysosome stain70086-T10 uL
7008650 uL
*Each vial of LysoView™ 633 makes 100 uL of 1000X dye after reconstitution.
** LysoView™ 540 has limited photostability and may not be suitable for all microcopy applications.
Cy Dye is a registered trademark of Cytiva; Pacific Blue is a trademark and Texas Red is a registered trademark of Thermo Fisher Scientific.

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LIPID DROPLETS

LipidSpot™ Lipid Droplet Stains

Intracellular lipid droplets are cytoplasmic organelles involved in the storage and regulation of triglycerides and cholesterol esters. LipidSpot™ dyes are fluorogenic neutral lipid stains that rapidly stain lipid droplets. The dyes can be used to stain lipid droplets in both live and fixed cells, with no wash step required. Cells also can be fixed and permeabilized after staining. LipidSpot™ stains show minimal background staining of cellular membranes or other organelles, unlike dyes like Nile Red. Available with green or deep-red/far-red fluorescence.

Oleic acid treated HeLa cells were fixed then stained with LipidSpot 488 (lipid droplets, green), CF594 WGA (cell surface, red), and Hoechst 33342 (nuclei, blue).
HeLa cells were treated with or without oleic acid treatment to induce lipid droplet formation, then stained with LipidSpot™ 610. The dye stained live and formaldehyde-fixed cells, and live cell staining was retained after fixation and Triton® X-100 permeabilization.
LipidSpot™ StainsFeatures
LipidSpot™ Lipid Droplet Stains• Rapidly and specifically stain lipid droplets
• Stain live or fixed cells, or fix and permeabilize after staining
• Available with green or deep-red/far-red fluorescence

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Cytoskeleton

ViaFluor® Live Cell Microtubule Stains
ViaFluor® Live Cell Microtubule Stains bind to polymerized tubulin and may be used to visualize microtubules in live cells for up to 24 hours in immortalized cell types. ViaFluor® Microtubule Stains are supplied with a vial of VRP, an efflux pump inhibitor that may improve probe retention and staining in certain cell types. Biotium offers green fluorescent ViaFluor® 488 and far-red fluorescent ViaFluor® 647 Live Cell Microtubule Stains. ViaFluor® 647 Microtubule Stain is compatible with super-resolution imaging by STED.

Phalloidin conjugates
Phalloidin is a toxin isolated from the Amanita phalloides mushroom. It is a bicyclic peptide that binds specifically to F-actin. Fluorescently labeled phalloidins stain F-actin at nanomolar concentrations. Unlike antibodies, the binding of phalloidin is not species-specific, and non-specific staining is negligible. Biotium offers Phalloidin Conjugates with a wide selection of CF® Dyes as well as traditional dyes and biotin. CF® Dyes have advantages in brightness, photostability, and water solubility compared to other commercially available fluorescent dyes. A number of our CF® Dyes have been validated in super-resolution imaging by STORM, STED, SIM, and other methods. Download the CF® Dye Selection Guide for more information on CF® Dyes.

Live HeLa cells stained with ViaFluor® 488 Microtubule Stain (green) and LysoView™ 650 (magenta).
Mitotic spindle in live MCF-7 cell stained with ViaFluor® 647 Microtubule Stain (magenta) and Hoechst 33342 (blue).
Fixed and permeabilized HeLa cells stained with CF®405M phalloidin (blue) and RedDot™ 2 (magenta)

Cytoskeletal Probes

Features

ViaFluor® Live Cell Microtubule Stains• Live cell microtubule stains
• Blue fluorescent ViaFluor® 405, green fluorescent ViaFluor® 488 and far-red fluorescent ViaFluor® 647 Microtubule Stains
• ViaFluor® 647 is compatible with super-resolution imaging by STED
Phalloidin Conjugates • Actin filament stains for fixed and permeabilized cell
• Choice of 16 bright & photostable CF® dyes
• Also available with biotin, FITC, and rhodamine dyes

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Vesicles & membrane trafficking

Fluid phase tracers
Dextrans are water soluble branched-chain polysaccharides. Fluorescently labeled dextrans are used as markers for trafficking of fluid phase endocytic cargo to lysosomes. They are also used as tracers for epithelial and endothelial permeability. They are also used as microinjected tracers for neuronal morphology. CF® Dye Dextrans are available with a variety of dye colors and molecular weights ranging from 3,500 to 250,000.

While formaldehyde fixation preserves the localization of dextrans in endosomes, the labeling generally cannot tolerate detergent permeabilization, likely because the dextran molecules remain in the fluid phase and do not associate closely enough with cellular proteins to become cross-linked by fixative.

CF® Dye Bovine Serum Albumin (BSA) can be used as a tracer for protein uptake and permeability.

Fluorescent hydrazides are formaldehyde-fixable, water soluble fluid phase tracers. They are also used as injectable tracers for neuronal morphology. We offer a wide selection of bright and photostable CF® Dye Hydrazides. Download the CF® Dye Selection Guide for more information on CF® Dyes.

CF® Dye Transferrin Conjugates
Transferrin is a glycoprotein iron carrier that delivers iron to vertebrate cells via receptor-mediated endocytosis. After binding to its receptor on the cell surface, transferrin is rapidly internalized by invagination of clathrin-coated pits. Inside endocytic vesicles, the acidic environment favors dissociation of iron from the transferrin-receptor complex. Following the release of iron, the apotransferrin is recycled to the plasma membrane where it is released from its receptor to scavenge more iron. We offer human transferrin labeled with a selection of our bright and photostable CF® Dyes for imaging of recycling endosomes by microscopy.

CF® Dye Conjugated Cholera Toxin Subunit B
Cholera toxin subunit B binds to ganglioside GM1 in lipid rafts on the plasma membrane. Cholera toxin subunit B is reported to be internalized by clathrin-dependent and independent pathways depending on cell type. The conjugates also can be used to label lipid rafts on the cell surface and as a retrograde neuronal tracer. Download the CF® Dye Selection Guide for more information on CF® Dyes.

Nerve Terminal Dyes
SynaptoGreen™ and SynaptoRed™ nerve terminal probes (originally called FM® dyes) are membrane dyes used to trace endocytic vesicles. While traditionally used to monitor activity dependent vesicle release at synapses, they can be used to label and track vesicles in non-neuronal cells types as well. See the Nerve Terminal Probes Flyer for more information.


HeLa cells were incubated with CF®568 dextran (red) and Hoechst 33342 (blue) at 37°C for 30 minutes, washed, and imaged.
U87-MG cells were incubated with CF®680R transferrin (red). CF®488A dextran (green), and Hoechst 33342 (blue) for 30 minutes at 37°C, washed and imaged.
HeLa cells were incubated with CF®488A cholera toxin subunit B (green) for 30 minutes at 37°C, then fixed, permeabilized, and stained with DAPI.

Vesicle and Membrane Trafficking Probes

Features

CF® Dye Dextrans• Water-soluble fluid phase markers for tracking bulk endocytosis and cell permeability
• Available with molecular weights of 10 kD, 40 kD, 70 kD, 150 kD, or 250 kD
• Choose from a selection of bright & photostable CF® dyes
Bovine Serum Albumin CF® Dye Conjugates• Fluorescent BSA for endocytosis and permeability studies
• Choice of 4 bright & photostable CF® dyes
CF® Dye Hydrazides• Bright & photostable fixable polar tracers
• Wide choice of dye colors
CF® Dye Human Transferrin Conjugates• Recycling endosome tracers with a selection of bright & photostable CF® dyes
CF® Dye Cholera Toxin Subunit B • Lipid raft and endocytosis probe
• Withstands fixation and permeabilization
• Choice of 6 CF® dye colors
Nerve Terminal Staining Kits • SynaptoGreen™ & SynaptoRed™ can be used label vesicles in both neuronal and non-neuronal cells
• AM1-43 and other AM dyes are fixable
• Available in kits with background reducing agents

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Extracellular Vesicles

Extracellular vesicles (EVs) are small, membrane-bound particles secreted from cells and thought to function as cellular messengers, carrying cargo from one cell to another. The small size of EVs makes them challenging to handle and analyze. Biotium has validated several products and technologies specifically for detection of extracellular vesicles and exosomes.

ExoBrite™ CTB EV Staining Kits

ExoBrite™ CTB EV Staining Kits were designed to overcome some of the challenges of EV detection, particularly in flow cytometry. ExoBrite™ stains bind to molecules in the EV membrane for bright, specific staining, with little to no background. A key advantage of ExoBrite™ stains is that, unlike many other dyes, they show little to no background aggregates of a similar size as exosomes or EVs. Also, unlike most membrane dyes, ExoBrite™ stains do not bind non-specifically to polystyrene beads, meaning that they can be used to stain bead-bound EVs.

ExoBrite™ CTB EV Stain advantages:

  • Designed for EV detection by flow cytometry
  • Bright fluorescence and low background for excellent signal-to-noise
  • Compatible with antibody co-staining (unlike CellBrite® Fix or MemBrite® Fix)
  • Stain purified or bead-bound EVs
  • Available in 4 colors for flexible experimental design

Validated EV Antibodies
Biotium offers several different antibody clones against EV marker proteins CD9, CD63, and CD81, each available in a wide variety of conjugate choices such as bright CF® Dyes and biotin.

View our full list of validated antibodies for EV and exosome detection

SEC-purified MCF-7-derived EVs were stained with ExoBrite™ CTB EV Stains.

Learn more about our products validated for EV and exosome detection:

Extracellular Vesicle Labeling

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Cytoplasm

ViaFluor® SE Cell Proliferation Dyes
Viafluor® SE dyes are amine-reactive dyes that covalently label proteins throughout the cell. They are membrane permeable compounds that are non-fluorescent until they enter viable cells. In the cytoplasm, they are hydrolyzed by cytoplasmic esterase enzymes to releases fluorescent amine-reactive dyes that react with proteins throughout the cytoplasm and intracellular compartments. ViaFluor® SE dyes can be used for dye dilution of cell division by flow cytometry or as cell-filling cytoplasmic stains. The staining also can withstand fixation and permeabilization for subsequent immunostaining. We offer blue fluorescent ViaFluor® 405, green fluorescent ViaFluor® CFSE, and red fluorescent ViaFluor® 568.

Calcein AM
Calcein AM is a cell-filling green fluorogenic cytoplasmic stain that uniformly labels the entire cell. It works by a similar mechanism as ViaFluor® CFSE, but the dye is not reactive, so labeling is not covalent. Only viable cells generate and retain the dye. Calcein AM is widely used as a viability stain to quantitate live cells, detect cell lysis, or monitor dye efflux by transporter proteins.

HeLa cells labeled with calcein AM.

Membrane-Permeant Cytoplasm Stains

Features

ViaFluor® SE Dyes• Membrane-permeable compound is hydrolyzed in live cells to release amine-reactive dye
• Covalent, fixable intracellular labeling
• Can be used for flow cytometry analysis of cell division by dye dilution
• For long-term labeling (days to weeks)
• Choice of blue or green fluorescence
Calcein AM• Membrane-permeable compound is hydrolyzed in live cells to release green fluorescent dye
• Uniform intracellular labeling
• Dead cells don’t retain dye, for true endpoint viability assay
• For short term labeling (≤24 hours)

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Neuronal stains

Anterograde & Retrograde Tracers

Hydroxystilbamidine (also called Fluoro-Gold™) has been used extensively as a retrograde tracer for neurons and also a histochemical stain. Fluoro-Gold™ is used for retrograde tracing and dendrite filling. Biotin ethylenediamine is equivalent to Neurobiotin™, a useful anterograde and transneuronal tracer.

Cholera toxin subunit B binds GM1 ganglioside in lipid rafts, and is used as a retrograde neuronal tracer. Available with a wide selection of bright and photostable CF® Dyes. See Cholera Toxin Conjugates.

WGA is a glycoprotein-binding lectin that has been used for retrograde and anterograde neuronal tracing. We offer WGA CF® Dye conjugates with fluorescence from UV to near-IR, plus HRP. See WGA Conjugates.

Labeled dextran amine can be used for both retrograde and anterograde tracing. CF® Dye dextrans are anionic with an aldehyde-fixable free amine group, and are available with a wide selection of colors and a range of molecular weights. See Dextran Conjugates.

Cultured rat hippocampal neurons microinjected with CF®647 hydrazide (red) and stained with SynaptoGreen™ (FM®1-43, green, synaptic vesicles). Image courtesy of Professor Guosong Liu, Tsinghua University, Beijing, China.

Cytosolic Tracers

CF® Dye Hydrazides
We offer a wide selection of our bright and photostable CF® Dye Hydrazides. Water-soluble and aldehyde-fixable, hydrazides can be microinjected as neuronal morphology tracers. Download the CF® Dye Selection Guide for more information on CF® Dyes.

Calcein, Calcein AM, and ViaFluor® SE Dyes
Calcein is a water soluble green fluorescent FITC derivative widely used for the study of cell membrane integrity that can be introduced by microinjection. Calcein-AM is a membrane-permeant AM ester of calcein that can be loaded into cells by incubation, where it is cleaved by esterases to release the dye. Calcein AM also can be used to quantitate cell viability, because dead cells will not hydrolyze the ester or retain the hydrolyzed dye. ViaFluor® SE Dyes work similarly to Calcein AM, except the hydrolyzed dyes are amine-reactive and covalently label cytoplasmic proteins, so staining can withstand fixation and permeabilization.

Other Classic Fluorescent Tracers
Lucifer Yellow CH and Lucifer Yellow Cadaverine are aldehyde-fixable fluorescent cytosolic and gap junction tracers. Hydroxystilbamidine (equivalent to Fluoro-Gold) is a UV-excitable green fluorescent dye that has been used extensively as a retrograde tracer for neurons and as a histochemical stain. It is available in solid form and as a ready-to-use 4% solution. A number of other classic hydrophilic dyes have been used as microinjectable tracers as well. See the Cellular Stains Brochure for more information.

Biotin Derivatives
Biocytin (e-biotinoyl-L-lysine) is a cellular tracer that can be introduced by microinjection; biocytin-hydrazide is an aldehyde-fixable analog. We also offer bright, photostable, and pH-independent CF® Dye biotin and CF® Dye biocytin, as well as classic fluorescent biotin derivatives. See the Cellular Stains Brochure to learn more.

Anterograde & Retrograde Tracers

ProductEx/EmCatalog number
Hydroxystilbamidine (Fluoro-Gold™)361/536 nm80014
Hydroxystilbamidine (Fluoro-Gold™), 4% in H2O361/536 nm80023
Biotin ethylenediamine, hydrobromide (Neurobiotin™)N/A90057
Biotin ethylenediamine, hydrochlorideN/A90075
Cholera Toxin ConjugatesChoice of 10 colors
Wheat Germ Agglutinin ConjugatesChoice of 13 colors, plus HRP
Dextran ConjugatesChoice of 11 colors & 5 molecular weights

Cytosolic Tracers

Cytosolic Tracers

Features

CF® Hydrazides• Microinjectable, fixable fluorescent tracers
• Wide selection of bright & photostable CF® dyes
Calcein • Water soluble green fluorescent tracer for microinjection
Calcein AM• Membrane-permeable compound is hydrolyzed in live cells to release water soluble green fluorescent dye
• Uniform intracellular labeling
• Dead cells don’t retain dye, for true endpoint viability assay
ViaFluor® SE Dyes• Membrane-permeable compound is hydrolyzed in live cells to release amine-reactive dye
• Covalent, fixable intracellular labeling
• Choice of blue or green fluorescence
Lucifer Yellow Derivatives• Widely used green fluorescent tracers for neuronal morphology and gap junction studies
• Fixable Lucifer Yellow CH and Lucifer Yellow Cadaverine
• Lucifer Yellow Cadaverin Biotin-X for secondary detection with streptavidin
Hydroxystilbamidine (equivalent to Fluoro-Gold™)• Widely used UV-excitable green fluorescent retrograde neuronal tracer
• Available in solid and 4% solution in water
Biotin Derivatives• Biotin-based tracers for secondary detection with streptavidin
CF® Dye Biotin• Biotin conjugates of our bright & photostable CF® dyes
CF® Biocytin• Aldehyde-fixable biocytin conjugated with bright & photostable CF® dyes.

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Synaptic Probes and Nerve Terminal Dyes

α-Bungarotoxin Conjugates

Fluorescent α-bungarotoxin can be used for labeling of nicotinic acetylcholine receptors in sections of neuromuscular junction. We offer α-bungarotoxin conjugates with a selection of our CF® Dyes and other labels. CF® Dyes offer advantages in brightness and photostability compared to other commercially available fluorescent dyes. Download the CF® Dye Selection Guide for more information on CF® Dyes.

Fluorescent Bungarotoxins

Features

CF® Dye α-Bungarotoxin Conjugates• Fluorescent probe for nicotinic acetylcholine receptors
• Stains neuromuscular junction endplates
• Wide selection of bright & photostable CF® dyes
Neuromuscular junction endplate in a rat skeletal muscle cryosection stained with CF®594 α-bungarotoxin (red). Nuclei are stained with DAPI (blue).


Nerve Terminal Dyes

SynaptoGreen™ and SynaptoRed™ nerve terminal probes (originally called FM® dyes) are membrane dyes used to trace endocytic vesicles. They are a series of fluorescent cationic styryl dyes developed to follow synaptic activity at neuromuscular junctions or synapses. The dyes label synaptic vesicles in neuronal tissues and cultured neurons in an activity-depending fashion. They also can be used to label endocytic vesicles in other cell types.

Nerve terminal dyes have highly hydrophilic, cationically charged head group at one end with lipophilic tails at the other end. They are virtually non-fluorescent in aqueous solution, but become intensely fluorescent in membranes. Following nerve stimulation, the dye molecules are internalized in newly formed endocytic vesicles. During exocytosis, the dyes are released from the vesicles along with neurotransmitters, causing a decrease in fluorescence signal. As a result, the change in fluorescent intensity reflects the amount of endocytosis/exocytosis or synaptic activity. The rate of fluorescence increase during endocytosis (on-rate), and the rate of fluorescence decrease during exocytosis (off-rate) vary from dye to dye. AM dyes and HM dyes are fixable nerve terminal dyes. After staining with these dyes, cells can be fixed and permeabilized for subsequent immunostaining. See the table below for a list of nerve terminal dyes and their properties.

General structure of SynaptoGreen™ and SynaptoRed™ dyes.
General structure of AM fixable nerve terminal dyes.

A common problem encountered with nerve terminal dyes is background fluorescence due to residual membrane staining, even after extensive washing. To reduce this background fluorescence, we offer three quencher or dye-clearing agents. ADVASEP-7, a sulfonated β-cyclodextrin, forms a water soluble inclusion complex with SynaptoGreen™ C4 that can be removed more effectively by washing. Biotium’s unique quencher, SCAS, reduces background fluorescence as soon as it is added to the preparation without the need for washing. Sulforhodamine 101 quenches SynaptoGreen™ background staining via fluorescent resonance energy transfer (FRET). We offer these reagents as individual products and in kits with dyes and the quencher/dye-clearing agents.

AM1-43 dorsal root ganglia
Neurons in mouse dorsal root ganglia (DRG) labeled with AM1-43. Image courtesy of Dr. David Corey, Harvard Medical School.

Properties of Nerve Terminal Dyes

Nerve Terminal Dyem*n*Fixable?SizeCatalog numberFeatures
SynaptGreen™ Dyes (Ex/Em ~480/660 nm in membranes)
SynaptoGreen™ C101No5 mg, 5 x 1 mg70042, 70043• Green nerve terminal probe
• Shortest tail for slowest on-rate & fastest off-rate
SynaptoGreen™ C2 (equivalent to FM®2-10)11No70044, 70045• Equivalent to FM®2-10
SynaptoGreen™ C321No70023, 70026• Green nerve terminal probe
SynaptoGreen™ C4 (equivalent to FM®1-43)31No70020, 70022• Equivalent to FM®1-43
SynaptoGreen™ C5 (equivalent to FM®1-84)41No70046, 70047• Equivalent to FM®1-84
SynaptoGreen™ C18 (equivalent to FM®3-25)171No70048, 70049• Equivalent to FM®3-25
AM1-4331Yes1 mg70024• Fixable version of SynaptoGreen C4
• Equivalent to FM®1-43FX
AM1-4441Yes70038• Improved fixability over AM1-43
AM2-1011Yes70036• Fixable analog of SynaptoGreen™ C2
AM3-25171Yes70051• Fixable far-red nerve terminal probe
HM1-4331Yes70053• Fixable red nerve terminal probe
SynaptoRed Dyes™ (Ex/Em ~510/750 nm in membranes)
SynaptoRed™ C103No5 mg, 5 x 1 mg70040, 70041• One carbon shorter than SynaptoRed™ C2
SynaptoRed™ C2 (equivalent to FM®4-64)13No70021, 70027• Equivalent to FM®4-64
SynaptoRed™ C2M** (equivalent to FM®5-95)13No70019, 70028• More water soluble than SynaptoRed™ C2
• Equivalent to FM®5-95
AM4-6413Yes1 mg70025• Fixable version of SynaptoRed™ C2
AM4-6533Yes70039• Fixable version of SynaptoRed™ C2
AM4-6643Yes70050• Fixable and spectrally identical to SynaptoRed™ C2
*m is the number of carbons in the lipophilic tail and n is the number of double bonds linking the two aromatic rings in the dye.
**The positively-charged end of SynaptoRed C2M is a trimethylammonium group.
FM is a registered trademark of Thermo Fisher Scientific.

Nerve Terminal Staining Kits

Nerve Terminal Staining KitNerve Terminal DyeBackground ReducerCatalog number
Nerve Terminal Staining Kit ISynaptoGreen™ C4 (5 x 1 mg )ADVASEP-7 (250 mg)70030
Nerve Terminal Staining Kit II (A)AM1-43 (1 mg)ADVASEP-7 (100 mg)70031
Nerve Terminal Staining Kit II (B)AM1-43 (1 mg)SCAS (100 mg)70031-1
Nerve Terminal Staining Kit IIISynaptoGreen™ C4 (5 x 1 mg)Sulforhodamine 101 (100 mg)70032
Nerve Terminal Staining Kit VSynaptoRed™ C2 (5 x 1 mg)ADVASEP-7 (250 mg)70034

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