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CELLULAR STAINS

Stains & Dyes for Live & Fixed Cells

 

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

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

 

Nucleus

NucSpot® 470
NucSpot® 470 is cell membrane-impermeant green fluorescent DNA stain. It is virtually non-fluorescent in the absence of DNA, but fluoresces bright green upon DNA binding. While other green nucleic acid stains like TOTO®, TO-PRO®, or SYTOX® dyes stain both the nucleus and cytoplasm, NucSpot® 470 specifically stains the nucleus of fixed and permeabilized cells. It also can be used to selectively stain dead cells in living cultures. NucSpot® 470 has green fluorescence that can be imaged using standard settings for FITC. With excitation at 460 nm, it also is an excellent match for instruments with blue LED excitation sources.

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.

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: High Affinity DNA/RNA Stains
Biotium offers a selection of cyanine dyes for nucleic acids (see the table below for more information). 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.

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

Features

NucSpot® 470• Nuclear-specific green counterstain for fixed cells
• Selectively stains dead cells in live cultures
• Excellent match for blue LED excitation sources
NucSpot® Live Nuclear Stains• Low-toxicity nuclear stains
• Fix before or after labeling
• Green fluorescent NucSpot™ Live 488 and far-red fluorescent NucSpot™ Live 650
RedDot™1 Far-Red Nuclear Stain• Membrane-permeant far-red nuclear stain for live cells
• Analzye DNA content for cell cycle analysis by flow cytometry
• Useful for cell number normalization for In Cell Western®
RedDot™2 Far-Red Nuclear Stain• Membrane impermeant far-red nuclear stain
• Selectively stains dead cells
• Specific nuclear counterstain for fixed cells
Live-or-Dye NucFix™ Red• Reactive membrane-impermeant nuclear stain
• Specifically stains nuclei of dead cells
• Cells can be fixed and permeabilized without dye transfer between cells
Hoechst Dyes • Membrane-permeable blue nuclear counterstains
• For live or fixed cells
DAPI• Classic blue nuclear counterstain for fixed cells
• Can be used at higher concentrations to stain live cells
EverBrite™ Mounting Media• Hardset and wet-set mounting media
• Broad dye compatibility
• Available with DAPI for one-step mounting and counterstaining

Asymmetric Cyanine Dyes

Catalog number

Unit Size

Ex/Em*

Replacement for

Features

Oxazole Yellow, 1 mM in DMSO400891 mL491/509 nmYO-PRO®-1 Iodide• Green fluorescent cell impermeant dye
• Selectively stains early apoptotic cells
TO Iodide, 1 mM in DMSO400881 mL515/531 nmTO-PRO®-1 Iodide• Green fluorescent cell impermeant dye
• Selectively stains dead cells
Thiazole Orange, 10 mM in DMSO400771 mL512/533 nmN/A• Green fluorescent membrane permeable dye
• Commonly used in reticulocyte analysis and electrophoresis
• Fixable live yeast cell stain
Thiazole Orange Homodimer, 1 mM in DMSO40079200 uL514/533 nmTOTO®-1 Iodide• Green fluorescent cell-impermeant dye
• High affinity dimeric cyanine dye
• Dead cell stain and electrophoresis dye
Thiazole Red, 1 mM in DMSO400871 mL642/661 nmTO-PRO®-3 Iodide• Far-red fluorescent cell impermeant dye for the Cy®5 channel
• Highly sensitive dead cell stain
Thiazole Red Homodimer, 1 mM in DMSO40080200 uL642/660 nmTOTO®-3 Iodide• Far-red fluorescent cell impermeant dye for the Cy®5 channel
• High affinity dimeric cyanine dye
• Useful dead cell stain
Thiazole Green, 1000X in DMSO40086-0.5mL500 uL497/520 nmSYBR® Green I• Structurally identical to SYBR® Green I
• Highly sensitive dsDNA detection by gel staining or qPCR
• Cell membrane-permeant
40086-1mL1 mL
* With DNA
SYBR, TOTO, TO-PRO, and YO-PRO are registered trademarks of Thermo Fisher Scientific

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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 aqueous phase, but become highly fluorescent in lipid bilayers. Staining is highly 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. Double labeling can identify cells that have fused or formed stable clusters. When live cells are stained, the dyes label plasma membranes and also are taken up into endocytic compartments. 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 delivery solutions that can be added directly to normal culture media to label suspended or adherent cells in culture. We offer a selection of dyes with fluorescence ranging from blue to near-infrared. The near-infrared CellBrite™ NIR dyes can be detected by either confocal microscopy or near-infrared in vivo imaging systems. We also offers a selection of stand-alone carbocyanine dyes.

Phospholipid Probes and Membrane Potential Dyes
Biotium offers a selection of fluorescent phospholipids for membrane labeling and trafficking studies, as well as fast responding and translational membrane potential dyes. See the Cellular Stains Brochure for more information.

CF® Dye Conjugated Cholera Toxin Subunit B
Cholera toxin subunit B binds to ganglioside GM1 in lipid rafts on the plasma membrane, and can withstand fixation and permeabilization. Cholera toxin subunit B conjugates also have been used as endocytic tracers and retrograde neuronal tracers. We offer CF® Dye Cholera Toxin Subunit B with a selection of our bright photostable, and pH-independent CF® dyes.

CellBrite™ Fix: Novel membrane dyes that can tolerate permeabilization
CellBrite™ Fix dyes are a new class 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 are not compatible with detergent permeabilization or methanol fixation. In contrast, CellBrite™ Fix Membrane Stains are unique in that their surface staining can withstand permeabilization and methanol fixation, allowing membrane staining to be combined with intracellular staining with antibodies. Unlike lectins such as WGA, which bind specific targets, CellBrite™ Fix dyes are general membrane stains. However, unlike original CellBrite™ dyes and lectins, CellBrite™ Fix dyes cannot be used to stain cells that are already fixed.

CellBrite™ Fix dyes are amine-reactive and are designed to accumulate at the cell membrane, where they become covalently attached to membrane proteins. As a result, surface staining is well-retained after permeabilization or methanol fixation, with only a slight increase in intracellular fluorescence compared to formaldehyde fixation alone. CellBrite™ Fix dyes are only weakly fluorescent in aqueous media but become intensely fluorescent upon membrane staining. This fluorogenic property of the dyes makes the staining very specific with low background. Due to their better water solubility, CellBrite™ Fix dyes yield much more uniform staining compared to lipophilic carbocyanine dyes like DiO and DiI. The dyes are non-cytotoxic and do not readily transfer between cells. They also can be used to stain yeast and bacteria (gram-positive or gram-negative).

Lectin Conjugates: CF® Dye Labeled WGA, Con A, PNA
Lectins are carbohydrate binding proteins that recognized specific sugar moieties on cell surface 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. Biotium offers CF® dye conjugates of WGA, Con A, and PNA in a variety of colors. WGA also is a live cell Gram stain for bacteria, while Concanavalin A and WGA conjugates can be used to stain cell wall and bud scars in yeast. See our Microbiology Technology Page or our Bacteria Stains Flyer and Yeast Stains Flyer for more information.

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 labeled at 4°C with CF®488A cholera toxin B (green), then fixed, permeabilized, and stained with DAPI.

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 membrane dyes that withstand detergent permeabilization or methanol fixation
• Bright, uniform cell surface staining in 15 minutes
• Available with green, red, and far-red fluorescence
• Can’t be used to stain after fixation
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 mammalian and yeast cells
• 10 CF® dye colors from UV to NIR
CF® Dye PNA Conjugates• Binds galactosyl (β-1,3) N-acetylgalactosamine
• Choice of 4 bright & photostable CF® dyes
CF® Dye Cholera Toxin Subunit B • Labels lipid rafts
• Withstands fixation and permeabilization
• Choice of 6 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, a membrane potential-sensitive dye for mitochondria in mammalian cells and yeast, stains 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 mitochondrial dye that can be used to image mitochondria following mitochondrial depolarization, or after fixation. MitoView™ 405 and MitoView™ 720 dyes localize to the cytoplasm after mitochondrial depolarization, but still retain fluorescence.

JC-1 and Other Classic Mitochondrial Membrane Potential 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 of cell viability to be assessed by measuring the ratio of red to green fluorescence by flow cytometry or fluorescence microplate reader.

Red fluorescent TMRM and TMRE are the preferred dyes for quantitative membrane potential measurements. We also offer a number of other classic mitochondrial dyes. See the Cellular Stains Brochure for more information.

MitoView™ 405.
MitoView™ Green.
MitoView™ 633
MitoView™ 720 in the Cy®5 channel.

ProductAbs/EmDetection channelPotential-dependent?Catalog no.Size
MitoView™ 405398/440 nmDAPIPartial†70070-T50 ug
7007020×50 ug
MitoView™ Green490/523 nmFITC, GFPNo70054-T50 ug
7005420×50 ug
MitoView™ 633622/648 nm*Cy®5, APC*Yes70055-T50 ug
7005520×50 ug
MitoView™ 720720/758 nm**Cy®5, Cy®7**Partial†70068-T50 ug
7006820×50 ug

* MitoView™ 633 also has red fluorescence in the Cy®3 channel and is not recommended for use with other red dyes.
**While optimal for Cy®7 settings, MitoView™ 720 is bright enough to be imaged in the Cy®5 channel.
† Dyes localize to the cytoplasm after mitochondrial depolarization, but still retain fluorescence.

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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.
ProductAbs/EmDetection channelCatalog numberSize
LysoView™ 405,
1000X in DMSO
318, 400/464 nmDAPI70066-T10 uL
7006650 uL
LysoView™ 488,
1000X in DMSO
506/532 nmGFP, FITC70067-T10 uL
7006750 uL
LysoView™ 540,
1000X in DMSO
541/634 nm (pH ≤5)Rhodamine, Cy®370061-T10 uL
7006150 uL
LysoView™ 633
(lyophilized solid)
634/659 nm (pH ≤5)Cy®570058-T1 vial*
7005810 vials*
LysoView™ 650,
1000X in DMSO
650/675 nmCy®570059-T10 uL
7005950 uL

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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 are fluorescent cell-permeable taxol probes for imaging the microtubule cytoskeleton in live cells. They are simple, rapid and sensitive stains. Taxol binds to polymerized tubulin and stabilizes microtubules, resulting in inhibition of mitosis. However, fluorescent taxol compounds like ViaFluor® stains are less disruptive of microtubule dynamics and cell division, presumably due to lower binding affinity of the fluorescent probe compared to taxol itself. ViaFluor® stains do not show cytotoxicity with up to 24 hours of exposure in immortalized cell types. ViaFluor® Microtubule Stains are supplied with a vial of verapamil, 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 dyes like Alexa Fluor®, DyLight®, and Cy® 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 10,000 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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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
Calciein 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.

Cytoplasm

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
• 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

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Nerve terminal & Neuronal stains

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 fluorescein 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. We offer blue, green, and red ViaFluor® SE dyes.

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 classic hydrophilic dyes have been used as microinjectable tracers. 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. Biotin ethylenediamine (equivalent to Neurobiotin™) is used as an anterograde and transneuronal tracer.

We offer bright, photostable, and pH-independent CF® dye biotin and CF® dye biocytin derivatives in a selection of colors for cell tracing or detection of biotin binding sites. We also offer a number of classic fluorescent biotin derivatives. See the Cellular Stains Brochure for more information.

Neuronal 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 Alexa Fluor® dyes and other next generation 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.

Cultured rat hippocampal neurons microinjected with CF®647 hydrazide (red) and stained with SynaptoGreen™ C4 (green). Image courtesy of Professor Guosong Liu, Tsinghua University.

Properties of Nerve Terminal Dyes

Nerve Terminal Dyem*n*Ex/Em in membranesFixable?Catalog No.Size
SynaptoGreen™ C101~480/600 nmNo70042, 700435 mg, 5 x 1 mg
SynaptoGreen™ C2 (equivalent to FM®2-10)11No70044, 70045
SynaptoGreen™ C321No70023, 70026
SynaptoGreen™ C4 (equivalent to FM®1-43)31No70020, 70022
SynaptoGreen™ C5 (equivalent to FM®1-84)41No70046, 70047
SynaptoGreen™ C18 (equivalent to FM®3-25)171No70048, 70049
AM1-4331Yes700241 mg
AM1-4441Yes70038
AM2-1011Yes70036
AM3-25171Yes70051
HM1-4331Yes70053
SynaptoRed™ C103~510/750 nmNo70040, 700415 mg, 5 x 1 mg
SynaptoRed™ C2 (equivalent to FM®4-64)13No70021, 70027
SynaptoRed™C2M** (equivalent to FM®5-95)13No70019, 70028
AM4-6413Yes700251 mg
AM4-6533Yes70039
AM4-6643Yes70050
*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

Catalog no.Nerve Terminal Staining KitNerve Terminal DyeBackground Reducing Agent
70030Nerve Terminal Staining Kit I5 x 1 mg SynaptoGreen™ C4250 mg ADVASEP-7
70031Nerve Terminal Staining Kit II (A)1 mg AM1-43100 mg ADVASEP-7
70031-1Nerve Terminal Staining Kit II (B)1 mg AM1-43100 mg SCAS
70032Nerve Terminal Staining Kit III5 x 1 mg SynaptoGreen™ C4100 mg Sulforhodamine 101
70034Nerve Terminal Staining Kit IV5 x 1 mg SynaptoRed™ C2250 mg ADVASEP-7

 

 

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