Fixable Polar Tracers

We prepare a wide variety of highly water-soluble dyes and other detectable probes that can be used as cell tracers. In most cases, the polarity of these water-soluble probes is too high to permit them to passively diffuse through cell membranes. Consequently, special methods for loading the dyes into cells must be employed, including microinjection, pinocytosis or techniques that temporarily permeabilize the cell's membrane ref (Techniques for loading molecules into the cytoplasm—Table 14.1). Our Influx pinocytic cell-loading reagent (I14402, see below and Chelators, Calibration Buffers, Ionophores and Cell-Loading Reagents—Section 19.8) is particularly useful for loading many of the polar tracers in this section—as well as the dextrans and fluorescent proteins described in Fluorescent and Biotinylated Dextrans—Section 14.5 and Protein Conjugates—Section 14.7—into many types of cells. Permeabilization of cells with staphylococcal α-toxin ref or the saponin ester β-escin is reported to make the membrane of smooth muscle cells permeable to low molecular weight (<1000 daltons) molecules, while retaining high molecular weight compounds.ref Electroporation has been used to transport several of the polar tracers through the skin ref and into cells.ref Many of these tracers can also be loaded into cells noninvasively as their cell-permeant acetoxymethyl (AM) esters, which are discussed in more detail in Viability and Cytotoxicity Assay Reagents—Section 15.2.


Alexa Fluor Hydrazides and Hydroxylamines

Molecular Probes fluorescent hydrazide and hydroxylamine derivatives continues to expand (Reagents for Modifying Aldehydes and Ketones—Section 3.3, Molecular Probes hydrazine, hydroxylamine and amine derivatives—Table 3.2). The blue-fluorescent Alexa Fluor 350 hydrazide and Alexa Fluor 350 hydroxylamine (A10439, A30627), green-fluorescent Alexa Fluor 488 hydrazide and Alexa Fluor 488 hydroxylamine (A10436, A30629; photo), orange-fluorescent Alexa Fluor 555 and Alexa Fluor 568 hydrazides (A20501MP, A10437, A10441; Figure 14.3.1), red-fluorescent Alexa Fluor 594 hydrazide ref (A10438, A10442) and far-red–fluorescent Alexa Fluor 633 hydrazide, Alexa Fluor 647 hydrazide and Alexa Fluor 647 hydroxylamine (A30634, A20502, A30632) are likely the best overall polar tracers in each of their various spectral ranges.ref These low molecular weight, cell membrane–impermeant molecules (Alexa Fluor 350 hydrazide, 349 daltons; Alexa Fluor 350 hydroxylamine, 585 daltons; ~570–760 daltons for the Alexa Fluor 488, 568 and 594 hydrazides and hydroxylamine; and about 1200 daltons for the Alexa Fluor 555 and 647 hydrazides and hydroxylamine) possess several properties that are superior to those of the widely used neuronal tracer lucifer yellow CH (L453, L682, L1177, L12926). Like lucifer yellow CH, the hydrazide moiety of the Alexa Fluor derivatives makes these tracers fixable by common aldehyde-based fixatives. We have determined that Alexa Fluor 594 hydrazide has a water solubility of ~84 mg/mL and the other Alexa Fluor hydrazides are likely to have comparable or higher water solubility.

Our rabbit polyclonal antibody to the Alexa Fluor 488 fluorophore (A11094, Anti-Dye and Anti-Hapten Antibodies—Section 7.4) quenches the fluorescence of the Alexa Fluor 488 dye (Anti–Lucifer Yellow Dye, Anti–Alexa Fluor 405/Cascade Blue Dye and Anti–Alexa Fluor 488 Dye Antibodies—Note 14.1) and, following cell fixation and permeabilization, can be used in conjunction with the reagents in our Tyramide Signal Amplification (TSA) Kits (TSA and Other Peroxidase-Based Signal Amplification Techniques—Section 6.2) to amplify the signal or with the anti–rabbit IgG conjugate of NANOGOLD or Alexa Fluor FluoroNanogold 1.4 nm gold clusters (N24916, A24926, A24927; Secondary Immunoreagents—Section 7.2) and the associated LI Silver Enhancement Kit (L24919, Secondary Immunoreagents—Section 7.2) for correlated fluorescence and light microscopy studies.

Although lucifer yellow CH can be used for confocal laser-scanning microscopy, its extinction coefficient at the 488 nm spectral line of the argon-ion laser (~700 cm-1M-1) is only about 1% of that of Alexa Fluor 488 hydrazide and Alexa Fluor 488 hydroxylamine (≥71,000 cm-1M-1) (Figure 14.3.2). Furthermore, the high photostability of the Alexa Fluor dyes permits their detection in very fine structures that cannot be seen with lucifer yellow CH staining. All of these Alexa Fluor dyes are remarkably bright and photostable. In addition, the Alexa Fluor hydrazide salts have high water solubility (typically greater than 8%). We offer the Alexa Fluor 568 and Alexa Fluor 594 hydrazides either as solids (A10437, A10438) or as 10 mM solutions in 200 mM KCl (A10441, A10442). The 10 mM solutions have been filtered through a 0.2 µm filter to remove any insoluble material prior to packaging. The Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 633 and Alexa Fluor 647 hydrazides (A10436, A20501MP, A30634, A20502) and Alexa Fluor 350, Alexa Fluor 488 and Alexa Fluor 647 hydroxylamines (A30627, A30629, A30632) are available only as solids. Our Alexa Fluor 350 hydrazide and Alexa Fluor 350 hydroxylamine, which are sulfonated coumarin derivatives (structure), are some of the few polar tracers that exhibit bright blue fluorescence.

 

Calcium Green dextran–labeled  


Figure 14.3.1 Confocal image stack of a 10,000 MW Calcium Green dextran–labeled (C3713, Fluorescent Ca2+ Indicator Conjugates—Section 19.4) climbing fiber in a sagittal cerebellar slice, showing incoming axon and terminal arborization (in yellow). The Purkinje cell innervated by this climbing fiber was labeled with Alexa Fluor 568 hydrazide (A10437, A10441) via a patch pipette and visually identified using bright-field microscopy. Image contributed by Anatol Kreitzer, Department of Neurobiology, Harvard Medical School.

 

Alexa Fluor 488 hydrazide  


Figure 14.3.2 Absorption spectra showing that the molar extinction coefficient (EC) at 488 nm of Alexa Fluor 488 hydrazide (A10436) in water (green line) is approximately 100-fold greater than that of lucifer yellow CH (L453, L682, L1177, L12926) in water (blue line).

 

Other Alexa Fluor Derivatives

To allow amplification of signals, especially in the finer processes of dye-filled neurons, we also offer Alexa Fluor 488 biocytin (A12924), Alexa Fluor 546 biocytin (A12923) and Alexa Fluor 594 biocytin (A12922). These unique probes combine our Alexa Fluor 488, Alexa Fluor 546 and Alexa Fluor 594 fluorophores with biotin and an aldehyde-fixable primary amine (see "Fluorescent Biotin Derivatives," below). In addition, we offer the bright blue-fluorescent Alexa Fluor 405 cadaverine (A30675, see below) as well as several other Alexa Fluor cadaverines (Reagents for Modifying Aldehydes and Ketones—Section 3.3, Molecular Probes hydrazine, hydroxylamine and amine derivatives—Table 3.2), all of which should be useful as tracing molecules because they are exceptionally bright, small and water soluble, and they each contain an aldehyde-fixable functional group. Alexa Fluor 546 biocytin has been used to label streptavidin-coated particles in order to quantitate fluorescence signals in an automated imaging system designed for analyzing immobilized particle arrays.ref


Lucifer Yellow CH

Lucifer yellow CH (LY-CH or LY, structure) has long been a favorite tool for studying neuronal morphology because it contains a carbohydrazide (CH) group that allows it to be covalently linked to surrounding biomolecules during aldehyde-based fixation.ref Loading of this polar tracer and other similar impermeant dyes is usually accomplished by microinjection,ref pinocytosis,ref scrape loading,ref ATP-induced permeabilization ref or osmotic shock ref (Techniques for loading molecules into the cytoplasm—Table 14.1), but can also be accomplished in cell suspensions or with adherent cells by using our Influx pinocytic cell-loading reagent (I14402, see below). Lucifer yellow CH localizes in the plant vacuole when taken up either through what is thought to be anion-transport channels ref or by fluid-phase endocytosis.ref Upon injection into the epidermal cells of Egeria densa leaves, lucifer yellow CH reportedly moved into the cytoplasm of adjacent cells, localized in the plant vacuole or moved in and out of the nucleus.ref The lithium salt of lucifer yellow CH is widely used for microinjection because of its relatively high water solubility (~8%). In addition to the solid (L453), we offer the lithium salt of lucifer yellow CH as a filtered 100 mM solution (L12926), ready for microinjection. The potassium salt (L1177, solubility ~1%) or the ammonium salt of lucifer yellow CH (L682, solubility ~6%) may be preferred in applications where lithium ions interfere with biological function.

Although its weak absorption at 488 nm (EC ~700 cm-1M-1) (Figure 14.3.2) makes it inefficiently excited with the argon-ion laser, lucifer yellow CH has been used as a neuronal tracer in some confocal laser-scanning microscopy studies.ref For electron microscopy studies, lucifer yellow CH can be used to photoconvert diaminobenzidine (DAB) into an insoluble, electron-dense reaction product ref (Fluorescent Probes for Photoconversion of Diaminobenzidine Reagents—Note 14.2). Alternatively, rabbit anti–lucifer yellow dye antibodies (Anti-Dye and Anti-Hapten Antibodies—Section 7.4) can be used in conjunction with our goat anti–rabbit IgG antibody conjugated to either NANOGOLD or Alexa Fluor FluoroNanogold 1.4 nm gold clusters (Secondary Immunoreagents—Section 7.2) and the LI Silver (LIS) Enhancement Kit (L24919, Secondary Immunoreagents—Section 7.2) to develop a more permanent, fade-free colorimetric or electron-dense signal from dye-filled neurons that is suitable for light or electron microscopy ref (Anti–Lucifer Yellow Dye, Anti–Alexa Fluor 405/Cascade Blue Dye and Anti–Alexa Fluor 488 Dye Antibodies—Note 14.1).

Intracellular injection of lucifer yellow CH has been extensively employed to delineate neuronal morphology in live neurons ref (photo) and in fixed brain slices,ref as well as to investigate intercellular communication through gap junctions.ref Lucifer yellow CH can also be used to label neurons by using dye-filled electrodes during electrophysiological recording in order to correlate neuronal function with structure and connectivity.


Other Lucifer Yellow Derivatives

Like lucifer yellow CH, lucifer yellow ethylenediamine (A1339) is fixable with standard aldehyde-based fixatives and can be used as a building block for new lucifer yellow derivatives.ref The thiol-reactive lucifer yellow iodoacetamide (L1338) can also be used as a microinjectable polar tracer, as well as for preparing fluorescent liposomes ref and for detecting the accessibility of thiols in membrane-bound proteins.ref In addition to these lucifer yellow derivatives, we offer a lucifer yellow–conjugated 10,000 MW dextran (D1825, Fluorescent and Biotinylated Dextrans—Section 14.5).


Cascade Blue Hydrazide

Molecular Probes Cascade Blue hydrazide is a fixable analog of the nonfixable, bright blue-fluorescent tracer methoxypyrenetrisulfonic acid ref (MPTS). All of the Cascade Blue hydrazide derivatives have reasonable water solubility, ~1% for the sodium and potassium salts (C687, C3221) and ~8% for the lithium salt (C3239). They also exhibit a stronger absorption (EC400 nm >28,000 cm-1M-1) and quantum yield (~0.54 in water) than lucifer yellow CH. In addition, Cascade Blue derivatives have good photostability and emissions that are well resolved from those of fluorescein and lucifer yellow CH.ref Cascade Blue hydrazide, which readily passes through gap junctions, and lucifer yellow derivatives can be simultaneously excited at 405 nm (Figure 14.3.3) for two-color detection at about 430 and 530 nm.ref Cascade Blue dyes, lucifer yellow CH and sulforhodamine 101 can be used in combination for three-color mapping of neuronal processes (Figure 14.3.4). We also offer anti–Alexa Fluor 405/Cascade Blue dye antibodies (Anti-Dye and Anti-Hapten Antibodies—Section 7.4) for localizing Cascade Blue dye–filled cells following fixation (Anti–Lucifer Yellow Dye, Anti–Alexa Fluor 405/Cascade Blue Dye and Anti–Alexa Fluor 488 Dye Antibodies—Note 14.1). Like lucifer yellow CH, Cascade Blue hydrazide and some other polar tracers are taken up by plants and sequestered into their central vacuoles. In onion epidermal cells, this uptake of Cascade Blue hydrazide is blocked by probenecid (P36400, Chelators, Calibration Buffers, Ionophores and Cell-Loading Reagents—Section 19.8), indicating that transfer may be through anion-transport channels.ref

 

Cascade Blue hydrazide  


Figure 14.3.3
Absorption spectra for equal concentrations of Cascade Blue hydrazide (C687, C3221, C3239) and lucifer yellow CH (L453, L682, L1177, L12926) in water.

 

Cascade Blue hydrazide  


Figure 14.3.4 Normalized fluorescence emission spectra for Cascade Blue hydrazide (C687, C3221, C3239), lucifer yellow CH (L453, L682, L1177, L12926) and sulforhodamine 101 (S359) in water.

 

Other Cascade Blue and Alexa Fluor 405 Derivatives

Cascade Blue acetyl azide (C2284) and Alexa Fluor 405 succinimidyl ester (A30000, A30100) are water-soluble, amine-reactive tracers that can be introduced either by microinjection or by fusion of dye-filled liposomes with cells. Once inside the cell, these derivatives will react with the amine groups of intracellular proteins. Cascade Blue ethylenediamine (C621) and Alexa Fluor 405 cadaverine (previously called Cascade Blue cadaverine, A30675) are aldehyde-fixable fluorophores with reactive properties similar to those of the ethylenediamine derivative of lucifer yellow (A1339). A Cascade Blue dye–labeled 10,000 MW dextran (D1976, Fluorescent and Biotinylated Dextrans—Section 14.5) is also available.


Biocytin and Other Biotin Derivatives

Biocytin (ε-biotinoyl-L-lysine, B1592, structure) and biotin ethylenediamine (A1593, structure) are microinjectable anterograde and transneuronal tracers.ref Retrograde transport of biocytin and biotin ethylenediamine in neurons has also been reported.ref These water-soluble tracers are often used to label neurons during electrophysiological measurements in order to correlate neuronal function with structure and connectivity.ref Biotin cadaverine (A1594) and biotin-X cadaverine (B1596) have slightly longer spacers than their ethylenediamine counterparts, making the hapten more accessible to the deep biotin-binding site in avidins.ref

Biocytin, biotin ethylenediamine, biotin cadaverine and biotin-X cadaverine all contain primary amines and can therefore be fixed in cells with formaldehyde or glutaraldehyde and subsequently detected using fluorescent- or enzyme-labeled avidin or streptavidin second-step reagents or with NANOGOLD and Alexa Fluor FluoroNanogold streptavidin ref (Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6). Biocytin hydrazide (B1603) and DSB-X biotin hydrazide (D20653) can serve as aldehyde-fixable tracers ref and as reactive probes for labeling glycoproteins and nucleic acids (Biotinylation and Haptenylation Reagents—Section 4.2).

As with the reactive lucifer yellow, and Cascade Blue and Alexa Fluor 405 derivatives discussed above, amine- or thiol-reactive biotin derivatives are useful for intracellular labeling applications. The succinimidyl esters of biotin and biotin-X (B1513, B1582) have been used to trace retinal axons in avian embryos.ref Because they are more water soluble, the sulfosuccinimidyl esters of biotin-X and biotin-XX (B6353, B6352) or the thiol-reactive biocytin maleimide (M1602) may be preferred for these applications.


Fluorescent Biotin Derivatives

Fluorescence of the finer processes of dye-filled neurons may fade rapidly or be obscured by the more intensely stained portions of the neuron, necessitating further amplification of the signal or other ultrastructural detection methods. Lucifer yellow biocytin (L6950), Alexa Fluor 488 biocytin (A12924), Alexa Fluor 546 biocytin (A12923), Alexa Fluor 594 biocytin (A12922), Oregon Green 488 biocytin (O12920) and tetramethylrhodamine biocytin (T12921) each incorporate a fluorophore, biotin and an aldehyde-fixable primary amine into a single molecule, thus enabling researchers to amplify the signals of these tracers with fluorescent or enzyme-labeled avidin or streptavidin conjugates (Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6). Although our lucifer yellow cadaverine biotin-X (L2601) lacks a primary amine, it was reported that this tracer was well retained in aldehyde-fixed tissues, even after sectioning, extraction with detergents and several washes.ref Because fluorescent biocytin derivatives contain free primary amines, they should be even more efficiently fixed by formaldehyde or glutaraldehyde.

Nonfixable Polar Tracers

Polar fluorescent dyes are commonly used to investigate fusion, lysis and gap-junctional communication and to detect changes in cell or liposome volume. These events are primarily monitored by following changes in the dye's fluorescence caused by interaction with nearby molecules. For example, because the fluorescence of many dyes at high concentrations is quenched, various processes that result in a dilution of the dyes, such as lysis or fusion of fluorescent dye–filled cells or liposomes, can produce an increase in fluorescence, thereby providing an easy method for monitoring these events. Cell–cell and cell–liposome fusion, as well as membrane permeability and transport through gap junctions, can all be monitored using these methods. Furthermore, a fluorogenic substrate such as fluorescein diphosphate (FDP, F2999; Detecting Enzymes That Metabolize Phosphates and Polyphosphates—Section 10.3) can be incorporated within a cell or vesicle that lacks the enzymatic activity to generate a fluorescent product; subsequent fusion with a cell or vesicle that contains the appropriate enzyme will generate a fluorescent product.ref

An ultrasensitive fusion assay that can be used to follow fusion of single vesicles utilizes an almost nonfluorescent potassium salt of an ion-sensitive indicator such as fluo-3 (F1240, F3715; Fluorescent Ca2+ Indicators Excited with Visible Light—Section 19.3) in one vesicle and a polyvalent ion such as Ca2+—or perhaps better La3+, which causes greater enhancement of the fluorescence of fluo-3—in a second vesicle.ref


Fluorescein Derivatives

The self-quenching of fluorescein derivatives provides a means of determining their concentration in dynamic processes such as lysis or fusion of dye-filled cells or liposomes (Assays of Volume Change, Membrane Fusion and Membrane Permeability—Note 14.3). Calcein (C481)—a polyanionic fluorescein derivative that has about six negative and two positive charges at pH 7 (structure)—as well as BCECF (B1151, structure), carboxyfluorescein (C194, C1904), the 5-isomer of Oregon Green 488 carboxylic acid (O6146) and fluorescein-5-(and 6-)sulfonic acid (F1130) are all soluble in water at >100 mM at pH 7. Unlike the other fluorescein derivatives, both calcein and Oregon Green 488 carboxylic acid exhibit fluorescence that is essentially independent of pH between 6.5 and 12.

These green-fluorescent polar tracers are widely used for investigating:

  • Cell volume changes in neurons and other cells ref
  • Gap junctional communication ref
  • Liposome formation, fusion and targeting ref
  • Membrane integrity and permeability ref

Fluorescence of calcein (but not of carboxyfluorescein or fluorescein sulfonic acid) is strongly quenched by Fe3+, Co2+, Cu2+ and Mn2+ at physiological pH but not by Ca2+ or Mg2+ ions.ref Monitoring the fluorescence level of cells that have been loaded with calcein (or its AM ester, see below) may provide an easy means for following uptake of Fe3+, Co2+, Cu2+, Mn2+ and certain other metals through ion channels.ref Increases in the internal volume of lipid vesicles and virus envelopes cause a decrease in Co2+-induced quenching of calcein, a change that can be followed fluorometrically.ref In addition, the Co2+-quenched calcein complex is useful for both lysis and fusion assays ref (Assays of Volume Change, Membrane Fusion and Membrane Permeability—Note 14.3). Calcein is a preferred reagent for following volume changes because its fluorescence is not particularly sensitive to either pH or physiological concentrations of other ions.ref

We prepare a high-purity grade of calcein (C481) that is generally >97% pure by HPLC. The chemical structure assigned to "calcein" in various literature references and by commercial sources has been inconsistent;ref our structure (structure) has been confirmed by NMR spectroscopy, and we believe that several past assignments of other structures to calcein were incorrect. We also offer a high-purity grade of 5-(and 6-)carboxyfluorescein (C1904) that contains essentially no polar or nonpolar impurities that might alter transfer rates of the dye between vesicles and cells.ref


Cell-Permeant Fluorescein Derivatives

Cell-permeant versions of carboxyfluorescein, fluorescein sulfonic acid, calcein and the Oregon Green dyes permit passive loading of cells (Viability and Cytotoxicity Assay Reagents—Section 15.2). Acid hydrolysis of nonfluorescent carboxyfluorescein diacetate (CFDA; C195, C1361, C1362; Viability and Cytotoxicity Assay Reagents—Section 15.2) to fluorescent carboxyfluorescein has been used to detect the fusion of dye-loaded clathrin-coated vesicles with lysosomes.ref CFDA has also been used to investigate cell–cell communication in plant cells.ref A probenecid-inhibitable anion-transport mechanism permits loading of carboxyfluorescein diacetate and Oregon Green 488 carboxylic acid diacetate (O6151, structure; Viability and Cytotoxicity Assay Reagents—Section 15.2) into hyphal tip-cells of some fungi.ref

Calcein AM (structure), but not the AM or acetate esters of BCECF or CFDA, is reported to differentially label lymphocytes, permitting their resolution into two populations based on fluorescence intensity, only one of which is taken up by lymphoid organs. This unique property makes calcein AM a useful probe for determining the lymph node homing potential of lymphocytes.ref

In an important technique for studying gap junctional communication, cells are simultaneously labeled with calcein AM (C1430, C3099, C3100MP) and DiI (D282, D3911, V22885; Tracers for Membrane Labeling—Section 14.4) and then mixed with unlabeled cells (Figure 14.3.5). When gap junctions are established, only the cytosolic calcein tracer (but not the DiI membrane probe) is transferred from the labeled cell to the unlabeled cell. Thus, after gap-junctional transfer, the initially unlabeled cells exhibit the green fluorescence of calcein but not the red fluorescence of DiI.ref This assay can be followed by either imaging or flow cytometry.ref In addition, calcein AM and DiI have been combined for use in following cell fusion ref and for analysis of cholesterol processing by macrophages following ingestion of apoptotic cells.ref


Study of gap junctional communication

Figure 14.3.5 A simple technique for the study of gap junctional communication. A population of cells are labeled simultaneously with DiI (D282, D3911) and calcein AM (C1430, C3099, C3100MP), and then mixed with an unlabeled cell population (panel A). The formation of gap junctions allows the cytosolic tracer calcein to cross into the unlabeled cell, while the membrane-bound DiI does not (panel B). Cells from the initial unlabeled population that have taken part in gap junctional communication will therefore display the green fluorescence of calcein while lacking the red-fluorescent signal of DiI.


Fluorescein Substitutes

Fluorophores and Their Amine-Reactive Derivatives—Chapter 1 describes several of our proprietary green-fluorescent dyes that have exceptional optical properties, including our Alexa Fluor 488 dye (Alexa Fluor Dyes Spanning the Visible and Infrared Spectrum—Section 1.3), BODIPY FL dye (BODIPY Dye Series—Section 1.4) and Oregon Green dyes (Fluorescein, Oregon Green and Rhodamine Green Dyes—Section 1.5). Not only do these innovative fluorescein substitutes exhibit high quantum yields in aqueous solution, but the dyes are significantly more photostable than fluorescein (Figure 14.3.6) and their fluorescence is less sensitive to pH (Figure 14.3.7). Their greater photostability makes them the preferred green-fluorescent dyes for fluorescence microscopy. In addition to their membrane-permeant versions, which are described in Viability and Cytotoxicity Assay Reagents—Section 15.2, highly water-soluble derivatives of these fluorescein substitutes are available for use as polar tracers:

  • Oregon Green 514 carboxylic acid (O6138, structure), which is highly photostable and has little pH sensitivity at near-neutral pH
  • BODIPY 492/515 disulfonic acid ref (D3238), which has narrow spectral bandwidths and bright green, pH-independent fluorescence
  • Carboxy-2',7'-dichlorofluorescein (C368), which has a lower pKa than fluorescein

 

Photostability of green-fluorescent antibody  

 

Figure 14.3.6 Comparison of photostability of green-fluorescent antibody conjugates. The following fluorescent goat anti–mouse IgG antibody conjugates were used to detect mouse anti–human IgG antibody labeling of human anti-nuclear antibodies in HEp-2 cells on prefixed test slides (INOVA Diagnostics Corp.): Oregon Green 514 (O6383, filled square), Alexa Fluor 488 (A11001, open circle), BODIPY FL (B2752, open triangle), Oregon Green 488 (O6380, open square) or fluorescein (F2761, filled circle). Samples were continuously illuminated and viewed on a fluorescence microscope using a fluorescein longpass filter set. Images were acquired every 5 seconds. For each conjugate, three data sets, representing different fields of view, were averaged and then normalized to the same initial fluorescence intensity value to facilitate comparison.

 

pH-dependent fluorescence of the Oregon Green 488  

 

Figure 14.3.7 Comparison of pH-dependent fluorescence of the Oregon Green 488 (filled circle), carboxyfluorescein (open circle) and Alexa Fluor 488 (open square) fluorophores. Fluorescence intensities were measured for equal concentrations of the three dyes using excitation/emission at 490/520 nm.

 

Sulforhodamines

Sulforhodamine 101 (S359, structure) and sulforhodamine B (S1307, structure) are orange- to red-fluorescent, very water-soluble sulfonic acid tracers with strong absorption and good photostability. Sulforhodamine 101—the precursor to reactive Texas Red derivatives—has been the preferred red-fluorescent polar tracer for use in combination with lucifer yellow CH, carboxyfluorescein or calcein ref (photo). Activity-dependent uptake of sulforhodamine 101 during nerve stimulation has been reported.ref Sulforhodamine 101 specifically labels astrocytes both in vivo ref and in acute brain slice preparations.ref Labeling is accomplished by application of a sulforhodamine 101 solution (1–25 µM in artificial cerebrospinal fluid) to the tissue for 1–5 minutes and is stable for several hours.ref This technique is particularly useful for cellular-context identification in conjunction with calcium imaging using Oregon Green 488 BAPTA-1, fluo-4 and related fluorescent indicators ref (Fluorescent Ca2+ Indicators Excited with Visible Light—Section 19.3). Because it is chemically stable, can be prepared in high purity and has a fluorescence quantum yield of nearly 1.0, we have included sulforhodamine 101 in our Reference Dye Sampler Kit (R14782, Fluorescence Microscopy Accessories and Reference Standards—Section 23.1), along with four other dyes whose spectra cover the visible wavelengths.

Sulforhodamine B is an alternative to sulforhodamine 101 for investigating neuronal morphology,ref preparing fluorescent liposomes,ref studying cell–cell communications ref and labeling elastic and collagen fibers.ref


Hydroxycoumarins

7-Hydroxycoumarin-3-carboxylic acid (H185) is a blue-fluorescent polar tracer (excitation/emission maxima of ~388/445 nm) with uses that complement those of calcein and the other green-fluorescent polar tracers.ref The membrane-permeant AM ester of calcein blue (C1429, Viability and Cytotoxicity Assay Reagents—Section 15.2), another coumarin-based tracer, can be used for passive loading of cells.ref


Polysulfonated Pyrenes

HPTS (8-hydroxypyrene-1,3,6-trisulfonic acid, also known as pyranine, H348; structure) is a unique pH-sensitive tracer. It fluoresces blue in acidic solutions and in acidic organelles,ref but fluoresces green in more basic organelles.ref In addition to its use as a probe for proton translocation,ref HPTS has been employed for intracellular labeling of neurons ref and as a fluid-phase endocytic tracer in catecholamine-secreting PC12 rat pheochromocytoma cells.ref HPTS forms a nonfluorescent complex with the cationic quencher DPX (X1525), and several assays have been described that monitor the increase in HPTS fluorescence that occurs upon lysis or fusion of liposomes or cells containing this quenched complex ref (Assays of Volume Change, Membrane Fusion and Membrane Permeability—Note 14.3). HPTS has also been used as a viscosity probe in unilamellar phospholipid vesicles.ref

The pH-insensitive 8-aminopyrene-1,3,6-trisulfonic acid (APTS, A6257) and 1,3,6,8-pyrenetetrasulfonic acid (P349) are extremely soluble in water (>25%); they have been utilized as blue-fluorescent tracers.ref As with HPTS, the fluorescence of APTS and 1,3,6,8-pyrenetetrasulfonic acid is quenched by DPX ref (Assays of Volume Change, Membrane Fusion and Membrane Permeability—Note 14.3) and by the cationic spin label CAT 1 ref (T506, see below). These quenched-fluorophore complexes are useful for following lysis of cells and liposomes.


ANTS–DPX

The polyanionic dye ANTS (A350) is often used in combination with the cationic quencher DPX (X1525) for membrane fusion or permeability assays, including complement-mediated immune lysis ref (Assays of Volume Change, Membrane Fusion and Membrane Permeability—Note 14.3). Thallium (Tl+) and cesium (Cs+) ions quench the fluorescence of ANTS, pyrenetetrasulfonic acid and some other polyanionic fluorophores.ref A review by Garcia ref describes how this quenching effect can be utilized to determine transmembrane ion permeability. The unusually high Stokes shift of ANTS in water (>150 nm) separates its emission from much of the autofluorescence of biological samples. An approximately fourfold enhancement of the quantum yield of ANTS is induced by D2O—a spectral characteristic that has been used to determine water permeability in red blood cell ghosts and kidney collecting tubules.ref ANTS has also been employed as a neuronal tracer.ref


Lanthanide Chelates

Terbium ion (Tb3+ from TbCl3, T1247) forms a chelate with dipicolinic acid (DPA) that is ~10,000 times more fluorescent than free Tb3+. Fusion of vesicles that have been separately loaded with DPA and Tb3+ results in enhanced fluorescence, providing the basis for liposome fusion assays (Assays of Volume Change, Membrane Fusion and Membrane Permeability—Note 14.3). The fluorescence emission spectrum of the Tb3+/DPA complex exhibits two sharp spectral peaks at 491 nm and 545 nm and a lifetime of several milliseconds. Because DPA is a major constituent of bacterial spores, Tb3+/DPA complex luminescence provides a straightforward yet sensitive method for their detection.ref


TOTO, YOYO and SYTO Nucleic Acid Stains

Our high-affinity nucleic acid stains, including TOTO-1 and YOYO-1 (T3600, Y3601; Nucleic Acid Detection and Genomics Technology—Chapter 8), form tight complexes with nucleic acids with slow off-rates for release of the dye. Consequently, nucleic acids that have been prelabeled with these dyes can be traced in cells following microinjection or during gene transfer ref (photo). The cell-to-cell transport via plasmodesmata of TOTO-1 dye–labeled RNA, single-stranded DNA and double-stranded DNA has been determined following microinjection of the labeled nucleic acids into plant cells.ref

Khoobehi and Peyman have demonstrated the use of our cell-permeant SYTO nucleic acid stains as ophthalmological tracers of blood flow. White blood cells were passively loaded with the green-fluorescent SYTO 16 dye (S7578, Nucleic Acid Stains—Section 8.1) or the red-fluorescent SYTO 59 dye (S11341, Nucleic Acid Stains—Section 8.1). Red blood cell membranes were labeled with DiD (D307, Tracers for Membrane Labeling—Section 14.4). By using an argon-ion laser to excite SYTO 16 and a red light–emitting He-Ne laser to excite both SYTO 59 and DiD, they were able to follow the relative mobility of the two types of blood cells.ref

Caged Fluorescent Dye Tracers

UV photolysis of photoactivatable fluorescent dyes provides a means of controlling—both spatially and temporally—the release of fluorescent tracers (Photoactivatable Reagents, Including Photoreactive Crosslinkers and Caged Probes—Section 5.3). Thus, caged dyes enable researchers to follow the movement of individual molecules and cellular structures,ref as well as to study cell lineage in live organisms (photo). CMNB-caged fluorescein ref (F7103, structure) is colorless and nonfluorescent until it is photolyzed at <365 nm to the intensely green-fluorescent free dye. Movement of the liberated fluorophore from the site of photolysis can then be followed. The succinimidyl ester of CMNB-caged carboxyfluorescein (C20050, Fluorescein, Oregon Green and Rhodamine Green Dyes—Section 1.5), which is water soluble at pH 7, is useful for preparation of other caged dye tracers, including those of proteins.

Fluorescent Retrograde Tracers

Anterograde and retrograde tracing of neurons has utilized a wide variety of fluorescent and nonfluorescent probes ref (Figure 14.3.8). Among the retrograde and anterograde tracers are biotin derivatives and polar fluorescent dyes (described in this section), lipophilic tracers such as DiI (D282, Tracers for Membrane Labeling—Section 14.4), dextran conjugates (Fluorescent and Biotinylated Dextrans—Section 14.5), fluorescent microspheres (Microspheres and Qdot Nanocrystals for Tracing—Section 14.6) and protein conjugates, including lectins (Protein Conjugates—Section 14.7).


Figure 14.3.8 Structural schematic of a neuron indicating the directions of retrograde and anterograde transport.


True Blue and Nuclear Yellow

The popular retrograde tracer true blue (T1323) is a UV light–excitable, divalent cationic dye that stains the cytoplasm with blue fluorescence.ref For two-color neuronal mapping, true blue has been combined with longer-wavelength tracers such as nuclear yellow ref (Hoechst S769121, N21485) or diamidino yellow,ref which primarily stain the neuronal nucleus with yellow fluorescence (photo, photo, photo). Fluorescent microspheres have also been used as a counterstain with true blue.ref True blue is reported to be a less cytotoxic retrograde tracer than Fluoro-Gold ref and to be a more efficient retrograde tracer than diamidino yellow.ref Both true blue and nuclear yellow are stable when subjected to immunohistochemical processing and can be used to photoconvert DAB into an insoluble, electron-dense reaction product.ref


Hydroxystilbamidine and Aminostilbamidine

Hydroxystilbamidine methanesulfonate (H22845, structure) was originally developed as a trypanocide and has been identified by Wessendorf ref as the active component of a dye that was named Fluoro-Gold by Schmued and Fallon ref and later sold for retrograde tracing under that trademark by Fluorochrome, Inc. The use of hydroxystilbamidine as a histochemical stain and as a retrograde tracer for neurons apparently goes back to the work of Snapper and collaborators in the early 1950s who, while studying the effects of hydroxystilbamidine on multiple myeloma, showed that therapeutically administered hydroxystilbamidine gives selective staining of ganglion cells.ref The comprehensive article by Wessendorf ref also describes several other early applications of hydroxystilbamidine that do not include its therapeutic uses:

The weakly basic properties of hydroxystilbamidine that result in its uptake by lysosomes are reportedly important for its mechanism of retrograde transport.ref We developed a product that we called hydroxystilbamidine (formerly catalog number H7599) in April 1995; however, we subsequently discovered that the chemical structure of our original "hydroxystilbamidine" corresponded to a novel dye that we now call aminostilbamidine (A22850, structure). Apparently, aminostilbamidine functions at least as well as authentic hydroxystilbamidine as a tracer.ref Aminostilbamidine, however, does not show the spectral shifts with DNA and RNA that are observed with authentic hydroxystilbamidine.


Propidium Iodide and DAPI for Retrograde Tracing

A variety of other low molecular weight dyes have been used as fluorescent retrograde neuronal tracers.ref These include propidium iodide ref (P1304MP, P21493) and DAPI.ref Both propidium iodide and DAPI can be used to photoconvert DAB into an insoluble, electron-dense product.ref The lactate salt of DAPI (D3571) has much higher water solubility than the chloride salt (D1306, D21490), making it the preferred form for microinjection.

NeuroTrace Fluorescent Nissl Stains

The Nissl substance, described by Franz Nissl more than 100 years ago, is unique to neuronal cells.ref Composed of an extraordinary amount of rough endoplasmic reticulum, the Nissl substance reflects the unusually high protein synthesis capacity of neuronal cells. Various fluorescent or chromophoric "Nissl stains" have been used for this counterstaining, including acridine orange,ref ethidium bromide,ref neutral red (N3246, Viability and Cytotoxicity Assay Reagents—Section 15.2), cresyl violet,ref methylene blue, safranin-O and toluidine blue-O.ref We have developed five fluorescent Nissl stains (Fluorescence characteristics of NeuroTrace fluorescent Nissl stains—Table 14.2) that not only provide a wide spectrum of fluorescent colors for staining neurons, but also are far more sensitive than the conventional dyes:

  • NeuroTrace 435/455 blue-fluorescent Nissl stain (N21479, photo)
  • NeuroTrace 500/525 green-fluorescent Nissl stain (N21480; photo, photo, photo)
  • NeuroTrace 515/535 yellow-fluorescent Nissl stain (N21481, photo)
  • NeuroTrace 530/615 red-fluorescent Nissl stain (N21482; photo, photo)
  • NeuroTrace 640/660 deep red–fluorescent Nissl stain (N21483

In addition, the Nissl substance redistributes within the cell body in injured or regenerating neurons. Therefore, these Nissl stains can also act as markers for physically or chemically induced neurostructural damage.ref Staining by the Nissl stains is completely eliminated by pretreatment of tissue specimens with RNase; however, these dyes are not specific stains for RNA in solutions. The strong fluorescence (emission maximum ~515–520 nm) of NeuroTrace 500/525 green-fluorescent Nissl stain (N21480) makes it the preferred dye for use as a counterstain in combination with orange- or red-fluorescent neuroanatomical tracers such as DiI ref (D282, D3911, V22885; Tracers for Membrane Labeling—Section 14.4; photo).

Polar Spin Label

The highly water-soluble cationic spin label 4-trimethylammonium-2,2,6,6-tetramethylpiperidine-1-oxyl iodide (CAT 1, T506) has been used to:

  • Quench fluorescent dyes in solutions, cells and cell membranes ref
  • Detect oxygen gradients and oxidation–reduction properties of cells ref
  • Study liposome permeability ref

Signal Amplification of Polar Tracers

Polar tracers such as Cascade Blue hydrazide, lucifer yellow CH, Alexa Fluor 488 hydrazide and biocytin penetrate even the finest structures of neurons and other cells; however, as the thickness of the sample is decreased, the fluorescence signal is reduced. Consequently, it may be necessary to further amplify the signal by secondary detection methods.

We provide rabbit polyclonal antibodies to the Alexa Fluor 488, Alexa Fluor 405/Cascade Blue, lucifer yellow, fluorescein, BODIPY FL, tetramethylrhodamine and Texas Red fluorophores (Anti-Dye and Anti-Hapten Antibodies—Section 7.4, Anti-fluorophore and anti-hapten antibodies—Table 7.19) and a vast number of dye- or enzyme-labeled anti-rabbit antibodies (Secondary Immunoreagents—Section 7.2, Summary of Molecular Probes secondary antibody conjugates—Table 7.1). Our polyclonal and monoclonal antibodies to fluorescein crossreact strongly with the Oregon Green dyes and somewhat with Rhodamine Green fluorophores, and our anti-tetramethylrhodamine and anti–Texas Red antibodies crossreact with tetramethylrhodamine, Lissamine rhodamine B, Rhodamine Red and Texas Red dyes.

Our Tyramide Signal Amplification (TSA) Kits (TSA and Other Peroxidase-Based Signal Amplification Technology—Section 6.2, Tyramide Signal Amplification (TSA) Kits—Table 6.1) can also be utilized to detect aldehyde-fixed biotin derivatives or, in combination with antibodies to aldehyde-fixed  fluorophore-labeled polar tracers, to further amplify the signal. Following fixation, biotinylated tracers such as biocytin and biotin ethylenediamine, can be detected using the reagents in our TSA Kits that contain horseradish peroxidase streptavidin and a fluorescent tyramide.

The relatively small size and easy penetration into tissues makes the streptavidin conjugates of the NANOGOLD, Alexa Fluor 488 FluoroNanogold and Alexa Fluor 594 FluoroNanogold 1.4 nm gold clusters (N24918, A24926, A24927; Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6) useful for ultrastructural studies of biotinylated tracers, particularly in combination with the LI Silver (LIS) Enhancement Kit (L24919, Secondary Immunoreagents—Section 7.2). Biotinylated polar tracers can also be detected with light microscopy using the Diaminobenzidine (DAB) Histochemistry Kit #3 (D22187, TSA and Other Peroxidase-Based Signal Amplification Techniques—Section 6.2).

Influx Pinocytic Cell-Loading Reagent

Our Influx pinocytic cell-loading reagent (I14402) works via a rapid and simple technique based on the osmotic lysis of pinocytic vesicles, an approach introduced by Okada and Rechsteiner.ref The probe is simply mixed at high concentration with the Influx reagent blended into growth medium, then incubated with live cells to allow pinocytic uptake of the surrounding solution. Subsequent transfer of the cells to a slightly hypotonic medium results in bursting of the pinocytic vesicles within the cells and the release of the probe into the cytosol (Figure 14.3.9, photo, photo).

The Influx pinocytic cell-loading reagent is highly effective for loading a diverse array of probes—including calcein (photo), Alexa Fluor hydrazides (photo), dextran conjugates of fluorophores ref and ion indicators (photo), fura-2 salts, Oregon Green 514 dye–labeled tubulin, Alexa Fluor 488 dye–labeled actin, heparin, hydroxyurea,ref DNA, oligonucleotides,ref and Qdot nanocrystals ref—into a variety of cell lines. We (or other researchers) have successfully tested the reagent and loading method with:

  • Bovine pulmonary artery endothelial cells (BPAEC)
  • Human epidermoid carcinoma cells (A431)
  • Human T-cell leukemia cells (Jurkat)
  • Murine fibroblasts (NIH 3T3 and CRE BAG 2)
  • Murine monocyte-macrophages (RAW264.7 and J774A.1)
  • Murine myeloma cells (P3x63AG8)
  • Rat basophilic leukemia cells (RBL)

More than 80% of the cells remained viable, as determined by subsequent exclusion of propidium iodide.

In addition to the Influx pinocytic cell-loading reagent and cell growth medium, all that is required to perform the loading procedure is sterile deionized water and the fluorescent probe or other polar molecule of interest. Cell labeling can be accomplished in a single 30-minute loading cycle and may be enhanced by repetitive loading. Although most types of cells load quickly and easily, optimal conditions for loading must be determined for each cell type. It is also important to note that cell-to-cell variability in the degree of loading is typical (photo) and that higher variability is generally observed when using large compounds, such as >10,000 MW dextrans and proteins.

The Influx pinocytic cell-loading reagent is packaged as a set of 10 tubes (I14402), each containing sufficient material to load 50 samples of cells grown on coverslips following the protocol provided (Influx Pinocytic Cell-Loading Reagent). Cells in suspension or in culture flasks may also be easily loaded; however, the number of possible cell loadings will depend on the cell suspension volume or size of culture flask used. The information provided with the Influx reagent includes general guidelines and detailed suggestions for optimizing cell loading. Use of the custom coverslip mini-rack or coverslip maxi-rack (C14784, C24784; Fluorescence Microscopy Accessories and Reference Standards—Section 23.1) facilitates cell loading and slide handling when using the Influx reagent.


Influx reagent pinocytic cell-loading method

Figure 14.3.9 Principle of the Influx reagent pinocytic cell-loading method (I14402). Cultured cells are placed in hypertonic Influx loading reagent (panel A), along with the material to be loaded into the cells (yellow fluid, panel B), allowing the material to be carried into the cells via pinocytic vesicles. When the cells are placed in hypotonic medium, the pinocytic vesicles burst (panel C), releasing their contents into the cytosol (panel D).

Loading P2X7 Receptor–Expressing Cells

P2X7 receptor–expressing cells such as macrophages and thymocytes exhibit reversible pore opening that can be exploited to provide an entry pathway for intracellular loading of both cationic and anionic fluorescent dyes with molecular weights of up to 900 daltons.ref Pore opening is induced by treatment with 5 mM ATP for five minutes and subsequently reversed by addition of divalent cations (Ca2+ or Mg2+). Dyes that have been successfully loaded into macrophage cells by this method include:

One of the most potent and widely used P2X receptor agonists, BzBzATP (2'-(or 3'-)O-(4-benzoylbenzoyl)adenosine 5'-triphosphate, B22358; Probes for Protein Kinases, Protein Phosphatases and Nucleotide-Binding Proteins—Section 17.3), is available.ref BzBzATP has more general applications for site-directed irreversible modification of nucleotide-binding proteins via photoaffinity labeling;ref see Probes for Protein Kinases, Protein Phosphatases and Nucleotide-Binding Proteins—Section 17.3 for more information on our nucleotide analogs.

Data Table

Cat # Links MW Storage Soluble Abs EC Em Solvent Notes
A350 icon 427.33 L H2O 353 7200 520 H2O  
A1339 icon icon 491.57 L H2O 425 12,000 532 H2O  
A1340 icon icon 533.65 L H2O 426 11,000 531 H2O  
A1593 icon 367.30 NC DMF, DMSO <300   none    
A1594 icon 442.50 NC DMF, DMSO <300   none    
A6257 icon 523.39 D,L H2O 424 19,000 505 pH 7  
A10436 icon icon 570.48 D,L H2O 493 71,000 517 pH 7  
A10437 icon 730.74 D,L H2O 576 86,000 599 pH 7 1
A10438 icon icon 758.79 D,L H2O 588 97,000 613 pH 7 1
A10439 icon 349.29 L H2O, DMSO 345 13,000 445 pH 7  
A10441 icon 730.74 FF,L H2O 576 86,000 599 pH 7 2
A10442 icon icon 758.79 FF,L H2O 588 97,000 613 pH 7 2
A12922 icon 1141.31 D,L DMSO, H2O 591 80,000 618 pH 7  
A12923 icon 1209.66 D,L DMSO, H2O 556 99,000 572 pH 7  
A12924 icon 974.98 D,L DMSO, H2O 494 62,000 520 pH 7  
A20501MP   ~1150 D,L H2O 554 150,000 567 pH 7  
A20502   ~1200 D,L H2O 649 250,000 666 pH 7  
A22850 icon 471.55 F,D,L H2O, DMSO 361 17,000 536 pH 7  
A30000 icon icon 1028.26 F,DD,L H2O, DMSO 400 35,000 424 pH 7 3, 4, 5
A30627 icon 584.52 F,D,L H2O, DMSO 353 20,000 437 MeOH 6
A30629 icon 895.07 F,D,L H2O, DMSO 494 77,000 518 pH 7 6, 7, 8
A30632   ~1220 F,D,L H2O, DMSO 651 250,000 672 MeOH 6
A30634   ~950 D,L H2O, DMSO 624 110,000 643 pH 7  
A30675 icon icon 666.58 F,D,L H2O 399 29,000 422 H2O  
B1151 icon icon 520.45 L pH >6 503 90,000 528 pH 9 9
B1370 icon 831.01 L DMF, pH >6 494 75,000 518 pH 9 9
B1513 icon 341.38 F,D DMF, DMSO <300   none    
B1582 icon 454.54 F,D DMF, DMSO <300   none    
B1592 icon 372.48 NC H2O <300   none    
B1596 icon 555.65 NC DMF, DMSO <300   none    
B1603 icon 386.51 D pH >6, DMF <300   none    
B6352 icon 669.74 F,D DMF, pH >6 <300   none   4
B6353 icon 556.58 F,D DMF, pH >6 <300   none   4
B10570 icon 644.70 L DMSO 494 68,000 523 pH 9 9
C194 icon icon 376.32 L pH >6, DMF 492 75,000 517 pH 9 9
C368 icon icon 445.21 L pH >6, DMF 504 107,000 529 pH 8 10
C481 icon icon 622.54 L pH >5 494 77,000 517 pH 9 11, 12
C621 icon icon 624.49 L H2O 399 30,000 423 H2O 3
C687 icon icon 596.44 L H2O 399 30,000 421 H2O 3, 13
C1430 icon icon 994.87 F,D DMSO <300   none   14
C1904 icon icon 376.32 L pH >6, DMF 492 78,000 517 pH 9 9, 15
C2284 icon icon 607.42 F,D,LL H2O, MeOH 396 29,000 410 MeOH 3, 16
C3099 icon icon 994.87 F,D DMSO <300   none   2, 14
C3100MP icon icon 994.87 F,D DMSO <300   none   14
C3221 icon icon 644.77 L H2O 399 31,000 419 H2O 3, 13
C3239 icon icon 548.29 L H2O 399 29,000 419 H2O 3, 13
D1306 icon icon 350.25 L H2O, DMF 342 28,000 450 pH 7  
D3238 icon 466.19 F,D,L H2O 490 97,000 515 H2O 17
D3571 icon icon 457.49 L H2O, MeOH 342 28,000 450 pH 7  
D20653 icon 341.45 D DMSO <300   none   18
D21490 icon icon 350.25 L H2O, DMF 342 28,000 450 pH 7 15
F1130 icon 478.32 D,L H2O, DMF 495 76,000 519 pH 9 9
F7103 icon icon 826.81 FF,D,LL H2O, DMSO 333 15,000 none DMSO 16, 19, 20
H185 icon 206.15 L pH >6, DMF 386 29,000 448 pH 10 21
H348 icon 524.37 D,L H2O 454 24,000 511 pH 9 22
H22845 icon 472.53 F,D,L H2O, DMSO 345 31,000 450 pH 5 23
L453 icon icon 457.24 L H2O 428 12,000 536 H2O 24, 25
L682 icon icon 479.44 L H2O 428 12,000 533 H2O 24, 25
L1177 icon icon 521.56 L H2O 427 12,000 535 H2O 24, 25
L1338 icon icon 659.51 F,D,L H2O 426 11,000 531 pH 7 26
L2601 icon icon 873.10 D,L H2O 428 11,000 531 H2O  
L6950 icon icon 850.03 D,L H2O 428 11,000 532 pH 7  
L12926 icon icon 457.24 FF,L H2O 428 12,000 536 H2O 2
M395 icon 538.40 L H2O 404 29,000 435 pH 8 27
M1602 icon 523.60 F,D pH >6, DMF <300   none    
N21479   see Notes F,D,L DMSO 435 see Notes 457 H2O/RNA 2, 28, 29
N21480   see Notes F,D,L DMSO 497 see Notes 524 H2O/RNA 2, 28, 29
N21481   see Notes F,D,L DMSO 515 see Notes 535 H2O/RNA 2, 28, 29
N21482   see Notes F,D,L DMSO 530 see Notes 619 H2O/RNA 2, 28, 29
N21483   see Notes F,D,L DMSO 644 see Notes 663 H2O/RNA 2, 28, 29
O6138 icon 512.36 L pH >6, DMF 506 86,000 526 pH 9 30
O6146 icon 412.30 L pH >6, DMF 492 85,000 518 pH 9 31
O12920 icon 887.39 L DMSO, H2O 495 66,000 522 pH 9 31
P349 icon 610.42 L H2O 374 51,000 403 H2O  
P1304MP icon icon 668.40 L H2O, DMSO 493 5900 636 H2O  
P21493 icon icon 668.40 L H2O, DMSO 493 5900 636 H2O 15
S359 icon icon 606.71 L H2O 586 108,000 605 H2O  
S1129 icon 518.43 F,D DMSO <300   none   32
S1307 icon 558.66 L H2O 565 84,000 586 H2O  
T506 icon 341.25 F,D H2O, MeOH <300   none    
T1247 icon 373.38 D H2O 270 4700 545 H2O 33, 34
T1323 icon 417.29 L DMSO 375 68,000 403 H2O  
T12921 icon 869.09 D,L DMSO 554 103,000 581 pH 7  
X1525 icon 422.16 D H2O 259 8800 none H2O  
  1. Maximum solubility in water is ~8% for A10437 and A10438.
  2. This product is supplied as a ready-made solution in the solvent indicated under "Soluble."
  3. The Alexa Fluor 405 and Cascade Blue dyes have a second absorption peak at about 376 nm with EC ~80% of the 395–400 nm peak.
  4. This sulfonated succinimidyl ester derivative is water-soluble and may be dissolved in buffer at ~pH 8 for reaction with amines. Long-term storage in water is NOT recommended due to hydrolysis.
  5. A30100 is an alternative packaging of A30000 but is otherwise identical.
  6. Aqueous stock solutions should be used within 24 hours; long-term storage is NOT recommended.
  7. The fluorescence lifetime (τ) of the Alexa Fluor 488 dye in pH 7.4 buffer at 20°C is 4.1 nanoseconds. Data provided by the SPEX Fluorescence Group, Horiba Jobin Yvon Inc.
  8. Abs and Em of the Alexa Fluor 488 dye are red-shifted by as much as 16 nm and 25 nm respectively on microarrays relative to aqueous solution values. The magnitude of the spectral shift depends on the array substrate material.ref
  9. Absorption and fluorescence of fluorescein derivatives are pH-dependent. Extinction coefficients and fluorescence quantum yields decrease markedly at pH <7.
  10. Absorption and fluorescence of dichlorofluorescein derivatives are pH-dependent. Extinction coefficients and fluorescence quantum yields decrease markedly at pH <5.
  11. C481 fluorescence is strongly quenched by micromolar concentrations of Fe3+, Co2+, Ni2+ and Cu2+ at pH 7.ref
  12. Kd(Co2+) for calcein is 120 nM, determined in 10 mM HEPES, 1 µM Ca2+, 1 mM Mg2+, 100 mM KCl.ref
  13. Maximum solubility in water is ~1% for C687, ~1% for C3221 and ~8% for C3239.
  14. Calcein AM is converted to fluorescent calcein (C481) after acetoxymethyl ester hydrolysis.
  15. This product is specified to equal or exceed 98% analytical purity by HPLC.
  16. Unstable in water. Use immediately.
  17. The absorption and fluorescence spectra of BODIPY derivatives are relatively insensitive to the solvent.
  18. The dissociation constant (Kd) for desthiobiotin binding to streptavidin is 1.9 nM.ref
  19. All photoactivatable probes are sensitive to light. They should be protected from illumination except when photolysis is intended.
  20. This product is colorless and nonfluorescent until it is activated by ultraviolet photolysis. Photoactivation generates a fluorescein derivative with spectral characteristics similar to 5-carboxyfluorescein (C1359).
  21. Spectra of hydroxycoumarins are pH-dependent. Below the pKa (~7.5), Abs shifts to shorter wavelengths (325–340 nm) and fluorescence intensity decreases.
  22. H348 spectra are pH-dependent.
  23. Hydroxystilbamidine in H2O has a wide emission bandwidth, with a second peak at ~600 nm.
  24. The fluorescence quantum yield of lucifer yellow CH in H2O is 0.21.ref
  25. Maximum solubility in water is ~8% for L453, ~6% for L682 and ~1% for L1177.
  26. Iodoacetamides in solution undergo rapid photodecomposition to unreactive products. Minimize exposure to light prior to reaction.
  27. Maximum solubility for M395 in water is ~25%.
  28. This product is essentially nonfluorescent except when bound to DNA or RNA.
  29. The active ingredient of this product is an organic dye with MW <1000. The exact MW and extinction coefficient values for this dye are proprietary.
  30. Absorption and fluorescence of Oregon Green 514 derivatives are pH-dependent only in moderately acidic solutions (pH <5).
  31. Absorption and fluorescence of Oregon Green 488 derivatives are pH-dependent only in moderately acidic solutions (pH <5).
  32. S1129 is converted to a fluorescent product (fluorescein-5-sulfonic acid, F1130) after acetate hydrolysis.
  33. Absorption and luminescence of T1247 are extremely weak unless it is chelated. Data are for dipicolinic acid (DPA) chelate. The luminescence spectrum has secondary peak at 490 nm.
  34. MW is for the hydrated form of this product.

Ordering Information

Sku Name Size Price Qty
A12924 Biocytin Alexa Fluor® 488 (Alexa Fluor® 488 Biocytin, Disodium Salt) 250 µg USD 253.00
A12923 Biocytin Alexa Fluor® 546 (Alexa Fluor® 546 Biocytin, Sodium Salt) 250 µg USD 253.00
A12922 Alexa Fluor® 594 Biocytin, Sodium Salt (Biocytin Alexa Fluor® 594) 250 µg USD 253.00
A30675 Alexa Fluor® 405 Cadaverine, Trisodium Salt 1 mg USD 231.00
A30627 Alexa Fluor® 350 hydroxylamine (Alexa Fluor® 350 C5-Aminooxyacetamide, trifluoroacetate Salt) 1 mg USD 800.00
A30629 Alexa Fluor® 488 Hydroxylamine (Alexa Fluor® 488 C5-Aminooxyacetamide, Bis(Triethylammonium) Salt) 1 mg USD 800.00
A30632 Alexa Fluor® 647 C5-Aminooxyacetamide, Bis(Triethylammonium) Salt (Alexa Fluor® 647 Hydroxylamine) 1 mg USD 800.00
A30000 Alexa Fluor® 405 Carboxylic Acid, Succinimidyl Ester 1 mg USD 292.00
A30100 Alexa Fluor® 405 Carboxylic Acid, Succinimidyl Ester 5 mg USD 1,058.00
A10439 Alexa Fluor® 350 hydrazide, Sodium Salt 5 mg USD 226.00
A10436 Alexa Fluor® 488 hydrazide, Sodium Salt, 1 mg 1 mg USD 226.00
A20501MP Alexa Fluor® 555 Hydrazide, Tris(Triethylammonium) Salt 1 mg USD 226.00
A10437 Alexa Fluor® 568 Hydrazide, Sodium Salt 1 mg USD 226.00
A10438 Alexa Fluor® 594 Hydrazide, Sodium Salt 1 mg USD 226.00
A30634 Alexa Fluor® 633 Hydrazide, Bis(Triethylammonium) Salt 1 mg USD 226.00
A20502 Alexa Fluor® 647 Hydrazide, Tris(triethylammonium) Salt 1 mg USD 225.00
A10441 Alexa Fluor® 568 Hydrazide, Sodium Salt, for microinjection 125 µL USD 226.00
A10442 Alexa Fluor® 594 Hydrazide, Sodium Salt, for microinjection 125 µL USD 225.00
A1339 Lucifer Yellow Ethylenediamine (N-(2-Aminoethyl)-4-Amino-3,6-Disulfo-1,8-Naphthalimide, Dipotassium Salt) 25 mg USD 84.00
A1593 Biotin Ethylenediamine (N-(2-Aminoethyl)Biotinamide, Hydrobromide) 25 mg USD 123.00
A350 ANTS (8-Aminonaphthalene-1,3,6-Trisulfonic Acid, Disodium Salt) 1 g USD 315.00
A1594 Biotin Cadaverine (N-(5-Aminopentyl)Biotinamide, Trifluoroacetic Acid Salt) 25 mg USD 159.00
A6257 APTS (8-Aminopyrene-1,3,6-Trisulfonic Acid, Trisodium Salt) 10 mg USD 205.00
A22850 Aminostilbamidine, Methanesulfonate 10 mg USD 165.00
B1592 Biocytin (ε-Biotinoyl-L-Lysine) 100 mg USD 132.00
B1603 Biocytin Hydrazide 25 mg USD 170.00
B10570 Biotin-4-Fluorescein 5 mg USD 205.00
B1582 Biotin-X, SE (6-((Biotinoyl)Amino)Hexanoic Acid, Succinimidyl Ester (Biotinamidocaproate, N-Hydroxysuccinimidyl Ester)) 100 mg USD 178.00
B6353 Sulfo-NHS-LC-Biotin (Biotin-X, SSE, 6-((Biotinoyl)Amino)Hexanoic Acid, Sulfosuccinimidyl Ester, Sodium Salt) 25 mg USD 157.00
B6352 Biotin-XX, SSE (6-((6-((Biotinoyl)Amino)Hexanoyl)amino)Hexanoic Acid, Sulfosuccinimidyl Ester, Sodium Salt) 25 mg USD 157.00
B1370 Fluorescein biotin, 5-((N-(5-(N-(6-(biotinoyl)amino)hexanoyl)amino)pentyl)thioureidyl)fluorescein 5 mg USD 199.00
B1513 D-Biotin, Succinimidyl Ester (succinimidyl D-Biotin) 100 mg USD 104.00
B1596 Biotin-X Cadaverine (5-(((N-(Biotinoyl)Amino)Hexanoyl)Amino)Pentylamine, Trifluoroacetic Acid Salt) 10 mg USD 168.00
B1151 BCECF Acid (2',7'-Bis-(2-Carboxyethyl)-5-(and-6)-Carboxyfluorescein), mixed isomers 1 mg USD 149.00
C481 Calcein, high purity 100 mg USD 132.00
C1430 Calcein, AM, cell-permeant dye 1 mg USD 213.00
C3099 Calcein, AM 1 mL USD 231.00
C3100MP Calcein, AM, cell-permeant dye 20 x 50 µg USD 261.00
C368 5-(and-6)-Carboxy-2',7'-Dichlorofluorescein, mixed isomers 100 mg USD 117.00
C1904 5(6)-FAM (5-(and-6)-Carboxyfluorescein) (FluoroPure™ grade), mixed isomers 100 mg USD 138.00
C194 5-(and-6)-Carboxyfluorescein, mixed isomers 5 g USD 153.00
C2284 Cascade Blue® Acetyl Azide, Trisodium Salt 5 mg USD 263.00
C621 Cascade Blue® Ethylenediamine, Trisodium Salt 10 mg USD 209.00
C3239 Cascade Blue® hydrazide, Trilithium Salt 10 mg USD 205.00
C687 Cascade Blue® hydrazide, Trisodium Salt 10 mg USD 209.00
D1306 DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) 10 mg USD 89.00
D21490 DAPI, FluoroPure™ grade 10 mg USD 133.00
D3571 DAPI (4',6-Diamidino-2-Phenylindole, Dilactate) 10 mg USD 89.00
D3238 BODIPY® 492/515 Disulfonate (4,4-Difluoro-1,3,5,7,8-Pentamethyl-4-Bora-3a,4a-Diaza-s-Indacene-2,6-Disulfonic Acid, Disodium Salt) 10 mg USD 94.00
F7103 Fluorescein bis-(5-Carboxymethoxy-2-Nitrobenzyl) Ether, Dipotassium Salt (CMNB-Caged Fluorescein) 5 mg USD 288.00
F1130 Fluorescein-5-(and-6)-Sulfonic Acid, Trisodium Salt 100 mg USD 138.00
H185 7-Hydroxycoumarin-3-Carboxylic Acid, reference standard 100 mg USD 178.00
H348 HPTS; pyranine (8-Hydroxypyrene-1,3,6-Trisulfonic Acid, Trisodium Salt) 1 g USD 132.00
H22845 Hydroxystilbamidine, methanesulfonate 10 mg USD 159.00
I14402 Influx™ Pinocytic Cell-Loading Reagent 1 set USD 129.00
L6950 Lucifer Yellow Biocytin, Potassium Salt (Biocytin Lucifer Yellow) 5 mg USD 209.00
L682 Lucifer Yellow CH, Ammonium Salt 25 mg USD 98.00
L453 Lucifer Yellow CH, Lithium Salt, 25 mg 25 mg USD 97.00
L12926 Lucifer Yellow CH, Lithium Salt, for microinjection 100 µL USD 181.00
L1177 Lucifer Yellow CH, Potassium Salt 25 mg USD 101.00
M1602 Nα-(3-Maleimidylpropionyl)Biocytin 25 mg USD 150.00
M395 MPTS (8-Methoxypyrene-1,3,6-Trisulfonic Acid, Trisodium Salt) 100 mg USD 176.00
N21479 NeuroTrace® 435/455 Blue Fluorescent Nissl Stain 1 mL USD 156.00
N21480 NeuroTrace® 500⁄525 Green Fluorescent Nissl Stain - Solution in DMSO 1 mL USD 161.00
N21481 NeuroTrace® 515⁄535 Yellow Fluorescent Nissl Stain - Solution in DMSO 1 mL USD 161.00
N21482 NeuroTrace® 530⁄615 Red Fluorescent Nissl Stain - Solution in DMSO 1 mL USD 161.00
N21483 NeuroTrace® 640⁄660 Deep-Red Fluorescent Nissl Stain - Solution in DMSO 1 mL USD 161.00
O6146 Oregon Green® 488 Carboxylic Acid, 5-isomer 5 mg USD 166.00
P1304MP Propidium Iodide 100 mg USD 129.00
P21493 Propidium Iodide - FluoroPure™ Grade 100 mg USD 154.00
S359 Sulforhodamine 101 25 mg USD 97.50
S1307 Sulforhodamine B 5 g USD 389.00
T12921 Biocytin TMR (5-(and-6)-Tetramethylrhodamine Biocytin) 5 mg USD 204.00
T506 CAT 1 (4-Trimethylammonium-2, 2,6,6-Tetramethylpiperidine- 1-Oxyl Iodide), spin trap 100 mg USD 696.00
X1525 DPX (p-Xylene-Bis-Pyridinium Bromide) 1 g USD 140.00
For Research Use Only. Not for use in diagnostic procedures.