Polar Tracers—Section 14.3

Page Contents

• Fixable Polar Tracers
• Nonfixable Polar Tracers
• Caged Fluorescent Dye Tracers
• Fluorescent Retrograde Tracers
• NeuroTrace Fluorescent Nissl Stains
• Polar Spin Label
• Signal Amplification of Polar Tracers
• Influx Pinocytic Cell-Loading Reagent
• Loading P2X7 Receptor–Expressing Cells
• Data Table
• Ordering Information

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 (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 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. Electroporation has been used to transport several of the polar tracers through the skin and into cells. 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; ), orange-fluorescent Alexa Fluor 555 and Alexa Fluor 568 hydrazides (A20501MP, A10437, A10441; Figure 14.3.1), red-fluorescent Alexa Fluor 594 hydrazide (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. 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 (), are some of the few polar tracers that exhibit bright blue fluorescence.

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.

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.

Lucifer Yellow CH

Lucifer yellow CH (LY-CH or LY, ) 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. Loading of this polar tracer and other similar impermeant dyes is usually accomplished by microinjection, pinocytosis, scrape loading, ATP-induced permeabilization or osmotic shock (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 or by fluid-phase endocytosis. 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. 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. For electron microscopy studies, lucifer yellow CH can be used to photoconvert diaminobenzidine (DAB) into an insoluble, electron-dense reaction product (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 (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 () and in fixed brain slices, as well as to investigate intercellular communication through gap junctions. 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. The thiol-reactive lucifer yellow iodoacetamide (L1338) can also be used as a microinjectable polar tracer, as well as for preparing fluorescent liposomes and for detecting the accessibility of thiols in membrane-bound proteins. 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 (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 (EC_{400 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. 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. 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.

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.

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, ) and biotin ethylenediamine (A1593, ) are microinjectable anterograde and transneuronal tracers. Retrograde transport of biocytin and biotin ethylenediamine in neurons has also been reported. These water-soluble tracers are often used to label neurons during electrophysiological measurements in order to correlate neuronal function with structure and connectivity. 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.

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 (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 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. 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. Because fluorescent biocytin derivatives contain free primary amines, they should be even more efficiently fixed by formaldehyde or glutaraldehyde.

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.

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.

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 ()—as well as BCECF (B1151, ), 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
• Gap junctional communication
• Liposome formation, fusion and targeting
• Membrane integrity and permeability

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. 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. 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. In addition, the Co²⁺-quenched calcein complex is useful for both lysis and fusion assays (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.

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; our 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.

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. CFDA has also been used to investigate cell–cell communication in plant cells. A probenecid-inhibitable anion-transport mechanism permits loading of carboxyfluorescein diacetate and Oregon Green 488 carboxylic acid diacetate (O6151, ; Viability and Cytotoxicity Assay Reagents—Section 15.2) into hyphal tip-cells of some fungi.

Calcein AM (), 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.

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. This assay can be followed by either imaging or flow cytometry. In addition, calcein AM and DiI have been combined for use in following cell fusion and for analysis of cholesterol processing by macrophages following ingestion of apoptotic cells.

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

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, ), Alexa Fluor 488 (A11001, ), BODIPY FL (B2752, ), Oregon Green 488 (O6380, ) or fluorescein (F2761, ). 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.

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

Sulforhodamines

Sulforhodamine 101 (S359, ) and sulforhodamine B (S1307, ) 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 (). Activity-dependent uptake of sulforhodamine 101 during nerve stimulation has been reported. Sulforhodamine 101 specifically labels astrocytes both in vivo and in acute brain slice preparations. 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. 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 (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, preparing fluorescent liposomes, studying cell–cell communications and labeling elastic and collagen fibers.

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. 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.

Polysulfonated Pyrenes

HPTS (8-hydroxypyrene-1,3,6-trisulfonic acid, also known as pyranine, H348; ) is a unique pH-sensitive tracer. It fluoresces blue in acidic solutions and in acidic organelles, but fluoresces green in more basic organelles. In addition to its use as a probe for proton translocation, HPTS has been employed for intracellular labeling of neurons and as a fluid-phase endocytic tracer in catecholamine-secreting PC12 rat pheochromocytoma cells. 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 (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.

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. As with HPTS, the fluorescence of APTS and 1,3,6,8-pyrenetetrasulfonic acid is quenched by DPX (Assays of Volume Change, Membrane Fusion and Membrane Permeability—Note 14.3) and by the cationic spin label CAT 1 (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 (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. A review by Garcia 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. ANTS has also been employed as a neuronal tracer.

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.

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 (). 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.

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.

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, as well as to study cell lineage in live organisms (). CMNB-caged fluorescein (F7103, ) 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.

Anterograde and retrograde tracing of neurons has utilized a wide variety of fluorescent and nonfluorescent probes (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. For two-color neuronal mapping, true blue has been combined with longer-wavelength tracers such as nuclear yellow (Hoechst S769121, N21485) or diamidino yellow, which primarily stain the neuronal nucleus with yellow fluorescence (, , ). Fluorescent microspheres have also been used as a counterstain with true blue. True blue is reported to be a less cytotoxic retrograde tracer than Fluoro-Gold and to be a more efficient retrograde tracer than diamidino yellow. 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.

Hydroxystilbamidine and Aminostilbamidine

Hydroxystilbamidine methanesulfonate (H22845, ) was originally developed as a trypanocide and has been identified by Wessendorf as the active component of a dye that was named Fluoro-Gold by Schmued and Fallon 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. The comprehensive article by Wessendorf also describes several other early applications of hydroxystilbamidine that do not include its therapeutic uses:

• As an AT-selective, nonintercalating nucleic acid stain for discriminating between DNA and RNA (Nucleic Acid Stains—Section 8.1)
• For histochemical staining of DNA, mucosubstances and elastic fibers, as well as mast cells
• As a ribonuclease inhibitor
• For lysosomal staining of live cells (Probes for Lysosomes, Peroxisomes and Yeast Vacuoles—Section 12.3)

The weakly basic properties of hydroxystilbamidine that result in its uptake by lysosomes are reportedly important for its mechanism of retrograde transport. 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, ). Apparently, aminostilbamidine functions at least as well as authentic hydroxystilbamidine as a tracer. 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. These include propidium iodide (P1304MP, P21493) and DAPI. Both propidium iodide and DAPI can be used to photoconvert DAB into an insoluble, electron-dense product. The lactate salt of DAPI (D3571) has much higher water solubility than the chloride salt (D1306, D21490), making it the preferred form for microinjection.

The Nissl substance, described by Franz Nissl more than 100 years ago, is unique to neuronal cells. 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, ethidium bromide, neutral red (N3246, Viability and Cytotoxicity Assay Reagents—Section 15.2), cresyl violet, methylene blue, safranin-O and toluidine blue-O. 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, )
• NeuroTrace 500/525 green-fluorescent Nissl stain (N21480; , , )
• NeuroTrace 515/535 yellow-fluorescent Nissl stain (N21481, )
• NeuroTrace 530/615 red-fluorescent Nissl stain (N21482; , )
• 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. 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 (D282, D3911, V22885; Tracers for Membrane Labeling—Section 14.4; ).

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
• Detect oxygen gradients and oxidation–reduction properties of cells
• Study liposome permeability

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 7.8) 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).

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. 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, , ).

The Influx pinocytic cell-loading reagent is highly effective for loading a diverse array of probes—including calcein (), Alexa Fluor hydrazides (), dextran conjugates of fluorophores and ion indicators (), fura-2 salts, Oregon Green 514 dye–labeled tubulin, Alexa Fluor 488 dye–labeled actin, heparin, hydroxyurea, DNA, oligonucleotides, and Qdot nanocrystals —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 () 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.

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).

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. 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:

• Ca²⁺ indicator: fura-2 (F1200, F6799; Fluorescent Ca2+ Indicators Excited with UV Light—Section 19.2)
• pH indicator: HPTS (H348)
• Aqueous tracers: lucifer yellow CH (L453, L682, L1177, L12926) and 6-carboxyfluorescein (C1360, Fluorescein, Oregon Green and Rhodamine Green Dyes—Section 1.5)
• Nucleic acid stain: YO-PRO-1 (Y3603, Nucleic Acid Stains—Section 8.1)

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. BzBzATP has more general applications for site-directed irreversible modification of nucleotide-binding proteins via photoaffinity labeling; see Probes for Protein Kinases, Protein Phosphatases and Nucleotide-Binding Proteins—Section 17.3 for more information on our nucleotide analogs.