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Section 14.5 — Fluorescent and Biotinylated Dextrans

Dextrans are hydrophilic polysaccharides characterized by their moderate to high molecular weight, good water solubility and low toxicity. They are widely used as both anterograde and retrograde tracers in neurons ref and for numerous other applications (Bibliography for D-8998). Dextrans are biologically inert due to their uncommon poly-(alpha-D-1,6-glucose) linkages, which render them resistant to cleavage by most endogenous cellular glycosidases. They also usually have low immunogenicity. Molecular Probes offers more than 100 fluorescent and biotinylated dextran conjugates in several different molecular weight ranges.

Properties of Our Dextran Conjugates

A Wide Selection of Substituents

Molecular Probes' dextrans are conjugated to biotin, dinitrophenyl (DNP) or a wide variety of fluorophores, including with four of our Alexa Fluor dyes (Table 14.3, Dextran Conjugates). In particular, we would like to highlight the dextran conjugates of our Alexa Fluor 488, Oregon Green and Rhodamine Green dyes, which are significantly brighter and more photostable than most fluorescein dextrans. Dextran-conjugated fluorescent indicators for calcium and magnesium ions (Section 20.4) and of pH indicators (Section 21.4) are described with their corresponding ion indicators in other chapters.

Dextran Size

Molecular Probes' dextrans include those with nominal molecular weights (MW) of 3000; 10,000; 40,000; 70,000; 500,000; and 2,000,000 daltons (Table 14.3). Because unlabeled dextrans are polydisperse — and may become more so during the chemical processes required for their modification and purification — the actual molecular weights present in a particular sample may have a broad distribution. For example, our "3000 MW" dextran preparations contain polymers with molecular weights predominantly in the range of ~1500–3000 daltons, including the dye or other label.

Degree of Substitution of Our Dextrans

Dextrans from other sources usually have a degree of substitution of 0.2 or fewer dye molecules per dextran molecule for dextrans in the 10,000 MW range. Our dextrans, however, typically contain 0.3–0.7 dyes per dextran in the 3000 MW range, 0.5–2 dyes per dextran in the 10,000 MW range, 2–4 dyes in the 40,000 MW range and 3–6 dyes in the 70,000 MW range. The actual degree of substitution is indicated on the product's label. If too many fluorophores are conjugated to the dextran molecule, quenching and undesired interactions with cellular components may occur. We have found our degree of substitution to be optimal for most applications, yielding dextrans that typically are much more fluorescent than are the labeled dextrans available from other sources, thus permitting lower quantities to be used for intracellular tracing. It has been reported that some commercially available fluorescein isothiocyanate (FITC) dextrans yield spurious results in endocytosis studies because of the presence of free dye or metal contamination.ref To overcome this problem, Molecular Probes removes as much of the free dye as possible by a combination of precipitation, dialysis, gel filtration and other techniques. The fluorescent dextran is then assayed by thin-layer chromatography (TLC) to ensure that it is free of low molecular weight dyes. Molecular Probes prepares several unique products that have two or even three different labels, including our fluoro-ruby, mini-ruby and micro-ruby products, described below. Not all of the individual dextran molecules of these products are expected to have all the substituents, or to be equally fixable, particularly in conjugates of the lowest molecular weight dextrans.

Dextran Net Charge and Method of Substitution

The net charge on the dextran depends on the fluorophore and the method of preparing the conjugate. Molecular Probes prepares most of its dextrans by reacting a water-soluble amino dextran (D-1860, D-1861, D-1862, D-3330, D-7144) with the succinimidyl ester of the appropriate dye, rather than with isothiocyanate derivatives such as FITC. This method provides superior amine selectivity and yields an amide linkage, which is somewhat more stable than the corresponding thioureas formed from isothiocyanates. Except for the Rhodamine Green and Alexa Fluor 488 conjugates, once the dye has been added, the unreacted amines on the dextran are capped to yield a neutral or anionic dextran. In the case of the Rhodamine Green and Alexa Fluor 488 dextrans, the unreacted amines on the dextran are not capped after dye conjugation. Thus these dextran conjugates are either neutral or cationic. The Alexa Fluor, Cascade Blue, lucifer yellow, fluorescein, Oregon Green and 4',5'-dichloro-2',7'-dimethoxyfluorescein (JOE) dextrans are intrinsically anionic, whereas most of the dextrans labeled with the zwitterionic rhodamine B, tetramethylrhodamine and Texas Red dyes are essentially neutral. To produce more highly anionic dextrans, Molecular Probes has developed a proprietary procedure for adding negatively charged groups to the dextran carriers; these products are designated "polyanionic" dextrans.

Dextran Fixability

Some applications require that the dextran tracer be subsequently treated with formaldehyde or glutaraldehyde for analysis.ref For these applications, Molecular Probes offers "lysine-fixable" versions of most of our dextran conjugates of fluorophores or biotin. These dextrans have covalently bound lysine residues that permit dextran tracers to be conjugated to surrounding biomolecules by aldehyde-mediated fixation for subsequent detection by immunohistochemical and ultrastructural techniques. We have also shown that all of our Alexa Fluor dextran conjugates can be fixed with aldehyde-based fixatives.

Loading Cells with Dextrans and Subsequent Tissue Processing

Unless taken up by an endocytic process, dextran conjugates are membrane impermeant and usually must be loaded by relatively invasive techniques (Table 14.1). As with the lipophilic tracers in Section 14.4, crystals of the dextran conjugates have been successfully loaded by simply placing them directly on some kinds of samples.ref We have found our Influx pinocytic cell-loading reagent (I-14402, Section 20.8) to be particularly useful for loading dextrans into a variety of adherent and nonadherent cells.photo Sterile filtration of dextran solutions before use with live cells is highly recommended.ref Biotin and biotinylated biomolecules with molecular weights up to >100,000 daltons are taken up by some plant cells through an endocytic pathway.ref

Our lysine-fixable dextrans and Alexa Fluor dextrans can be fixed in place with formaldehyde or glutaraldehyde, allowing subsequent tissue processing, such as sectioning. A protocol has been published for embedding tissues in plastic for high-resolution characterization of neurons filled with lysine-fixable fluorescent dextrans.ref Fixation of biotinylated or fluorescent dextrans also permits the use of fluorescent- or enzyme-labeled conjugates of avidin and streptavidin (Section 7.5, Table 7.2) or of anti-dye antibodies (Section 7.3), respectively. These techniques can amplify the signal, which is important for detecting fine structure in sections or for changing the detection mode.ref We provide antibodies to the Alexa Fluor 488, Cascade Blue, lucifer yellow, fluorescein, BODIPY FL, tetramethylrhodamine and Texas Red fluorophores (Section 7.3). Our antibodies to fluorescein crossreact strongly with the Oregon Green dyes and somewhat with the Rhodamine Green fluorophore, and our anti-tetramethylrhodamine and anti–Texas Red antibodies crossreact with tetramethylrhodamine, Lissamine rhodamine B, Rhodamine Red and Texas Red dyes. Molecular Probes' ELF 97 Immunohistochemistry Kit and ELF 97 Cytological Labeling Kits (E-6600, E-6602, E-6603; Section 6.2) and several of our TSA (Tyramide Signal Amplification) Kits (Section 6.3) can be utilized directly with biotinylated dextrans or combined with antibodies to the fluorophore-labeled dextrans to further amplify the signal, making it possible to detect ultrafine structures accessible to these dextran conjugates. The NANOGOLD conjugates of secondary antibodies (N-24915, N-24916, N-24917; Section 7.2) and streptavidin (N-24918, Section 7.5) can be utilized to convert fluorescence detection of labeled dextrans in fixed-cell preparations to light microscopic visualization or, following silver enhancement with the LI Silver Enhancement Kit (L-24919, Section 7.2), detection by electron microscopy.

Photoconversion of neurons labeled with lysine-fixable fluorescent dextrans in the presence of diaminobenzidine (DAB) can be used to produce electron-dense products for electron microscopy.ref Electron-dense products can also be generated from peroxidase or colloidal gold conjugates of avidin, streptavidin or anti-dye antibodies.ref

Neuronal Tracing with Dextrans

Fluorescent and biotinylated dextrans are routinely employed to trace neuronal projections. Dextrans can function efficiently as anterograde or retrograde tracers,ref depending on the study method and tissue type used. Active transport of dextrans occurs only in live, not fixed tissue.ref Comparative studies of rhodamine isothiocyanate, rhodamine B dextran (D-1824) and lysinated tetramethylrhodamine dextran (fluoro-ruby, D-1817) have shown that the dextran conjugates produce less diffusion at injection sites and more permanent labeling than do the corresponding free dyes.ref Dextran conjugates with molecular weights up to 70,000 daltons have been employed as neuronal tracers in a wide variety of species. The availability of fluorescent dextran conjugates with different sizes and charges permitted the analysis of direction and rate of axonal transport in the squid giant axon.ref

Multilabeled Dextrans

Our fixable dextrans, most of which are lysinated dextrans (see the products marked by a single dagger (dagger) in Table 14.3), are generally preferred for neuronal tracing because they may transport more effectively and can be fixed in place with aldehydes after labeling. Molecular Probes prepares a number of multilabeled dextrans that are fixable, including some that have acquired the distinction of unique names in various publications:

Fluoro-ruby and fluoro-emerald photo have been extensively employed for retrograde and anterograde neuronal tracing,ref transplantation ref and cell-lineage tracing.ref Both products can be used to photoconvert DAB into an insoluble, electron-dense reaction product.ref Like fluoro-ruby and fluoro-emerald, micro-ruby and mini-ruby are brightly fluorescent, making it easy to visualize the electrode during the injection process. DiI (D-282, Section 14.4) or other lipophilic probes in Section 14.4 can be used to mark the sites of microinjection.ref In addition, because these dextrans include a covalently linked biotin, filled cells can be probed with standard enzyme-labeled avidin or streptavidin conjugates or with NANOGOLD streptavidin (Section 7.5, Table 7.2) to produce a permanent record of the experiment.ref Mini-ruby has proven useful for intracellular filling in fixed brain slices ref and has been reported to produce staining comparable to that achieved with lucifer yellow CH (L-453, L-12926; Section 14.3).ref Moreover, the use of mini-ruby in conjunction with standard peroxidase-mediated avidin–biotin methods does not cause co-conversion of lipofuscin granules found in adult human brain, a common problem during photoconversion of lucifer yellow CH.ref The lysine-fixable micro-emerald and mini-emerald — dextrans that are triply labeled with fluorescein, biotin and lysine — provide a contrasting color that is better excited by the argon-ion laser of confocal laser scanning microscopes; they have uses similar to micro-ruby and mini-ruby, respectively.

3000 MW Dextrans

The nominally 3000 MW dextrans offer several advantages over higher molecular weight dextrans, including faster axonal diffusion and greater access to peripheral cell processes.refphoto Our "3000 MW" dextran preparations contain polymers with molecular weight predominantly in the range of ~1500–3000 daltons, including the dye or other label. Our list of 3000 MW dextrans includes fluorescein, Rhodamine Green, tetramethylrhodamine, Texas Red and biotin conjugates. We also offer lysine-fixable 3000 MW dextrans that are simultaneously labeled with both fluorescein and biotin (micro-emerald, D-7156) or tetramethylrhodamine and biotin (micro-ruby, D-7162).

The 3000 MW fluorescein dextran and tetramethylrhodamine dextran photophoto (D-3306, D-3308) have been observed to readily undergo both anterograde and retrograde movement in live cells.ref These 3000 MW dextrans appear to passively diffuse within the neuronal process, as their intracellular transport is not effectively inhibited by colchicine or nocodazole, both of which disrupt active transport by depolymerizing microtubules.ref Moreover, these small dextrans diffuse at rates equivalent to those of smaller tracers such as sulforhodamine 101 and biocytin (~2 millimeters/hour at 22°C) and about twice as fast as 10,000 MW dextrans. The relatively low molecular weight of the dextrans may result in transport of some labeled probes through gap junctions (see below). By use of anti-tetramethylrhodamine (A-6397, Section 7.3) and peroxidase–anti-peroxidase complex staining, the signal from tetramethylrhodamine-conjugated dextrans can be detected in the fine dendrite configuration of cortical projection neurons.ref

NeuroTrace BDA-10,000 Neuronal Tracer Kit

Designed for both the first-time user and the experienced neuroscientist, our NeuroTrace BDA-10,000 Neuronal Tracer Kit (N-7167) contains convenient amounts of each of the components required for neuroanatomical tracing using BDA methods,ref including:

The neuronal tracer BDA-10,000 is transported over long distances and fills fine processes bidirectionally, including boutons in the anterograde direction and dendritic structures in the retrograde direction.ref Two days to two weeks after BDA-10,000 is injected into the desired region of the brain, the brain tissue can be fixed and sectioned. BDA-10,000 can also be applied to cut nerves and allowed to transport. Following incubation with avidin–HRP and then DAB, the electron-dense DAB reaction product can be viewed by either light or electron microscopy ref (Figure 14.5). The NeuroTrace BDA-10,000 labeling method can be readily combined with other anterograde or retrograde labeling methods or with immunohistochemical techniques. BDA-10,000 is available as a separate product (D-1956), as are BDA derivatives with other molecular weights — BDA-3000 (D-7135),ref BDA-70,000 (D-1957) and BDA-500,000 (D-7142).

DNP-X Dextrans

Our lysine-fixable 3000 MW and 10,000 MW 2,4-dinitrophenyl-X dextrans (D-12982, D-12983) are expected to have applications similar to those of the BDA products. The nonfluorescent dinitrophenyl group can be detected in fixed-cell preparations by our polyclonal antibody to the DNP hapten (A-6430, Section 7.3) and developed with either fluorescent or electron dense conjugates of anti-rabbit IgG antibodies. DNP dextran conjugates are also useful for study of the immune response to this widely used hapten.ref

Cell Lineage Tracing with Dextrans

Fluorescent dextrans — particularly the fluorescein and rhodamine conjugates — have been used extensively for tracing cell lineage.ref In this technique, the dextran is microinjected into a single cell of the developing embryo, and the fate of that cell and its daughters can be followed in vivo.photo Examples using this method include studies of:

Developmental studies show that the lysinated fluorescent dextrans are also suitable for cell ablation studies, presumably through the generation of oxygen radicals.ref

The lysine-fixable tetramethylrhodamine and Texas Red dextran conjugates (Table 14.3) are most frequently cited for lineage tracing studies; they are often preferred over other conjugates because they have bright fluorescence and are relatively photostable. Our Alexa Fluor 546, Alexa Fluor 568 and Alexa Fluor 594 dextrans (D-22911, D-22912, D-22913) are likely to provide equal or superior performance as orange- to red-fluorescent polar tracers. As a second color, particularly in combination with the Texas Red dextrans, people have most often used our lysine-fixable fluorescein dextrans ref (e.g., D-3306, D-1820, D-1822). However, the photostability of fluorescein conjugates is not as high as that of the tetramethylrhodamine and Texas Red conjugates. Consequently, we recommend our green-fluorescent Alexa Fluor 488 dextran conjugate (D-22910) or, alternatively, our lysine-fixable Oregon Green 488 (D-7171, D-7173), Oregon Green 514 (D-7175) and Rhodamine Green (D-7153) dextran conjugates; see Figure 1.6, Figure 7.1 and Figure 11.1 for a comparison of the photostability of the Alexa Fluor 488, Oregon Green and Rhodamine Green dyes and fluorescein. The more photostable dextrans may also be less phototoxic in cells. While these fixable conjugates can be employed with long-term preservation of the tissue, some researchers prefer to co-inject a fluorescent, nonlysinated dextran along with a nonfluorescent, lysine-fixable biotin dextran (BDA, Table 14.3). The nonfluorescent BDA can then be fixed in place with aldehyde-based fixatives and probed with any of our fluorescent or enzyme-labeled streptavidin and avidin conjugates described in Section 7.5 (Table 7.2).

Vybrant Cell Lineage Tracing Kit

The Vybrant Cell Lineage Tracing Kit (V-22915) combines an aldehyde-fixable Cascade Blue dextran with the superior brightness of our orange-red–fluorescent Alexa Fluor 546 dye to permit the detection of widely differentiated cell lineages. The Cascade Blue dextran is first injected into the desired parent cells. After differentiation, the cells are fixed and the signal is amplified using a rabbit IgG antibody against the Cascade Blue dye and an Alexa Fluor 546 conjugate of goat anti-rabbit IgG as the detection reagent. The orange-red–fluorescent Alexa Fluor 546 dye can be viewed using filters appropriate for tetramethylrhodamine (Table 24.2). The Vybrant Cell Lineage Tracing Kit contains

High MW Dextran Conjugates

Our 500,000 and 2,000,000 MW fluorescent dextrans may be particularly useful for lineage tracing at early stages of development, although these biopolymers have lower water solubility and a greater tendency to precipitate or clog microinjection needles than our lower molecular weight dextrans. Some studies suggest that lower molecular weight dextrans may leak from blastomeres, complicating analysis. Injection of 2,000,000 MW fluorescein- and Texas Red dye–conjugated dextrans into separate cells of the two-cell stage zebrafish embryo allowed the construction of a fate map.ref The 500,000 MW and 2,000,000 MW dextrans are labeled with fluorescein, tetramethylrhodamine or Texas Red dyes or with biotin, and all contain aldehyde-fixable lysine groups. The nonfluorescent 500,000 MW amino dextran (D-7144) can be conjugated with the researcher's choice of amine-reactive reagents.

Caged Fluorophore Dextrans

Dextrans with caged fluorophores are of particular interest to developmental biologists, because they can be injected early in development when the cells are large, and then later activated with UV illumination when the cells of interest may be small or buried in tissue.refphoto A caged-fluorescein dextran conjugate has been used in this way to demonstrate lineage restriction boundaries in the early Drosophila embryo.ref Two-photon excitation has been used to photoactivate a caged fluorescein dextran at the two-cell stage of sea urchin embryo development.ref A 10,000 MW dextran conjugate of DMNB-caged fluorescein photo (D-3310) is available (Table 14.3), as is a DMNB-caged fluorescein dextran (D-7146) that has been further modified with both lysine and biotin to make it fixable, as well as detectable by avidin conjugates. See Chapter 17 for a discussion of caged probes and Section 14.3 for a description of our lower molecular weight caged tracers for microinjection.

Studying Intercellular Communication with Dextrans

The size of dextrans may be exploited to study connectivity between cells. Examples include studies of the passage of 3000 MW dextrans through plasmodesmata ref and modulation of gap junctional communication by transforming growth factor–beta1 and forskolin.ref However, the dispersion of molecular weights in our "3000 MW" dextran preparations, which contain polymers with total molecular weights predominantly in the range of ~1500–3000 daltons but may also contain molecules <1500 daltons, may complicate such analyses.

An important experimental approach to identifying cells that form gap junctions makes use of simultaneous introduction of the polar tracer lucifer yellow CH (~450 daltons) and a tetramethylrhodamine 10,000 MW dextran. Because low molecular weight tracers like lucifer yellow CH (L-453, L-12926; Section 14.3) pass through gap junctions and dextrans do not, the initially labeled cell exhibits red fluorescence, whereas cells connected through gap junctions have yellow fluorescence.ref This technique has been used to follow the loss of intercellular communication in adenocarcinoma cells,ref to show the re-establishment of communication during wound healing in Drosophila ref and to investigate intercellular communication at different stages in Xenopus embryos.ref Similar experiments could employ our lucifer yellow 10,000 MW dextran (D-1825) and a low molecular weight red-fluorescent tracer such as sulforhodamine 101 (S-359, Section 14.3) or a blue-fluorescent dye such as Cascade Blue hydrazide (C-687, Section 14.3).

Simultaneous loading of cells with two (or more) dextrans that differ in both their molecular weight and in the dye's fluorescence properties has been used to assess subcellular heterogeneities in the submicroscopic structure of cytoplasm.ref

Probing Membrane Permeability with Dextrans

Labeled dextrans are often used to investigate the exclusion or transfer of macromolecules across cell membranes. For example, fluorescent dextrans have been used to monitor the effectiveness of electroporation, a technique that produces pores in the cell membrane, thus providing a convenient method for introducing materials such as exogenous DNA. Fluorescein dextrans with molecular weights ranging from 4000 to 150,000 daltons were used to determine the effect of electroporation variables — pulse size, shape and duration — on plasma-membrane pore size in chloroplasts,ref red blood cells ref and fibroblasts.ref Fluorescence recovery after photobleaching (FRAP) techniques have been used to monitor nucleocytoplasmic transport of fluorescent dextrans of various molecular weights, allowing the determination of the size-exclusion limit of the nuclear pore membrane,ref as well as to study the effect of epidermal growth factor and insulin on the nuclear membrane and on nucleocytoplasmic transport.ref

Microinjected 3000 MW fluorescent dextrans concentrate in interphase nuclei of Drosophila embryos, whereas 40,000 MW dextrans remain in the cytoplasm and enter the nucleus only after breakdown of the nuclear envelope during prophase. This size-exclusion phenomenon was used to follow the cyclical breakdown and reformation of the nuclear envelope during successive cell divisions.ref Similarly, our 10,000 MW Calcium Green dextran conjugate (Section 20.4) was shown to diffuse across the nuclear membrane of isolated nuclei from Xenopus laevis oocytes, but the 70,000 MW and 500,000 MW conjugates could not.ref Significantly, depletion of nuclear Ca2+ stores by inositol 1,4,5-triphosphate (Ins 1,4,5-P3, I-3716; Section 18.2) or by calcium chelators (Section 20.8), blocked nuclear uptake of the 10,000 MW Calcium Green dextran conjugate but not entry of lucifer yellow CH. Our 3000 MW Calcium Green dextran conjugate (C-6765) is actively transported in adult nerve fibers over a significant distance and is retained in presynaptic terminals in a form that allows monitoring of presynaptic Ca2+ levels.ref Fluorescent dextrans with molecular weights up to 20,000 daltons are reported to be taken up by the feeding tubes of nematodes but 40,000 MW and 70,000 MW dextrans are not.ref

Following Endocytosis and Fusion with Dextrans

Fluorescence of some of the dyes that Molecular Probes uses to prepare its dextran conjugates is sensitive to the pH of the medium (Chapter 21). Consequently, internalization of labeled dextrans into acidic organelles of cells can often be tracked by measuring changes in the fluorescence of the dye.ref Fluorescence of fluorescein-labeled dextrans is strongly quenched upon acidification (Figure 21.2); however, fluorescein's lack of a spectral shift in acidic solution makes it difficult to discriminate between internalized probe that is quenched and the residual fluorescence of the external medium. Dextran conjugates that either shift their emission spectra, such as the SNARF and SNAFL dextrans, or undergo significant shifts of their excitation spectra, such as BCECF, Oregon Green, HPTS and 4',5'-dichloro-2',7'-dimethoxyfluorescein (JOE) dextrans, are much more useful for following the internalization by ratio imaging (Loading and Calibration of Intracellular Ion Indicators). Our pH indicator conjugates and their optical responses are described in Section 21.4.

Discrimination of internalized fluorescent dextrans from external dextrans can be improved by adding a reagent that quenches the fluorescence of the external probe. For example, our anti-dye antibodies (Section 7.3) usually quench the fluorescence of their conjugate dyes and may be useful for discriminating between externally bound dextrans and internalized dextrans. Trypan blue can also be used as a quencher for some of the external dextran conjugates. Fluorescent dextrans can also be encapsulated in liposomes.ref Using Texas Red– and fluorescein-labeled dextrans encapsulated in liposomes, researchers have obtained evidence that antigen processing occurs within dense lysosomes, rather than in earlier endocytic compartments.ref Researchers have also used liposome-encapsulated fluorescent dextrans to investigate liposome fusion with isolated nuclei ref and the effect of additives on vesicle size.ref Intracellular fusion of endosomes has been followed by using the fluorescence enhancement of BODIPY FL avidin (A-2641, Section 7.5) that occurs when it complexes with a biotinylated dextran.ref

Tracing Fluid Transport with Dextrans

Fluorescent dextrans are important tools for studying the hydrodynamic properties of the cytoplasmic matrix. The intracellular mobility of these fluorescent tracers can be investigated using fluorescence recovery after photobleaching (FRAP) techniques. We offer a range of dextran sizes, thus providing a variety of hydrodynamic radii for investigating both the nature of the cytoplasmic matrix and the permeability of the surrounding membrane. Because of their solubility and biocompatibility, fluorescent dextrans have been used to monitor in vivo tissue permeability and flow in the uveoscleral tract,ref capillaries ref and proximal tubules,ref as well as diffusion of high molecular weight substances in the brain's extracellular environment.ref Fluorescent dextrans have also been used to assess permeability of the blood–brain barrier ref and to monitor blood flow.ref

Our 10,000 MW DMNB-caged fluorescein dextran photo (D-3310) and the corresponding triple-labeled DMNB-caged fluorescein, biotin and lysine dextran (D-7146) are fluorescent only after UV photolysis,ref enabling researchers to conduct photoactivation of fluorescence (PAF) experiments analogous to FRAP experiments in which the fluorophore is photoactivated upon illumination rather than bleached. Measuring the bright signal of the photoactivated fluorophore against a dark background should be intrinsically more sensitive than measuring a dark (photobleached) region against a bright field. In a collaboration with Walter Lempert of Princeton University, we have shown caged fluorescein dextran (D-3310) and caged HPTS (D-7060, Section 14.3) to be excellent probes for tracing vortices in water using a technique called photoactivated nonintrusive tracking of molecular motion (PHANTOMM) ref (Figure 14.6). Furthermore, diffusional coupling between dendritic spines and shafts was measured both by FRAP experiments with fluorescein dextran and by PAF experiments with DMNB-caged fluorescein dextran.ref DMNB-caged fluorescein was also employed to evaluate a system that combined confocal laser scanning microscopy with local photolysis of caged compounds.ref

Our Bibliography of Dextran Applications

Our bibliography of dextran applications (Bibliography for D-8998) contains over 950 references. It includes references in which dextrans from several different sources were used. Because the source, molecular weight of the dextran, net charge, degree of substitution and nature of the dye may significantly affect the application, the methods described in this section and in the references in our bibliography should be considered guides rather than definitive protocols. In most cases, however, our fluorescent dextrans are much brighter and have higher negative charge than dextrans available from other sources. Furthermore, we use rigorous methods for removing as much unconjugated dye as practical, and then assay our dextran conjugates by thin-layer chromatography to ensure the absence of low molecular weight contaminants.

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