Section 14.5 - Fluorescent and Biotinylated Dextrans · 14.5 Fluorescent and Biotinylated Dextrans...

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14.5 Fluorescent and Biotinylated DextransDextrans are hydrophilic polysaccharides characterized by

their moderate to high molecular weight, good water solubilityand low toxicity. They are widely used as both anterograde andretrograde tracers in neurons 1,2 and for numerous other applica-tions. Dextrans are biologically inert due to their uncommonpoly-(α-D-1,6-glucose) linkages, which render them resistant tocleavage by most endogenous cellular glycosidases. They alsousually have low immunogenicity. Molecular Probes offers al-most 100 fluorescent and biotinylated dextran conjugates inseveral molecular weight ranges.

Properties of Our Dextran Conjugates

A Wide Selection of SubstituentsMolecular Probes’ dextrans are conjugated to biotin or a wide

variety of fluorophores, including five of our Alexa Fluor dyes(Table 14.4). In particular, we would like to highlight the dextranconjugates of our Alexa Fluor 488, Oregon Green and Rhoda-mine Green dyes, which are significantly brighter and morephotostable than most fluorescein dextrans. Dextran-conjugatedfluorescent indicators for calcium and magnesium ions (Section

20.4) and for pH (Section 21.4) are described with their corre-sponding ion indicators in other chapters.

Dextran SizeMolecular Probes’ dextrans include those with nominal molec-

ular weights (MW) of 3000; 10,000; 40,000; 70,000; 500,000;and 2,000,000 daltons (Table 14.4). Because unlabeled dextransare polydisperse — and may become more so during the chemicalprocesses required for their modification and purification — theactual molecular weights present in a particular sample may havea broad distribution. For example, our “3000 MW” dextran prepa-rations contain polymers with molecular weights predominantlyin the range of ~1500–3000 daltons, including the dye or otherlabel.

Degree of Substitution of Our DextransDextrans from other commercial sources usually have a degree

of substitution of 0.2 or fewer dye molecules per dextran mole-cule for dextrans in the 10,000 MW range. Our dextrans, how-ever, typically contain 0.3–0.7 dyes per dextran in the 3000 MWrange, 0.5–2 dyes per dextran in the 10,000 MW range, 2–4 dyes

Section 14.5

D-7756 1,1′-dilinoleyl-3,3,3′,3′-tetramethylindocarbocyanine, 4-chlorobenzenesulfonate(FAST DiI™ solid; DiI∆9,12-C18(3), CBS) ................................................................................................................................................................ 5 mg

D-3899 1,1′-dilinoleyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (FAST DiI™ oil; DiI∆9,12-C18(3), ClO4) .............................................................. 5 mgD-7779 1,1′-dioctadecyl-5,5′-diphenyl-3,3,3′,3′-tetramethylindocarbocyanine chloride (5,5′-Ph2-DiIC18(3)) ................................................................... 5 mgD-7778 3,3′-dioctadecyl-5,5′-di(4-sulfophenyl)oxacarbocyanine, sodium salt (SP-DiOC18(3)) ......................................................................................... 5 mgD-7777 1,1′-dioctadecyl-6,6′-di(4-sulfophenyl)-3,3,3′,3′-tetramethylindocarbocyanine (SP-DiIC18(3)) ............................................................................ 5 mgD-275 3,3′-dioctadecyloxacarbocyanine perchlorate (‘DiO’; DiOC18(3)) .......................................................................................................................... 100 mgD-7776 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine-5,5′-disulfonic acid (DiIC18(3)-DS) ................................................................................ 5 mgD-282 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (‘DiI’; DiIC18(3)) .......................................................................................... 100 mgD-3911 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (‘DiI’; DiIC18(3)) *crystalline* ..................................................................... 25 mgD-7757 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt

(‘DiD’ solid; DiIC18(5) solid) .................................................................................................................................................................................. 10 mgD-12730 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine-5,5′-disulfonic acid (DiIC18(5)-DS) ............................................................................. 5 mgD-307 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate (‘DiD’ oil; DiIC18(5) oil) ............................................................................ 25 mgD-12731 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide (‘DiR’; DiIC18(7)) .............................................................................................. 10 mgD-3886 1,1′-dioleyl-3,3,3′,3′-tetramethylindocarbocyanine methanesulfonate (∆9-DiI) .................................................................................................... 25 mgL-7781 Lipophilic Tracer Sampler Kit ............................................................................................................................................................................... 1 kitN-22883 NeuroTrace™ CM-DiI tissue-labeling paste .......................................................................................................................................................... 100 mgN-22882 NeuroTrace™ DiD tissue-labeling paste ................................................................................................................................................................ 500 mgN-22880 NeuroTrace™ DiI tissue-labeling paste ................................................................................................................................................................. 500 mgN-22881 NeuroTrace™ DiO tissue-labeling paste ................................................................................................................................................................ 500 mgN-22884 NeuroTrace™ Multicolor Tissue-Labeling Kit *DiO, DiI, DiD pastes, 500 mg each* ............................................................................................. 1 kitO-246 octadecyl rhodamine B chloride (R18) ................................................................................................................................................................. 10 mgT-3163 N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide (FM® 1-43) ............................................................................. 1 mgT-1111 N-(3-triethylammoniumpropyl)-4-(4-(4-(diethylamino)phenyl)butadienyl)pyridinium dibromide (RH 414) ......................................................... 5 mgT-3166 N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide (FM® 4-64) .................................................... 1 mgT-13320 N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide

(FM® 4-64) *special packaging* .......................................................................................................................................................................... 10 x 100 µgT-23360 N-(3-trimethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide (FM® 5-95) ................................................. 1 mgV-22888 Vybrant® CM-DiI cell-labeling solution ................................................................................................................................................................. 1 mLV-22887 Vybrant® DiD cell-labeling solution ...................................................................................................................................................................... 1 mLV-22885 Vybrant® DiI cell-labeling solution ........................................................................................................................................................................ 1 mLV-22886 Vybrant® DiO cell-labeling solution ...................................................................................................................................................................... 1 mLV-22889 Vybrant® Multicolor Cell-Labeling Kit *DiO, DiI, DiD solutions, 1 mL each* ........................................................................................................ 1 kit

Cat # Product Name Unit Size

582 Chapter 14 — Fluorescent Tracers of Cell Morphology and Fluid Flow www.probes.com

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’slabel. If too many fluorophores are conjugated to the dextranmolecule, quenching and undesired interactions with cellularcomponents may occur. We have found our degree of substitutionto be optimal for most applications, yielding dextrans that aretypically much more fluorescent than the labeled dextrans avail-able from other sources, thus permitting lower quantities to beused for intracellular tracing. It has been reported that somecommercially available fluorescein isothiocyanate (FITC) dex-trans yield spurious results in endocytosis studies because of thepresence of free dye or metal contamination.3,4 To overcome thisproblem, Molecular Probes removes as much of the free dye aspossible by a combination of precipitation, dialysis, gel filtrationand other techniques. The fluorescent dextran is then assayed bythin-layer chromatography (TLC) to ensure that it is free of lowmolecular weight dyes. Molecular Probes prepares several uniqueproducts that have two or even three different labels, includingour fluoro-ruby, mini-ruby and micro-ruby products, describedbelow. Not all of the individual dextran molecules of these prod-ucts are expected to have all the substituents, or to be equallyfixable, particularly in conjugates of the lowest molecular weightdextrans.

Table 14.4 Molecular Probes’ selection of dextran conjugates.

Label(s)(Absorption/Emission Maxima) *

3000 MW 10,000 MW 40,000 MW 70,000 MW 500,000 MW 2,000,000 MW

Cascade Blue (400/420) D-7132 † D-1976 †

Lucifer yellow (428/532) D-1825 †

Alexa Fluor 488 (494/521) D-22910 ‡

Oregon Green 488 (496/524) D-7170, D-7171 † D-7172, D-7173 †

Fluorescein (494/518) D-3305, D-3306 † D-1821, D-1820 † D-1844, D-1845 † D-1823, D-1822 † D-7136 † D-7137 †

Fluorescein + biotin (494/518) D-7156 † D-7178 †

DMNB-caged fluorescein (494/518) § D-3310

DMNB-caged fluorescein + biotin(494/518) §

D-7146 †

Rhodamine Green (502/527) D-7163 D-7153 †

BODIPY FL (505/513) D-7168 ‡

Oregon Green 514 (511/530) D-7175 † D-7176

Tetramethylrhodamine (555/580) D-3307, D-3308 † D-1816, D-1817, †D-1868 ‡

D-1842 D-1819, D-1818 † D-7139 †

Alexa Fluor 546 (556/573) D-22911 ‡

Tetramethylrhodamine + biotin(555/580)

D-7162 † D-3312 †

Rhodamine B (570/590) D-1824 D-1841

Alexa Fluor 568 (578/603) D-22912 ‡

Alexa Fluor 594 (590/617) D-22913 ‡

Texas Red (595/615) D-3329, D-3328 † D-1828, D-1863 † D-1829 D-1830, D-1864 †

Alexa Fluor 647 (650/668) D-22914 ‡

Biotin (<300/none) D-7135 † D-1956 † D-1957 † D-7142 †

Amino (<300/none) D-3330 D-1860 D-1861 D-1862 D-7144

* Approximate absorption and emission maxima, in nm, for conjugates. † “Lysine-fixable” dextrans contain lysines and can therefore be fixed in place with formaldehyde orglutaraldehyde. ‡ “Fixable” dextrans contain free amines (but not lysines) and can be fixed in place with formaldehyde or glutaraldehyde. § Absorption and emission maximaare for the conjugate following photolysis.

Dextran Net Charge and Method of SubstitutionThe net charge on the dextran depends on the fluorophore and

the method of preparing the conjugate. Molecular Probes preparesmost of its dextrans by reacting a water-soluble amino dextran(D-1860, D-1861, D-1862, D-3330, D-7144) with the succinim-idyl ester of the appropriate dye, rather than reacting a nativedextran with isothiocyanate derivatives such as FITC. This meth-od provides superior amine selectivity and yields an amide link-age, which is somewhat more stable than the corresponding thio-ureas formed from isothiocyanates. Except for the RhodamineGreen and Alexa Fluor 488 conjugates, once the dye has beenadded, the unreacted amines on the dextran are capped to yield aneutral or anionic dextran. In the case of the Rhodamine Greenand Alexa Fluor 488 dextrans, the unreacted amines on the dex-tran are not capped after dye conjugation. Thus, these dextranconjugates may be either neutral, anionic or cationic. The AlexaFluor, Cascade Blue, lucifer yellow, fluorescein and OregonGreen dextrans are intrinsically anionic, whereas most of thedextrans labeled with the zwitterionic rhodamine B, tetramethyl-rhodamine and Texas Red dyes are essentially neutral. To producemore highly anionic dextrans, Molecular Probes has developed aproprietary procedure for adding negatively charged groups to the

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dextran carriers; these products are designated “polyanionic”dextrans.

Dextran FixabilitySome applications require that the dextran tracer be subsequent-

ly treated with formaldehyde or glutaraldehyde for analysis.5,6 Forthese applications, Molecular Probes offers “lysine-fixable” ver-sions of most of our dextran conjugates of fluorophores or biotin.These dextrans have covalently bound lysine residues that permitdextran tracers to be conjugated to surrounding biomolecules byaldehyde-mediated fixation for subsequent detection by immuno-histochemical and ultrastructural techniques. We have also shownthat all of our Alexa Fluor dextran conjugates can be fixed withaldehyde-based fixatives.

Loading Cells with Dextrans and SubsequentTissue Processing

Unless taken up by an endocytic process, dextran conjugatesare membrane impermeant and usually must be loaded by rela-tively invasive techniques (Table 14.1). As with the lipophilictracers in Section 14.4, crystals of the dextran conjugates havebeen successfully loaded by simply placing them directly onsome kinds of samples.7 We have found our Influx pinocytic cell-loading reagent (I-14402, Section 14.3, Section 20.8) to be partic-ularly useful for loading dextrans into a variety of adherent andnonadherent cells (Figure 20.94). Sterile filtration of dextransolutions before use with live cells is highly recommended.8

Biotin and biotinylated biomolecules with molecular weights upto >100,000 daltons are taken up by some plant cells through anendocytic pathway.9,10

Our lysine-fixable dextrans and Alexa Fluor dextrans can befixed in place with formaldehyde or glutaraldehyde, allowingsubsequent tissue processing, such as sectioning. A protocol hasbeen published for embedding tissues in plastic for high-resolu-tion characterization of neurons filled with lysine-fixable fluores-cent dextrans.11 Fixation of biotinylated or fluorescent dextransalso permits the use of fluorescent- or enzyme-labeled conjugatesof avidin and streptavidin (Section 7.6, Table 7.17) or of anti-dyeantibodies (Section 7.4, Table 7.13), respectively. These tech-niques can amplify the signal, which is important for detectingfine structure in sections or for changing the detection mode.12

We provide antibodies to the Alexa Fluor 488, Cascade Blue,lucifer yellow, fluorescein, BODIPY FL, tetramethylrhodamineand Texas Red fluorophores and to the 2,4-dinitrophenyl (DNP)and nitrotyrosine haptens (Section 7.4). Our antibodies to fluores-cein crossreact strongly with the Oregon Green dyes and some-what with the Rhodamine Green fluorophore, and our anti-tetra-methylrhodamine and anti–Texas Red antibodies crossreact withtetramethylrhodamine, Lissamine rhodamine B, Rhodamine Redand Texas Red dyes. Molecular Probes’ ELF 97 Immunohisto-chemistry Kit and ELF 97 Cytological Labeling Kits (E-6600,E-6603; Section 6.3) and several of our TSA (Tyramide Signal-Amplification) Kits (Section 6.2) can be utilized directly withbiotinylated dextrans or combined with antibodies to the fluoro-phore-labeled dextrans to further amplify the signal, making itpossible to detect ultrafine structures accessible to these dextranconjugates. The NANOGOLD and Alexa Fluor FluoroNanogold

conjugates of secondary antibodies (Section 7.3) and streptavidin(Section 7.6) can be utilized to allow detection of labeled dextransin fixed-cell preparations by light microscopy or, following silverenhancement with the LI Silver Enhancement Kit (L-24919,Section 7.3), by electron microscopy.

Photoconversion of neurons labeled with lysine-fixable fluo-rescent dextrans in the presence of diaminobenzidine (DAB)using our Diaminobenzidine (DAB) Histochemistry Kits (Section7.3, Section 7.6) can be used to produce electron-dense productsfor electron microscopy 13 (see Fluorescent Probes for Photocon-version of Diaminobenzidine Reagents in Section 1.5). Electron-dense products can also be generated from peroxidase or colloidalgold conjugates of avidin, streptavidin or anti-dye antibodies.14

Neuronal Tracing with Dextrans

Fluorescent and biotinylated dextrans are routinely employedto trace neuronal projections. Dextrans can function efficiently asanterograde or retrograde tracers,2 depending on the study meth-od and tissue type used. Active transport of dextrans occurs onlyin live, not fixed tissue.15 Comparative studies of rhodamineisothiocyanate, rhodamine B dextran (D-1824) and lysinatedtetramethylrhodamine dextran (fluoro-ruby, D-1817) have shownthat the dextran conjugates produce less diffusion at injectionsites and more permanent labeling than do the corresponding freedyes.5 Dextran conjugates with molecular weights up to 70,000daltons have been employed as neuronal tracers in a wide varietyof species. The availability of fluorescent dextran conjugates withdifferent sizes and charges permitted the analysis of direction andrate of axonal transport in the squid giant axon.16

Multilabeled DextransOur fixable dextrans, most of which are lysinated dextrans

(see the products marked by a single dagger (†) in Table 14.4),are generally preferred for neuronal tracing because they maytransport more effectively and can be fixed in place with alde-hydes after labeling. Molecular Probes prepares a number ofmultilabeled dextrans that are fixable, including some that haveacquired the distinction of unique names in various publications:

• Fluoro-ruby 5,13,17–22 — a red-orange–fluorescent, aldehyde-fixable 10,000 MW dextran labeled with both tetramethyl-rhodamine and lysine (D-1817; Figure 14.63). 3000 MW,70,000 MW and 2,000,000 MW versions of fluoro-ruby arealso available (D-3308, D-1818, D-7139).

• Fluoro-emerald 18,22,23 — a green-fluorescent, aldehyde-fixable10,000 MW dextran labeled with both fluorescein and lysine(D-1820; Figure 14.64, Figure 14.65, Figure 14.66). 3000MW, 40,000 MW, 70,000 MW, 500,000 MW and 2,000,000MW versions of fluoro-emerald are also available 24,25

(D-3306, D-1845, D-1822, D-7136, D-7137).• Micro-ruby (D-7162) and mini-ruby 26–30 (D-3312) —

red-orange–fluorescent, aldehyde-fixable 3000 MW and10,000 MW dextrans simultaneously labeled with tetra-methylrhodamine, biotin and lysine.

• Micro-emerald (D-7156) and mini-emerald (D-7178) —green-fluorescent, aldehyde-fixable dextrans simultaneouslylabeled with fluorescein, biotin and lysine.

Section 14.5


• Biotinylated dextran amine (BDA) 1,8,31–38 — nonfluorescent, aldehyde-fixable dex-trans simultaneously labeled with both biotin and lysine. These are available in severalmolecular weights (D-1956, D-1957, C-7135, D-7142; Table 14.4). A useful reviewhas been published on the BDA products.39

Fluoro-ruby and fluoro-emerald (Figure 14.66) have been extensively employed forretrograde and anterograde neuronal tracing,5 transplantation 20 and cell-lineage trac-ing.2,19,21,22,40 Both products can be used to photoconvert DAB into an insoluble, electron-dense reaction product.13 Like fluoro-ruby and fluoro-emerald, micro-ruby and mini-rubyare brightly fluorescent, making it easy to visualize the electrode during the injectionprocess. DiI (D-282, Section 14.4) or other lipophilic probes in Section 14.4 can be usedto mark the sites of microinjection.41 In addition, because these dextrans include a co-valently linked biotin, filled cells can be probed with standard enzyme-labeled avidin orstreptavidin conjugates or with NANOGOLD and FluoroNanogold streptavidin (Section7.6) to produce a permanent record of the experiment.42 Mini-ruby has proven useful forintracellular filling in fixed brain slices 27 and has been reported to produce stainingcomparable to that achieved with lucifer yellow CH 26 (L-453, L-682, L-1177, L-12926;Section 14.3). Moreover, the use of mini-ruby in conjunction with standard peroxidase-mediated avidin–biotin methods does not cause co-conversion of lipofuscin granulesfound in adult human brain, a common problem during photoconversion of lucifer yellowCH.26 The lysine-fixable micro-emerald and mini-emerald — dextrans that are triplylabeled with fluorescein, biotin and lysine — provide a contrasting color that is betterexcited by the argon-ion laser of confocal laser-scanning microscopes; they have usessimilar to micro-ruby and mini-ruby, respectively.

3000 MW DextransThe nominally 3000 MW dextrans offer several advantages over higher molecular

weight dextrans, including faster axonal diffusion and greater access to peripheral cellprocesses 24 (Figure 14.67). Our “3000 MW” dextran preparations contain polymers withmolecular weight predominantly in the range of ~1500–3000 daltons, including the dyeor other label. Our list of 3000 MW dextrans includes fluorescein, Rhodamine Green,tetramethylrhodamine, Texas Red and biotin conjugates. We also offer lysine-fixable3000 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 (D-3306,D-3308; Figure 14.68, Figure 14.69) have been observed to readily undergo both antero-grade and retrograde movement in live cells.18,24 These 3000 MW dextrans appear topassively diffuse within the neuronal process, as their intracellular transport is not effec-tively inhibited by colchicine or nocodazole, both of which disrupt active transport bydepolymerizing microtubules.24 Moreover, these small dextrans diffuse at rates equivalentto those of smaller tracers such as sulforhodamine 101 and biocytin (~2 millimeters/hourat 22°C) and about twice as fast as 10,000 MW dextrans. The relatively low molecularweight of the dextrans may result in transport of some labeled probes through gap junc-tions (see below). By use of anti-tetramethylrhodamine antibodies (A-6397, Section 7.4)and peroxidase–anti-peroxidase complex staining, the signal from tetramethylrhodamine-conjugated dextrans can be detected in the fine dendrite configuration of cortical projec-tion neurons.12

NeuroTrace BDA-10,000 Neuronal Tracer KitDesigned for both the first-time user and the experienced neuroscientist, our Neuro-

Trace BDA-10,000 Neuronal Tracer Kit (N-7167) contains convenient amounts of eachof the components required for neuroanatomical tracing using BDA methods,39 including:

• Lysine-fixable, biotinylated 10,000 MW dextran amine (BDA-10,000)• Avidin–horseradish peroxidase (avidin–HRP)• 3,3′-Diaminobenzidine (DAB)• A rigorously tested protocol that ensures fast and simple tracing experiments

The neuronal tracer BDA-10,000 is transported over long distances and fills fineprocesses bidirectionally, including boutons in the anterograde direction and dendritic

Figure 14.64 Lineage tracing of three dorsal blas-tomeres of 32-cell Xenopus laevis embryos inject-ed with Molecular Probes’ fluorescent dextran con-jugates. The tier 1 dorsal (A1) blastomere wasinjected with 10,000 MW lysine-fixable, fluoresceindextran (D-1820), the tier 2 dorsal (B1) blastomerewith lysine-fixable Texas Red 10,000 MW dextran(D-1863), and the tier 3 dorsal (C3) blastomerewith lysine-fixable Cascade Blue 10,000 MW dex-tran (D-1976). Embryos were fixed in formalde-hyde, embedded and sectioned. The image on theleft shows a 13 µm–thick section of a stage 6 (32-cell) embryo fixed right after injection; this sectionexhibits significant autofluorescence due to thepresence of residual yolk. The image on the right isa stage 10 (early gastrula) embryo. Triple-exposurephotographs of the sectioned embryos were takenon a Zeiss Axiophot with a 10X objective. Imagescontributed by Marie Vodicka, Department of Mo-lecular and Cell Biology, University of California,Berkeley.

Figure 14.63 Fluorescence excitation and emissionspectra of tetramethylrhodamine-labeled 10,000molecular weight dextran (fluoro-ruby, D-1817) inpH 7.0 buffer.

Our Influx pinocytic cell-loading re-agent (I-14402, Section 20.8) is usefulfor loading dextran conjugates intosome live cells.

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structures in the retrograde direction.8,35–38 Two days to two weeks after BDA-10,000 isinjected 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 incu-bation with the avidin–HRP conjugate and then DAB, the electron-dense DAB reactionproduct can be viewed by either light or electron microscopy 39,42 (Figure 14.70). TheNeuroTrace BDA-10,000 labeling method can be readily combined with other antero-grade or retrograde labeling methods or with immunohistochemical techniques. BDA-10,000 is available as a separate product (D-1956), as are BDA derivatives with othermolecular weights — BDA-3000 43 (D-7135), BDA-70,000 (D-1957) and BDA-500,000(D-7142). A detailed protocol that utilizes our BDA-10,000 probe, streptavidin–HRPconjugate (S-911, Section 7.6) and tetramethylbenzidine to anterogradely label fineprocesses in neurons has been published.1

Cell Lineage Tracing with Dextrans

Fluorescent dextrans — particularly the fluorescein and rhodamine conjugates —have been used extensively for tracing cell lineage.21,44,45 In this technique, the dextran ismicroinjected into a single cell of the developing embryo, and the fate of that cell and itsdaughters can be followed in vivo (Figure 14.64). Examples using this method includestudies of:

• Dorsoventral axis determination in zebrafish mutants 46

• Early cell-fate commitment and lineage restrictions in developing zebrafish 47–49

• Lineage and dopamine phenotype in tadpole hypothalamus 50

• Migration of neural crest cells in Xenopus 51

• Neural crest cell fate in zebrafish 52

• Progeny tracing in the grasshopper neuroblast 8

Developmental studies show that the lysinated fluorescent dextrans are also suitablefor cell ablation studies, presumably through the generation of oxygen radicals.53

The lysine-fixable tetramethylrhodamine and Texas Red dextran conjugates (Table14.4) are most frequently cited for lineage tracing studies; they are often preferred overother conjugates because they have bright fluorescence and are relatively photostable.Our Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594 and Alexa Fluor 647 dextrans(D-22911, D-22912, D-22913, D-22914) are likely to provide equal or superior perfor-mance as orange- to red-fluorescent polar tracers. As a second color, particularly incombination with the Texas Red dextrans, people have most often used our lysine-fixablefluorescein dextrans (e.g., D-3306, D-1820, D-1822). However, the photostability offluorescein conjugates is not as high as that of the tetramethylrhodamine and Texas Redconjugates. Consequently, we recommend our green-fluorescent Alexa Fluor 488 dextranconjugate (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 conju-gates; see Figure 1.42, Figure 7.19 and Figure 11.8 for a comparison of the photostabilityof the Alexa Fluor 488, Oregon Green and Rhodamine Green dyes and fluorescein. Themore photostable dextrans may also be less phototoxic in cells. Although these fixableconjugates can be employed with long-term preservation of the tissue, some researchersprefer to co-inject a fluorescent, nonlysinated dextran along with a nonfluorescent,lysine-fixable biotin dextran (BDA, Table 14.4). The nonfluorescent BDA can then befixed in place with aldehyde-based fixatives and probed with any of our fluorescent orenzyme-labeled streptavidin and avidin conjugates described in Section 7.6 (Table 7.17).

Vybrant Cell Lineage Tracing KitThe Vybrant Cell Lineage Tracing Kit (V-22915) combines an aldehyde-fixable

Cascade Blue dextran with the superior brightness of our red-orange–fluorescent AlexaFluor 546 dye to permit the detection of widely differentiated cell lineages. The CascadeBlue dextran is first injected into the desired parent cells. After differentiation, the cellsare fixed and the signal is amplified using a rabbit IgG antibody against the CascadeBlue dye and an Alexa Fluor 546 conjugate of goat anti–rabbit IgG antibody as thedetection reagent (Figure 7.65). The red-orange–fluorescent Alexa Fluor 546 dye can be

Figure 14.65 Development of the leech centralnervous system into segments was characterizedwith the cell lineage tracer, 10,000 MW lysine-fix-able, fluorescein dextran (fluoro-emerald, D-1820).Fluoro-emerald was injected into a neuroectoder-mal cell of a leech embryo. The teloblast eventuallydifferentiated into five segmentally iterated ganglialabeled with fluoro-emerald (pseudocolored blue inthis image). Associated muscle fibers were identi-fied with a Lan 3-14 monoclonal antibody and vi-sualized with a Cy3 antibody (pseudocolored red).Nuclei were counterstained with TOTO-3 dye(T-3604, pseudocolored green). Image reprintedfrom the cover of Development 127 (4) (2000),used with the permission of The Company of Biol-ogists Ltd.

Figure 14.66 A whole-mount of the embryonicbrain of Xenopus laevis that has been double-labeled with our 10,000 MW lysine-fixable, fluores-cein dextran and tetramethylrhodamine dextran(fluoro-emerald, D-1820; fluoro-ruby, D-1817).The tetramethylrhodamine dextran was used to la-bel the neurons projecting from the retina, whereasthe fluorescein dextran was applied to the transect-ed spinal cord, thus allowing the detailed evalua-tion of the topological relationship of these twopopulations of neurons (Neurosci Lett 127, 150(1991)). Image contributed by Martina Manns andBernd Fritzsch, Department of Biomedical Scienc-es, Creighton University.

Section 14.5


viewed using filters appropriate for tetramethylrhodamine (Table 24.8). The Vybrant CellLineage Tracing Kit contains:

• Lysine-fixable, 10,000 MW, Cascade Blue dextran (3 vials)• Rabbit IgG anti–Cascade Blue antibody• Alexa Fluor 546 goat anti–rabbit IgG antibody conjugate• A detailed experimental protocol

High MW Dextran ConjugatesOur 500,000 and 2,000,000 MW fluorescent dextrans (Table 14.4) may be particularly

useful for lineage tracing at early stages of development, although these biopolymershave lower water solubility and a greater tendency to precipitate or clog microinjectionneedles than our lower molecular weight dextrans. Some studies suggest that lower mo-lecular weight dextrans may leak from blastomeres, complicating analysis. Injection of2,000,000 MW fluorescein- and Texas Red dye–conjugated dextrans into separate cellsof the two-cell stage zebrafish embryo allowed the construction of a fate map.54 The500,000 MW and 2,000,000 MW dextrans are labeled with fluorescein, tetramethyl-rhodamine or Texas Red dyes or with biotin, and all contain aldehyde-fixable lysinegroups. The nonfluorescent 500,000 MW aminodextran (D-7144) can be conjugated withthe researcher’s choice of amine-reactive reagents.

Caged Fluorophore DextransDextrans with caged fluorophores are of particular interest to developmental biolo-

gists, because they can be injected early in development when the cells are large, andthen later activated with UV illumination when the cells of interest may be small orburied in tissue 55–57 (Figure 17.7). A caged-fluorescein dextran conjugate has been usedin this way to demonstrate lineage restriction boundaries in the early Drosophila em-bryo.58 Two-photon excitation has been used to photoactivate a caged-fluorescein dextranat the two-cell stage of sea urchin embryo development.59 A 10,000 MW dextran conju-gate of DMNB-caged fluorescein (D-3310, Figure 17.7) is available (Table 14.4), as is aDMNB-caged fluorescein dextran (D-7146) that has been further modified with bothlysine and biotin to make it fixable, as well as detectable by avidin conjugates. See Chap-ter 17 for a discussion of caged probes and Section 14.3 for a description of our lowermolecular weight caged tracers for microinjection.

Studying Intercellular Communication with Dextrans

The size of dextrans may be exploited to study connectivity between cells. Examplesinclude studies of the passage of 3000 MW dextrans through plasmodesmata 25 and mod-ulation of gap junctional communication by transforming growth factor–β1 and forsko-lin.60 However, the dispersion of molecular weights in our “3000 MW” dextran prepara-tions, which contain polymers with total molecular weights predominantly in the range of~1500–3000 daltons but may also contain molecules <1500 daltons, may complicatesuch analyses.

An important experimental approach to identifying cells that form gap junctions makesuse of simultaneous introduction of the polar tracer lucifer yellow CH (~450 daltons) anda tetramethylrhodamine 10,000 MW dextran. Because low molecular weight tracers likelucifer yellow CH (L-453, L-12926; Section 14.3) pass through gap junctions and dex-trans do not, the initially labeled cell exhibits red fluorescence, whereas cells connectedthrough gap junctions have yellow fluorescence 60 (Figure 14.71) This technique has beenused to follow the loss of intercellular communication in adenocarcinoma cells,61 to showthe re-establishment of communication during wound healing in Drosophila 62 and toinvestigate intercellular communication at different stages in Xenopus embryos.63,64

Similar experiments could employ our lucifer yellow 10,000 MW dextran (D-1825) and alow molecular weight red-fluorescent tracer such as sulforhodamine 101 (S-359, Section14.3) 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 theirmolecular weight and in the dye’s fluorescence properties has been used to assess sub-cellular heterogeneities in the submicroscopic structure of cytoplasm.65

Figure 14.68 The attached eighth nerve from thevestibular labyrinth of a turtle, Pseudemys scripta,exposed to 3000 MW lysine-fixable, tetramethyl-rhodamine dextran (D-3308) and incubated withF-actin–specific BODIPY FL phallacidin (B-607).F-actin–labeled ciliary bundles were stained greenby the phallacidin, and the calyceal and boutonendings of their primary afferents were stained redby the dextran. This image is a projection of 40confocal images. Image contributed by Laura Di-Caprio and Ellengene Peterson, Department of Bio-logical Sciences, Ohio University.

Figure 14.67 Secondary motor neurons in a spinalcord whole mount of a male western mosquitofish(Gambusia affinis affinis) that have been labeledwith 3000 MW lysine-fixable, Texas Red dextran(D-3328). The dextran crystals were applied to thebipinnate inclinator muscles of the anal appendicu-lar support fin, and the dye was transported fromthe axons to cell body and dendrites. Motor neu-rons were visualized and photographed through abandpass optical filter appropriate for Texas Reddye, by epifluorescence microscopy. Image con-tributed by E. Rosa-Molinar, Department of Cell Bi-ology and Anatomy, University of Nebraska Medi-cal Center, and Bernd Fritzsch, Department ofBiomedical Sciences, Creighton University.

Caged fluorophore dextrans haveparticular utility for developmentalbiology studies. The fluorescence canbe “turned on” with UV light at anystage of development and the fluoro-phore becomes a hapten for antibodiesto the dye.

587

Probing Membrane Permeability with Dextrans

Labeled dextrans are often used to investigate the exclusion or transfer of macromole-cules across cell membranes. For example, fluorescent dextrans have been used to moni-tor the effectiveness of electroporation, a technique that produces pores in the cell mem-brane, thus providing a convenient method for introducing materials such as exogenousDNA. Fluorescein dextrans with molecular weights ranging from 4000 to 150,000 dal-tons were used to determine the effect of electroporation variables — pulse size, shapeand duration — on plasma-membrane pore size in chloroplasts,66 red blood cells 67 andfibroblasts.68 Fluorescence recovery after photobleaching (FRAP) techniques have beenused to monitor nucleocytoplasmic transport of fluorescent dextrans of various molecularweights, allowing the determination of the size-exclusion limit of the nuclear pore mem-brane,69–71 as well as to study the effect of epidermal growth factor and insulin on thenuclear membrane and on nucleocytoplasmic transport.72,73

Microinjected 3000 MW fluorescent dextrans concentrate in interphase nuclei ofDrosophila embryos, whereas 40,000 MW dextrans remain in the cytoplasm and enter thenucleus only after breakdown of the nuclear envelope during prophase. This size-exclu-sion phenomenon was used to follow the cyclical breakdown and reformation of the nucle-ar envelope during successive cell divisions.74 Similarly, our 10,000 MW Calcium Greendextran conjugate (C-3713, Section 20.4) was shown to diffuse across the nuclear mem-brane of isolated nuclei from Xenopus laevis oocytes, but the 70,000 MW and 500,000 MWconjugates could not.75 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 entryof lucifer yellow CH. Our 3000 MW Calcium Green dextran conjugate (C-6765) is active-ly transported in adult nerve fibers over a significant distance and is retained in presynap-tic terminals in a form that allows monitoring of presynaptic Ca2+ levels.76 Fluorescentdextrans with molecular weights up to 20,000 daltons are reported to be taken up by thefeeding tubes of nematodes, but 40,000 MW and 70,000 MW dextrans are not.77

Following Endocytosis and Fusion with Dextrans

Some of the dyes that Molecular Probes uses to prepare its dextran conjugates exhibitfluorescence that is sensitive to the pH of the medium (Chapter 21). Consequently, inter-nalization of labeled dextrans into acidic organelles of cells can often be tracked by mea-suring changes in the fluorescence of the dye.78,79 Fluorescence of fluorescein-labeleddextrans is strongly quenched upon acidification (Figure 21.2); however, fluorescein’s lackof a spectral shift in acidic solution makes it difficult to discriminate between internalizedprobe that is quenched and the residual fluorescence of the external medium. Dextranconjugates that either shift their emission spectra, such as the SNARF and SNAFL dex-trans, or undergo significant shifts of their excitation spectra, such as BCECF, OregonGreen and HPTS dextrans, are much more useful for following the internalization by ratioimaging (see Loading and Calibration of Intracellular Ion Indicators in Section 20.1). OurpH indicator conjugates and their optical responses are described in Section 21.4.

Discrimination of internalized fluorescent dextrans from external dextrans can beimproved by adding a reagent that quenches the fluorescence of the external probe. Forexample, our anti-dye antibodies (Section 7.4) usually quench the fluorescence of theircognate dyes and may be useful for discriminating between externally bound dextrans andinternalized dextrans. In addition, trypan blue can also be used as a quencher for some ofthe external dextran conjugates.

Fluorescent dextrans can also be encapsulated in liposomes.80–82 Using Texas Red–and fluorescein-labeled dextrans encapsulated in liposomes, researchers have obtainedevidence that antigen processing occurs within dense lysosomes, rather than in earlierendocytic compartments.83 Researchers have also used liposome-encapsulated fluorescentdextrans to investigate liposome fusion with isolated nuclei 84 and the effect of additives onvesicle size.85 Intracellular fusion of endosomes has been followed by using the fluores-cence enhancement of BODIPY FL avidin that occurs when it complexes with a biotiny-lated dextran 86 (Figure 16.24). We have found our Oregon Green 514 streptavidin(S-6369, Section 7.6) to have an over 15-fold increase in fluorescence intensity upon

Figure 14.69 The antero-ventral and antero-dor-sal lateral line nerves in an Ambystoma mexi-canum whole-mounted brain after labeling with3000 MW Rhodamine Green dextran (D-7163)and 3000 MW lysine-fixable, tetramethylrho-damine dextran (D-3308), respectively. In bothcases, the dextran was applied to the respectivecut cranial nerves. This photomicrograph showsthe four segregated ventral fascicles of the mech-anosensory lateral line fibers and the intermin-gling of the dorsal electrosensory fibers. Imagecontributed by Bernd Fritzsch, Department of Bio-medical Sciences, Creighton University.

Figure 14.70 A mitral cell in the olfactory bulb of achinook salmon that has been retrogradely labeledusing our NeuroTrace BDA-10,000 Neuronal TracerKit (N-7167).

Section 14.5

The combination of low molecularweight fluorescent dyes and dextransis extensively utilized to study gapjunction formation.


upon illumination rather than bleached (Figure 7.93). Measuringthe bright signal of the photoactivated fluorophore against a darkbackground should be intrinsically more sensitive than measuringa dark (photobleached) region against a bright field. In a collabo-ration with Walter Lempert of Princeton University, we haveshown caged fluorescein dextran (D-3310) to be an effectiveprobe for tracing vortices in water using a technique called photo-activated nonintrusive tracking of molecular motion (PHAN-TOMM).99 Furthermore, diffusional coupling between dendriticspines and shafts was measured both by FRAP experiments withfluorescein dextran and by PAF experiments with DMNB-cagedfluorescein dextran.100 DMNB-caged fluorescein was also em-ployed to evaluate a system that combined confocal laser-scan-ning microscopy with local photolysis of caged compounds.95

Our Bibliography of Dextran Applications

Our bibliography of dextran applications contains over 1100references. It includes references in which dextrans from severaldifferent sources were used. Because the source, molecularweight of the dextran, net charge, degree of substitution andnature of the dye may significantly affect the application, themethods described in this section and in the references in ourbibliography should be considered guides rather than definitiveprotocols. In most cases, however, our fluorescent dextrans aremuch brighter and have higher negative charge than dextransavailable from other sources. Furthermore, we use rigorous meth-ods for removing as much unconjugated dye as practical, andthen assay our dextran conjugates by thin-layer chromatographyto ensure the absence of low molecular weight contaminants.

References

1. J Neurosci Methods 109, 81 (2001); 2. Brain Res Bull 51, 11 (2000); 3. JCell Sci 96, 721 (1990); 4. J Cell Biol 105, 1981 (1987); 5. Brain Res 526,127 (1990); 6. Brain Res Bull 25, 139 (1990); 7. J Neurosci Methods 81, 169(1998); 8. J Neurosci 14, 5766 (1994); 9. Plant Physiol 98, 673 (1992);10. Plant Physiol 93, 1492 (1990); 11. Biotech Histochem 67, 153 (1992);12. J Neurosci Methods 65, 157 (1996); 13. J Histochem Cytochem 41, 777(1993); 14. Brain Res Bull 30, 115 (1993); 15. Trends Neurosci 13, 14 (1990);16. Proc Natl Acad Sci U S A 92, 11500 (1995); 17. Mol Biol Cell 6, 1491(1995); 18. J Neurosci Methods 53, 35 (1994); 19. Nature 363, 630 (1993);20. Proc Natl Acad Sci U S A 90, 1310 (1993); 21. Nature 344, 431 (1990);22. Dev Biol 109, 509 (1985); 23. J Cell Biol 128, 293 (1995); 24. J NeurosciMethods 50, 95 (1993); 25. Plant J 4, 567 (1993); 26. J Neurosci Methods 55,105 (1994); 27. Brain Res 608, 78 (1993); 28. J Neurosci Methods 74, 9(1997); 29. Glia 20, 145 (1997); 30. Hippocampus 8, 57 (1998); 31. J Neuros-ci 15, 5222 (1995); 32. J Neurosci 15, 5139 (1995); 33. J Neurosci Methods53, 23 (1994); 34. J Neurosci Methods 52, 153 (1994); 35. Brain Res 607, 47(1993); 36. J Neurosci Methods 48, 75 (1993); 37. J Neurosci Methods 45, 35(1992); 38. J Neurosci Methods 41, 239 (1992); 39. J Neurosci Methods 103,23 (2000); 40. Development 104 Suppl, 231 (1988); 41. Biophys J 75, 2558(1998); 42. Neuroscience Protocols, Wouterlood FG, Ed. 93-050-14, pp. 01–14 (1993); 43. J Neurosci Methods 76, 167 (1997); 44. Science 252, 569(1991); 45. Development 108, 581 (1990); 46. Nature 370, 468 (1994);47. Development 120, 483 (1994); 48. Science 265, 517 (1994); 49. Science261, 109 (1993); 50. J Neurosci 12, 1351 (1992); 51. Development 118, 363(1993); 52. Development 120, 495 (1994); 53. Dev Biol 120, 520 (1987);54. Nature 361, 451 (1993); 55. Dev Biol 201, 247 (1998); 56. Methods MolBiol 135, 349 (2000); 57. Biochem Cell Biol 75, 551 (1997); 58. Cell 68, 923(1992); 59. Dev Biol 175, 177 (1996); 60. J Neurosci 15, 262 (1995); 61. ProcNatl Acad Sci U S A 85, 473 (1988); 62. Dev Biol 127, 197 (1988); 63. J CellBiol 110, 115 (1990); 64. Dev Biol 129, 265 (1988); 65. J Immunol 157, 3396

Figure 14.71 Dual tracer technique for identification of gap junction–coupled cells. A) Cells are labeled with a mixture of a small polar tracersuch as lucifer yellow CH (green circles) and a relatively large tetrameth-ylrhodamine-labeled dextran (red circles). B) Adjoining gap junction–coupled cells are accessible to the low molecular weight tracer whereasthe much larger dextran conjugate is excluded. Coupled cells with sin-gle-color lucifer yellow CH labeling are readily distinguished from initiallylabeled cells with dual fluorescence.

A

B

binding free biotin, which may make it the preferred probe for thisapplication (Figure 16.24).

Tracing Fluid Transport with Dextrans

Fluorescent dextrans are important tools for studying thehydrodynamic properties of the cytoplasmic matrix. The intra-cellular mobility of these fluorescent tracers can be investigatedusing fluorescence recovery after photobleaching (FRAP) tech-niques. We offer a range of dextran sizes, thus providing a varietyof hydrodynamic radii for investigating both the nature of thecytoplasmic matrix and the permeability of the surroundingmembrane. Because of their solubility and biocompatibility,fluorescent dextrans have been used to monitor in vivo tissuepermeability and flow in the uveoscleral tract,87,88 capillaries 89,90

and proximal tubules,91 as well as diffusion of high molecularweight substances in the brain’s extracellular environment.92

Fluorescent dextrans have also been used to assess permeabilityof the blood–brain barrier 93 and to monitor blood flow.94

Our 10,000 MW DMNB-caged fluorescein dextran (D-3310,Figure 17.7) and the corresponding triple-labeled DMNB-cagedfluorescein, biotin and lysine dextran (D-7146) are fluorescentonly after UV photolysis,95–98 enabling researchers to conductphotoactivation of fluorescence (PAF) experiments analogous toFRAP experiments in which the fluorophore is photoactivated

589

(1996); 66. Biophys J 58, 823 (1990); 67. Bio-electrochem Bioenerg 20, 57 (1988); 68. Biotech-niques 6, 550 (1988); 69. J Cell Biol 102, 1183(1986); 70. EMBO J 3, 1831 (1984); 71. J BiolChem 258, 11427 (1983); 72. J Cell Biol 110, 559(1990); 73. Biochemistry 26, 1546 (1987);74. Biotechniques 17, 730 (1994); 75. Science270, 1835 (1995); 76. Neurosci Lett 258, 124(1998); 77. Parasitology 109, 249 (1994);

References — continued

78. FASEB J 8, 573 (1994); 79. J Cell Sci 105,861 (1993); 80. Agr Biol Chem 50, 399 (1986);81. FEBS Lett 179, 148 (1985); 82. J Cell Biol99, 1989 (1984); 83. Cell 64, 393 (1991);84. Biochemistry 26, 765 (1987); 85. Biochemis-try 29, 4582 (1990); 86. Biophys J 71, 487(1996); 87. Proc Natl Acad Sci U S A 85, 2315(1988); 88. Arch Ophthalmol 105, 844 (1987);89. Microvasc Res 36, 172 (1988); 90. Am J

Physiol 245, H495 (1983); 91. Am J Physiol 253,F366 (1987); 92. Biophys J 65, 2277 (1993);93. Pflugers Arch 427, 86 (1994); 94. J CerebBlood Flow Metab 13, 359 (1993); 95. Neuron15, 755 (1995); 96. Anal Chem 70, 2459 (1998);97. Biophys J 74, 3302 (1998); 98. AIAA Journal34, 449 (1996); 99. Exp Fluids 18, 249 (1995);100. Science 272, 716 (1996).

Section 14.5

Product List — 14.5 Fluorescent and Biotinylated Dextrans

Cat # Product Name Unit SizeD-22910 dextran, Alexa Fluor® 488; 10,000 MW, anionic, fixable ....................................................................................................................................... 5 mgD-22911 dextran, Alexa Fluor® 546; 10,000 MW, anionic, fixable ....................................................................................................................................... 5 mgD-22912 dextran, Alexa Fluor® 568; 10,000 MW, anionic, fixable ....................................................................................................................................... 5 mgD-22913 dextran, Alexa Fluor® 594; 10,000 MW, anionic, fixable ....................................................................................................................................... 5 mgD-22914 dextran, Alexa Fluor® 647; 10,000 MW, anionic, fixable ....................................................................................................................................... 2 mgD-3330 dextran, amino, 3000 MW .................................................................................................................................................................................... 100 mgD-1860 dextran, amino, 10,000 MW ................................................................................................................................................................................. 1 gD-1861 dextran, amino, 40,000 MW ................................................................................................................................................................................. 1 gD-1862 dextran, amino, 70,000 MW ................................................................................................................................................................................. 1 gD-7144 dextran, amino, 500,000 MW ............................................................................................................................................................................... 100 mgD-7135 dextran, biotin, 3000 MW, lysine fixable (BDA-3000) ........................................................................................................................................... 10 mgD-1956 dextran, biotin, 10,000 MW, lysine fixable (BDA-10,000) ..................................................................................................................................... 25 mgD-1957 dextran, biotin, 70,000 MW, lysine fixable (BDA-70,000) ..................................................................................................................................... 25 mgD-7142 dextran, biotin, 500,000 MW, lysine fixable (BDA-500,000) ................................................................................................................................. 10 mgD-7168 dextran, BODIPY® FL, 10,000 MW, fixable ........................................................................................................................................................... 5 mgD-7132 dextran, Cascade Blue®, 3000 MW, anionic, lysine fixable ................................................................................................................................... 10 mgD-1976 dextran, Cascade Blue®, 10,000 MW, anionic, lysine fixable ................................................................................................................................ 25 mgD-3310 dextran, DMNB-caged fluorescein, 10,000 MW, anionic ...................................................................................................................................... 5 mgD-7146 dextran, DMNB-caged fluorescein and biotin, 10,000 MW, lysine fixable ............................................................................................................. 5 mgD-3305 dextran, fluorescein, 3000 MW, anionic ............................................................................................................................................................... 10 mgD-3306 dextran, fluorescein, 3000 MW, anionic, lysine fixable ......................................................................................................................................... 10 mgD-1821 dextran, fluorescein, 10,000 MW, anionic ............................................................................................................................................................ 25 mgD-1820 dextran, fluorescein, 10,000 MW, anionic, lysine fixable (fluoro-emerald) ........................................................................................................... 25 mgD-1844 dextran, fluorescein, 40,000 MW, anionic ............................................................................................................................................................ 25 mgD-1845 dextran, fluorescein, 40,000 MW, anionic, lysine fixable ...................................................................................................................................... 25 mgD-1823 dextran, fluorescein, 70,000 MW, anionic ............................................................................................................................................................ 25 mgD-1822 dextran, fluorescein, 70,000 MW, anionic, lysine fixable ...................................................................................................................................... 25 mgD-7136 dextran, fluorescein, 500,000 MW, anionic, lysine fixable .................................................................................................................................... 10 mgD-7137 dextran, fluorescein, 2,000,000 MW, anionic, lysine fixable ................................................................................................................................. 10 mgD-7156 dextran, fluorescein and biotin, 3000 MW, anionic, lysine fixable (micro-emerald) ............................................................................................. 5 mgD-7178 dextran, fluorescein and biotin, 10,000 MW, anionic, lysine fixable (mini-emerald) ............................................................................................. 10 mgD-1825 dextran, lucifer yellow, 10,000 MW, anionic, lysine fixable .................................................................................................................................. 25 mgD-7170 dextran, Oregon Green® 488; 10,000 MW, anionic ............................................................................................................................................... 5 mgD-7171 dextran, Oregon Green® 488; 10,000 MW, anionic, lysine fixable ........................................................................................................................ 5 mgD-7172 dextran, Oregon Green® 488; 70,000 MW, anionic ............................................................................................................................................... 5 mgD-7173 dextran, Oregon Green® 488; 70,000 MW, anionic, lysine fixable ........................................................................................................................ 5 mgD-7175 dextran, Oregon Green® 514; 10,000 MW, anionic, lysine fixable ........................................................................................................................ 5 mgD-7176 dextran, Oregon Green® 514; 70,000 MW, anionic ............................................................................................................................................... 5 mgD-1824 dextran, rhodamine B, 10,000 MW, neutral .......................................................................................................................................................... 25 mgD-1841 dextran, rhodamine B, 70,000 MW, neutral .......................................................................................................................................................... 25 mgD-7163 dextran, Rhodamine Green™, 3000 MW .............................................................................................................................................................. 5 mgD-7153 dextran, Rhodamine Green™, 10,000 MW, lysine fixable ..................................................................................................................................... 10 mgD-3307 dextran, tetramethylrhodamine, 3000 MW, anionic .............................................................................................................................................. 10 mgD-3308 dextran, tetramethylrhodamine, 3000 MW, anionic, lysine fixable ........................................................................................................................ 10 mgD-1868 dextran, tetramethylrhodamine, 10,000 MW, anionic, fixable ............................................................................................................................... 25 mgD-1817 dextran, tetramethylrhodamine, 10,000 MW, lysine fixable (fluoro-ruby) ............................................................................................................ 25 mgD-1816 dextran, tetramethylrhodamine, 10,000 MW, neutral ........................................................................................................................................... 25 mgD-1842 dextran, tetramethylrhodamine, 40,000 MW, neutral ........................................................................................................................................... 25 mgD-1818 dextran, tetramethylrhodamine, 70,000 MW, lysine fixable .................................................................................................................................. 25 mgD-1819 dextran, tetramethylrhodamine, 70,000 MW, neutral ........................................................................................................................................... 25 mgD-7139 dextran, tetramethylrhodamine, 2,000,000 MW, lysine fixable ............................................................................................................................. 10 mgD-7162 dextran, tetramethylrhodamine and biotin, 3000 MW, lysine fixable (micro-ruby) ............................................................................................... 5 mg


14.6 FluoSpheres and TransFluoSpheresMicrospheres for Tracing

FluoSpheres and TransFluoSpheres polystyrene microspheres 1 satisfy several pre-requisites of ideal long-term biological tracers. Because the dyes in our microspheres areincorporated throughout the microsphere rather than just on its surface, the fluorescenceoutput per microsphere is significantly greater than that obtained from protein or dextranconjugates (Table 6.6) and is relatively immune to photobleaching and other environ-ment-dependent effects. FluoSpheres and TransFluoSpheres microspheres are also bio-logically inert and physically durable, and they are available from Molecular Probes witha large number of uniform sizes and surface properties. Furthermore, their spectral prop-erties can be freely manipulated during manufacture without altering their surface proper-ties. Our current carboxylate-modified fluorescent microspheres have much higher sur-face charges than were previously available, making them more hydrophilic and thusmore useful as tracers. The additional carboxylate groups also make it easier to couplethe microspheres to proteins and other biomolecules. See Section 6.5 for an extensivediscussion of the properties of our FluoSpheres and TransFluoSpheres polystyrene beads.Molecular Probes also makes numerous fluorescent microsphere products that are usefulas standards for imaging (Section 24.1) and flow cytometry (Section 24.2).

Availability of intensely fluorescent, highly uniform microspheres in different colorsand sizes permits diverse applications in tracking particles and cells, tracing fluid dynam-ics and amplifying signals. In addition, measuring the effect of various interventions onregional blood flow is an important quantitative application of fluorescent microspheres.Using a mixture of beads of different sizes, each labeled with a different fluorescent color,researchers can discriminate the size dependence of uptake or transport of microspheresin vivo in cells, capillaries, lung or other tissues.2 Our smallest microspheres can be mi-croinjected into cells (see below) or are actively taken up by phagocytosis (Section 16.1).

Fluorescent Microspheres for RegionalBlood Flow Studies

Relatively large radiolabeled microspheres (10–15 µm in diameter) have long beenused for regional blood flow studies in tissues and organs. However, fluorescent micro-spheres have been shown to be superior to radioactive microspheres in chronic bloodflow measurements.3 In most cases, the microspheres are injected at desired locations inthe circulatory system and eventually lodge in the capillaries, where they can later becounted in dissected tissue sections. To eliminate the hazards, expense and disposalproblems of the radiolabeled microspheres,4 researchers have turned to fluorescent andcolored microspheres for measuring myocardial and cortical blood flow.5–11 In addition,blood flow in brain cortex has been measured using much smaller microspheres (1.3 µm)by determining velocities of individual fluorescent beads with strobe epi-illumination.12

Blood flow measurements using fluorescent microspheres in other organs, including the

Figure 14.72 Normalized fluorescence emissionspectra of the dyes contained in FluoSpheres poly-styrene microspheres for blood flow determination,after extraction into 2-ethoxyethyl acetate (Cello-solve acetate). The eleven colors of fluorescent mi-crospheres represented are: 1) blue, 2) blue-green,3) green, 4) yellow-green, 5) yellow, 6) orange, 7)red-orange, 8) red, 9) carmine, 10) crimson and11) scarlet (Table 14.5).

Product List — 14.5 Fluorescent and Biotinylated Dextrans — continued

Molecular Probes’Microsphere Productsand Prices

The catalog numbers and prices ofall of our FluoSpheres microsphereproducts for tracing are listed in thetables of this section. Our completeline of luminescent microspheres isdiscussed in Section 6.5.

D-3312 dextran, tetramethylrhodamine and biotin, 10,000 MW, lysine fixable (mini-ruby) .............................................................................................. 10 mgD-3328 dextran, Texas Red®, 3000 MW, lysine fixable ..................................................................................................................................................... 10 mgD-3329 dextran, Texas Red®, 3000 MW, neutral ............................................................................................................................................................... 10 mgD-1863 dextran, Texas Red®, 10,000 MW, lysine fixable .................................................................................................................................................. 25 mgD-1828 dextran, Texas Red®, 10,000 MW, neutral ............................................................................................................................................................ 25 mgD-1829 dextran, Texas Red®, 40,000 MW, neutral ............................................................................................................................................................ 25 mgD-1864 dextran, Texas Red®, 70,000 MW, lysine fixable .................................................................................................................................................. 25 mgD-1830 dextran, Texas Red®, 70,000 MW, neutral ............................................................................................................................................................ 25 mgN-7167 NeuroTrace™ BDA-10,000 Neuronal Tracer Kit .................................................................................................................................................... 1 kitV-22915 Vybrant® Cell Lineage Tracing Kit ......................................................................................................................................................................... 1 kit

Cat # Product Name Unit Size

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