tritc-dextran - tdbcons.comtdbcons.com/images/pdf/tritc_dextran.pdf · fig. 3 dependence of...

$: TRITC-Dextran - tdbcons.comtdbcons.com/images/pdf/tritc_dextran.pdf · Fig. 3 Dependence of ˜uorescence of TRITC-dextran with pH in the range 4-9. Selected dextran fractions prepared$
Fig. 3 Dependence of �uorescence of TRITC-dextran with pH in the range 4-9.

Selected dextran fractions prepared from native Dextran B512F are labelled with tetramethyl-rhodamine B by a procedure similar to that described by de Belder and Granath. The rhodamine moiety is attached by a stable thiocarbamoyl linkage and the labelling procedure does not lead to any depolymerisation of the dextran. The TRITC-dextrans carry from 0.001-0.008 mol. TRITC per glucose unit and at these low levels of substitution confer minimal charges to the dextran, which is an essential requirement for permeability studies .

References:1. de Belder AN and Granath K. Preparation and properties of �uorescein labelled dextrans. Carbohydr Res. 1973;30:375-378.

TRITC-DextranTetramethyl-rhodamine isothiocyanate-dextran

Trade name: TRITC- dextran

Chemical names:

CASnr:

Structure:

Fig. 1 Structural representation of fragment of TRITC-dextran molecule.

Properties

Spectral data

Fig. 2 Fluorescence scan of TRITC-dextran 40 in 0.025M borate pH 9.0 (9.9mg in 50 ml bu�er) Excitation 550nm; Emission 571.5nm.

TdB ConsultancyAB, Virdings Allé 28 SE-754 50 Uppsala. Phone 46 187001204 Fax 46 18 7001206 www.tdbcons.se. Version TD010AA, Date: 12/2010

Dextran 3´, 6´bis(tetramethylamino) -3-oxospiro(isobenzofuran-1(3H),9´-[9H]xanthen]-5(or 6)- yl)carbamothioate.Tetramethyl-rhodamine B-dextranTetramethyl-rhodamine B thiocarbamoyl-dextran.

O

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OHO

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OOHH

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HOHO

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HOHO

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OHHH

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OOHH

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N(Me)2O(Me)2N

COOH

Synthesis

TRITC-dextran is supplied as a red powder which dissolves freely in water or salt solutions giving a red solution. The product also dissolves in DMSO, formamide and certain other polar organic solvents but is insoluble in lower aliphatic alcohols, acetone, chloroform, dimethylformamide.

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TD40_20239(EM)

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Excitation is best performed at 550nm and �uorescence measured at 572 nm (see Fig.2.). Studies in our laboratories have shown that the �uorescence from a TRITC-dextran solution shows only slight changes over the range pH 3-9 (see Fig.3). Similar results have been reported earlier . This is of interest when making quantita-tive measurements. Measurements in biological media may signi�cantly a�ect the �uorescence intensity which may be enhanced or depressed.

References:2. Geisow MJ. Fluorescein conjugates as indicators of subcellular pH. A critical evaluation. Exp Cell Res. 1984 Jan: 150(1): 29-35.

TRITC-dextran is supplied as a red powder.

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Physical chemical properties of TRITC-dextransThe dextran molecule at molecular weights greater than 5 000 Daltons behaves as a �exible and extended coil in solution. Table 1 (below) shows the molecular dimensions at various molecular weights.

Dextrans and TRITC-dextrans will exhibit Newtonian �ow characteris-tics i.e. the viscosity is independent of shear rate. Studies in the range pH 4-10 establish that the viscosity is independent of pH. The viscosities of dextran fractions at various concentrations is shown in Fig. 4.

2 million 270500 000 147 100 000 6970 5840 44.510 23.6Albumin 35

Dextran (Mw) Stoke´s radius

Fig. 4. The dependence of viscosity on concentration for various dextran fractions.


Table 1. Molecular dimensions of dextran expressed as Stokes Radii (Å)

Storage and stability

FITC-dextrans are stable for 3 days at 37°C in rabbit plasma, muscle homogenate, liver homogenate and urine. No changes in the mol.wt. and no release of �uorescein moieties were noted. Further FITC-dextran was stable in 6% trichloroacetic acid at room temperature for 3 days .

Hydrolysis of the thiocarbamoyl linkage would give rise to 4- or 5-aminorhodamine which is readily determined by HPLC. In unpublished studies, the stability of an autoclaved FITC-dextran 70 solution was studied at temperatures from 8 to 50 C over a period of 5 months. Only at 50 C could a slight increase (1%) in free amino�uorescein be noted. In other unpublished studies, FITC-dextran was found to be stable at pH 4 for up to 1 month at temperatures up to 35°C. At 80°C and pH 4, the thiocarbamoyl linkage was stable for 30min. - however the dextran may start show signs of degradation. At pH 9, considerable (24% decrease in �uorescence) took place at 36°C over 1 month.

References:3. Kurtzhals P, Larsen C and Johansen M. High performance size-exclusion chromatographic procedure for the determination of �uoresceinyl isothiocya-nate dextrans of various molecular masses inbiological media,. J Chromatogr. 1989:491;117-127.4. Thorball N. FITC-dextran tracers in microcirculatory and permeability studies using combined �uorescence Stereo Microscopy. Fluorescence Stereo microscopy and electron microscopy. Histochemistry. 1981:71;209-233.

ToxicityTheir toxicity patterns follow those of the parent dextrans. Clinical dextrans fractions have been employed for over 50 years as plasma volume expanders. Dextran induced anaphylactoid reations (DIARs) have been observed in humans after injection of clinical dextran solutions .The reactions vary from mild skin reactions to severe shock states. The incidence of severe reactions is about 1 in 2000. TRITC-dextrans are also likely to display this type of behaviour but few reports of problems with experimental animals have appeared.

References:5. Ljungström KG, Rench H, Strandberg K. et al. Adverse reations to dextran in Sweden 1970-1979. Acta Chir Scand. 1983; 149: 253-262.6. Hedin H, Richter W and Ring J. Dextran-induced anaphylactoid reations in man: role of dextran reactive antibodies. Int Arch Allergy appl Immun. 1976; 52:145-159.

The stability of TRITC-dextrans has not been investigated in detail but it is presumed to be similar to that of FITC-dextrans (see data-�le). Only at elevated pH (>9) and elevated temperatures is there a risk for hydrolysis of the thiocarbamoyl linkage.

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Applications

The microvasculature of the hamster cheek pouch has proved to be a useful model for studying plasma leakage in di�erent experi-mental conditions, e.g. following ischemia/reperfusion, topical application of a whole range of in�ammatory mediators, parasites and bacteria. With this technique, vascular permeablility changes can be studied in real time and be related to other microvascular events such as leukocyte adhesion and activation. The cheek pouches are examined by intravital �uorescence microscopy using suitable �lter (490/520nm) and images are captured with a digital camera. A 5% TRITC-dextran 150 solution in normal saline is administred i.v. (approx. 100mg/kg bodyweight) .

Permeability studies using combined �uorescence stereo-microscopy, �uorescence light microscopy and electron microscopy were reported by Thorball . Extensive studies of tissue �xation techniques in the presence of FITC-dextrans are described. Details of the microscopy set-up (�lters, illumination) may be found in this article .

Mullick and co-workers have described a new method for sequen-tial quantitation of endothelial layer permeability using TRITC-dextran 4. The perfusate contained 42µg/ml TD4-more details can be found in this article .

References:9. Hultström D and Svensjö E. Intravital and electron microscopy study of bradykinin induced vascular permeability changes using FITC-dextran as a tracer. Journal of Pathology. 1979; 129:125-133.10. Svensjö E. The hamster cheek pouch as a research model in in�ammation. Chapter 30 In: David Shepro (Editor), Microvasular Research-Biology and Pathology. p. 195-207, 2006, Elsevier Academic Press.11. Svensjö E, Saraiva EM, Bozza MT, et al. Salivary gland homogenates of Lutzomyia longipalpis and its vasodilatory peptide maxadilan cause plasma leakage via PAC1 receptor activation. J Vasc Res. 2009;46:435-446.12. Jonsson J, Arfors KE, and Hint HC. Studies on relationships between blood and lymphatic systems within the microcirculation, 6 Europ.Conf.Microcirculation

An alternative procedure using rabbit ear chambers has been described. The regenerative titanium ear-chambers (rabbits) were used to study the blood/lymph systems in the microcirculation with �uorescent-dextrans. Lymph ingrowth is seen after 4-8 weeks of implantation .

Aalborg. 1970; 214-218 (Karger, Basel 1971).13. Mullick AE, Walsh BA, Reiser KM et al. Chronic estradiol treatment attenuates sti�ening, glycoxidation and permeability of rat carotid arteries. Am J Physiol Heart Circ Physiol., 2001; 281: H2204-H2210.

General procedures

TRITC-dextrans are primarily used for studying permeability and transport in cells and vessels and tissues. An added bene�t is that measurements of the �uorescence provide quantitative data on transport and permeability of healthy and diseased tissues. Such studies can be performed in real time by intravital �uorescence microscopy. The technique o�ers high sensitivity and concentra-tions down to 1µg/ml can be detected in tissue �uids. Unlike FITC-dextrans, the �uorescence of TRITC-dextrans is not depen-dent on pH in the range 4 to 9. A further important property is that TRITC-dextran does not bind to artery walls .

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TRITC-dextranTetramethyl-rhodamine isothiocyanate-dextran

References:7. Walsh BA, Mullick AE, Banka CE et al. Estradiol acts separately on the LDL-particle and artery walls to reduce LDL-accumulation. J Lipid Res. 2000; 41: 134-141.8. Gardner G, Banka CE, Roberts KA et al. Modi�ed LDL-mediated increase in endothelial-layer permeability are attenuated with 17B-estradiol. Arterscler Thromb Vasc Biol.,1999; 19: 854-861.

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TRITC-dextran 150 injected in a hamster cheek pounch 15min after histamine challenge. (by kind permission of E.Svensjö).

Before histamine

List of products available

TD4-100mg TRITC-dextran 4 4000TD4-1g TRITC-dextran 4 4000TD20-100mg TRITC-dextran 20 20000 TD20-1g TRITC-dextran 20 20000TD40-100mg TRITC-dextran 40 40000TD40-1g TRITC-dextran 40 40000 TD70-100g TRITC-dextran 70 700000TD70-1g TRITC-dextran 70 700000TD150-100mg TRITC-dextran 150 1500000TD150-1g TRITC-dextran 150 1500000

Catalog no Name Mol.wt


1. Permeability studies in arteries and microvasculature TRITC-dextran 4 and 70 were used to study permeability in carotid arteries in folate deplete rats - concentrations of 42 µg/ml were injected at 7 ml/min .

References:22. Symons JD, Mullick AE, Ensunsa JL et al. Hyperhomocysteinemia evoked by folate depletion; e�ects on coronary and carotid arterial function, Arterioscler Thromb Vasc Biol. 2002 May; 22: 772-80.

The subcellular �uorescence of FITC- and TRITC-dextran was studied in mouse macrophage lysosomes. A comparison of the values enabled estimates of the intracellular pH. Cultures were incubated with 1mg/ml dextran concentrations . Other studies on proton accumulation in living cells using these �uorescent probes are reported by S.Okhuma and B.Poole . Uptake of TRITC-dextran 10 in response to osmotic cell swelling was studied in intestine 407 cells (0.5mg/ml) - details of the quantitation method are given . A further example of the use of TRITC-dextran as a �uid phase marker is found in the paper by M.Kauppi and colleagues . Cells were exposed to TRITC-dextran 10 (5mg/ml) in air-medium for 1 hr at 37 C. TRITC-dextran 4 (1mg/ml in Hank´s balanced salt solution) was used to study the barrier function of tight junctions in prostate cancer cell cultures .M.Mairhofer and colleagues have studied the late endosomal localisation of SLP-1 in perinuclear bodies with TRITC-dextran 4 (5mg/ml) . For studies of trans-cellular protein delivery, a TRITC-labelled dextran 10 (0.1mg/ml) was used to examine the kinetics of internalization of transactivator fusions .

W.Tong and colleagues studied the permeability of arti�cial polyelctrolyte microcapsules with both FITC- and TRITC dextrans(4 and 70) . Interestingly whereas FITC-dextrans did not penetrate the membrane, the slightly polycationic TRITC-dextrans penetrated the membrane. These e�ects were dependent on pH and salt concentration in the media.

Permeability studies in cells

References:14. Geisow MJ. Fluorescein conjugates as indicators of subcellular pH. Exp Cell Res. 1984, Jan;150:29-35.15. Ohkuma S and Poole B. Fluorescence probe measurement of the intralyso-somal pH in living cells and the perturbation of pH by various agents. Proc Natl Acad Sci. 1978 Jul;75:3327-31.16. van der Wijk T, Tomassen SF, Houtsmuller AB et al. Increased vesicle recycling in reponse to osmotic cell swelling. Cause and consequence of hypotonicity-provoked ATP release. J Biol Chem. 2003 Oct; 278: 40020-5.17. Kauppi M, Simonsen A, Bremnes B et al. The small GTPase Rab22 interacts with EEA1 and controls endosomal membrane tra�cking. J Cell Sci. 2002; 115: 899-911.18. Shah GV, Muralidharan A, Gokulgandhi M et al. Cadherin switching and activation of B-catenin signaling underlie proinvasive actions of calcitonin-calcitonin receptor axis in prostate cancer. J Biol Chem. 2009; 284: 1018-1030.19. Mairhofer M, Steiner M, Salzer U et al. Stomatin-like protein-1 interacts with stomatin and is targeted to late endosomes. J Biol Chem. 2009; 42: 29218-29.20. Fittipaldi A, Ferrari A, Zoppé et al. Cell membrane lipid rafts mediate caveolar endocytosis in HIV-1 Tat fusion proteins. J Biol Chem. 2003; 278: 34141-9.21. Tong W, Dong W, Gao C et al. Charge-controlled permeability of polyelectro-lyte microcapsules. J Phys Chem B. 2005 Jul; 109: 13159-65.

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