Dextran label

The primary advantage of MSC for utilization in cell therapy is the ease with which they can be harvested from the bone marrow, isolated by plastic adherence, expanded in culture, genetically engineered, differentiated, and handled in vitro. The ease with which mesenchymal stem cells can be iron-dextran labeled (Ittrich et ah, 2005 Arab et al, 2004) and monitored by MRl... 

Fig. 3 Polypeptide vesicle with endocytosis capability, (a) Vesicles formed from poly(L-arginme)6o-h-poly(L-leucme)2o- The poly(L-arginme) block provides an added cell-penetrating feature to the vesicles, (b, c) LCSM images of internalized vesicles (green) containing Texas-Red-labeled dextran (red) in (b) epithelial and (c) endothelial cells. Colocalization of the vesicles and Texas-Red-labeled dextran appears as a yellow fluorescent signal. Adapted from [44] with permission.Copyright 2007 Macmillan Publishers...

Maximum labelling of heparin with F-D was achieved at 5 hours at 25 °C, pH 8.4. In the case of heparin, the efficiency of labelling was not dependent on molecular weight, but solely a function of the ratio of the concentrations of labelling reagent to monosaccharide subunit in the reaction mixture. Similar results were encountered in the labelling of dextrans of different molecular weight (9). 

Elimination from the vitreous occurs by one of two pathways. This can be visualized by injecting fluorescent compounds and examining the concentration distribution in frozen sections obtained after a steady state has been established [230]. If the major route of elimination is by means of the re-tina/choroid, at steady state the lowest concentration would be in the vicinity of the retina. The contours observed in frozen sections of the rabbit eye obtained after intravitreal injection of fluorescein exhibit this pattern, with the highest concentration immediately behind the lens (Fig. 16A). Compounds not chiefly eliminated through the retina exit the vitreous by passive diffusion and enter the posterior aqueous, where they are eliminated by the natural production and outflow of aqueous humor. In such a situation, the contours would be perpendicular to the retina, with the highest concentration towards the rear of the vitreous cavity. This appears to be the case for fluorescently labeled dextran polymer, whose contours decrease in concentration toward the hyaloid membrane (Fig. 16B). 

M.D. Gulcev, G.LG. Goring, M. Rakic, and J.D. Brennan, Reagentless pH-based biosensing using a fluorescently-labelled dextran co-entrapped with a hydrolytic enzyme in sol-gel derived nanocomposite films. Anal. Chim. Acta 457, 47-59 (2002). 

Fluorescence resonance energy transfer has also been used for ionic strength measurements.(95) Fluorescein labeled dextran (donor) and polyethyleneimine-Texas Red (acceptor) were placed behind a dialysis membrane. The polymer association is ionic strength dependent and the ratio of intensities (F o/Fw) was used as the measured parameter. Since both the donor and acceptor are fluorescent, this kind of sensor may allow expand the sensitive ionic strength range by shifts in observation wavelength, as was discussed for pH probe Carboxy SNAFL-2 (see Section 10.3). 

If the soluble protein that specifically adsorbs to the fiber can be extrinsi-cally labeled, the background problem can be avoided. Of course, in vivo proteins cannot be labeled. However, it is conceivable that a protein labeled with a bulky extrinsic group (e.g., fluorescent dextrans) could be confined by a molecular sieve membrane (e.g., a dialysis membrane) within a closed volume surrounding the specifically derivatized optical fiber. When exposed to the (unlabeled) protein in the biological fluid under investigation, the membrane-clad fiber would allow some unlabeled protein to permeate in and... 

Nielsen HM, Verhoef JC, Ponec M, Rassing MR (1999b) TR146 cells grown on filters as a model of human buccal epithelium Permeability of fluorescein isothiocyanate-labelled dextrans in the presence of sodium glycocholate. J Control Release 60 223-233... 

Figure 11.3 Permeability to FITC-labelled dextrans across rat (O, [94]) and human ( , [66]) alveolar type I (ATI) cell-like...

A further proof of principle was conducted by Cooney et al. who demonstrated the feasibility of the Andersen cascade impactor as a cell compatible deposition device [90], Permeability coefficients of fluorescent isothiocyanate-labeled dextrans after impaction as aerosols on Calu-3 cells were calculated. Deposition did not negatively affect the cell monolayer integrity. 

Liposomes (without peptide) were labelled with rhodamine-PE in the lipid bilayer and in the inner compartment using FITC-dextrane 9000 and incubated with DCs for one hour. Figure 3 shows clearly that only in the case of AVE 3 and AVE 43 could a significant uptake be observed. The fluorescence inside the DCs is in the case of AVE 3 homogenously distributed in the cytosol, whereas in the case of AVE 43 the liposomes seem to be caught in granular structures, presumably endosomes. The PS causes the liposomes... 

Figure 2 Principle of spectral bio-imaging HUVEC incubated with pH-sensitive DOPE CHEMS liposomes (loaded with fluorescein isothiocyanate-dextran), cholera toxin subunit B (Alexa Fluor 594 labeled), and diamidino-phenylindole-dihydrochloride.

No specific cargo molecules have been identified to track macropinocytosis, which makes it difficult to follow the intracellular fate of macropinosomes. However, one substance that is applied to study macropinocytosis and to track macropinocytic vesicles is labeled high-molecular-weight dextran (molecular weight 70,000) (77). 

Starvation and incubation with labeled dextran For this method, cells are incubated under starvation [Earle s balanced salt solution (BBSS) medium] conditions for two hours. To label early endocytic compartments, cells are subsequently incubated with 0.5 mg/mL dextran tetramethylrhoda-mine for five minutes. To label late endosomes, cells are washed and further incubated for an additional 10 minutes (130). 

The quantification of fluorescent particles in cellular systems is difficult because several aspects such as autofluorescence, bleaching (see below), and quenching hamper analysis. Keep in mind that many fluorophores show a pH-dependent change in emission spectrum and intensity fluorescein-labeled dextrans (FITC-dextran) and calcein are strongly quenched upon acidification. If available, one should read the fluorescence intensity at its isosbestic point, where the intensity is not pH dependent. 

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