Molecular weight considerations

Below you will find some advice on how to choose the optimal molecular weight for your research area.

Lysosome labelling

Cell culture studies undertaken to label lysosomes can utilize a wide variety of sizes. Dextrans will traffic to the lysosomes when taken up via fluid phase endocytosis from the surrounding cell culture media.

Size determination for intravital imaging studies

Endocytosis
Depending on the target cells or tissue, a variety of sizes are applicable in these cases with the right selection of fluorophores. Systemic endothelial cells surrounding the lumen of blood vessels are best labeled using a larger molecular weight dextrans in sizes at or greater than 70kDa. This allows for a long resident time within the plasma to allow for uptake by these cell types. Proximal tubules in kidneys (which first see all of the material filtered by the kidney glomeruli) are best labeled with smaller molecular weight dextrans 4kDa to 40kDa.

3 panel test 2

Still photomicrographs show the distribution of a large 150 Da-TRITC-dextran and a 4 kDa FITC-dextran at the surface of a living kidney. Panel A show an image taken prior to infusion of the dextrans. A glomerulus is seen in the center, surrounded by proximal tubules. The nuclei of all cells are seen in cyan. Panel B shows a 2-fold zoomed image of a glomerulus. The 150 kDa-TRITC-dextran is maintained in the circulating plasma which highlights the capillary loops of the glomerulus as well as the rapidly moving red blood cells (RBCs), which appear as dark streaks due to their rapid velocity. Panel C vividly shows two distal tubules (center) with the small 4k Da FITC-dextran filling the lumen of those tubules. In stark contrast, the lumens of the surrounding proximal tubules are largely devoid of the small dextran, instead showing accumulation of the 4k Da FITC-dextran in the early endosomes. Due to the FITC’s decreased fluorescence at low pH’s, there is little to no fluorescence emanating from late endosomes and lysosomes.

Microvascular flow

Determination of microvascular flow can be determined by utilizing a large molecular weight dextran to fill the plasma space within the blood and outline the red blood cells (RBCs). Once this is achieved, labeled RBCs can be injected into the vasculature to track them as they flow along blood vessels. For these studies, microscope systems with very high acquisition rates (in the 10’s of frames/second) are necessary. Alternatively, slower scanning systems can utilize a parallel linescan bisecting a blood vessel to produce a columnar image with RBC shadows appearing down the image. The angle of the slope can be used to determine speed by rationing distance and acquisition time.

Vascular permeability

The maintenance of vascular integrity under physiologic conditions, pathology, or in response to a therapeutic agent can be assessed by examining the time and degree a larger dextran will cross the vascular barrier into the interstitial space. This is best done with two larger size dextrans, a 150kDa dextran that will only leak into the interstitial space under more severe injury, and a moderately sized dextran such a 70kDa dextran which will naturally leak into the interstitial space slowly. Acquiring images of the same region at various time points will help asses leakage by ratioing the intensity of interstitial fluorescence to vascular fluorescence. In studies such as these, it is essential the dextrans have a narrow dispersion around the average molecular weight. Similarly, sized dextrans having a broad dispersion characteristic will erroneously appear to have vascular injury as the smaller polymers more readily cross into the interstitial space.

Hamster cheek 2

Histamine induced macromolecular (FITC-dextran) leakage in hamster cheek pouch. To the left: Control (1 min after FITC-dextran injection). To the right: Histamine infused leakage of 150 kDa FITC-dextran.

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