Preparation of Fluorescently Labeled Dextrans and Ficolls

This chapter describes the preparation of fluorescently labeled dextrans and ficolls. The fact that dextran and Ficoll cannot cross biological membranes makes them ideal as markers and probes of specific subcellular compartments. Fluorescein dextran was first reported as a marker for fluid phase pinocytosis. A red-fluorescent, cyanine derivative of dextran has been used to mark the pinosome compartment in a multiparameter study of living Swiss 3T3 cells. The dibutyl tin dilaurate method is convenient and suitable for studies in which the degree of substitution doesn't need to be controlled accurately and detectability of small amounts is the limiting factor. It is found that when the method is used to prepare tetramethylrhodamine (TRTC-) derivatives, a minor complication results, apparently due to the hydrophobicity of the fluorophore. TRTC-dextran is slightly soluble in ethanol, so that repeated washing in ethanol sometimes results in progressive loss of labeled material. The aminoethylcarboxymethyl method involves conversion of hydroxyl groups to O-carboxy-methyl ethers and subsequent amidation of these groups by ethylene diamine in the presence of a water soluble carbodiimide. It is found that the degree of substitution can be determined with reasonable accuracy by reading the absorbance of the fluorophore for a known concentration of fluorescent polysaccharide.