Cell sorting using fluorescently-labeled

The labeled glycans may be analyzed by cell sorting after staining with fluorescently modified streptavidin. Alternatively, the cells may be lysed and the labeled glycans isolated using immobilized streptavidin. 

Antibodies labeled with fluorescent molecules have several applications, particularly in cytochemistry and cell sorting. There are many fluorochromes used in labeling (1), such as coumarin derivatives, phycobiliproteins, and rare earth chelates however, fluorescein and rhodamine (Table 1) are the most commonly used. 

In this chapter, we provide protocols to determine the ability of a peptide to mediate DNA internalization in cultured human tumor cells. Fluorescence-assisted cell sorting (FACS) analysis is used to obtain quantitative data on the time and temperature dependence of macromolecular delivery. Confocal microscopy is used to study the subcellular localization in both fixed and live cells. Fluorescently labeled transferrin and dextran are used to label the clathrin-dependent (15) and the non-clathrin, non-caveolar (16) endocytic compartments, respectively. Expression of a caveolin-l-YFP fusion protein is used to label cell surface caveolae and intracellular caveosomes (17). Finally a protocol, for the overexpression of dominant-negative dynamin [GTPase deficient dynamin-2 containing the amino acid substitution K44A (18)] is provided to evaluate the dynamin dependence of the uptake mechanism. 

The solid-bound library is treated with a labeled soluble biological target. For many studies a fluorescent label has been employed because of the high sensitivity of fluorescence detection. The labeled receptor binds to those resin beads that are derivatized with compounds that have the highest affinity to the biological receptor.The labeled beads are then selected followed by structural elucidation of the support-bound compound (see Section 1.4.1).The identity of the bioactive substance can be limited to a few alternative structures by mass spectrometric determination of the molecular mass. Highly efficient, automated methods have been developed to isolate the labeled beads, for example, by use of a fluorescence-activated cell sorting instrument [96]. 

This chapter provides an introduction to the use of fluorescent probes in flow cytometry. Sample preparation for the use of surface labeling with antibodies as well as for the use of nucleic acid probes is discussed. The utility of cell sorting is also discussed. 

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