Chapter 6 Fluorescent Labeling of Cell Surfaces

Abstract

This chapter discusses the different aspects of fluorescent labeling of cell surfaces. Fluorescence microscopy has been used in the study of cell surfaces. Surface organization, physical state, dynamics, and function are all accessible through appropriate labels of the surface, but these labels must be applied intelligently if they are to yield valid information. Most fluorophores reacting with proteins in solution ought to react with cell surfaces. The coupling of commonly used fluorophores to cells is limited then, by the reaction conditions required and by the hydrophobicity of the fluorophore. Hydrophobic fluorescent molecules such as pyrene, diphenylhexatriene, and the carbocyanine dyes readily label lipid bilayers, in liposomes or in cells. The diverse proteins of cell surfaces bind or are bound by ligands ranging from peptide hormones, toxins, through antibodies and their fragments and lectins, to large particles-low density lipoprotein particles, and even viruses. Cells labeled for microscopy must be washed after labeling, otherwise the high concentration on fluorescent ligand in the medium will swamp signals from the surface. This washing also removes some nonspecifically bound ligand and, by disrupting the equilibrium between free and bound ligand, some specifically bound ligand as well.