Imaging membrane lipid domains to characterize their organization and function has been hindered by the lack of reliable lipid-specific probes. In this paper, we provide detailed methods to investigate, mainly by confocal microscopy, the distribution and dynamics of two components of the “lipid rafts,” sphingomyelin (SM) and cholesterol, using two specific lipid probes that have been extensively studied in the laboratory: lysenin, a SM-binding toxin and the fluorescent esters of poly(ethylene glycol) cholesteryl ether (PEG-Chol) that label cholesterol-rich domains. The production of nontoxic forms of lysenin as well as its specific binding behavior have allowed monitoring the distribution and the dynamics of SM-rich domains in living cell membranes. Because of its water-solubility and low toxicity, the fluorescent PEG-Chol can be used to follow the reorganization of cell surface cholesterol-rich domains as well as intracellular cholesterol dynamics in living cells. These probes can thus provide important informations on lipid distribution and traffic in living cell membranes.
