Single Cell Analysis of Lipid Rafts

Lipid rafts are plasma membrane microdomains that serve as platforms for the assembly of proteins involved in signal transduction pathways. Given that lipid rafts are relatively resistant to cold extraction with nonionic detergents, lipid raft associated and nonassociated proteins have been identified using biochemical methods such as sucrose-gradient density centrifugation. For identification of raft-associated proteins in individual cells, imaging methods, such as fluorescence microscopy, can be used. Detergent solubilization of non-raft regions of the plasma membrane and extraction of non-raft associated proteins are done on cells affixed to microscope slides and prior to immunostaining. This methodology has the advantages of requiring smaller cell numbers than traditional biochemical methods and also permits the study of migration of signaling proteins into and out of rafts during cell activation. An additional adaptation of the method allows identification of lipid raft-associated proteins during cognate interactions between cells. Here, as an example, we describe the methodology used in our laboratory to study lipid raft-associated molecules during T lymphocyte interactions with antigen-presenting cells.