Abstract

Lipid rafts are specialized micro-domains of the plasma membrane enriched in glycosphingolipid and cholesterol that play important role in signal transduction, membrane trafficking, and cell adhesion. A distinct feature of lipid rafts is their resistance to solubilization with non-ionic detergent Triton X-100 (TX-100). In this study, we used flow cytometry to evaluate TX-100 resistance of 74 cell membrane molecules expressed on normal human peripheral blood lymphocytes (PBL), thymocytes, and 12 lymphoid cell lines. Resistance of membrane molecules to solubilization with TX-100 was determined by comparing the intensities of fluorescence of cells treated with TX-100 or left untreated. The majority of antigens analyzed were easily solubilized with TX-100 that resulted in decreased fluorescence intensity. However, a group of antigens showed TX-100 resistance in the range of 20-100%. These included all glycosylphosphatidylinositol (GPI)-anchored antigens under study, as well as some glycolipid and trans-membrane antigens. With the few exceptions, antigen resistance to solubilization with TX-100 was stable parameter, which did not depend on cell type in which it was analyzed. There was a good correspondence between the antigens showing resistance to solubilization with TX-100 as evaluated by our flow cytometry method, and the antigens that were previously demonstrated in detergent-resistant membranes using a more standard method of physical fractionation. Taken collectively, our data suggest that flow cytometry is a useful method for rapid evaluation of the possible association of a membrane antigen with lipid rafts.