Resistance of cellular membrane antigens to solubilization with Triton X-100 as a marker of their association with lipid rafts--analysis by flow cytometry.
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.
Filatov
Alexander V
AV
Institute of Immunology, Kashirskoye Shosse 24-2, 115478 Moscow, Russia. sorben@online.ru
Shmigol
Irina B
IB
Kuzin
Igor I
II
Sharonov
George V
GV
Feofanov
Alexei V
AV
D000906:Antibodies / Q000276:immunology; D000954:Antigens, Surface / Q000187:drug effects / Q000276:immunology; D015415:Biomarkers; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D008214:Lymphocytes / Q000187:drug effects*; D021962:Membrane Microdomains / Q000378:metabolism*; D018613:Microscopy, Confocal; D017830:Octoxynol / Q000494:pharmacology*; D012995:Solubility; D013501:Surface-Active Agents / Q000494:pharmacology*
D000906:Antibodies; D000954:Antigens, Surface; D015415:Biomarkers; D013501:Surface-Active Agents; D017830:Octoxynol