title: A flow-cytometry method for analyzing the composition of membrane rafts.
abstract: Membrane rafts are involved in a broad variety of biological processes. Their protein composition under growth factor stimulation, anti-inflammatory or proinflammatory microenvironments, or in the course of pathogenic infections still remains to be determined. However, current techniques aimed at the identification of particular proteins on membrane rafts are not devoid of pitfalls. Membrane rafts were obtained by detergent-free based differential centrifugation from Jurkat T cells and J774 macrophages. Membrane rafts were labeled with fluorochrome-labeled antibodies directed against different cell membrane molecules, and with fluorochrome-labeled cholera toxin B that targets GM1 and analyzed by flow cytometry. CD3, CD11a, and GM1 were shown to be differentially expressed on Jurkat T cell-derived membrane rafts, indicating heterogeneity in membrane rafts composition. On the other hand, it was shown in J774 cell-derived membrane rafts that most but not all CD14 is present in the GM1-containing membrane fragments, thus confirming the heterogeneity of membrane rafts composition in other cell lines. The method described here allows the fluorometric assessment of the relative expression of more than one membrane raft component at a time, and at a single vesicle level in a fast and sensitive manner. This method seems to be a suitable approach to evaluate the molecular heterogeneity in membrane rafts composition.
authors: Departamento de Inmunología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, México DF, México.
authors: M Guadalupe
authors: MG
authors: Morales-García
authors: Jean-Jacques
authors: JJ
authors: Fournié
authors: M Maximina Bertha
authors: MM
authors: Moreno-Altamirano
authors: Gabriela
authors: G
authors: Rodríguez-Luna
authors: Ricardo-Mondragón
authors: RM
authors: Flores
authors: F Javier
authors: FJ
authors: Sánchez-García
mesh_terms: D000818:Animals; D040881:CD11a Antigen / Q000032:analysis; D017252:CD3 Complex / Q000032:analysis; D002458:Cell Fractionation; D002469:Cell Separation / Q000379:methods; D005434:Flow Cytometry / Q000379:methods*; D005677:G(M1) Ganglioside / Q000032:analysis; D006801:Humans; D019169:Jurkat Cells; D021962:Membrane Microdomains / Q000737:chemistry* / Q000648:ultrastructure; D051379:Mice; D008854:Microscopy, Electron
chemical_list: D040881:CD11a Antigen; D017252:CD3 Complex; D005677:G(M1) Ganglioside