TY - JOUR
T1 - A flow-cytometry method for analyzing the composition of membrane rafts
AU - Morales-García, M. Guadalupe
AU - Fournié, Jean Jacques
AU - Moreno-Altamirano, M. Maximina Bertha
AU - Rodríguez-Luna, Gabriela
AU - Ricardo-Mondragón- Flores, Flores
AU - Sánchez-García, F. Javier
PY - 2008/10
Y1 - 2008/10
N2 - 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.
AB - 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.
KW - Detergent-free isolation
KW - Flow cytometry analysis
KW - Membrane rafts
UR - http://www.scopus.com/inward/record.url?scp=53049099931&partnerID=8YFLogxK
U2 - 10.1002/cyto.a.20630
DO - 10.1002/cyto.a.20630
M3 - Artículo
C2 - 18727060
SN - 1552-4922
VL - 73
SP - 918
EP - 925
JO - Cytometry Part A
JF - Cytometry Part A
IS - 10
ER -