Detection of receptor trimers on the cell surface by flow cytometric fluorescence energy homotransfer measurements.
Fluorescence energy homotransfer offers a powerful tool for the investigation of the state of oligomerization of cell surface receptors on a cell-by-cell basis by measuring the polarized components of fluorescence intensity of cells labeled with fluorescently stained antibodies. Here we describe homotransfer-based methods for the flow cytometric detection and analysis of hetero- and homo-associations of cell surface receptors. Homotransfer efficiencies for two- and three-body energy transfer interactions are defined and their frequency distribution curves are computed from the fluorescence anisotropy distributions of multiple-labeled cells. The fractions of receptors involved in homo-clustering is calculated based on the dependence of the fluorescence anisotropy on the surface concentration of the fluorescently stained antibodies. A homotransfer analysis of the homo- and hetero-clustering of the MHCI and MHCII glycoproteins, the cytokine receptor IL-2Ralpha, transferrin receptor and the receptor-type tyrosine phosphatase CD45 on JY B and Kit-225-K6 T cells is presented. We investigated how various factors such as the type of dye, rotational mobility of the dye and dye-targeting antibody, as well as the wavelength of the exciting light affect the homotransfer. We show that the homotransfer technique combined with the high statistical resolution of flow cytometry is an effective tool for detecting different oligomeric states of receptors by using fluorophores having restricted rotational mobility on the time scale of fluorescence.
Bene
László
L
Department of Biophysics and Cell Biology, Medical and Health Science Center, Research Center for Molecular Medicine, University of Debrecen, Hungary. bene@jaguar.dote.hu
Szöllosi
János
J
Szentesi
Gergely
G
Damjanovich
László
L
Gáspár
Rezso
R
Waldmann
Thomas A
TA
Damjanovich
Sándor
S
D000911:Antibodies, Monoclonal / Q000378:metabolism; D001402:B-Lymphocytes / Q000378:metabolism; D002462:Cell Membrane; D002478:Cells, Cultured; D004735:Energy Transfer; D005434:Flow Cytometry; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D015395:Histocompatibility Antigens Class I / Q000276:immunology; D000949:Histocompatibility Antigens Class II / Q000276:immunology; D006801:Humans; D053645:Interleukin-2 Receptor alpha Subunit; D011946:Receptors, Antigen / Q000276:immunology; D011956:Receptors, Cell Surface / Q000032:analysis* / Q000737:chemistry*; D018123:Receptors, Interleukin / Q000276:immunology; D011990:Receptors, Transferrin / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology
D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes; D015395:Histocompatibility Antigens Class I; D000949:Histocompatibility Antigens Class II; C508017:IL2RA protein, human; D053645:Interleukin-2 Receptor alpha Subunit; D011946:Receptors, Antigen; D011956:Receptors, Cell Surface; D018123:Receptors, Interleukin; D011990:Receptors, Transferrin