Detection of receptor trimers on the cell surface by flow cytometric fluorescence energy homotransfer measurements. 2005

László Bene, and János Szöllosi, and Gergely Szentesi, and László Damjanovich, and Rezso Gáspár, and Thomas A Waldmann, and Sándor Damjanovich
Department of Biophysics and Cell Biology, Medical and Health Science Center, Research Center for Molecular Medicine, University of Debrecen, Hungary. bene@jaguar.dote.hu

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.

UI MeSH Term Description Entries
D011946 Receptors, Antigen Molecules on the surface of B- and T-lymphocytes that recognize and combine with specific antigens. Antigen Receptors,Antigen Receptor,Receptor, Antigen
D011956 Receptors, Cell Surface Cell surface proteins that bind signalling molecules external to the cell with high affinity and convert this extracellular event into one or more intracellular signals that alter the behavior of the target cell (From Alberts, Molecular Biology of the Cell, 2nd ed, pp693-5). Cell surface receptors, unlike enzymes, do not chemically alter their ligands. Cell Surface Receptor,Cell Surface Receptors,Hormone Receptors, Cell Surface,Receptors, Endogenous Substances,Cell Surface Hormone Receptors,Endogenous Substances Receptors,Receptor, Cell Surface,Surface Receptor, Cell
D011990 Receptors, Transferrin Membrane glycoproteins found in high concentrations on iron-utilizing cells. They specifically bind iron-bearing transferrin, are endocytosed with its ligand and then returned to the cell surface where transferrin without its iron is released. Transferrin Receptors,Transferrin Receptor,Receptor, Transferrin
D002462 Cell Membrane The lipid- and protein-containing, selectively permeable membrane that surrounds the cytoplasm in prokaryotic and eukaryotic cells. Plasma Membrane,Cytoplasmic Membrane,Cell Membranes,Cytoplasmic Membranes,Membrane, Cell,Membrane, Cytoplasmic,Membrane, Plasma,Membranes, Cell,Membranes, Cytoplasmic,Membranes, Plasma,Plasma Membranes
D002478 Cells, Cultured Cells propagated in vitro in special media conducive to their growth. Cultured cells are used to study developmental, morphologic, metabolic, physiologic, and genetic processes, among others. Cultured Cells,Cell, Cultured,Cultured Cell
D004735 Energy Transfer The transfer of energy of a given form among different scales of motion. (From McGraw-Hill Dictionary of Scientific and Technical Terms, 6th ed). It includes the transfer of kinetic energy and the transfer of chemical energy. The transfer of chemical energy from one molecule to another depends on proximity of molecules so it is often used as in techniques to measure distance such as the use of FORSTER RESONANCE ENERGY TRANSFER. Transfer, Energy
D005434 Flow Cytometry Technique using an instrument system for making, processing, and displaying one or more measurements on individual cells obtained from a cell suspension. Cells are usually stained with one or more fluorescent dyes specific to cell components of interest, e.g., DNA, and fluorescence of each cell is measured as it rapidly transverses the excitation beam (laser or mercury arc lamp). Fluorescence provides a quantitative measure of various biochemical and biophysical properties of the cell, as well as a basis for cell sorting. Other measurable optical parameters include light absorption and light scattering, the latter being applicable to the measurement of cell size, shape, density, granularity, and stain uptake. Cytofluorometry, Flow,Cytometry, Flow,Flow Microfluorimetry,Fluorescence-Activated Cell Sorting,Microfluorometry, Flow,Cell Sorting, Fluorescence-Activated,Cell Sortings, Fluorescence-Activated,Cytofluorometries, Flow,Cytometries, Flow,Flow Cytofluorometries,Flow Cytofluorometry,Flow Cytometries,Flow Microfluorometries,Flow Microfluorometry,Fluorescence Activated Cell Sorting,Fluorescence-Activated Cell Sortings,Microfluorimetry, Flow,Microfluorometries, Flow,Sorting, Fluorescence-Activated Cell,Sortings, Fluorescence-Activated Cell
D005454 Fluorescence Polarization Measurement of the polarization of fluorescent light from solutions or microscopic specimens. It is used to provide information concerning molecular size, shape, and conformation, molecular anisotropy, electronic energy transfer, molecular interaction, including dye and coenzyme binding, and the antigen-antibody reaction. Anisotropy, Fluorescence,Fluorescence Anisotropy,Polarization, Fluorescence,Anisotropies, Fluorescence,Fluorescence Anisotropies,Fluorescence Polarizations,Polarizations, Fluorescence
D005456 Fluorescent Dyes Chemicals that emit light after excitation by light. The wave length of the emitted light is usually longer than that of the incident light. Fluorochromes are substances that cause fluorescence in other substances, i.e., dyes used to mark or label other compounds with fluorescent tags. Flourescent Agent,Fluorescent Dye,Fluorescent Probe,Fluorescent Probes,Fluorochrome,Fluorochromes,Fluorogenic Substrates,Fluorescence Agents,Fluorescent Agents,Fluorogenic Substrate,Agents, Fluorescence,Agents, Fluorescent,Dyes, Fluorescent,Probes, Fluorescent,Substrates, Fluorogenic
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man

Related Publications

László Bene, and János Szöllosi, and Gergely Szentesi, and László Damjanovich, and Rezso Gáspár, and Thomas A Waldmann, and Sándor Damjanovich
August 2000, Cytometry,
László Bene, and János Szöllosi, and Gergely Szentesi, and László Damjanovich, and Rezso Gáspár, and Thomas A Waldmann, and Sándor Damjanovich
January 2001, Methods in cell biology,
László Bene, and János Szöllosi, and Gergely Szentesi, and László Damjanovich, and Rezso Gáspár, and Thomas A Waldmann, and Sándor Damjanovich
March 1987, Cytometry,
László Bene, and János Szöllosi, and Gergely Szentesi, and László Damjanovich, and Rezso Gáspár, and Thomas A Waldmann, and Sándor Damjanovich
October 1987, Immunology letters,
László Bene, and János Szöllosi, and Gergely Szentesi, and László Damjanovich, and Rezso Gáspár, and Thomas A Waldmann, and Sándor Damjanovich
March 1984, Cytometry,
László Bene, and János Szöllosi, and Gergely Szentesi, and László Damjanovich, and Rezso Gáspár, and Thomas A Waldmann, and Sándor Damjanovich
February 2007, Biophysical journal,
László Bene, and János Szöllosi, and Gergely Szentesi, and László Damjanovich, and Rezso Gáspár, and Thomas A Waldmann, and Sándor Damjanovich
August 1998, Biophysical journal,
László Bene, and János Szöllosi, and Gergely Szentesi, and László Damjanovich, and Rezso Gáspár, and Thomas A Waldmann, and Sándor Damjanovich
July 1987, Cytometry,
László Bene, and János Szöllosi, and Gergely Szentesi, and László Damjanovich, and Rezso Gáspár, and Thomas A Waldmann, and Sándor Damjanovich
May 1984, Biophysical journal,
László Bene, and János Szöllosi, and Gergely Szentesi, and László Damjanovich, and Rezso Gáspár, and Thomas A Waldmann, and Sándor Damjanovich
October 2005, Cytometry. Part A : the journal of the International Society for Analytical Cytology,
Copied contents to your clipboard!