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Proceedings Paper

Distance calibration of fluorescence energy-transfer values on cell surfaces
Author(s): Peter Salga; Andrea Bodnar; Sandor Damjanovich; Laszlo Matyus
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Paper Abstract

Different kinds of cell surface receptor clusters have been discovered recently using fluorescence resonance energy transfer (FRET) measurements. This method is capable for identifying molecular interactions, however the exact distances remain obscure, because the classical Foerster efficiency-distance relationship is valid only in the case of one donor one acceptor systems. This condition can not be fulfilled when cell surface molecules are labeled with monoclonal antibodies carrying different number of fluorescent donor and acceptor molecules. Our aim was to carry out FRET measurements on such cell surface receptors, where the distances are constant, and the only changing parameter is the donor-acceptor ratio of the used labels. For our experiments we used JY B lymphoblastoid cells, and we labeled the MHC class I heavy chain with KE-2 or W6/32 monoclonal antibodies and the length chain with L-368 monoclonal antibody tagged with different numbers of donor or acceptor molecules. The FRET efficiencies were measured either in a microscope using the photobleaching method or in a fluorescence activated cell sorter. We changed the donor acceptor ratio in a wide range in order to make a suitable calibration curve for other FRET experiments. The obtained calibration curve gives us the possibility to relate FRET efficiencies to real distances of cell surface receptors. Another source of deviation in the FRET efficiencies arise from the selected method. There was a marked difference between the FRET efficiencies measured by flow cytometry and with the photobleaching method even on same cells and between same epitopes.

Paper Details

Date Published: 9 June 1998
PDF: 6 pages
Proc. SPIE 3261, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing V, (9 June 1998); doi: 10.1117/12.310559
Show Author Affiliations
Peter Salga, Univ. Medical School of Debrecen (Hungary)
Andrea Bodnar, Univ. Medical School of Debrecen (Hungary)
Sandor Damjanovich, Univ. Medical School of Debrecen (Hungary)
Laszlo Matyus, Univ. Medical School of Debrecen (Hungary)

Published in SPIE Proceedings Vol. 3261:
Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing V
Thomas Taiwei Lu; Carol J. Cogswell; Jeremy M. Lerner; Jose-Angel Conchello; Jeremy M. Lerner; Thomas Taiwei Lu; Tony Wilson, Editor(s)

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