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Proceedings Paper

Measurement of receptor cross-linking at the cell surface via multiparameter flow cytometry
Author(s): Richard G. Posner; Jennifer Bold; Yael Bernstein; Joe Rasor; Joshua Braslow; William S. Hlavacek; Alan S. Perelson
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Paper Abstract

Many cellular responses, particularly in the immune system, are triggered by ligand binding to a cell-surface receptor. However, as indicated by bell-shaped dose-response curves, ligand binding alone is sometimes insufficient to trigger a response. Often, ligand binding must also induce the aggregation of cell-surface receptors through crosslinking, which occurs when a ligand binds simultaneously to two or more receptors. Thus, an important goal in cell biology has been to establish quantitative relationships between the amount of ligand present on a cell surface and the number of crosslinked ligand-specific cell-surface receptors. To better understand ligand-induced receptor aggregation, we have been investigating the binding of a model multivalent antigen (DNP25PE) to cell-surface anti-DNP FITC-labeled IgE (FITC- IgE). To determine the kinetic and equilibrium parameters that characterize crosslinking in this system, we have developed a combined theoretical and experimental approach that is based on multiparameter flow cytometry. With this approach, we can measure both the average number of ligand molecules that are bound per cell and the average number of receptor binding sites that are bound per cell. The average number of DNP25PE per cell is determined by measuring the fluorescence of phycoerythrin. The average number of occupied IgE sites per cell is determined by measuring the fluorescence of FITC, which is quenched upon ligand binding. This novel approach, together with conventional methods for changes in intracellular calcium, allows us to correlate for the first time the dynamics of IgE crosslinking with cell activation.

Paper Details

Date Published: 1 May 1998
PDF: 12 pages
Proc. SPIE 3256, Advances in Optical Biophysics, (1 May 1998); doi: 10.1117/12.307056
Show Author Affiliations
Richard G. Posner, Northern Arizona Univ. (United States)
Jennifer Bold, Northern Arizona Univ. (United States)
Yael Bernstein, Northern Arizona Univ. (United States)
Joe Rasor, Northern Arizona Univ. (United States)
Joshua Braslow, Northern Arizona Univ. (United States)
William S. Hlavacek, Los Alamos National Lab. (United States)
Alan S. Perelson, Los Alamos National Lab. (United States)

Published in SPIE Proceedings Vol. 3256:
Advances in Optical Biophysics
Joseph R. Lakowicz; J. B. Alexander Ross, Editor(s)

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