Measurement of receptor crosslinking at the cell surface via multiparameter flow cytometry

Richard G. Posner, Jennifer Bold, Yael Bernstein, Joe Rasor, Joshua Braslow, William S. Hlavacek, Alan S. Perelson

Research output: Contribution to journalConference article

5 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)132-143
Number of pages12
JournalProceedings of SPIE - The International Society for Optical Engineering
Volume3256
DOIs
StatePublished - Dec 1 1998
EventAdvances in Optical Biophysics - San Jose, CA, United States
Duration: Jan 25 1998Jan 26 1998

Keywords

  • Aggregation
  • Crosslinking
  • FcεRI
  • Flow Cytometry
  • IgE
  • Receptor

ASJC Scopus subject areas

  • Electronic, Optical and Magnetic Materials
  • Condensed Matter Physics
  • Computer Science Applications
  • Applied Mathematics
  • Electrical and Electronic Engineering

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