skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Scatchard analysis of fluorescent concanavalin A binding to lymphocytes

Abstract

Standard Scatchard analysis of ligand binding to cell receptors requires the use of isotopes and is imprecise at low ligand concentrations. To evaluate the feasibility of Scatchard analysis via fluorescence flow cytometry, the binding of fluorescein isothio-cyanate-derivatized concanavalin A (FITC-ConA) to murine lymphocytes at 4{degrees}C was compared to {sup 125}I-ConA binding. A FACS IV flow cytometer was used for analysis of cells after fluorescent ligand binding. A simple spectrophotometric technique was used to calibrate the relation between cytometer-determined fluorescence and ligand binding per cell. As FITC-ConA binding showed a quasi-Gaussian distribution, the mean number of molecules bound per cell was easily calculated. Scatchard analysis of FITC-ConA binding yielded results (1.9 x 10{sup 6} receptors/cell, K = 3.6 x 10{sup -15}) similar to those obtained With {sup 125}I-ConA (1.4 x 10{sup 6} receptors/cell, K = 5.2 x 10{sup -15}). Cytometric Scatchard plots showed less scatter and seemed more precise, suggesting superiority to radioactive ligand measurements, particularly at low ligand concentrations. 32 refs., 4 figs., 1 tab.

Authors:
 [1]
  1. Univ. of California Irvine, Long Beach, CA (United States)
Publication Date:
Sponsoring Org.:
USDOE
OSTI Identifier:
443835
Resource Type:
Journal Article
Journal Name:
Cytometry
Additional Journal Information:
Journal Volume: 20; Journal Issue: 3; Other Information: PBD: 1 Jul 1995
Country of Publication:
United States
Language:
English
Subject:
55 BIOLOGY AND MEDICINE, BASIC STUDIES; CONCANAVALIN A; RECEPTORS; MITOGENS; CELL FLOW SYSTEMS; ACCURACY; COMPARATIVE EVALUATIONS; RADIOASSAY; LECTINS; LIGANDS; ISOTOPES; LYMPHOCYTES; FLUORESCEIN; IODINE 125

Citation Formats

Gordon, I L. Scatchard analysis of fluorescent concanavalin A binding to lymphocytes. United States: N. p., 1995. Web. doi:10.1002/cyto.990200307.
Gordon, I L. Scatchard analysis of fluorescent concanavalin A binding to lymphocytes. United States. https://doi.org/10.1002/cyto.990200307
Gordon, I L. 1995. "Scatchard analysis of fluorescent concanavalin A binding to lymphocytes". United States. https://doi.org/10.1002/cyto.990200307.
@article{osti_443835,
title = {Scatchard analysis of fluorescent concanavalin A binding to lymphocytes},
author = {Gordon, I L},
abstractNote = {Standard Scatchard analysis of ligand binding to cell receptors requires the use of isotopes and is imprecise at low ligand concentrations. To evaluate the feasibility of Scatchard analysis via fluorescence flow cytometry, the binding of fluorescein isothio-cyanate-derivatized concanavalin A (FITC-ConA) to murine lymphocytes at 4{degrees}C was compared to {sup 125}I-ConA binding. A FACS IV flow cytometer was used for analysis of cells after fluorescent ligand binding. A simple spectrophotometric technique was used to calibrate the relation between cytometer-determined fluorescence and ligand binding per cell. As FITC-ConA binding showed a quasi-Gaussian distribution, the mean number of molecules bound per cell was easily calculated. Scatchard analysis of FITC-ConA binding yielded results (1.9 x 10{sup 6} receptors/cell, K = 3.6 x 10{sup -15}) similar to those obtained With {sup 125}I-ConA (1.4 x 10{sup 6} receptors/cell, K = 5.2 x 10{sup -15}). Cytometric Scatchard plots showed less scatter and seemed more precise, suggesting superiority to radioactive ligand measurements, particularly at low ligand concentrations. 32 refs., 4 figs., 1 tab.},
doi = {10.1002/cyto.990200307},
url = {https://www.osti.gov/biblio/443835}, journal = {Cytometry},
number = 3,
volume = 20,
place = {United States},
year = {Sat Jul 01 00:00:00 EDT 1995},
month = {Sat Jul 01 00:00:00 EDT 1995}
}