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:
-
- 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}
}