SCATCHARD ANALYSIS OF FLUORESCENT CONCANAVALIN-A BINDING TO LYMPHOCYTES

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 now cytometry, the binding of fluorescein isothiocyanate-derivatized concanavalin A (FITC-ConA) to murine lymphocytes at 4 degrees C was compared to I-125-ConA binding. A FACS IV now cytometer (Becton-Dickinson, Mountain View, CA) 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 (1.3 x 10(6) receptors/cell, K = 3.6 x 10(-15)) similar to those obtained with I-125-ConA(1.4 x 10(6) receptors/cell, K = 5.2 x 10(-15)). Cytometric Scatchard plots showed less scatter and seemed more precise, suggesting superiority to radioactive ligand measurements, particularly at low Ligand concentrations. (C) 1995 Wiley-Liss, Inc.