Scatchard Analysis by Flow Cytometry

Abstract

The goal of the protocols described here is to measure the affinity of a ligand for cell surface receptor(s) and to determine the number of receptors per cell, following the classic method of Scatchard [1]. The preferred method for analysis of ligand binding is to measure ligand bound to cells at equilibrium without removing unbound ligand, since removing unbound ligand allows ligand dissociation to begin and may result in underestimation of the equilibrium value. As first described by Bohn [2] and Steinkamp and Kraemer [3], the small diameters of the sample stream and excitation beam used in most flow cytometers provide excellent discrimination between cell-associated and free ligand. Further discussion of the basis of this discrimination and analysis of the effects of ligand affinity, number of receptors per cell, and sample stream diameter on expected signal (fluorescence from bound ligand) to noise (fluorescence from free ligand) may be found elsewhere [4]. When the equilibrium method (Sect. 6.3.1) is not suitable, the rapid dilution method (Sect. 6.3.2) is frequently an acceptable substitute.