Calibration of flow cytometric fluorescence standards using the isoparametric analysis of ligand binding

To extract ligand-cell binding parameters from flow cytometric fluorescence data, one needs a method of converting the measured cellular fluorescence to the actual number of bound molecules producing that fluorescence. Bead standards containing a known number of fluorophores do not necessarily provide the correct conversion factor. We therefore present a method for calibrating flow cytometric bead standards. The technique uses the isoparametric analysis (Chatelier et al: EMBO J 5:1181-1186, 1986) to construct a plot of fluorescence per cell versus the number of bound ligands per cell, thus allowing a direct comparison of the quantum yields of the bead-associated fluorophore with that of the cell-bound fluorophore. The potential of this analysis is demonstrated by contrasting the fluorescent brightness of fluorescein isothiocyanate associated with thymocyte nuclei and synthetic polymer beads with that of fluoresceinated epidermal growth factor bound to A431 cells. When the standards are improperly calibrated, then the number of ligand-binding sites is incorrectly reported, and the derived Scatchard plot may exhibit an apparent positive or negative cooperativity as well as a strong dependence on cell concentration.