ANALYSIS OF FLUORESCENCE LIFETIME AND QUENCHING OF FITC-CONJUGATED ANTIBODIES ON CELLS BY PHASE-SENSITIVE FLOW-CYTOMETRY

Fluorescent antibodies are often used to measure the number of recepto r sites on cells, The quantitative estimate of the number of receptor sites using this procedure assumes that the fluorescence intensity on a cell is proportional to the number of bound antibodies, Quenching ma y invalidate this assumption, For many fluorophores, intermolecular in teractions and energy transfer between molecules in close proximity to one another result in self-quenching, This effect can occur in antibo dy probes with a high fluorochrome to protein (F/P) ratio. It can also occur due to close proximity of antibodies relative to one another on a highly labelled cell surface, Since self-quenching is accompanied b y a change in the fluorescence decay and a decrease in the fluorescenc e lifetime, it may be conveniently identified using fluorescence lifet ime spectroscopy, In this paper we apply the phase-sensitive detection method to investigate the impact of self-quenching on fluorescence li fetimes by flow cytometry, using a model system consisting of FITC con jugated anti-mouse Thy1.2 antibodies bound to murine thymus cells, We show that in addition to the expected variation of lifetimes as a func tion of F/P ratio of the probes, the fluorescence lifetime diminishes also as a function of antibody labelling concentration on the cell sur face, This is consistent with self-quenching effects expected at high densities of FITC molecules. (C) 1996 Wiley-Liss, Inc.