Analysis of fluorescence lifetime and quenching of FITC-conjugated antibodies on cells by phase-sensitive flow cytometry.

Abstract

Fluorescent antibodies are often used to measure the number of receptor 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 may invalidate this assumption. For many fluorophores, intermolecular interactions and energy transfer between molecules in close proximity to one another results in self-quenching. This effect can occur in antibody probes with a high fluorochrome to protein (F/P) ratio. It can also occur due to close proximity antibodies relative to one another on a highly labeled cell surface. Since self-quenching is accompanied by a change in the fluorescence decay and a decrease in the fluorescence lifetime, it may be conveniently identified using fluorescence lifetime spectroscopy. In this paper we apply the phase-sensitive detection method to investigate the impact of self-quenching on fluorescence lifetimes by flow cytometry, using a model system consisting of FITC conjugated anti-mouse Thy1.2 antibodies bound to murine thymus cells. We show that in addition to the expected variation of lifetimes as a function of F/P ratio of the probes, the fluorescence lifetime diminishes also as a function of antibody labeling concentration on the cell surface. This is consistent with self-quenching effects expected at high densities of FITC molecules.