Fluorescence quenching; a practical problem in flow cytometry

Dual fluorescence analysis with a single-laser fluorescence-activated cell sorter is dependent on the use of two fluorochromes with similar excitation wavelengths but different emission wavelengths. The dye pair fluorescein and R-phycoerythrin (RPE) have been widely employed for this purpose and interaction between the two dyes has not been observed. Here evidence is presented to show that at high concentrations RPE can completely quench the fluorescein signal in dual fluorescence analysis of human tonsil lymphocytes labelled with pairs of monoclonal antibodies. Reduction in the fluorescein signal correlated with the intensity of red (RPE) staining. This phenomenon can seriously compromise interpretation of dual immunofluorescence carried out on a single laser instrument and can best be avoided by careful analysis of single colour controls.