Fluorescence quenching of fluorescein by R-phycoerythrin. A pitfall in dual fluorescence analysis.

The use of a single laser fluorescence-activated cell sorter (FACS) to analyse cellular subpopulations by immunochemical staining requires an alternative dye to fluorescein with appropriate spectral characteristics. R-phycoerythrin (RPE) is widely employed for this purpose. In this study the ability of RPE to quench the fluorescein emission when both are attached to the same cell has been demonstrated by dual labelling of tonsil lymphocytes with pairs of monoclonal antibodies. Reduction of the fluorescein signal correlated with the amount of RPE attached and the relative intensity of emission from the two fluorochromes. The possible photochemical mechanisms which result in a reduction of the fluorescein signal by RPE are discussed. The inclusion of control tests, in which RPE is omitted, is recommended in order to avoid misinterpretation of the results of subpopulation analysis by single laser FACS - especially when low levels of fluorescein staining are obtained.