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Fluorescence quenching; a practical problem in flow cytometry
Author(s) -
Chapple M. R.,
Johnson G. D.,
Davidson R. S.
Publication year - 1990
Publication title -
journal of microscopy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.569
H-Index - 111
eISSN - 1365-2818
pISSN - 0022-2720
DOI - 10.1111/j.1365-2818.1990.tb03030.x
Subject(s) - fluorescein , fluorescence , flow cytometry , phycoerythrin , excitation wavelength , signal (programming language) , chemistry , wavelength , laser , biophysics , immunofluorescence , quenching (fluorescence) , laser induced fluorescence , staining , optics , microbiology and biotechnology , antibody , biology , physics , pathology , medicine , immunology , computer science , programming language
SUMMARY 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.