Long wavelength fluorophores and cell-by-cell correction for autofluorescence significantly improves the accuracy of flow cytometric energy transfer measurements on a dual-laser benchtop flow cytometer

Authors

  • Zsolt Sebestyén,

    1. Department of Biophysics and Cell Biology, Faculty of Medicine, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary
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  • Péter Nagy,

    1. Cell Biophysics Workgroup of the Hungarian Academy of Sciences, University of Debrecen, Debrecen, Hungary
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  • Gábor Horváth,

    1. Department of Biophysics and Cell Biology, Faculty of Medicine, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary
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  • György Vámosi,

    1. Cell Biophysics Workgroup of the Hungarian Academy of Sciences, University of Debrecen, Debrecen, Hungary
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  • Reno Debets,

    1. Department of Medical Oncology, Clinical and Tumor Immunology, University Hospital Rotterdam–Daniel den Hoed Cancer Center, Rotterdam, The Netherlands
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  • Jan W. Gratama,

    1. Department of Medical Oncology, Clinical and Tumor Immunology, University Hospital Rotterdam–Daniel den Hoed Cancer Center, Rotterdam, The Netherlands
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  • Denis R. Alexander,

    1. Laboratory of Lymphocyte Signalling and Development, Programme of Molecular Immunology, The Babraham Institute, Cambridge, United Kingdom
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  • János Szöllősi

    Corresponding author
    1. Department of Biophysics and Cell Biology, Faculty of Medicine, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary
    • Department of Biophysics and Cell Biology, Faculty of Medicine, Medical and Health Science Center, University of Debrecen, Nagyerdei kert 98, H-4012 Debrecen, Hungary
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Abstract

Background

Flow cytometric fluorescence resonance energy transfer (FCET) is an efficient method to map associations between biomolecules because of its high sensitivity to changes in molecular distances in the range of 1–10 nm. However, the requirement for a dual-laser instrument and the need for a relatively high signal-to-noise system (i.e., high expression level of the molecules) pose limitations to a wide application of the method.

Methods

Antibodies conjugated to cyanines 3 and 5 (Cy3 and Cy5) were used to label membrane proteins on the cell surface. FCET measurements were made on a widely used benchtop dual-laser flow cytometer, the FACSCalibur, by using cell-by-cell analysis of energy transfer efficiency.

Results

To increase the accuracy of FCET measurements, we applied a long wavelength donor–acceptor pair, Cy3 and Cy5, which beneficially affected the signal-to-noise ratio in comparison with the classic pair of fluorescein and rhodamine. A new algorithm for cell-by-cell correction of autofluorescence further improved the sensitivity of the technique; cell subpopulations with only slightly different FCET efficiencies could be identified. The new FCET technique was tested on various direct and indirect immunofluorescent labeling strategies. The highest FCET values could be measured when applying direct labeling on both (donor and acceptor) sides. Upon increasing the complexity of the labeling scheme by introducing secondary antibodies, we detected a decrease in the energy transfer efficiency.

Conclusions

We developed a new FCET protocol by applying long wavelength excitation and detection of fluorescence and by refining autofluorescence correction. The increased accuracy of the new method makes cells with low receptor expression amenable to FCET investigation, and the new approach can be implemented easily on a commercially available dual-laser flow cytometer, such as a FACSCalibur. Cytometry 48:124–135, 2002. © 2002 Wiley-Liss, Inc.

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