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.
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.ResultsTo 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.
Department of Biophysics and Cell Biology, Faculty of Medicine, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary.
Zsolt
Z
Sebestyén
Péter
P
Nagy
Gábor
G
Horváth
György
G
Vámosi
Reno
R
Debets
Jan W
JW
Gratama
Denis R
DR
Alexander
János
J
Szöllosi
D000465:Algorithms; D002138:Calibration; D002461:Cell Line, Transformed; D004735:Energy Transfer; D005434:Flow Cytometry / Q000295:instrumentation* / Q000379:methods*; D005453:Fluorescence; D005456:Fluorescent Dyes; D006801:Humans; D007834:Lasers; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence / Q000379:methods; D013382:Subtraction Technique; D014407:Tumor Cells, Cultured
D005456:Fluorescent Dyes