1552-4930/asset/olbannerleft.gif?v=1&s=cf6fcbfdc8ffa722551b71f8be0977b5b04db6ee)
1552-4930/asset/olbannerright.gif?v=1&s=6d0ba4e64946e5d9d4ebba4b8395a68bf324cd0b)
Original Article
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
Article first published online: 27 JUN 2002
DOI: 10.1002/cyto.10121
Copyright © 2002 Wiley-Liss, Inc.
1552-4930/asset/cover.gif?v=1&s=3df33f34517aaa739d7876df137e2ab92c5f82e7 "Cytometry")
Additional Information
How to Cite
Sebestyén, Z., Nagy, P., Horváth, G., Vámosi, G., Debets, R., Gratama, J. W., Alexander, D. R. and Szöllősi, J. (2002), 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. Cytometry, 48: 124–135. doi: 10.1002/cyto.10121
Publication History
- Issue published online: 27 JUN 2002
- Article first published online: 27 JUN 2002
- Manuscript Accepted: 1 MAY 2002
- Manuscript Revised: 15 APR 2002
- Manuscript Received: 1 FEB 2002
Funded by
- European Commission. Grant Number: FP 5 QLRT-1999-3126
- Hungarian–British Joint Academic and Research Programme. Grant Number: 069
- Hungarian Academy of Sciences. Grant Number: OTKA 303999
- Hungarian Ministry of Health. Grant Numbers: ETT 07-461, ETT 117/2001
- Hungarian Ministry of Education. Grant Number: FKFP 0607
- Abstract
- Article
- References
- Cited By
Keywords:
- autofluorescence;
- flow cytometric resonance energy transfer (FCET);
- indirect immunofluorescence;
- cyanine 3 and 5 donor–acceptor pair;
- CD45 isoforms;
- HLA class I molecules
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.