Dear Richard! Yes, we have used the FacsCalibur to perform FRET measurements using Cy3 and Cy5 as donor acceptor pair. The 488 nm excitation is not optimal for Cy3 (543 would be better), 632 is optimal for Cy5, and with this setup we could obtain reasonable good FRET distribution curves (actually they are better than that obtained with FITC/TRITC pair because the autofluorescence is much less of a problem). In addition we could use the 488->520 band for autofluorescence correction on a cell-by-cell basis. The method was presented in the ISAC meeting in San Diego, and a manuscript has been accepted in the CYTOMETRY and will be published within a few month. If you are interested we can send the manuscript. 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 Zsolt Sebestyén1, Péter Nagy2, Gábor Horváth1, György Vámosi2, Reno Debets3, Jan W. Gratama3, Denis R. Alexander4, János Szöllősi1 In addition we have elaborated a software which can calculate the FRET efficiency on a cell-by-cell basis and made it available for the public. We have also included some help file and some sample data. The web page is: www.biophys.dote.hu/kutatas/research.htm and link to follow: Instruments, software. The FLEX program is on the side. We are still working on the help instruction, and as we proceed we constantly upgrade our web page. Pascal Batard has used Phycoerythrin and Allophycocyanin as a donor/acceptor pair in FacsCalibur, and has shown that the sizes of the fluorophores can decrease the maximum FRET efficiency obtainable. This manuscript has been also accepted in the Cytometry and will be published within a few month. You can approach Pascal Batard for preprints. Pascal Batard <Pascal.Batard@inst.hospvd.ch> The Use of Phycoerythrin and Allophycocyanin for Fluorescence Resonance Energy Transfer Analyzed by Flow Cytometry: Advantages and Limitations Pascal Batard1,*, Janos Szollosi2, Immanuel Luescher3, Jean-Charles Cerottini3, Rob MacDonald3, Pedro Romero1 Regards Janos At 08:52 AM 5/15/2002 -0400, you wrote: >Greetings everyone: > >Has anyone had any success doing FRET on a single or dual laser FACS >analyzer? [ie-Facscan or Facscalibur] > >Suggestions of FRET pairs and tips would be greatly appreciated. > >All my best, > >-Rich > > > >Richard Konz, Jr. >Scientist >Manager; Flow and Image Cytometry Facility >Department of Musculoskeletal Sciences >Wyeth >200 Cambridge Park Drive >Cambridge, MA 02140 >Office: 617.665.5522 >Lab: 617.665.5551 >rkonz@wyeth.com Janos Szollosi University of Debrecen Medical and Health Science Center Faculty of Medicine Department of Biophysics and Cell Biology P.O.Box 39, Nagyerdei krt. 98 H-4012 Debrecen Hungary Phone/Fax: (36) (52) 412-623 E-mail: szollo@jaguar.dote.hu
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