Selecting the right fluorophores and flow cytometer for fluorescence resonance energy transfer measurements.
BACKGROUND
Fluorescence resonance energy transfer applied in flow cytometry (FCET) is an excellent tool for determining supramolecular organization of biomolecules at the cell surface or inside the cell. Availability of new fluorophores and cytometers requires the establishment of fluorophore dye pairs most suitable for FCET measurements.


METHODS
A gastric tumor cell line (N87) was labeled for major histocompatibility complex class I heavy chain and beta2-microglobulin with antibodies conjugated with fluorescein- and indocarbocyanine-like fluorophores and analyzed in FCET measurements on a cell-by-cell basis using three flow cytometers: FACSCalibur, FACSDiVa, and FACSArray.


RESULTS
Normalized fluorescence intensity values were measured and normalized energy transfer efficiencies, spectral overlap integrals, and crucial dye- and instrument-dependent parameters were calculated for all matching pairs of seven fluorophores on the three commercial cytometers. The most crucial parameter in determining the applicability of the donor-acceptor pairs was the normalized fluorescence intensity and the least important one was the spectral overlap.


CONCLUSIONS
On the basis of available laser lines, the optimal dye pair for all three cytometers is the Alexa546-Alexa647 pair, which produces high energy transfer efficiency values and has the best spectral characteristics with regard to laser excitation, detection of emission, and spectral overlap.
Horváth
Gábor
G
Department of Biophysics and Cell Biology, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary.
Petrás
Miklós
M
Szentesi
Gergely
G
Fábián
Akos
A
Park
John W
JW
Vereb
György
G
Szöllosi
János
J
D045744:Cell Line, Tumor; D005434:Flow Cytometry / Q000295:instrumentation* / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation* / Q000379:methods*; D005456:Fluorescent Dyes / Q000494:pharmacology; D005805:Genes, MHC Class I; D006023:Glycoproteins / Q000378:metabolism; D006801:Humans; D007202:Indicators and Reagents / Q000494:pharmacology; D037102:Lectins; D011806:Quinolinium Compounds / Q000494:pharmacology
C481052:Alexa fluor 546; C109662:FCN3 protein, human; D005456:Fluorescent Dyes; D006023:Glycoproteins; D007202:Indicators and Reagents; D037102:Lectins; D011806:Quinolinium Compounds