Use of phycoerythrin and allophycocyanin for fluorescence resonance energy transfer analyzed by flow cytometry: advantages and limitations.
BACKGROUND
This study validates the use of phycoerythrin (PE) and allophycocyanin (APC) for fluorescence energy transfer (FRET) analyzed by flow cytometry.


METHODS
FRET was detected when a pair of antibody conjugates directed against two noncompetitive epitopes on the same CD8alpha chain was used. FRET was also detected between antibody conjugate pairs specific for the two chains of the heterodimeric alpha (4)beta(1) integrin. Similarly, the association of T-cell receptor (TCR) with a soluble antigen ligand was detected by FRET when anti-TCR antibody and MHC class I/peptide complexes (<<tetramers>>) were used.


RESULTS
FRET efficiency was always less than 10%, probably because of steric effects associated with the size and structure of PE and APC. Some suggestions are given to take into account this and other effects (e.g., donor and acceptor concentrations) for a better interpretation of FRET results obtained with this pair of fluorochromes.


CONCLUSIONS
We conclude that FRET assays can be carried out easily with commercially available antibodies and flow cytometers to study arrays of multimolecular complexes.
Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, Lausanne Branch, University Hospital (CHUV), Niveau 5, Avenue Pierre Decker 4, 1005 Lausanne, Switzerland. pascal.batard@inst.hospvd.ch
Pascal
P
Batard
Janos
J
Szollosi
Immanuel
I
Luescher
Jean-Charles
JC
Cerottini
Rob
R
MacDonald
Pedro
P
Romero
D016827:CD8 Antigens / Q000032:analysis; D002478:Cells, Cultured; D005434:Flow Cytometry / Q000379:methods*; D005453:Fluorescence; D005456:Fluorescent Dyes; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D010798:Phycocyanin; D010799:Phycoerythrin; D011948:Receptors, Antigen, T-Cell / Q000032:analysis; D013050:Spectrometry, Fluorescence; D013602:T-Lymphocytes, Cytotoxic / Q000378:metabolism
D016827:CD8 Antigens; C431698:CD8alpha antigen; D005456:Fluorescent Dyes; D011948:Receptors, Antigen, T-Cell; C002497:allophycocyanin; D010798:Phycocyanin; D010799:Phycoerythrin