Use of phycoerythrin and allophycocyanin for fluorescence resonance energy transfer analyzed by flow cytometry: Advantages and limitations

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Abstract
Abstract 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 CD8α chain was used. FRET was also detected between antibody conjugate pairs specific for the two chains of the heterodimeric α 4β1 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. Cytometry 48:97–105, 2002. © 2002 Wiley‐Liss, Inc.