This unit presents protocols describing the measurement of protein associations usingFRET by flow and image cytometry. The theoretical background of FRET is describedin detail inUNIT 1.12, and will not be discussed here. FRET is ideal for the investigation ofprotein associations, but can also be used for the sorting of cells in which interaction ofone protein with another is detected by FRET (Sz¨oll´o´si et al., 1998; M´atyus et al., 2001;Nagy et al., 2005; van Wageningen et al., 2006). The proteins under investigation canbe labeled by fluorescent antibodies or fluorescent protein (FP) variants. The protocolsdescribed are applicable to both situations, except where indicated. Four protocols will bepresented. Basic Protocol 1 describes flow cytometric FRET based on the measurementof donor quenching. This method provides a FRET value on a population basis. BasicProtocol 2 covers flow cytometric FRET based on the measurement of fluorescenceintensities in the donor, FRET, and acceptor channels, providing cell-by-cell FRETvalues. Alternate Protocol 1 is based on cell-by-cell correction for autofluorescence andrequires the measurement of four fluorescence intensities. The algorithm described canbe applied for image cytometric FRET as well. Alternate Protocol 2 is a procedurefor application of the FRET protocol to microscopy. Basic Protocol 3 describes imagecytometric FRET resolved by donor photobleaching. Consult Table 12.8.1 for applicablecombinations of donor and acceptor dye pairs
