title: Measuring FRET in flow cytometry and microscopy.
abstract: This unit presents protocols describing the measurement of protein associations using FRET as determined by flow and image cytometry. The proteins under investigation can be labeled by fluorescent antibodies or fluorescent protein (FP) variants. The flow cytometry protocols determine FRET based on the measurement of donor quenching, which provides a FRET value on a population basis, or based on the measurement of fluorescence intensities in the donor, FRET, and acceptor channels, which provides cell-by-cell FRET values. An extension of this protocol is based on cell-by-cell correction for autofluorescence and requires the measurement of four fluorescence intensities. The algorithm described can be applied in image cytometric FRET as well. The image protocol determines FRET resolved by donor photobleaching. The authors provide extensive discussion of pitfalls, limitations, and interpretation.
authors.affiliation: University of Debrecen, Debrecen, Hungary.
authors.forename: Péter
authors.initials: P
authors.lastname: Nagy
authors.forename: György
authors.initials: G
authors.lastname: Vereb
authors.forename: Sándor
authors.initials: S
authors.lastname: Damjanovich
authors.forename: László
authors.initials: L
authors.lastname: Mátyus
authors.forename: János
authors.initials: J
authors.lastname: Szöllõsi
mesh_terms: D005434:Flow Cytometry / Q000379:methods*; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000032:analysis; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000032:analysis*; D008853:Microscopy / Q000379:methods; D013050:Spectrometry, Fluorescence
chemical_list: D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins