Unit

UNIT 12.8 Measuring FRET in Flow Cytometry and Microscopy

  1. Péter Nagy,
  2. György Vereb,
  3. Sándor Damjanovich,
  4. László Mátyus,
  5. János Szöllősi

Published Online: 1 NOV 2006

DOI: 10.1002/0471142956.cy1208s38

Current Protocols in Cytometry

Current Protocols in Cytometry

How to Cite

Nagy, P., Vereb, G., Damjanovich, S., Mátyus, L. and Szöllősi, J. 2006. Measuring FRET in Flow Cytometry and Microscopy. Current Protocols in Cytometry. 38:12.8:12.8.1–12.8.13.

Author Information

  1. University of Debrecen, Debrecen, Hungary

Publication History

  1. Published Online: 1 NOV 2006
  2. Published Print: OCT 2006

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.

Keywords:

  • fluorescence resonance energy transfer;
  • Förster distance;
  • autofluorescence;
  • photobleaching;
  • cell surface mapping