Measuring FRET in Flow Cytometry and Microscopy

Péter Nagy1, György Vereb1, Sándor Damjanovich1, László Mátyus1, János Szöllősi1

1 University of Debrecen, Debrecen
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 12.8
DOI:  10.1002/0471142956.cy1208s38
Online Posting Date:  November, 2006
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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

     
 
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Table of Contents

  • Basic Protocol 1: Flow Cytometric FRET Resolved by Donor Quenching
  • Basic Protocol 2: Flow Cytometric FRET Measurements Based on Detection of Three Fluorescence Intensities
  • Alternate Protocol 1: Cell‐by‐Cell Correction for Autofluorescence
  • Alternate Protocol 2: Application of the FRET Protocol to Microscopy
  • Basic Protocol 3: Donor Photobleaching FRET Measurements in Microscopy
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Flow Cytometric FRET Resolved by Donor Quenching

  Materials
  • Cells of interest
  • Donor‐conjugated antibody
  • Acceptor‐conjugated antibody
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 1% to 3.7% (v/v) paraformaldehyde in PBS (optional)
  • Flow cytometer capable of detecting donor fluorescence intensity

Basic Protocol 2: Flow Cytometric FRET Measurements Based on Detection of Three Fluorescence Intensities

  Materials
  • Cells of interest
  • Donor‐conjugated antibody
  • Acceptor‐conjugated antibody
  • Phosphate buffered saline (PBS; appendix 2A)
  • 1% to 3.7% paraformaldehyde in PBS
  • Flow cytometer capable of detecting donor fluorescence and acceptor fluorescence intensities in the donor, FRET, and acceptor channels

Alternate Protocol 1: Cell‐by‐Cell Correction for Autofluorescence

  Materials
  • Cells labeled with donor‐conjugated antibody on coverslip
  • Cells double‐labeled with donor‐conjugated antibody and acceptor‐conjugated antibody on coverslip
  • Fluorescence microscope with filters appropriate for the fluorophores used
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Figures

Videos

Literature Cited

Literature Cited
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