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Rapid Imaging of BCL-2 Family Interactions in Live Cells Using FLIM-FRET

  • Elizabeth J. Osterlund
  • Nehad Hirmiz
  • Christian Tardif
  • David W. Andrews
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1877)

Abstract

The Bcl-2 proteins control cell death via interchanging interactions within the Bcl-2 family. Fluorescence lifetime imaging microscopy (FLIM) is used to detect Förster resonance energy transfer (FRET) between two fluorescent-fusion proteins in live cells. FLIM-FRET has been used to detect specific interactions and their disruption, for Bcl-2 family proteins. To date, this has been possible only in low throughput, due to the time required for serial data acquisition. We developed an automated optical system with eight parallel detectors for rapid and efficient data collection. Here we describe how to use this system for FLIM-FRET imaging of Bcl-2 family protein interactions in a 384-well plate format.

Key words

FLIM-FRET FLIM Hyperspectral High throughput Fluorescence lifetime imaging microscopy mCerulean3 Bcl-2 family BH3 mimetic 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Elizabeth J. Osterlund
    • 1
    • 2
  • Nehad Hirmiz
    • 2
    • 3
  • Christian Tardif
    • 4
  • David W. Andrews
    • 1
    • 2
  1. 1.Department of BiochemistryUniversity of TorontoTorontoCanada
  2. 2.Sunnybrook Research InstituteTorontoCanada
  3. 3.School of Biomedical EngineeringMcMaster UniversityHamiltonCanada
  4. 4.National Optics InstituteQuebec CityCanada

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