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Potential limitations in the use of KillerRed for fluorescence microscopy

M Nordgren1, B Wang, O Apanasets

  • 1Laboratory of Lipid Biochemistry and Protein Interactions, Department of Molecular Cell Biology, Katholieke Universiteit Leuven, Leuven, Belgium.

Journal of Microscopy
|November 19, 2011
PubMed

Insights

KillerRed protein generates reactive oxygen species upon illumination. Researchers found it also emits green light, potentially causing misinterpretation in biological imaging studies.

Area of Science:

  • Cell Biology
  • Biochemistry
  • Biophysics

Background:

  • KillerRed is a red fluorescent protein and photosensitizer.
  • It generates reactive oxygen species (ROS) upon green light exposure.
  • ROS can be used to study cellular processes and induce cell death.

Purpose of the Study:

  • To investigate if KillerRed-induced ROS in peroxisomes trigger autophagosome sequestration.
  • To assess KillerRed's utility as a tool for studying ROS-mediated cellular events.

Main Methods:

  • Utilized KillerRed protein as a photosensitizer within peroxisomes.
  • Employed green fluorescent protein-tagged microtubule-associated protein 1 light chain 3 (GFP-LC3) as an autophagosome marker.
  • Analyzed KillerRed's fluorescence properties upon different light excitations.

Main Results:

  • Observed that KillerRed emits weak green fluorescence when excited at 480 nm.
  • This dual fluorescence can lead to erroneous interpretations in experiments using green fluorophores.
  • The study did not confirm peroxisomal ROS sequestration into autophagosomes.

Conclusions:

  • KillerRed's unintended green fluorescence is a critical pitfall for biological imaging.
  • Recommendations are provided to prevent misinterpretation of data when using KillerRed.
  • Caution is advised when combining KillerRed with green fluorescent markers.

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