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Dual-color bioluminescence imaging assay using green- and red-emitting beetle luciferases at subcellular resolution

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Bioluminescence imaging is widely used to monitor cellular events, including gene expression in vivo and in vitro. Moreover, recent advances in luciferase technology have made possible imaging at the single-cell level. To improve the bioluminescence imaging system, we have developed a dual-color imaging system in which the green-emitting luciferase from a Brazilian click beetle (Emerald Luc, ELuc) and the red-emitting luciferase from a railroad worm (Stable Luciferase Red, SLR) were used as reporters, which were localized to the peroxisome and the nucleus, respectively. We clearly captured simultaneously the subcellular localization of ELuc in the peroxisome and SLR in the nucleus of a single cell using a high-magnification objective lens with 3-min exposure time without binning using a combination of optical filters. Furthermore, to apply this system to quantitative time-lapse imaging, the activation of nuclear factor triggered by tumor necrosis factor α was measured using nuclear-targeted SLR and peroxisome-targeted ELuc as the test and internal control reporters, respectively. We successfully quantified the kinetics of activation of nuclear factor κB using nuclear-targeted SLR and the transcriptional change of the internal control promoter using peroxisome-targeted ELuc simultaneously in a single cell, and showed that the activation kinetics, including activation rate and amplitude, differed among cells. The results demonstrated that this imaging system can visualize the subcellular localization of reporters and track the expressions of two genes simultaneously at subcellular resolution.

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We thank T. Yamazaki from Toyobo for providing the NF-κB reporter vector. We also thank S. Kumata and M. Sasao from the National Institute of Advanced Industrial Science and Technology for excellent technical assistance. This study was supported by a Grant-in-Aid for Scientific Research (no. 21590266 to Y.N.) from the Japan Society for the Promotion of Science.

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Correspondence to Yoshihiro Nakajima or Yoshihiro Ohmiya.

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Published in the topical collection Analytical Bioluminescence and Chemiluminescence with guest editors Elisa Michelini and Mara Mirasoli.

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Yasunaga, M., Nakajima, Y. & Ohmiya, Y. Dual-color bioluminescence imaging assay using green- and red-emitting beetle luciferases at subcellular resolution. Anal Bioanal Chem 406, 5735–5742 (2014). https://doi.org/10.1007/s00216-014-7981-7

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  • Dual-color bioluminescence imaging
  • Luciferase
  • Nuclear factor κB
  • Subcellular imaging
  • Time-lapse imaging