4.7 Article

A novel thermostable beetle luciferase based cytotoxicity assay

Journal

SCIENTIFIC REPORTS
Volume 11, Issue 1, Pages -

Publisher

NATURE PORTFOLIO
DOI: 10.1038/s41598-021-89404-z

Keywords

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Funding

  1. National Institutes of Health [S10 RR1796401, S10 OD2178501A1, R01DE025804]
  2. MIC at USC
  3. Ming Hsieh Institute for Research of Engineering-Medicine for Cancer at USC

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Cytotoxicity assays are crucial for testing novel immunotherapies for cancer treatment. The Matador-Glo assay utilizes a thermostable variant of Click Beetle Luciferase for luminescence detection and is simple, fast, specific, sensitive, cost-efficient, and not labor-intensive. This assay can also be combined with the marine luciferase-based Matador assay for dual luciferase detection of cell death, and the target cells expressing Luc146-1H2 can be used for in vivo bioluminescence imaging applications.
Cytotoxicity assays are essential for the testing and development of novel immunotherapies for the treatment of cancer. We recently described a novel cytotoxicity assay, termed the Matador assay, which was based on marine luciferases and their engineered derivatives. In this study, we describe the development of a new cytotoxicity assay termed 'Matador-Glo assay' which takes advantage of a thermostable variant of Click Beetle Luciferase (Luc146-1H2). Matador-Glo assay utilizes Luc146-1H2 and D-luciferin as the luciferase-substrate pair for luminescence detection. The assay involves ectopic over-expression of Luc146-1H2 in the cytosol of target cells of interest. Upon damage to the membrane integrity, the Luc146-1H2 is either released from the dead and dying cells or its activity is preferentially measured in dead and dying cells. We demonstrate that this assay is simple, fast, specific, sensitive, cost-efficient, and not labor-intensive. We further demonstrate that the Matador-Glo assay can be combined with the marine luciferase-based Matador assay to develop a dual luciferase assay for cell death detection. Finally, we demonstrate that the Luc146-1H2 expressing target cells can also be used for in vivo bioluminescence imaging applications.

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