Abstract
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.
Highlights
Cytotoxicity assays are essential for the testing and development of novel immunotherapies for the treatment of cancer
Encoding Luc146-1H2, a thermostable mutant of beetle luciferase luciferases from Photuris pennsylvanica (LucPpe), carrying a C-terminal Flag epitope tag into a lentiviral vector in which the expression of Luc146-1H2 was driven by constitutive active human EF1α promoter
Twenty-four hours after transfection, cell death was induced by treatment with digitonin (30 μg/ml for 90 min) and the luminescence activity was measured by the addition of D-luciferin containing assay buffer
Summary
Cytotoxicity assays are essential for the testing and development of novel immunotherapies for the treatment of cancer. 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. Cytotoxicity assays that are based on the release of constitutively expressed cellular endogenous enzymes such as lactate dehydrogenase (LDH)[3], glyceraldehyde 3-phosphate dehydrogenase (G3PDH)[4] or adenylate kinase (AK)[5] were developed These assays have poor sensitivity and do not distinguish between the death of target and effector cells in co-culture experiments. The Matador assay is highly sensitive and specific, marine luciferases are not ideal for in vivo bioluminescence imaging application due to the relatively high cost of their substrates (e.g., coelenterazine) and high background[9]. Cell lines engineered to express marine luciferases for their use in Matador assay cannot be utilized for in vivo bioluminescence experiments, thereby necessitating the need to generate two luciferase expressing stable cell lines; i.e., one expressing a marine luciferase for in vitro cytotoxicity assays and another expressing a firefly luciferase for in vivo bioluminescence applications
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