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ONE-Glo + Tox Luciferase Reporter and Cell Viability Assay

by Promega
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  • Overview
  • Protocols
  • Specifications
  • Resources
  • Understand Reporter Gene Activity in the Context of Cell Viability

    The ONE-Glo™ + Tox Assay combines luciferase assay chemistry with a cell viability marker to better understand reporter gene expression in the context of cell health. The assay uses a two-step, addition-only process to make these measurements in a single well of a plate, negating the need to run parallel assays. The simple "add-mix-read" assay format and optimized reagents combine to create a flexible, automation-friendly assay that can be scaled to meet your needs.

    Step 1: Cell Viability Assay

    In the first part of the assay, a nonlytic fluorescence-based method (CellTiter-Fluor™ Cell Viability Assay) is used to measure the relative number of live cells in culture. The CellTiter-Fluor™ Assay measures a conserved and constitutive protease activity within living cells. This live-cell protease activity is restricted to intact viable cells and is measured using a fluorogenic, cell-permeant peptide substrate (glycylphenylalanyl-aminofluorocoumarin; GF-AFC). The substrate enters intact cells where it is cleaved by the live-cell protease to generate a fluorescent signal proportional to the number of living cells. The live-cell protease becomes inactive upon loss of cell membrane integrity and leakage into the surrounding culture medium. Fluorescence of the free AFC fluorophore is measured with a microplate reader or CCD imager using an excitation wavelength of 380–400nm and emission wavelength of 505nm.

    Step 2: ONE-Glo™ Luciferase Assay

    The second part of the assay uses the ONE-Glo™ Luciferase Assay System to quantify firefly luciferase gene expression from cells expressing the reporter. Ideally suited for high- and ultrahigh-throughput applications, the ONE-Glo™ Assay Reagent contains a fluoroluciferin substrate that gives increased stability and greater tolerance of sample components, and has less odor than standard luciferase assay reagents. Luminescence is measured with a microplate reader or CCD imager.


    Niles, A.L. et al. (2007) A homogeneous assay to measure live and dead cells in the same sample by detecting different protease markers. Anal. Biochem. 366, 197–206.

    Schematic of the ONE-Glo™+Tox Luciferase Reporter and Cell Viability Assay.
  • Protocols

    Complete Protocol

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    ONE-Glo™ + Tox Luciferase Reporter and Cell Viability Assay Technical Manual

    PDF (524 KB)

    Quick Protocols

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    ONE-Glo™ + Tox Luciferase Reporter and Cell Viability Assay FB129

    PDF (124 KB)

  • Certificate of Analysis

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    Storage Conditions

    -30C TO -10C

    Use Restrictions

    For Research Use Only. Not for Use in Diagnostic Procedures.

  • Resources