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 first part of the assay is a nonlytic fluorescence assay (CellTiter-Fluor Cell Viability Assay) that measures the relative number of live cells in a culture population after experimental manipulation. The CellTiter-Fluor Assay measures a conserved and constitutive protease activity within live cells and therefore serves as a marker of cell viability. The 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. This 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. The second part of the assay uses the ONE-Glo Luciferase Assay System to quantify firefly luciferase reporter gene expression from cells made to express this reporter enzyme. The ONE-Glo Luciferase Assay Buffer and ONE-Glo Luciferase Assay Substrate, provided with this system, are combined to form the ONE-Glo Reagent. Ideally suited for high- and ultrahigh-throughput applications, the ONE-Glo Assay contains a new fluoroluciferin substrate, resulting in a more stable reagent that is more tolerant to sample components and has less odor than standard luciferase assay reagents. Luminescence is measured with a microplate reader or CCD imager.|
Acquire More Data: Measure cell viability and firefly luciferase expression in the same assay well.Better Biology: Understand reporter gene expression in the context of cell viability.Easy to Perform: The assay uses a simple sequential “add-mix-read” format.Flexible and Automation-Friendly: The volumes of each assay component can be scaled to meet throughput needs, up to 1,536-well format.