ONE-Glo™ + Tox Luciferase Reporter and Cell Viability Assay
Sensitive Detection of Reporter Activity and Cell Health
- Measures cell viability and firefly luciferase activity in the same assay well
- Easy to use—simply add reagent, mix and read
- Scalable to meet throughput needs, up to 1,536-well format
Catalog Number:
Size
Catalog Number: E7110
Catalog Number: E7120
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.
Reference
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.
Example ONE-Glo™ + Tox Assay Data
Ionomycin Titration in 96- and 384-well Plates
In the experiment shown here, 1 × 104 (96-well plate; Panel A) or 5 × 103 (384-well plate; Panel B) cells expressing NFAT response element were treated with serial titrations of ionomycin in the presence of PMA for 6 hours. At specific concentrations, ionomycin and PMA work cooperatively to stimulate NFAT-dependent gene expression. However, higher concentrations of ionomycin result in cytotoxicity seen as a decrease in viability (fluorescence). A decrease in reporter expression (luciferase) activity is also observed due to the increase in cytotoxicity.
Protocols
Specifications
Catalog Number:
Contenido
| Item | Part # | Presentación |
|---|---|---|
|
ONE-Glo™ Luciferase Assay Buffer |
E605A | 1 × 10ml |
|
ONE-Glo™ Luciferase Assay Substrate |
E606A | 1 × 1 vial |
|
GF-AFC Substrate |
G608A | 1 × 10μl |
|
Assay Buffer |
G610A | 1 × 10ml |
SDS
Search for SDSCertificado de Análisis
Use Restrictions
For Research Use Only. Not for Use in Diagnostic Procedures.Condiciones de Almacenaje
Contenido
| Item | Part # | Presentación |
|---|---|---|
|
ONE-Glo™ Luciferase Assay Buffer |
E605B | 1 × 100ml |
|
ONE-Glo™ Luciferase Assay Substrate |
E606B | 1 × 1 vial |
|
GF-AFC Substrate |
G608B | 2 × 50μl |
|
Assay Buffer |
G610B | 1 × 50ml |
SDS
Search for SDSCertificado de Análisis
Use Restrictions
For Research Use Only. Not for Use in Diagnostic Procedures.Condiciones de Almacenaje
Resources
No related resources available
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