Frequently Asked Questions

Most standard plate readers designed for measuring luminescence are suitable for this assay. Some instruments do not require gain adjustment, while others may require optimization of the gain settings to achieve sensitivity and dynamic range. An integration time of 0.3–1 second per well should serve as guidance. For exact instrument settings, consult your instrument manual.

The ROS-Glo™ Assay may be used to assess H₂O₂ in cells using either a lytic, homogeneous approach, or in cell culture media to allow for a non-lytic approach to the assay. We recommend assaying cells that are at <50% confluence.

The ROS-Glo ™ Assay may also be used as a defined, in vitro enzymatic assay.

Homogeneous (Lytic) Assay

• Test compounds may be added along with the H₂O₂ Substrate solution.
• If experimental treatment time is longer than 6 hours, add the test compound first, then add the H₂O₂ Substrate solution for the final 6 hours of treatment.

Non-Lytic Assay

• Media may be transferred to a separate plate after exposure to the H₂O₂
• Substrate solution, for processing separately. Culture media may not be stored or frozen prior to performing the ROS-Glo™ Assay.

Standard Curves

• For the purposes of quantifying the concentration of H₂O₂ absolutely, a H₂O₂ standard curve may be prepared. Guidelines may be found in Technical Manual #TM391.

Multiplexing Cell Health Assays

Live cell cytotoxicity assays such as the CellTox™ Green Cytotoxicity Assay (Cat.# G8731) may be multiplexed with the ROS-Glo™ Assay.  If the Non-Lytic protocol is used for the ROS-Glo™ Assay, then and endpoint lytic, homogeneous cell health assay such as the CellTiter-Glo^® 2.0 Luminescent Cell Viability Assay (Cat.# G9241) may be used with cells once the culture media supernatants have been reserved for the ROS asssay. Guidelines may be found in the Appendix of Technical Manual #TM391.

For Cell-Based Assays (Lytic or Non-Lytic)

The concentration of H₂O₂ intracellularly or extracellularly for cells in culture is dynamic and dependent upon ROS generation and consumption by the cells, the medium, the treatment applied to the cells, the vehicle that may be used with the treatment, and the effect of each of these upon the other. Promega recommends the following controls so that experimental signals are interpreted in the appropriate context:

1. Medium without cells, plus the vehicle used for test compounds (e.g., DMSO).
2. Medium with cells, plus the vehicle used for test compounds (e.g., DMSO).
3. Medium without cells, plus test compound.
4. Medium with cells, plus test compound.
5. Optional positive control H₂O₂ inducer: 50µM menadione in medium, plus and minus cells.

For Enzymatic Assays

1. Measure the enzyme-independent background signal by including samples that lack an essential reaction component (e.g., a no-enzyme control).
2. Compare active enzyme reactions with test compounds to active reactions in the presence of the treatment vehicle alone, without the drug or small molecule treatment (e.g., DMSO).
3. Some test compounds generate H₂O₂ in solution, in the absence of enzymatic activity. Control for these by comparing them with test compounds applied to inactivate enzyme assay samples. Consider applying this control only with compounds previously identified as H₂O₂ inducers in the enzyme assay.