Bioluminescent Assay for Enzymatic Screening Programs and Cell-based Assays

Promega offers the ROS-Glo H₂O₂ Assay. A non-horseradish peroxidase (HRP)-dependent, plate-based bioluminescent assay for detecting reactive oxygen species (ROS), ROS-Glo H₂O₂ Assay is designed to specifically detect hydrogen peroxide (H₂O₂) in both enzyme and cell-based applications. The ROS-Glo H₂O₂ Assay measures the activity of enzymes that generate or eliminate H₂O₂, and possesses many benefits over other commercially available assays for small molecule screening, including low false hit rate, signal stability and compatibility with liquid handling. Importantly for screening applications, the assay does not rely on a reaction catalyzed by HRP, which is known to cause a high number of false hits. The assay can also be used to measure changes in the level of ROS by directly detecting H₂O₂in cultured mammalian cells after two reagent additions, with no cell sample preparation required, reducing variability and the number of cells required.  

The new assays enable researchers to more effectively study the biology of ROS, and to screen large compound libraries for their capacity to alter H₂O₂levels in cultured cells or in enzymatic reactions. Although ROS can be beneficial to human cells as they are common byproducts of metabolism, they can also lead to cellular damage, such as oxidative stress, through environmental factors or aberrant metabolism.

The homogeneous ROS-Glo H₂O₂ Assay uses a modified luciferin substrate that reacts directly with H₂O₂ to generate a luciferin precursor. Upon addition of detection reagent, precursor is converted to luciferin and Ultra-Glo Recombinant Luciferase included in the detection reagent produces a light signal proportional to the level of H₂O₂ in the sample.

Due to the bioluminescent format and high sensitivity, ROS-Glo H₂O₂ Assay can be multiplexed with a variety of other cell-based assays to gain additional data from a sample. For example, CellTox Green Cytotoxicity Assay reagent can be added to the same well at cell plating or treatment to kinetically monitor cytotoxicity by measuring changes in fluorescence, followed by implementation of the ROS-Glo H₂O₂ Assay to measure changes in ROS levels by luminescence detection.

Promega Corp., 800-356-9526, www.promega.com/rosglo