Oxidative Stress 1 Reagent Kit

• Robust assays – Z’ values are >0.3 for multiplexed and singleplexed assays 
• Validated on Cellomics ArrayScan HCS readers: also works on other High Content Screening (HCS) platforms and fluorescent microscopes 
• Customized components and bulk quantities available 
• Optimized protocol and reagent preparations included
   

• HCS Reagent Kits have been validated in HCS assays for reliable and reproducible results 
• HCS Reagent Kits work on all HCS platforms offering flexibility in end-user instrumentation 
• Purchase only the reagents needed for research or for large screening applications 
• Offers ease-of-use, no optimization or assay development required which saves valuable research time

This kit can be used with other Cellomics BioApplications. Thus, automated plate handling, focusing, cell image acquisition/processing, and data analysis/management are combined in one HCS system to assay for test compounds regulating oxidative stress. In addition to HCS instruments, cells labeled by the kit reagents can be viewed and analyzed by fluorescence microscopy-based instruments.

The use of cell-based assays to assess mechanisms of cytotoxicity is becoming prevalent in both drug discovery and basic life sciences research. One of the several methods by which compounds can induce a toxic response in cells is via oxidative stress. Although numerous cytotoxicity mechanisms are investigated using HCS, oxidative stress is one of the most important. We have developed a method to quantify chemically induced oxidative stress by measuring the amount of DNA bound to ethidium, a product of dihydroethidium (DHE) oxidation. The Oxidative Stress I Kit contains DHE and Hoechst 33342 at optimized concentrations. The method’s principle is that reactive oxygen species convert non-fluorescent dihydroethidium to fluorescent ethidium, which then intercalates into DNA. The DNA binding dye, Hoechst 33342, is used to identify the nuclei of individual cells, and then the DHE fluorescence is measured to evaluate whether oxidative stress occurred.

Figure 1. Images from using Oxidative Stress 1 Kit. Superoxide formation in HepG2 cells was determined by DHE staining following treatment with rotenone. HepG2 cells were plated in triplicate in 96 well plates overnight and then treated with media alone or 350 μM rotenone for 24 hrs (min/max). The cells were stained with DHE and Hoechst dyes for 30 minutes and then the fixed cells were imaged and analyzed. Following treatment with rotenone we can see both nuclear condensation and also increased accumulation of ethidium following oxidation by ROS. The percentage of cells that were responders for oxidative stress (based on presence of DHE intensity) was automatically calculated and used to calculate the Z’ scores: Z’ from 3 min/max plates = 0.49 ± 0.06, and %COV from max wells = 7.8% ± 1.7%.

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