Cellomics HCS Reagent Kit for measuring oxidative stress.

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%.

Description

The use of cell-based assays to assess mechanisms of cytotoxicity is becoming prevalent in both drug discovery and basic life science 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 high-content screening (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 intercalates into DNA. The DNA binding dye, Hoechst 33342, is used to identify the nuclei of the individual cells, and then the DHE fluorescence is measured to evaluate whether oxidative stress occurred.

Related Poster Presentations

	Kay Opperman, Bhaskar S. Mandavilli, and Richik N. Ghosh (2008) Assessment of Oxidative Stress and DNA Damage in Individual Cells Using Automated, Quantitative Cell-Based Imaging.
	Jeffrey R. Haskins, Bhaskar Mandavilli, Megan Cuda, Chandrasekaran Vasudevan, Richik N. Ghosh (2007) New reagent kits for high content cell-based assays for cytotoxicity.
	Chandrasekaran Vasudevan, and Jeffrey A. Haskins (2006) Leveraging the Power of High Content Screening for Monitoring Oxidative Stress.
	Gareth W. Phillips, William A. Irwin, Elodie J. Howard-Cofield, Laura E. Randle, Vivek C. Abraham, Jeffrey R. Haskins and Peter J. O'Brien (2005) Value of Incorporation of an Oxidative Stress Biomarker into High Content Screening (HCS) for Human Toxicity Potential.
	Vivek C. Abraham and Jeffrey R. Haskins (2005) Measurement of Oxidative Stress using Kinetic High Content Cell-based Assays.

Key Contents

• Dihydroethidium (DHE)
• Hoechst 33342 Dye
• Wash Buffer (10X Dulbecco’s PBS)
• Thin Plate Seal Assembly

Ordering Information

Screening size kits and components (bulk and custom) available with special pricing. Please call.

To order Cellomics HCS Reagent Kits, please call 800-874-3723 in the U.S.
or contact your local distributor of Thermo Scientific Products.

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