Monday, August 24, 2009

Measurement of Reactive Oxygen Species (ROS) in the Microplate Format

Reactive Oxygen Species (ROS) is a phrase used to describe a number of reactive molecules and free radicals derived from molecular oxygen. Reactive oxygen species were originally thought to be only released by phagocytic cells as part of their role in host defense. Recent work has demonstrated that ROS have a role in cell signaling, including; apoptosis; gene expression; and the activation of cell signaling cascades.

The measurement of reactive oxygen species is dependent on the analytic target. At the cellular level specific reactive oxygen species can be individually assessed from tissue culture, while at the whole animal level typically the effects of oxidative stress are measured from blood product (e.g. serum or plasma) or from urine samples. Depending on the ROS species of interest, measurements can be made using any one of the three different read modalities: absorbance; fluorescence; and luminescence. For example, glutathione, the most significant non enzymatic oxidant defense mechanisms can be measured using absorbance [1] or luminescence [2]. Likewise lipid peroxidation can be assessed by the detection of detection of thiobarbituric acid (TBA) reactive compounds such as malondialdehyde in microplates colorimetrically at 532 nm or by fluorescence using a 530 nm excitation wavelength and a 550 nm emission wavelength [3].

Mitogenic stimulation of cells has been shown to result in an increase in hydrogen peroxide (H2O2), which can be detected by the production of fluorescence from dichlorofluorescein (DCF) [4] or Peroxy Green 1 (PG-1) [5]. These compounds are non-fluorescent until they are oxidized within the cell by reactive oxygen species.

The multimode readers from BioTek, such as the Synergy 2, Synergy 4 and Synergy Mx are ideal platforms for quantitation of these compounds and many others associated reactive oxygen species. In addition to the detection readers available from BioTek, the Precision pipettor and MicroFill or MicroFlo dispensers can be used to pipette samples or dispense reagents respectively.

Do you have any interest in measuring reactive oxygen species in microplates? Do you currently measure ROS using flow cytometry or by microscopy? What indicator dye do you use?


1. Baker, MA, G.J. Cerniglia, and A. Zaman. (1990) Microtiter plate Assay for the measurement of glutathione and glutathione disulfide in large numbers of Biological Samples. Anal. Biochem., 190:360-365
2. Promega GSH-Glo Glutathione Assay Technical Bulletin, TB369, Promega Corporation, Madison, WI.
3. Pryor, W.A., J.P. Stanley, and E. Blair. (1976) Autoxidation of polyunsaturated fatty acids: II. A Suggested mechanism for the Formation of TBA-reactive materials from prostaglandin-like Endoperoxides. Lipids, 11:370-379.
4. Tarpley, M.M., D.A. Wink, and M.B. Grisham (2004) Methods for detection of reactive Metabolites of Oxygen and Nitrogen: in vitro and in vivo considerations. Am . J. Physiol Regul Integr Comp Physiol. 286:R431-R444.
5. Miller, E.V., O. Tulyathan, E.Y. Isacoff, and C.J. Chang (2007). Molecular imaging of hydrogen peroxide produced for cell signaling. Nature Chemical Biology, 3(5):263-267.

By: BioTek Instruments

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