I have isolated mitochondria from heart tissue. I need to do ROS. I have used LeBel et al (1992) method to estimate ROS in cells, but I don't know which method will be good for mitochondrial fraction isolated from tissue.
Dear Dr. Shobana Ganesan,
The following publication depicts a protocol for measurements of ROS in tissue mitochondrial fraction
Methods Mol Biol. Author manuscript; available in PMC 2011 Mar 15.
Methods Mol Biol. 2010; 648: 245–255.
doi: 10.1007/978-1-60761-756-3_16
PMCID: PMC3057530
NIHMSID: NIHMS276265
Measurement of Mitochondrial ROS Production
Anatoly A. Starkov
Abstract
The significance of reactive oxygen species (ROS) as aggravating or primary factors in numerous pathologies is widely recognized, with mitochondria being considered the major intracellular source of ROS. It is not yet possible to routinely measure mitochondrial ROS in animals or cultured cells with a reasonable degree of certainty. However, at the level of isolated mitochondria, one can easily monitor and quantify the rate of ROS production, identify major sites of ROS production, and compare the rates of ROS production in mitochondria isolated from normal and diseased tissue. In this chapter, we describe in detail the most recent and reliable method to measure mitochondrial ROS as the rate of H2O2 emission. This method may be employed with minimal modifications to measure H2O2 production by mitochondria isolated from various tissues and under a wide variety of experimental conditions.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3057530/
Hoping this will be helpful,
Rafik
In addition to the paper suggested by Rafik you could think about measuring oxidative stress in submitochondrial particles. These are relatively easy to obtain and provide a reliable method of measuring superoxide anion production. If you are interested please have a look at the publication in the attachment.
Best, Christian
Article Increased oxidative stress in submitochondrial particles int...
fluorescent dyes can be used to measure ROS from mitochondria by real-time quantification. Scopoletin, Amplex Red are a few examples. You can refer the following article for more information.
Gusdon AM, Chen J, Votyakova TV, Mathews CE. Quantification, Localization, and Tissue Specificities of Mouse Mitochondrial Reactive Oxygen Species Production. Methods in enzymology. 2009;456:439-457. doi:10.1016/S0076-6879(08)04424-8.
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