What is MitoSOX?
MitoSOX™ Red reagent is a novel fluorogenic dye specifically targeted to mitochondria in live cells. Oxidation of MitoSOX™ Red reagent by superoxide produces red fluorescence. • Readily oxidized by superoxide but not by other ROS- or RNS-generating systems.
What does MitoSOX measure?
Abstract. MitoSOX-based assays are widely used to detect mitochondrial reactive oxygen species (ROS), especially superoxide.
How do you use MitoSOX?
Apply 1.0–2.0 mL of 5 μM MitoSOX™ reagent working solution (prepared in step 1.2) to cover cells adhering to coverslip(s). Incubate cells for 10 minutes at 37ГC, protected from light. 1.3 Wash cells. Wash cells gently three times with warm buffer.
Can MitoSOX fix?
As far as I know, MitoSOX is not fixable. Hey Bhabesh Kumar Tripathy , the uptake of MitoSOX into the mitochondria is dependent on the mitochondrial membrane potential. When there is no membrane potential anymore, MitoSOX will leak out.
How do you test for mitochondrial ROS?
Investigators have used redox-active probes that, upon oxidation by ROS, yield products exhibiting fluorescence, chemiluminescence, or bioluminescence. Mitochondria-targeted probes can be used to detect ROS generated in mitochondria.
How do mitochondria produce ROS?
Mitochondrial ROS (mtROS or mROS) are reactive oxygen species (ROS) that are produced by mitochondria. Generation of mitochondrial ROS mainly takes place at the electron transport chain located on the inner mitochondrial membrane during the process of oxidative phosphorylation.
How is ROS measured in cell culture?
In cultured cells, an increase in cellular levels of reactive oxygen species (ROS) can be detected using multiple techniques including colorimetric assays, immunoblotting, and immunofluorescence.
How do you find ROS?
ROS are highly reactive molecules and are extremely unstable, so detection of ROS relies on measuring the end products that are formed when they react with particular substances. The end products can be measured by changes in their fluorescence, color, or luminescence.
How does mitochondria get rid of ROS?
In eukaryotic cells, mitochondria are the main source of ROS. Mitochondrial antioxidant system involves proteins that decrease ROS formation, enzymes that directly react with ROS, and non-enzymatic antioxidants that also remove ROS and other oxygen derivatives.
How are ROS produced in the body?
Reactive oxygen species (ROS) are generated during mitochondrial oxidative metabolism as well as in cellular response to xenobiotics, cytokines, and bacterial invasion. Oxidative stress refers to the imbalance due to excess ROS or oxidants over the capability of the cell to mount an effective antioxidant response.
How are ROS formed in the body?
How are ROS formed?
Most reactive oxygen species are generated as by-products during mitochondrial electron transport. The sequential reduction of oxygen through the addition of electrons leads to the formation of a number of ROS including: superoxide; hydrogen peroxide; hydroxyl radical; hydroxyl ion; and nitric oxide.
How does mitosox work?
MitoSOX™ Red reagent permeates live cells where it selectively targets mitochondria. It is rapidly oxidized by superoxide but not by other reactive oxygen species (ROS) and reactive nitrogen species (RNS). The oxidized product is highly fluorescent upon binding to nucleic acid.
What is mitosox red reagent?
MitoSOX Red reagent is novel fluorogenic dye specifically targeted to mitochondria in live cells. Oxidation of MitoSOX Red reagent by superoxide produces red fluorescence.
How can the production of superoxide by mitochondria be seen?
The production of superoxide by mitochondria can be visualized in fluorescence microscopy using the MitoSOX™ Red reagent. MitoSOX™ Red reagent permeates live cells where it selectively targets mitochondria.
What are the controls for mitosox™ red superoxide detection assay?
Knockdown of the mitochondrial fluorescence signal by SOD mimetics such as FeTCPP and the manganese porphyrin MnTBAP provides valuable negative controls in MitoSOX™ Red superoxide detection assays. Useful positive controls (not shown) include treatment with antimycin A, doxorubicin, or high glucose.