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Cat. No.: MSAKMR-200 (for 20-200T)

Cat. No.: MSAKMR-1k (for 100-1000T)

Description

Mitochondrial Superoxide Assay Kit with MitoSOX Red is a reagent kit that uses MitoSOX Red as a fluorescent probe to quickly and sensitively detect changes in superoxide within mitochondria. This kit can be used with various fluorescence detection systems, including fluorescence microscopy, laser confocal microscopy, fluorescence microplate readers, and flow cytometry.

Superoxide generally refers to the superoxide anion (O2·-), which is a free radical form of oxygen. In the respiratory chain, when NADPH oxidase transfers electrons to oxygen molecules, it generates superoxide anions (O2·-). The superoxide anion (O2·-) is a potent oxidant that can be produced by stimulated white blood cells, among others, to defend against microbial infections. However, superoxide anions (O2·-) can also cause oxidative damage and are closely associated with the onset of various diseases.

MitoSOX Red, abbreviated as MSR and also known as MitoSOX or Mito-HE, has a molecular weight of 759.7 and a molecular formula of C43H43IN3P. It is a novel fluorescent probe for detecting superoxide within mitochondria, which can rapidly and specifically target mitochondria to selectively detect mitochondrial superoxide. MitoSOX Red is a dihydroethidium derivative with a cationic triphenylphosphonium group. Dihydroethidium (HE) can be oxidized by reactive species (such as superoxide) to ethidium, which then binds to DNA and produces fluorescence. MitoSOX Red exhibits similar properties, being rapidly oxidized by superoxide but not by other reactive oxygen species (ROS) or reactive nitrogen species (RNS). The oxidized product binds to nucleic acids and produces strong fluorescence.

Principle

The positive charge on the phosphate group of MitoSOX Red is surrounded by three lipophilic phenyl groups, facilitating its entry into live cells and selective targeting of mitochondria, where it can be oxidized by superoxide. The oxidized MitoSOX Red binds to mitochondrial nucleic acids, producing intense red fluorescence with an excitation wavelength of 510 nm and an emission wavelength of 580 nm.

Usage

Mitochondrial Superoxide Assay Kit provides MitoSOX Red as a 5mM stock solution. The recommended final concentration for use has been optimized for most cell types; however, to achieve the best results, it is advisable to conduct some optimization for different cell types. The typical final concentration of MitoSOX Red ranges from 1-5μM, with the most recommended final concentration being 5μM. Excessive accumulation of MitoSOX Red in mitochondria can cause cellular damage, so the working concentration of MitoSOX Red should not exceed 5μM to avoid potential cytotoxicity, such as altering mitochondrial morphology or causing MitoSOX Red to redistribute to the nucleus or cytoplasm. Additionally, the kit provides mSoxUp as a positive control for inducing superoxide production within mitochondria, with a typical final concentration range of 0.5-2X, and the most recommended final concentration being 1X.

This kit is available in small and medium packages. When MitoSOX Red (5mM) is diluted at a ratio of 1:1000, it can be used for 20 and 100 assays per well in a 6-well plate with a detection volume of 1 ml per well, respectively. For a 96-well plate with a detection volume of 100 μl per well, it can be used for 200 and 1000 assays, respectively.

Components

• MitoSOX Red (5mM)
• mSoxUp (1000X)

Storage

Store at -20ºC, valid for one year. MitoSOX Red (5mM) should be stored protected from light, and storage at -80ºC is more effective.

Precautions

• BSA and Phenol Red interfere with the detection of this fluorescent probe and should be avoided.
• Fluorescent dyes are prone to quenching; protect from light as much as possible to slow down fluorescence quenching.
• According to the literature, MitoSOX Red may redistribute after reacting with superoxide and bind to mitochondrial or nuclear DNA, which can result in red fluorescence being concentrated in the nucleus.
• This kit is only for detecting live cells or tissues and cannot be used for fixed or frozen cell or tissue samples.
• When using a fluorescence microplate reader, use a black or white plate suitable for fluorescence detection.
• For first-time use, aliquot and store at -20ºC to avoid repeated freeze-thaw cycles.
• This product is for scientific research use by professionals only and is not intended for clinical diagnosis or treatment, nor for use in food or drugs, and should not be stored in a household environment.
• For your safety and health, wear a lab coat and disposable gloves when handling.