Product Information

Revised: 03–March–2005

MitoSOX™ Red mitochondrial superoxide indicator *for live-cell

imaging* (M36008)
Quick Facts
Storage upon receipt:
• ≤ –20ºC
• Protect from light
• Desiccate
• Avoid freeze-thaw cycles

Ex/Em: 510/580 nm

Note: To preserve the activity of the MitoSOX™ Red

reagent, minimize exposure to air.

Figure 1. Selectivity of the MitoSOX™ Red mitochondrial superoxide indicator. Cell-

Introduction free systems were used to generate a variety of reactive oxygen species (ROS) and
Mitochondrial superoxide is generated as a byproduct of oxi- reactive nitrogen species (RNS); each oxidant was then added to a separate 10 µM
solution of MitoSOX™ Red reagent and incubated at 37˚C for 10 minutes. Excess DNA
dative phosphorylation. In an otherwise tightly coupled electron was added (unless otherwise noted) and the samples were incubated for an additional
transport chain, approximately 1–3% of mitochondrial oxygen 15 minutes at 37˚C before fluorescence was measured. The Griess nitrite determina-
consumed is incompletely reduced; those “leaky” electrons can tion kit (for nitric oxide, peroxynitrite, and nitrite standards only; gray bars) and
quickly interact with molecular oxygen to form superoxide anion, dihydrorhodamine 123 (DHR 123; white bars) were employed as positive controls for
the predominant reactive oxygen species (ROS) in mitochon- oxidant generation. Superoxide dismutase (SOD), a superoxide scavenger, was used as
a negative control for superoxide. The results show that the MitoSOX™ Red probe (black
dria.1–4 Increases in cellular superoxide production have been
bars) is readily oxidized by superoxide but not by the other oxidants.
implicated in cardiovascular diseases, including hypertension,
atherosclerosis, and diabetes-associated vascular injuries,5–7
as well as in neurodegenerative diseases such as Parkinsonʼs,
Alzheimerʼs, and amyotrophic lateral sclerosis (ALS).8–12
The assumption that mitochondria serve as the major intracel-
Materials
lular source of ROS has been based largely on experiments with
isolated mitochondria rather than direct measurements in living Contents
cells. MitoSOX™ Red mitochondrial superoxide indicator is a MitoSOX™ Red mitochondrial superoxide indicator (MW = 759)
novel fluorogenic dye for highly selective detection of super- is supplied in 10 vials, each containing 50 μg.
oxide (Figure 1) in the mitochondria of live cells. MitoSOX™
Red reagent is live-cell permeant and is rapidly and selec- Storage and Handling
tively targeted to the mitochondria. Once in the mitochondria, Upon receipt, the MitoSOX™ Red reagent should be stored
MitoSOX™ Red reagent is oxidized by superoxide and exhibits upright, desiccated, and protected from light at ≤–20˚C. Avoid
red fluorescence. MitoSOX™ Red reagent is readily oxidized by freeze-thaw cycles. MitoSOX™ Red reagent is packaged with
superoxide but not by other ROS- or reactive nitrogen species an oxygen scavenging pouch that will extend the shelf life of the
(RNS)–generating systems, and oxidation of the probe is pre- product. After removing vials for use, seal the remaining vials
vented by superoxide dismutase. The oxidation product becomes into the pouch to preserve the activity of the reagent. Vials
highly fluorescent upon binding to nucleic acids. should be allowed to warm to room temperature before opening.
This reagent may enable researchers to distinguish artifacts When stored properly, components should be stable for at least
of isolated mitochondrial preparations from direct measurements 6 months. Note: MitoSOX™ Red reagent is a derivative of ethid-
of superoxide generated in the mitochondria of live cells. It may ium bromide and should be treated with the appropriate caution.
also provide a valuable tool in the discovery of agents that modu-
late oxidative stress in various pathologies.

MP 36008 MitoSOX ™ Red mitochondrial superoxide indicator

Spectral Characteristics protocols should be used as a starting point, and optimal labeling
MitoSOX™ Red mitochondrial superoxide indicator has exci- conditions should be determined empirically.
tation/emission maxima of approximately 510/580 nm.
1.1 Prepare 5 μM MitoSOX™ reagent working solution.
Materials Recommended but Not Provided Dilute the 5 mM MitoSOX™ reagent stock solution (prepared
Hankʼs balanced salt solution with calcium and magnesium above) in HBSS/Ca/Mg or suitable buffer to make a 5 μM
(HBSS/Ca/Mg, available from Gibco (14025-092)). MitoSOX™ reagent working solution.
Note: The concentration of the MitoSOX™ reagent working so-
lution should not exceed 5 μM. Concentrations exceeding 5 μM
can produce cytotoxic effects, including altered mitochondrial
Experimental Protocol morphology and redistribution of fluorescence to nuclei and the
cytosol.
Reagent Preparation
Prepare 5 mM MitoSOX™ reagent stock solution. Dissolve 1.2 Load cells. Apply 1.0–2.0 mL of 5 μM MitoSOX™ reagent
the contents (50 μg) of one vial of MitoSOX™ mitochondrial working solution (prepared in step 1.2) to cover cells adhering
superoxide indicator (Component A) in 13 μL of dimethylsulfox- to coverslip(s). Incubate cells for 10 minutes at 37˚C, protected
ide (DMSO) to make a 5 mM MitoSOX™ reagent stock solution. from light.

Labeling Live Eukaryotic Cells 1.3 Wash cells. Wash cells gently three times with warm buffer.
This protocol was developed using live bovine pulmonary
epithelial (BPAE) cells, MRC5 human lung fibroblasts, and 1.4 Prepare cells for viewing. Stain cells with counterstains as
mouse 3T3 fibroblasts adhering to coverslips, but can be adapted desired and mount in warm buffer for imaging.
for use with other cell types. Recommendations for experimental

References
1. J Cell Mol Med 6, 175 (2002); 2. J Biol Chem 279, 4127 (2004); 3. J Neurochem 80, 780 (2002); 4. Bioorganic and Medicinal Chemistry 10, 3013
(2002); 5. Am J Physiol Heart Circ Physiol 284, H605 (2003); 6. Bioorganic and Medicinal Chemistry 10, 3013 (2002); 7. J Neurochem 89, 1283
(2004); 8. Trends Neurosci 24, S15 (2001); 9. J Cell Mol Med 6, 175 (2002); 10. J Neurosci 18, 923 (1998); 11. Neurochem Res 28, 1563 (2003);
12. IUBMB Life 55, 329 (2003).

Product List Current prices may be obtained from our Web site or from our Customer Service Department.

Cat # Product Name Unit Size

M36008 MitoSOX™ Red mitochondrial superoxide indicator *for live-cell imaging* ................................................................................................... 10 x 50 µg

MitoSOX ™ Red mitochondrial superoxide indicator 2

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MitoSOX ™ Red mitochondrial superoxide indicator 3