Product Information
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.
Quick Facts
Revised: 03–March–2005
Introduction
Mitochondrial superoxide is generated as a byproduct of oxi-dative phosphorylation. In an otherwise tightly coupled electron transport chain, approximately 1–3% of mitochondrial oxygen consumed is incompletely reduced; those “leaky” electrons can quickly interact with molecular oxygen to form superoxide anion, the predominant reactive oxygen species (ROS) in mitochon-dria.1–4 Increases in cellular superoxide production have been implicated in cardiovascular diseases, including hypertension, atherosclerosis, and diabetes-associated vascular injuries,5–7 as well as in neurodegenerative diseases such as Parkinson ʼs, Alzheimer ʼs, and amyotrophic lateral sclerosis (ALS).8–12
The assumption that mitochondria serve as the major intracel-lular source of ROS has been based la
rgely on experiments with isolated mitochondria rather than direct measurements in living cells. MitoSOX ™ Red mitochondrial superoxide indicator is a novel fluorogenic dye for highly selective detection of super-oxide (Figure 1) in the mitochondria of live cells. MitoSOX™ Red reagent is live-cell permeant and is rapidly and selec-tively targeted to the mitochondria. Once in the mitochondria, MitoSOX™ Red reagent is oxidized by superoxide and exhibits red fluorescence. MitoSOX™ Red reagent is readily oxidized by superoxide but not by other ROS- or reactive nitrogen species (RNS)–generating systems, and oxidation of the probe is pre-vented by superoxide dismutase. The oxidation product becomes highly fluorescent upon binding to nucleic acids.
This reagent may enable researchers to distinguish artifacts of isolated mitochondrial preparations from direct measurements of superoxide generated in the mitochondria of live cells. It may also provide a valuable tool in the discovery of agents that modu-late oxidative stress in various pathologies.
Materials
Contents
modulate
MitoSOX™ Red mitochondrial superoxide indicator (MW = 759) is supplied in 10 vials, each containi
ng 50 μg.
Storage and Handling
Upon receipt, the MitoSOX™ Red reagent should be stored upright, desiccated, and protected from light at ≤–20˚C. Avoid freeze-thaw cycles. MitoSOX™ Red reagent is packaged with an oxygen scavenging pouch that will extend the shelf life of the product. After removing vials for use, seal the remaining vials into the pouch to preserve the activity of the reagent. Vials
should be allowed to warm to room temperature before opening. When stored properly, components should be stable for at least 6 months. Note: MitoSOX™ Red reagent is a derivative of ethid-ium bromide and should be treated with the appropriate caution.
MitoSOX™ Red mitochondrial superoxide indicator *for live-cell imaging* (M36008)
Figure 1. Selectivity of the MitoSOX™ Red mitochondrial superoxide indicator. Cell-
free systems were used to generate a variety of reactive oxygen species (ROS) and 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 was added (unless otherwise noted)
and the samples were incubated for an additional 15 minutes at 37˚C before fluorescence was measured. The Griess nitrite determina-tion kit (for nitric oxide, peroxynitrite, and nitrite standards only; gray bars) and
dihydrorhodamine 123 (DHR 123; white bars) were employed as positive controls for 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 bars) is readily oxidized by superoxide but not by the other oxidants.
Spectral Characteristics
MitoSOX™ Red mitochondrial superoxide indicator has exci-tation/emission maxima of approximately 510/580 nm.
Materials Recommended but Not Provided
Hank ʼs balanced salt solution with calcium and magnesium (HBSS/Ca/Mg, available from Gibco (14025-092)).
Current pric e s may be ob t ained 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
protocols should be used as a starting point, and optimal labeling conditions should be determined empirically.
1.1 Prepare 5 μM MitoSOX™ reagent working solution. Dilute the 5 mM MitoSOX™ reagent stock solution (prepared above) in HBSS/Ca/Mg or suitable buffer to make a 5 μM MitoSOX™ reagent working solution.
Contact Information
Further information on Molecular Probes products, including product bibliographies, is available from your local distributor or directly from Molecular Probes. Customers in Europe, Africa and the Middle East should contact our office in Paisley, United Kingdom. All others should contact our Technical As s is t ance Department in Eugene, Oregon. Please visit our Web site — probes.invitrogen — for the most up-to-date information.
Molecular Probes, Inc.
29851 Willow Creek Road, Eugene, OR 97402
Phone: (541) 465-8300 • Fax: (541) 335-0504
Customer Service: 6:00 am to 4:30 pm (Pacific Time)
Phone: (541) 335-0338 • Fax: (541) 335-0305 • order@probes Toll-Free Ordering for USA:
Order Phone: (800) 438-2209 • Order Fax: (800) 438-0228 Technical Assistance: 8:00 am to 4:00 pm (Pacific Time) Phone: (541) 335-0353 • Toll-Free (800) 438-2209
Fax:  (541) 335-0238 • tech@probes Invitrogen European Headquarters
Invitrogen, Ltd.
3 Fountain Drive
Inchinnan Business Park
Paisley PA4 9RF, UK
Phone: +44 (0) 141 814 6100 • Fax: +44 (0) 141 814 6260 Email: euroinfo@invitrogen
Technical Services: eurotech@invitrogen
Molecular Probes products are high-quality reagents and materials intended for research pur p os e s only. These products must be used by, or directl y under the super v ision of, a tech n ically qual i f ied individual experienced in handling potentially hazardous chemicals. Please read the Material Safety Data Sheet pro v id e d for each prod u ct; other regulatory considerations may apply.
Several Molecular Probes products and product applications are covered by U.S. and foreign patents and patents pending. Our prod u cts are not available for resale or oth e r commercial uses without a specifi c agreement from Molecular Probes, Inc. We welcome inquiries about licensing the use of our dyes, trade m arks or technolo-gies. Please submit inquiries by e-mail to busdev@probes. All names con t ain i ng the des i g n a t ion ® are reg i s t ered with the U.S. Patent and Trade m ark Offi ce. Copyright 2005, Molecular Probes, Inc. All rights reserved. This information is subject to change without notice.

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