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| Product Name | Mitochondrial Superoxide Indicator MitoSOX Red, Fluorometric Detection |
| Catalog No. | CATR-HMM-0064 |
| Description | Fluorogenic probe MitoSOX Red reagent for selective detection of superoxide anion (O2·-) specifically within mitochondria of live cells. MitoSOX Red consists of a hydroethidine fluorophore conjugated to a triphenylphosphonium (TPP+) cation moiety that drives mitochondrial accumulation driven by the highly negative mitochondrial membrane potential (-150 to -180 mV). Once inside mitochondria, superoxide specifically oxidizes hydroethidine to 2-hydroxyethidium (Ex/Em 510/580 nm), distinct from nonspecific oxidation products. The red fluorescent signal is specific for mitochondrial superoxide and is not generated by cytoplasmic superoxide, hydrogen peroxide, peroxynitrite, or hydroxyl radical. |
| Intended Use | Specific detection and quantification of mitochondrial superoxide production in live cells by fluorescence microscopy, flow cytometry, or microplate reader for oxidative stress, ischemia-reperfusion, mitochondrial dysfunction, and aging research. |
| Principle / Technology | TPP+ cation targets dye to mitochondrial matrix (100-1000× cytoplasmic concentration); hydroethidine oxidation is superoxide-specific generating 2-hydroxyethidium with distinct excitation/emission from nonspecific ethidium oxidation products; mitochondrial localization confirmed by co-staining with MitoTracker Green. |
| Detection Method | Fluorescence microscopy (TRITC/Cy3 filter: Ex 510/550 nm, Em 580 nm); flow cytometry (488 or 514 nm excitation, 585/42 nm emission); microplate reader (Ex 510/Em 580 nm). |
| Sample Type | Live adherent and suspension cultured cells; primary cells; isolated mitochondria. |
| Performance Range / Specifications | Linear fluorescence response to superoxide levels over 10-1000 pmol O2·-/min/10^6 cells; signal increases 5-50 fold upon mitochondrial superoxide induction by antimycin A or rotenone. |
| Sensitivity / LOD | Detectable increase in mitochondrial superoxide upon treatment with 1 µM antimycin A (Complex III inhibitor) within 10 minutes; detection threshold ~0.5 pmol O2·-/min/10^6 cells. |
| Specificity | >100-fold selectivity for superoxide over hydrogen peroxide, peroxynitrite, hydroxyl radical, and nitric oxide; mitochondrial localization verified by >95% co-localization with MitoTracker Green FM. |
| Reaction Conditions / Protocol | Prepare 5 µM MitoSOX working solution in HBSS/Ca2+/Mg2+; incubate cells 10 minutes at 37 °C protected from light; wash 3× with warm buffer; analyze immediately. |
| Components / Formulation | MitoSOX Red reagent (50 µg, lyophilized), anhydrous DMSO (100 µL), detailed protocol. |
| Storage Conditions | Store MitoSOX at -20 °C desiccated and protected from light; DMSO at room temperature. |
| Shelf Life | 12 months from date of manufacture when stored as directed. |
| Package Specifications | 50 µg (sufficient for ~100 coverslip stains or ~50 flow cytometry samples at 1×10^6 cells each). |
| Product Form | Lyophilized deep purple powder; reconstitutes to purple DMSO solution. |
| Quality Control | Each lot tested for mitochondrial localization specificity using MitoTracker Green co-staining; superoxide specificity verified using superoxide dismutase (SOD) inhibition control; fluorescence intensity in antimycin A-treated vs. untreated cells. |
| Key Features | Mitochondria-specific superoxide detection; superoxide-specific oxidation product distinct from nonspecific oxidation; live-cell compatible; co-localization with MitoTracker validated; compatible with GFP and FITC channels. |
| Purity | MitoSOX Red reagent ≥90% by HPLC; 2-hydroxyethidium generation superoxide-specific verified. |
| Concentration | Prepare 5 mM stock in DMSO; use at 2.5-5 µM final concentration. |
| Activity / Unit Definition | Fluorescence enhancement >20-fold upon superoxide oxidation; 2-hydroxyethidium quantum yield ~0.3. |
| Molecular Weight | MitoSOX Red: approximately 760 g/mol (cationic hydroethidine-TPP+ conjugate). |
| Source / Origin | Synthetic fluorogenic probe manufactured under controlled laboratory environment. |
| pH Range / Optimal pH | Optimal staining in physiological pH 7.2-7.4; fluorescence is pH-independent from pH 6.8 to 7.8. |
| Shipping Conditions | Ambient or cold pack; protect from moisture and light. |
| Expiration Date / Stability | 12 months at -20 °C; reconstituted 5 mM DMSO stock stable for 1 week at -20 °C in single-use aliquots. |
| Regulatory / Compliance | For research use only; not for diagnostic procedures. Handle with appropriate chemical safety practices (potential mutagen). |
| Compatibility | Compatible with DMEM, RPMI-1640, HBSS, and phenol red-free media. Phenol red may cause background fluorescence at emission wavelengths — use phenol red-free formulations for microscopy. Compatible with GFP co-expression with appropriate filter selection (GFP: Ex 488/Em 510 nm; MitoSOX: Ex 510/Em 580 nm). |
| Recommended Buffer System | Use HBSS with Ca2+ and Mg2+ for staining; avoid phosphate buffers as they may chelate Ca2+ needed for mitochondrial function. |
| Application Notes / Precautions | Work under subdued lighting — MitoSOX is highly light-sensitive. Prepare working solution fresh and use within 1 hour. Do not exceed 5 µM concentration as higher doses may cause nonspecific mitochondrial staining. Include SOD (100 U/mL) or mitoTEMPO (10 µM) pretreatment as specificity controls. Optimize staining time (5-15 min) for each cell type; over-incubation increases background. For flow cytometry, gate on viable cell population by FSC/SSC before analyzing MitoSOX fluorescence. |
| Batch-to-Batch Consistency | MitoSOX content within ±5% of label; superoxide selectivity index (2-hydroxyethidium vs. ethidium) within ±15% of reference lot. |
For research use only, not for clinical use.
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