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| Product Name | Cellular ROS/RNS Multiplex Detection Assay Kit (DCFH-DA + DAF-FM Diacetate) |
| Catalog No. | CMTR-HMM-0075 |
| Description | Dual-probe multiplex assay for simultaneous detection of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in live cells. DCFH-DA reports broad-spectrum ROS through oxidation to fluorescent DCF (green), while DAF-FM diacetate reacts specifically with nitric oxide (NO) to form a fluorescent benzotriazole. Spectral separation enables simultaneous measurement by flow cytometry or fluorescence plate reader. |
| Intended Use | Multiplex intracellular ROS and NO detection; oxidative/nitrosative stress studies; inflammation research; antioxidant/NO-scavenger screening; ischemia-reperfusion models; endothelial function studies. |
| Principle / Technology | DCFH-DA deacetylated intracellularly, oxidized by ROS to fluorescent DCF (Ex/Em 485/535 nm); DAF-FM diacetate deacetylated, reacts with NO+O₂ to form fluorescent triazole (Ex/Em 495/515 nm) |
| Detection Method | Fluorometric; flow cytometry, microscopy, or plate reader; DCF: FL1 channel; DAF-FM: FL1/FL2 with compensation |
| Sample Type | Live mammalian cells; primary cells and cell lines; 10⁴-10⁵ cells per condition |
| Sensitivity / LOD | DCFH-DA detects H₂O₂ ≥10 nM; DAF-FM detects NO ≥3 nM; 20-50 fold fluorescence enhancement |
| Specificity | DCFH-DA: broad ROS; DAF-FM: highly NO-specific; minimal RNS/ROS cross-reactivity |
| Reaction Conditions / Protocol | Load cells with 10 µM DCFH-DA + 5 µM DAF-FM diacetate in serum-free medium (30-45 min, 37 °C); wash; treat; read fluorescence |
| Components / Formulation | DCFH-DA (50 mg), DAF-FM diacetate (1 mg), anhydrous DMSO, H₂O₂ positive control, DEA NONOate NO donor, PBS (10×) |
| Storage Conditions | Store at -20 °C; protect all probes from light; desiccate; DCFH-DA moisture-sensitive |
| Shelf Life | 12 months at -20 °C |
| Package Specifications | 100 assays (flow), 500 assays (plate reader), 50 assays (microscopy) |
| Product Form | Lyophilized probes with liquid controls |
| Key Features | Dual ROS/RNS detection; cell-permeant probes; spectral separation for multiplexing; positive controls included; compatible with flow, microscopy, and plate reader |
| Purity | DCFH-DA ≥97% HPLC; DAF-FM diacetate ≥95% HPLC; free dye <1% |
| Concentration | As specified per kit format; probe stock solutions in DMSO as specified |
| Activity / Unit Definition | Standard curve linearity R² ≥0.99 per lot; dynamic range as specified |
| Molecular Weight | Varies by probe and buffer component |
| Source / Origin | Synthetic fluorescent probes; recombinant enzymes; purified metabolite standards |
| pH Range / Optimal pH | pH 7.2–7.4 for cell-based assays; pH varies for biochemical metabolite detection |
| Shipping Conditions | Cold pack -20 °C; amber vials with desiccant |
| Expiration Date / Stability | 12 months at -20 °C; stock solutions 1 month at -20 °C aliquoted; working solutions 2 hr |
| Regulatory / Compliance | For laboratory and research use only; RUO; ISO 9001 |
| Compatibility | Compatible with fluorescence microplate readers, luminometers, and spectrophotometers |
| Recommended Buffer System | Assay-specific buffer optimized per product protocol |
| Application Notes / Precautions | Normalize signal to cell number or protein content; avoid probe photobleaching; include appropriate antioxidant controls for ROS assays |
| Batch-to-Batch Consistency | Standard curve parameters within ±15% of reference lot |
For research use only, not for clinical use.
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