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| Product Name | Reactive Oxygen Species Detection Assay Kit (DCFH-DA Probe) |
| Catalog No. | CMTR-HMM-0057 |
| Description | A fluorometric assay kit for detecting intracellular reactive oxygen species using the cell-permeable probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). The non-fluorescent DCFH-DA diffuses into cells, is deacetylated by intracellular esterases to DCFH, and is subsequently oxidized by ROS to the highly fluorescent 2',7'-dichlorofluorescein (DCF). |
| Intended Use | Measuring intracellular ROS levels as an indicator of oxidative stress in studies of inflammation, environmental toxicology, nanomaterial safety assessment, ischemia-reperfusion injury, and redox signaling pathway research. |
| Principle / Technology | DCFH-DA passively diffuses across the cell membrane; intracellular esterases hydrolyze the acetate groups to yield the polar, membrane-impermeable DCFH; DCFH is then oxidized by hydroxyl radicals (OH•), peroxynitrite (ONOO-), and other ROS to the highly fluorescent DCF (Ex/Em 495/529 nm); hydrogen peroxide oxidation of DCFH requires cellular peroxidase activity for efficient conversion. |
| Detection Method | Fluorescence microscopy with FITC filter set (Ex/Em 495/529 nm), flow cytometry (488 nm excitation, FL1/530 nm channel), or fluorescence microplate reader |
| Sample Type | Live adherent and suspension mammalian cells, primary cells, tissue explants, isolated leukocytes; may be adapted for C. elegans, zebrafish embryos, and other small model organisms |
| Performance Range / Specifications | DCFH-DA working concentration: 5–50 μM; incubation: 20–60 minutes at 37°C; fluorescence fold-increase of 10–200× over baseline upon ROS stimulation (cell type- and stimulus-dependent) |
| Sensitivity / LOD | Detection of ROS in cells treated with 100 μM H2O2 within 15 minutes; fluorescence increase of ≥2-fold distinguishable from unstained autofluorescence background |
| Specificity | DCFH-DA detects a broad spectrum of ROS including hydroxyl radical, peroxyl radical, peroxynitrite, and hydrogen peroxide (peroxidase-dependent); less reactive toward superoxide anion and hypochlorous acid; signal represents cumulative intracellular ROS without distinguishing individual species |
| Reaction Conditions / Protocol | Wash cells and resuspend in serum-free medium or assay buffer; add DCFH-DA probe at 10–20 μM final concentration; incubate 20–45 minutes at 37°C in the dark (loading phase); wash cells 2–3 times to remove extracellular probe; add ROS inducer (H2O2, rotenone, antimycin A) or test compounds; incubate for desired stimulation time; measure fluorescence by plate reader, microscopy, or flow cytometry; include unstained and probe-only controls, and positive control such as tert-butyl hydroperoxide or H2O2 |
| Components / Formulation | DCFH-DA probe (lyophilized or 20 mM DMSO stock), ROS Inducers: TBHP (tert-butyl hydrogen peroxide, 100 mM), H2O2 (positive control); ROS Inhibitor: NAC (N-acetyl-L-cysteine, antioxidant negative control); DMSO for probe reconstitution; Assay Buffer (HBSS or serum-free medium recommendation) |
| Storage Conditions | DCFH-DA lyophilized: -20°C protected from light and moisture, stable 24 months; DCFH-DA DMSO stock (20 mM): -20°C protected from light, desiccated, stable 6 months; working solution: prepare fresh and use within 1 hour |
| Shelf Life | 24 months for lyophilized probe |
| Package Specifications | 100 assays (96-well format), 500 assays; also available in 1 mg and 50 mg bulk |
| Product Form | Lyophilized probe with positive and negative control reagents |
| Quality Control | Each lot tested for oxidation-dependent fluorescence enhancement (≥20-fold with 100 μM H2O2 + HRP); basal probe auto-oxidation rate <5% per hour at 37°C; DCFH-DA purity ≥97% by HPLC |
| Key Features | Cell-permeable probe with esterase-dependent intracellular trapping; broad-spectrum ROS detection for comprehensive oxidative stress assessment; positive control (TBHP) and antioxidant control (NAC) included; compatible with multiple detection platforms (plate reader, microscope, flow cytometer); widely cited in oxidative stress literature |
For research use only, not for clinical use.
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