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| Product Name | Caspase-3/-7 Green Detection Reagent, Live Cell Imaging |
| Catalog No. | CCAT-HMM-0067 |
| Description | Cell-permeable fluorogenic substrate for real-time detection of activated caspase-3 and caspase-7 in live cells by fluorescence microscopy or high-content imaging. The reagent consists of a tetrapeptide DEVD recognition sequence conjugated to a DNA-binding fluorescent dye via a cleavable linker. In apoptotic cells, activated executioner caspases-3 and -7 cleave the DEVD sequence, releasing the DNA dye that translocates to the nucleus and fluoresces bright green upon DNA binding (Ex/Em 502/530 nm). The nuclear-restricted fluorescence provides a high signal-to-noise ratio and clear morphological distinction between apoptotic and non-apoptotic cells, suitable for kinetic apoptosis monitoring over 24-72 hours. |
| Intended Use | Real-time kinetic detection and quantification of caspase-3/-7 activation in live cells during apoptosis for drug screening, mechanism-of-action studies, time-lapse imaging, and high-content analysis. |
| Principle / Technology | DEVD peptide substrate is specific for caspase-3 and -7 cleavage; released DNA dye binds nuclear DNA with fluorescence enhancement; nuclear localization provides morphological confirmation of apoptosis; cell permeability enables live-cell kinetic monitoring without fixation or lysis. |
| Detection Method | Fluorescence microscopy (FITC/GFP filter set: Ex 488/Em 530 nm); high-content imaging systems; fluorescence microplate reader (Ex/Em 500/530 nm). |
| Sample Type | Live adherent and suspension cultured cells; primary cells; 3D spheroids and organoids with optimized imaging conditions. |
| Performance Range / Specifications | Fluorescence signal increases 10-100 fold upon caspase activation over 0-72 hours; detectable in 5-20% of cells within 2-4 hours of apoptosis induction by staurosporine (1 µM). |
| Sensitivity / LOD | Detection of caspase-3/-7 activation in as few as 200 cells per well (96-well); signal detectable within 30 minutes of strong apoptotic stimulus. |
| Specificity | Highly specific for caspase-3 and caspase-7 DEVDase activity; minimal cleavage by caspase-8 (<2% cross-reactivity) and caspase-6 (<1%); no cleavage by caspase-1, granzyme B, or cathepsins. |
| Reaction Conditions / Protocol | Add reagent directly to culture medium at 1:1000-1:2000 dilution; incubate for desired time course (30 min to 72 hours); image or read fluorescence without washing; reagent is non-toxic and compatible with long-term culture. |
| Components / Formulation | Caspase-3/-7 Green reagent (25 µL/vial, 2 mM DMSO solution), DMSO control (1 mL), detailed protocol. |
| Storage Conditions | Store at -20 °C, desiccated, protected from light; minimize freeze-thaw cycles. |
| Shelf Life | 6 months from date of manufacture. |
| Package Specifications | 25 µL (sufficient for ~50 assays in 96-well format at 1:1000 dilution), 5 × 25 µL multi-pack. |
| Product Form | Concentrated DMSO solution; pale yellow to colorless. |
| Quality Control | Each lot tested for caspase activation kinetics in staurosporine-treated HeLa cells vs. untreated and Z-VAD-FMK inhibited controls; fluorescence enhancement >10-fold in treated vs. untreated cells at 4 hours. |
| Key Features | Live-cell and real-time detection; no wash, no lysis, no fixation; nuclear-localized fluorescence for morphological confirmation; non-toxic for long-term imaging; compatible with time-lapse microscopy and high-content screening; caspase-3 and -7 dual detection. |
| Purity | DEVD-fluorophore conjugate ≥95% by HPLC; DMSO anhydrous grade. |
| Concentration | 2 mM stock in DMSO; use at 1-2 µM final in culture medium. |
| Activity / Unit Definition | DEVD cleavage rate: kcat/Km ~10^5 M-1s-1 for caspase-3 at 37 °C. |
| Molecular Weight | Proprietary; approximately 800-1000 Da (DEVD-DNA dye conjugate). |
| Source / Origin | Synthetic peptide-fluorophore conjugate manufactured under controlled conditions. |
| pH Range / Optimal pH | Optimal caspase activity at pH 7.2-7.4; fluorescence is pH-independent between pH 6.5-8.0 due to DNA binding. |
| Shipping Conditions | Ambient or cold pack; protect from light. |
| Expiration Date / Stability | 6 months at -20 °C; after opening, aliquot and store at -20 °C, avoiding moisture. |
| Regulatory / Compliance | For research use only; not for diagnostic procedures. DMSO-containing solution — handle with appropriate PPE. |
| Compatibility | Compatible with standard culture media (DMEM, RPMI-1640, MEM with or without phenol red). Phenol red may contribute to background in microscopy — use phenol red-free medium for imaging. Compatible with nuclear counterstains (Hoechst 33342, DAPI in fixed cells). GFP co-expression may cause spectral overlap — use sequential imaging or spectral unmixing. |
| Recommended Buffer System | No buffer addition needed; reagent is compatible with standard culture medium. |
| Application Notes / Precautions | Include pan-caspase inhibitor Z-VAD-FMK (20-50 µM) or caspase-3/-7 specific inhibitor as negative control. For kinetic imaging over >24 hours, consider using lower concentration (0.5 µM) to minimize potential phototoxicity. Protect plates from ambient light between image acquisitions. For endpoint analysis, add 1:1000 Hoechst 33342 for nuclear counterstaining and total cell count normalization. DMSO concentration in culture medium should not exceed 0.1% v/v. |
| Batch-to-Batch Consistency | DEVD conjugate purity within ±3% of reference lot; caspase activation kinetics within ±20% of reference lot at 4-hour time point. |
For research use only, not for clinical use.
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