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| Product Name | Caspase-8 Activity Fluorometric Assay Kit |
| Catalog No. | CCAT-HMM-0055 |
| Description | A fluorometric assay kit specifically designed to quantify caspase-8 enzymatic activity in cell and tissue lysates. The assay uses a synthetic tetrapeptide substrate conjugated to 7-amino-4-trifluoromethylcoumarin that is specifically cleaved by active caspase-8, releasing the fluorescent AFC moiety. Caspase-8 is a key initiator caspase in the extrinsic death receptor-mediated apoptosis pathway. An AFC standard curve enables quantitative activity determination. |
| Intended Use | Quantitative measurement of caspase-8 protease activity as an indicator of death receptor-mediated apoptotic pathway activation. |
| Principle / Technology | Enzymatic cleavage of IETD-AFC peptide substrate by active caspase-8 yielding fluorescent AFC |
| Detection Method | Fluorescence (excitation 400 nm, emission 505 nm) |
| Sample Type | Cell lysates, tissue homogenates from apoptosis-induced samples |
| Performance Range / Specifications | Linear range: 0.05–5 nmol AFC released; detection time: 30–120 min |
| Sensitivity / LOD | Detection limit: 0.02 nmol AFC |
| Specificity | IETD tetrapeptide sequence selectively cleaved by caspase-8 and granzyme B; minimal cleavage by caspase-3 or caspase-9 |
| Reaction Conditions / Protocol | Lyse cells with provided lysis buffer 10 min on ice; centrifuge; incubate lysate with reaction buffer plus IETD-AFC substrate at 37 °C; read fluorescence at 30–60 min intervals |
| Components / Formulation | IETD-AFC substrate (lyophilized), AFC standard (100 mM), caspase-8 reaction buffer, cell lysis buffer, DTT (1 M), caspase-8 inhibitor (IETD-fmk) for specificity control |
| Storage Conditions | –20 °C for substrate and inhibitor; 2–8 °C for buffers |
| Shelf Life | 12 months at –20 °C |
| Package Specifications | 100 tests |
| Product Form | Lyophilized substrate; liquid reagents |
| Quality Control | Validated with recombinant active caspase-8; inhibitor control blocks ≥90% of signal |
| Key Features | Included IETD-AFC substrate and IETD-fmk inhibitor pair confirms caspase-8 signal specificity |
| Purity | Reagent grade; fluorophore-labeled reagents ≥90% labeling efficiency |
| Concentration | As specified per kit; sufficient for stated number of tests |
| Activity / Unit Definition | Apoptosis induction confirmed by positive control treatment producing ≥5-fold signal increase |
| Molecular Weight | Varies by dye and enzyme component |
| Source / Origin | Recombinant annexin proteins; synthetic fluorophore conjugates; chromogenic/fluorogenic peptide substrates |
| pH Range / Optimal pH | Binding/washing buffer pH 7.2–7.4 |
| Shipping Conditions | Cold packs; protect fluorophores from light; some components may require –20 °C |
| Expiration Date / Stability | 6–12 months at recommended storage temperature |
| Regulatory / Compliance | Research use; manufactured under ISO 9001 |
| Compatibility | Compatible with flow cytometry and fluorescence microscopy platforms |
| Recommended Buffer System | Annexin binding buffer with calcium; cell lysis buffer with protease inhibitors for caspase assays |
| Application Notes / Precautions | Process samples promptly after staining; include unstained and single-stain controls for flow cytometry compensation; use appropriate apoptosis inducers as positive controls |
| Batch-to-Batch Consistency | Positive control signal within ±20% of reference lot; staining pattern consistent |
For research use only, not for clinical use.
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