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| Product Name | TUNEL Apoptosis Detection Kit (Fluorometric, dUTP-FITC) |
| Catalog No. | CCAT-HMM-0044 |
| Description | A terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling kit for the in situ detection of DNA fragmentation in apoptotic cells. TdT enzyme catalyzes the template-independent addition of FITC-labeled dUTP to the 3'-hydroxyl termini of fragmented DNA, enabling direct visualization and quantification of apoptotic cells by fluorescence microscopy or flow cytometry. |
| Intended Use | In situ detection and quantification of apoptosis in cultured cells, tissue cryosections, and formalin-fixed paraffin-embedded tissue samples for oncology research, developmental biology, and neurodegenerative disease studies. |
| Principle / Technology | During apoptosis, endonucleases cleave genomic DNA at internucleosomal linker regions, generating double-strand breaks with exposed 3'-OH ends; TdT enzyme adds multiple FITC-dUTP molecules to these free 3'-OH ends in a template-independent polymerization reaction, amplifying the fluorescent signal at DNA break sites. |
| Detection Method | Fluorescence microscopy with FITC filter set (Ex/Em: 488/520 nm) or flow cytometry (FL1 channel); counterstain with DAPI or Hoechst for nuclear localization |
| Sample Type | Cultured adherent cells grown on coverslips or chamber slides, cell suspensions, frozen or FFPE tissue sections fixed and permeabilized appropriately |
| Performance Range / Specifications | Positive labeling in >95% of cells treated with DNase I (positive control); minimal background labeling (<2%) in untreated healthy control cells |
| Sensitivity / LOD | Detection of single apoptotic cell among 1,000 normal cells by fluorescence microscopic examination |
| Specificity | TdT specifically recognizes free 3'-OH DNA ends generated by apoptosis-associated endonucleases; random nicks in non-apoptotic cells produce negligible signal under optimized conditions; signal intensity correlates with extent of DNA fragmentation |
| Reaction Conditions / Protocol | Fix cells or tissue sections with 4% paraformaldehyde (20 min); permeabilize with 0.2% Triton X-100 or proteinase K depending on sample type; wash with PBS; add 50 μL TUNEL reaction mixture (TdT enzyme + FITC-dUTP in reaction buffer); incubate 60 minutes at 37°C in humidified dark chamber; wash with PBS; counterstain with DAPI; mount with antifade medium; analyze by fluorescence microscopy; include DNase I-treated positive control and omit-TdT negative control |
| Components / Formulation | TdT enzyme (recombinant, in glycerol storage buffer), FITC-dUTP (in nucleotide buffer), 5× TdT Reaction Buffer (potassium cacodylate, Tris-HCl, BSA, cobalt chloride, pH 6.6), DNase I for positive control preparation, mounting medium with DAPI |
| Storage Conditions | -20°C for enzyme and nucleotide components protected from light; reaction buffer at -20°C; avoid repeated freeze-thaw cycles (<3); DAPI mounting medium at 2–8°C |
| Shelf Life | 12 months from date of manufacture |
| Package Specifications | 20 tests, 50 tests (slide format); 50 reactions for flow cytometry |
| Product Form | Concentrated enzyme and nucleotide solutions requiring dilution in reaction buffer before use |
| Quality Control | Each lot tested on DNase I-digested positive control and untreated negative control cells to verify labeling specificity; TdT activity verified; FITC-dUTP incorporation efficiency confirmed by fluorescence intensity measurement |
| Key Features | Direct fluorescent labeling eliminates secondary antibody amplification steps; broadly applicable to cells and tissue samples; compatible with simultaneous immunofluorescence co-staining; widely validated in apoptosis peer-reviewed publications |
For research use only, not for clinical use.
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