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| Product Name | Cell Proliferation ELISA Kit, BrdU Incorporation, Chemiluminescent Detection |
| Catalog No. | CATR-HMM-0060 |
| Description | Chemiluminescent ELISA-based cell proliferation assay kit that measures 5-bromo-2'-deoxyuridine (BrdU) incorporation into newly synthesized DNA of proliferating cells. BrdU, a thymidine analog, is added to culture medium and incorporated into DNA during S-phase of the cell cycle. After fixation and DNA denaturation, incorporated BrdU is detected by anti-BrdU antibody conjugated to horseradish peroxidase (HRP), with chemiluminescent substrate generating a signal proportional to the number of proliferating cells. Provides a non-radioactive alternative to [3H]-thymidine incorporation assays with equivalent sensitivity. |
| Intended Use | Quantitative measurement of cell proliferation in response to growth factors, cytokines, mitogens, and drug treatments; screening of anti-proliferative compounds; lymphocyte activation and mixed lymphocyte reaction (MLR) assays. |
| Principle / Technology | BrdU incorporation into replicating DNA during S-phase; anti-BrdU-HRP antibody detection after DNA denaturation; chemiluminescent HRP substrate produces light proportional to incorporated BrdU; non-radioactive alternative to 3H-thymidine. |
| Detection Method | Chemiluminescent microplate reader; luminometer (integration time 0.1-1 second per well). |
| Sample Type | Adherent and suspension cultured mammalian cells; primary lymphocytes, splenocytes, thymocytes; cancer cell lines. |
| Performance Range / Specifications | Linear detection range: 500-50,000 cells per well (96-well); dynamic range >2 orders of magnitude; correlation with [3H]-thymidine incorporation R² >0.95. |
| Sensitivity / LOD | Detection limit ~100 proliferating HeLa cells per well after 2-hour BrdU pulse labeling; signal-to-noise ratio >10:1 at 1,000 cells/well. |
| Specificity | Anti-BrdU antibody specifically recognizes BrdU in single-stranded DNA; minimal cross-reactivity with thymidine (<0.1%) or 5-iodo-2'-deoxyuridine (<1%). |
| Reaction Conditions / Protocol | Add BrdU labeling solution (10 µM final); incubate 2-24 hours; fix cells (30 min); denature DNA; add anti-BrdU-HRP antibody (90 min); wash; add chemiluminescent substrate; read immediately. |
| Components / Formulation | BrdU labeling reagent (1000× concentrate), FixDenat solution, anti-BrdU-HRP antibody concentrate, wash buffer concentrate (10×), chemiluminescent substrate (luminol + enhancer + peroxide), 96-well microplate, sealing tape, protocol. |
| Storage Conditions | Store all components at 2-8 °C; anti-BrdU-HRP antibody protect from light. |
| Shelf Life | 12 months from date of manufacture. |
| Package Specifications | 1 × 96-well plate kit (200 tests using 4 wells per condition), 5 × 96-well plate kit. |
| Product Form | Liquid reagents; microplate with clear flat-bottom wells. |
| Quality Control | Each lot tested for BrdU incorporation in PHA-stimulated human PBMCs vs. unstimulated control (≥5-fold signal increase); chemiluminescent substrate activity verified. |
| Key Features | Non-radioactive BrdU detection; chemiluminescent readout with high sensitivity; no cell lysis required; direct cell fixation preserves morphology; compatible with high-throughput screening; results within 4-5 hours. |
| Purity | BrdU ≥99% purity by HPLC; anti-BrdU antibody affinity purified; HRP conjugate RZ >3.0. |
| Concentration | BrdU labeling reagent: 10 mM (1000×); anti-BrdU-HRP: use at 1:100 dilution. |
| Activity / Unit Definition | Anti-BrdU antibody affinity: Kd <1 nM for BrdU in ssDNA. |
| Molecular Weight | BrdU: 307.10 g/mol (C9H11BrN2O5). |
| Source / Origin | Monoclonal anti-BrdU antibody produced in mouse hybridoma; HRP from horseradish; BrdU synthesized chemically. |
| pH Range / Optimal pH | All reagents pH 7.2-7.6; FixDenat solution is acidic for DNA denaturation. |
| Shipping Conditions | Cold pack (2-8 °C); chemiluminescent substrate component may be shipped separately on dry ice. |
| Expiration Date / Stability | 12 months at 2-8 °C; chemiluminescent substrate retains >90% activity for 6 months after opening. |
| Regulatory / Compliance | For research use only; not for diagnostic or therapeutic applications. Mouse monoclonal antibody produced under IACUC-approved protocols. |
| Compatibility | Compatible with most adherent and suspension mammalian cell types. Not recommended for cells with very slow proliferation rates (doubling time >72 hours) as BrdU incorporation signal may be low. |
| Recommended Buffer System | PBS-based wash buffer; denaturation solution contains HCl. |
| Application Notes / Precautions | Optimize BrdU labeling time for each cell type — 2-4 hours for rapidly dividing cells, 18-24 hours for slow-growing cells. Avoid over-confluence (>80%) at labeling start. Aspirate labeling medium carefully to prevent cross-well contamination. Denaturation step is critical — insufficient denaturation reduces antibody access to BrdU; excessive denaturation may detach cells. Protect chemiluminescent substrate from light; warm to room temperature before use. Read plate within 10 minutes of substrate addition for optimal signal. |
| Batch-to-Batch Consistency | Anti-BrdU-HRP titer within ±15% of reference lot; chemiluminescent substrate relative light units within specification. |
For research use only, not for clinical use.
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