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| Product Name | BrdU Cell Proliferation ELISA Kit (Colorimetric) |
| Catalog No. | CCAT-HMM-0049 |
| Description | A non-radioactive immunoassay kit for the quantitative detection of 5-bromo-2'-deoxyuridine (BrdU) incorporation into newly synthesized DNA of proliferating cells. BrdU (a thymidine analog) is added to cell culture and incorporated during the S-phase of the cell cycle, and subsequent detection by anti-BrdU antibody coupled to peroxidase enzyme provides a colorimetric readout. |
| Intended Use | Quantification of cell proliferation rates in growth factor and cytokine bioassays, anti-proliferative drug screening, and immunology research including lymphocyte activation studies. |
| Principle / Technology | Exogenously supplied BrdU is taken up by cells and phosphorylated to BrdU-triphosphate, which substitutes for dTTP during DNA replication; incorporated BrdU in genomic DNA is detected by a peroxidase-conjugated monoclonal anti-BrdU antibody following DNA denaturation to expose the epitope; TMB substrate generates a colorimetric signal proportional to DNA synthesis. |
| Detection Method | Colorimetric ELISA measured at 450 nm (reference 620 nm) using a microplate spectrophotometer |
| Sample Type | Adherent and suspension mammalian cells cultured in 96-well microplates; primary lymphocytes, hematopoietic cells, and established cell lines |
| Performance Range / Specifications | BrdU labeling period: 2–24 hours depending on cell doubling time; linear detection range: 500–100,000 cells per well; absorbance 0.1–2.5 OD at 450 nm |
| Sensitivity / LOD | Detection of BrdU incorporation in approximately 200–500 proliferating cells per well in optimized conditions |
| Specificity | BrdU monoclonal antibody shows negligible cross-reactivity with endogenous thymidine, EdU, or IdU; signal depends on active DNA synthesis during the labeling period, distinguishing cycling from quiescent cells |
| Reaction Conditions / Protocol | Add BrdU labeling reagent (10 μM final) to culture medium; incubate 2–24 hours at 37°C; remove medium and fix cells with FixDenat solution (30 min at RT); wash; add anti-BrdU-POD antibody (90 min at RT); wash 3 times; add TMB substrate; incubate 5–30 minutes until color development; stop with 1 M H2SO4; read absorbance at 450 nm against 620 nm reference |
| Components / Formulation | BrdU Labeling Reagent (1000× concentrate in sterile PBS), FixDenat Solution (formaldehyde-based fixation and DNA denaturation buffer), Anti-BrdU-POD (monoclonal antibody-peroxidase conjugate), Washing Buffer (10× PBS concentrate), TMB Substrate Solution, Stop Solution (1 M H2SO4) |
| Storage Conditions | All kit components at 2–8°C; BrdU labeling reagent at 2–8°C protected from light; stable for 12 months |
| Shelf Life | 12 months from date of manufacture |
| Package Specifications | 1 × 96-well plate (96 tests), 5 × 96-well plates (480 tests) |
| Product Form | Liquid ready-to-use reagent kit with all components pre-diluted or requiring simple dilution |
| Quality Control | Each lot validated with exponentially growing CHO and MCF-7 cell lines; background (no BrdU) control <0.1 OD; positive control (10% FBS proliferating cells) >1.0 OD; inter-assay CV <10% |
| Key Features | Non-radioactive alternative to [3H]-thymidine incorporation assays; ELISA format compatible with standard absorbance plate readers; pre-optimized fixation and denaturation step eliminates user optimization; provides total well-level proliferation data without single-cell resolution |
For research use only, not for clinical use.
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