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| Product Name | EdU Cell Proliferation Kit (Click Chemistry, Alexa Fluor 488) |
| Catalog No. | CCAT-HMM-0050 |
| Description | A copper(I)-catalyzed azide-alkyne cycloaddition (click chemistry) detection kit for the fluorescent labeling of 5-ethynyl-2'-deoxyuridine (EdU) incorporated into newly synthesized DNA. Unlike antibody-based BrdU detection, the small fluorescent azide probe freely diffuses through intact chromatin without the need for DNA denaturation. |
| Intended Use | Fluorescence-based detection of DNA replication activity for cell proliferation analysis in flow cytometry, fluorescence microscopy, and high-content imaging screening applications. |
| Principle / Technology | EdU (a nucleoside analog of thymidine containing an alkyne group) is fed to cells and incorporated into DNA during active replication (S-phase); a copper-catalyzed click reaction covalently couples a fluorescent Alexa Fluor azide to the alkyne group; the small molecule azide probe (~0.6 kDa) efficiently penetrates double-stranded DNA allowing detection without harsh denaturation conditions that could compromise simultaneous protein epitope detection. |
| Detection Method | Fluorescence microscopy with FITC/GFP filter set (Ex/Em: 495/519 nm for Alexa Fluor 488), flow cytometry (488 nm excitation, FL1/530/30 nm channel), or high-content imaging platforms |
| Sample Type | Proliferating mammalian cells in adherent or suspension culture, tissue sections, whole-mount embryos (zebrafish, Drosophila), organoid cultures |
| Performance Range / Specifications | EdU incorporation detectable after 30-minute pulse labeling; EdU concentration 10 μM for routine cell culture; click reaction time 30 minutes; compatible with cell densities 1,000–100,000 cells per sample |
| Sensitivity / LOD | S-phase cell detection sensitivity approaching 0.5–1% of the total cell population by flow cytometry analysis |
| Specificity | Alkyne-azide click reaction is highly specific; negligible background labeling from endogenous cellular alkynes; EdU does not cross-react with anti-BrdU antibodies enabling dual-pulse labeling with EdU and BrdU for cell cycle kinetics |
| Reaction Conditions / Protocol | Label cells with 10 μM EdU in culture medium for desired pulse duration (30 min to 24 hours); harvest, wash, and fix cells with 4% paraformaldehyde (15 min); permeabilize with 0.5% Triton X-100 (20 min); wash; add Click-iT reaction cocktail (copper sulfate, Alexa Fluor 488 azide, reaction buffer additive); incubate 30 minutes at RT protected from light; wash; counterstain with DAPI or Hoechst; analyze |
| Components / Formulation | EdU (10 mM stock in DMSO), Alexa Fluor 488 Azide, Copper(II) Sulfate Solution (CuSO4), Click-iT Reaction Buffer, Reaction Buffer Additive (reducing agent for Cu(I) generation), DAPI nuclear counterstain, detailed protocol |
| Storage Conditions | EdU: -20°C protected from light for 12 months; Alexa Fluor 488 Azide: -20°C protected from light; Copper Sulfate and buffer additives: 2–8°C; working click cocktail prepared fresh |
| Shelf Life | 12 months from date of manufacture |
| Package Specifications | 50 tests (coverslip format), 100 tests (flow cytometry format) |
| Product Form | Concentrated stock reagents requiring preparation of click reaction cocktail |
| Quality Control | Each lot validated with HeLa cells pulse-labeled with EdU; labeling index compared to parallel BrdU assay with ≥95% concordance; copper concentration optimized to avoid fluorescence quenching |
| Key Features | No DNA denaturation preserves antigenicity for multiplexed protein detection; small azide probe penetrates dense chromatin and tissue architecture; compatible with antibody co-staining for simultaneous proliferation and marker analysis; superior signal-to-noise compared to BrdU antibody methods |
For research use only, not for clinical use.
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