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| Product Name | Hoechst 33342 Nuclear Staining Reagent for Live-Cell Analysis |
| Catalog No. | CCAT-HMM-0048 |
| Description | A cell-permeable bisbenzimide fluorescent dye that binds to the minor groove of double-stranded DNA in live or fixed cells with minimal cytotoxicity at low concentrations. Hoechst 33342 is widely used for live-cell nuclear counterstaining, cell cycle analysis, and identification of condensed apoptotic nuclei by characteristic morphological changes. |
| Intended Use | Nuclear visualization in live-cell imaging, cell cycle DNA content quantitation, detection of apoptotic nuclear condensation and fragmentation, and identification of side-population stem cells via differential dye efflux. |
| Principle / Technology | Hoechst 33342 (Ex/Em: 350/461 nm bound to DNA) binds to consecutive A-T base pairs in the minor groove of B-form double-stranded DNA; undergoes a fluorescence enhancement of approximately 30-fold upon binding; cells with condensed apoptotic nuclei exhibit increased fluorescence intensity due to chromatin compaction, and fragmented nuclei display characteristic punctate staining pattern. |
| Detection Method | Fluorescence microscopy with DAPI/UV filter set (Ex 330–380 nm, Em 420–470 nm), flow cytometry with UV or violet laser (350 nm or 405 nm excitation), or fluorescence microplate reader |
| Sample Type | Live or fixed adherent and suspension mammalian cells; tissue sections; isolated nuclei; bacterial cells for DNA content quantification |
| Performance Range / Specifications | Staining concentrations: 0.1–10 μg/mL for live-cell imaging, 5–20 μg/mL for cell cycle analysis; effective staining within 5–15 minutes at 37°C; useful UV excitation 350 nm, emission maximum 461 nm |
| Sensitivity / LOD | Nuclear visualization in single cells at dye concentrations as low as 0.1 μg/mL; distinction of apoptotic nuclei with 2-fold increased fluorescence over normal nuclei |
| Specificity | Preferential binding to A-T rich DNA regions; does not intercalate but binds in the minor groove; minimal binding to single-stranded DNA or RNA; cell-permeable for live-cell compatibility unlike DAPI |
| Reaction Conditions / Protocol | For live-cell nuclear staining: add Hoechst 33342 to culture medium at 0.1–10 μg/mL final concentration; incubate 10–30 minutes at 37°C; image directly without washing. For fixed-cell staining: permeabilize cells with 0.1% Triton X-100 if needed; apply 1–5 μg/mL Hoechst; incubate 5–10 minutes; wash; mount in antifade medium |
| Components / Formulation | Hoechst 33342 trihydrochloride (2'-[4-ethoxyphenyl]-5-[4-methyl-1-piperazinyl]-2,5'-bi-1H-benzimidazole trihydrochloride) supplied as 10 mg/mL stock solution in water or as lyophilized powder |
| Storage Conditions | Stock solution: 2–8°C protected from light, stable 6 months; lyophilized powder: -20°C protected from light and moisture, stable 24 months |
| Shelf Life | 24 months from date of manufacture for lyophilized form |
| Package Specifications | 1 mL at 10 mg/mL, 10 mL at 10 mg/mL, 100 mg or 1 g lyophilized powder |
| Product Form | Aqueous stock solution or lyophilized powder requiring dissolution in water |
| Quality Control | Each lot tested for nuclear staining specificity and fluorescence intensity on HeLa and NIH/3T3 cells; purity ≥98% by HPLC; endotoxin tested |
| Key Features | Cell-permeable for live-cell applications; UV-excitable dye compatible with DAPI filter sets on standard fluorescence microscopes; dual-excitation capability with violet (405 nm) lasers on modern flow cytometers; widely cited for side-population stem cell identification |
For research use only, not for clinical use.
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