Hoechst 33258 (bisbenzimide, HOE 33258), CAS 23491-45-4, is a blue-emitting fluorescent dye that binds strongly to adenine-thymine (A-T) rich regions in the minor groove of double-stranded DNA (dsDNA). Though it binds to all nucleic acids, AT-rich dsDNA greatly enhances its fluorescence.
Spectral Properties:
- Excitation/Emission: 351/463 nm when bound to DNA. Its large Stokes shift is ideal for multicolor labeling.
- pH Sensitivity: Fluorescence intensity increases with higher pH.
- Unbound Dye: Emits green fluorescence (510–540 nm) if excess dye is present or washing is insufficient.
Applications:
- DNA Staining: Commonly used to stain chromosomes and nuclei in live and fixed cells.
- Fluorescence Microscopy & Flow Cytometry: Useful for nuclear visualization and cell sorting.
- Cell-Cycle Studies: Quenched by bromodeoxyuridine (BrdU), making it effective for tracking dividing cells.
Advantages:
- Cellular Permeability: Penetrates living cells, though with less permeability than Hoechst 33342.
- Lower Toxicity: Less toxic than DAPI, supporting higher cell viability.
Typical working concentrations are 0.1–10 μg/mL, making Hoechst 33258 a reliable tool in DNA visualization, cell sorting, and nuclear structure studies.
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