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Hoechst 33258 *CAS 23491-45-4*

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HeLa cells were seeded in 96-well microplates and incubated at 37 °C, 5% CO2 for 24 hours. Cells were fixed with 4% formaldehyde for 20 minutes at RT and then stained with 5 µM Hoechst 33258 for 30 minutes at 37 °C, washed, and imaged on a Keyence BZ-X microscope.
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Physical properties
Molecular weight533.88
Spectral properties
Extinction coefficient (cm -1 M -1)460001
Excitation (nm)352
Emission (nm)454
Quantum yield0.03401
Storage, safety and handling
Certificate of OriginDownload PDF
H-phraseH303, H313, H340
Hazard symbolT
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R68
StorageFreeze (< -15 °C); Minimize light exposure


Molecular weight
Extinction coefficient (cm -1 M -1)
Excitation (nm)
Emission (nm)
Quantum yield
The Hoechst stains are a family of fluorescent stains for labeling DNA in fluorescence microscopy. Because these fluorescent stains label DNA, they are also commonly used to visualize nuclei and mitochondria. Two of these closely related bis-benzimides are commonly used: Hoechst 33258 and Hoechst 33342. Both dyes are excited by ultraviolet light at around 350 nm, and both emit blue/cyan fluorescence light around an emission maximum at 461 nm. The Hoechst stains may be used on live or fixed cells, and are often used as a substitute for another nucleic acid stain, DAPI. The key difference between them is that the additional ethyl group of Hoechst 33342 renders it more lipophilic, and thus more able to cross intact cell membranes. In some applications, Hoechst 33258 is significantly less permeant. These dyes can also be used to detect the contents of a sample DNA by plotting a standard emission-to-content curve.


Common stock solution preparation

Table 1. Volume of Water needed to reconstitute specific mass of Hoechst 33258 *CAS 23491-45-4* to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.

0.1 mg0.5 mg1 mg5 mg10 mg
1 mM187.308 µL936.54 µL1.873 mL9.365 mL18.731 mL
5 mM37.462 µL187.308 µL374.616 µL1.873 mL3.746 mL
10 mM18.731 µL93.654 µL187.308 µL936.54 µL1.873 mL

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Spectral properties

Extinction coefficient (cm -1 M -1)460001
Excitation (nm)352
Emission (nm)454
Quantum yield0.03401



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View all 42 references: Citation Explorer
DNA sequence recognition by Hoechst 33258 conjugates of hairpin pyrrole/imidazole polyamides
Authors: Correa BJ, Canzio D, Kahane AL, Reddy PM, Bruice TC.
Journal: Bioorg Med Chem Lett (2006): 3745
Recognition of B-DNA by neomycin--Hoechst 33258 conjugates
Authors: Willis B, Arya DP.
Journal: Biochemistry (2006): 10217
Design of new bidentate ligands constructed of two Hoechst 33258 units for discrimination of the length of two A3T3 binding motifs
Authors: Tanada M, Tsujita S, Sasaki S.
Journal: J Org Chem (2006): 125
The Hoechst 33258 Covalent Dimer Covers a Total Turn of the Double-stranded DNA
Authors: Streltsov SA, Gromyko AV, Oleinikov VA, Zhuze AL.
Journal: J Biomol Struct Dyn (2006): 285
Amine terminated G-6 PAMAM dendrimer and its interaction with DNA probed by Hoechst 33258
Authors: Choi YS, Cho TS, Kim JM, Han SW, Kim SK.
Journal: Biophys Chem (2006): 142
Unique cooperative binding interaction observed between a minor groove binding Pt antitumor agent and Hoechst dye 33258
Authors: Harris A, Qu Y, Farrell N.
Journal: Inorg Chem (2005): 1196
Hoechst 33258 as a pH-sensitive probe to study the interaction of amine oxide surfactants with DNA
Authors: Goracci L, Germani R, Savelli G, Bassani DM.
Journal: Chembiochem (2005): 197
Recognition of a 10 base pair sequence of DNA and stereochemical control of the binding affinity of chiral hairpin polyamide-Hoechst 33258 conjugates
Authors: Reddy PM, Toporowski JW, Kahane AL, Bruice TC.
Journal: Bioorg Med Chem Lett (2005): 5531
Hydration changes in the association of Hoechst 33258 with DNA
Authors: Kiser JR, Monk RW, Smalls RL, Petty JT.
Journal: Biochemistry (2005): 16988
Activity of Hoechst 33258 against Pneumocystis carinii f. sp. muris, Candida albicans, and Candida dubliniensis
Authors: Disney MD, Stephenson R, Wright TW, Haidaris CG, Turner DH, Gigliotti F.
Journal: Antimicrob Agents Chemother (2005): 1326