bisBenzimide H 33258

for fluorescence, ≥98.0% (HPLC)

2′-(4-Hydroxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5′-bi(1H-benzimidazole) trihydrochloride, BXI-72, BBIH, 2-[2-(4-Hydroxyphenyl)-6-benzimidazoyl]-6-(1-methyl-4-piperazyl)benzimidazole trihydrochloride, Hoechst 33258, HOE 33258, NSC334072
Empirical Formula (Hill Notation):
C25H24N6O · 3HCl
CAS Number:
Molecular Weight:
Beilstein/REAXYS Number:
EC Number:
MDL number:
PubChem Substance ID:
Pricing and availability is not currently available.

Quality Level


for fluorescence


≥98.0% (HPLC)




water and ethanol: 10 mg/mL
H2O: soluble
ethanol: soluble


λex 338 nm; λem 505 nm (pH 7.0)
λex 355 nm; λem 465 nm in TE buffer; DNA

storage temp.


SMILES string




InChI key


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bisBenzimide H 33258 is useful for staining DNA, chromosomes and nuclei. bisBenzimide H 33258 may be used for fluorescence microscopy or flow cytometry.
Excitation max. = 346 nm
Emission max. = 460 nm
A bibenzimidazole dye that has been utilized as a fluorescent stain. It is membrane-permeable and selectively binds to adenine-thymine regions in the minor groove of B-form DNA with a high binding constant, making it a useful stain for DNA, chromosomes, and nuclei . It has been shown that the fluorescence is relatively insensitive to the presence of various solution reagents (i.e., BSA, RNA, Tris, sucrose, TEA/EDTA, Triton X-100, and SDS). Furthermore, the solution ionic strength and pH has been shown to be independent of the Stokes′ shift of bisBenzimide H 33258 . The properties of this dye make it useful in several applications such as detecting mycoplasma contamination in tissue culture , indicating early onset of apoptosis, sorting living cells based on DNA content, and acting as a probe for DNA-Drug interactions. Utilized in the design and development of a fluorimetric assay to quantitate cell numbers in microtiter plates. The methodology of the assay is based on Hoechst 33258 binding to DNA and is sensitive for a thousand cells. One application of this is assay is the normalization of ELISA data. The illumination of Hoechst 33253 coupled with Merocyanine 540 can be utilized as an effective method to eradicate bacterial infections (i.e., A. laidlawii contaminated corneal endothelial cell cultures) . BisBenzimide H 33258 is commonly used in many established staining protocols (David M. Terrian, Cancer Cell Signaling – Methods and Protocols,
Springer, Volume 218 ).

Biochem/physiol Actions

bisBenzimide H 33258 is a potent and selective Bcl-XL inhibitor. bisBenzimide H 33258 is a membrane-permeable, fluorescent DNA stain with low cytotoxicity that intercalate in A-T regions of DNA.

Other Notes

Useful reagent for cytogenetic studies; for the fluorescent staining of DNA in cells; simple two-step fluorimetric assay of DNA in isolated nuclei, cell extracts, or crude homogenates using Hoechst 33258


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Hazard Statements

Precautionary Statements

Personal Protective Equipment

dust mask type N95 (US),Eyeshields,Gloves


NONH for all modes of transport

WGK Germany


Flash Point(F)

Not applicable

Flash Point(C)

Not applicable

Certificate of Analysis
Certificate of Origin
F Oberhammer et al.
The EMBO journal, 12(9), 3679-3684 (1993-09-01)
To date, apoptosis has been characterized biochemically by the production of 180-200 bp internucleosomal DNA fragments resulting from the activation of an endonuclease(s). The principal morphological feature of apoptosis is the condensation of chromatin and it has been assumed that...
M Battaglia et al.
Biotechnic & histochemistry : official publication of the Biological Stain Commission, 69(3), 152-156 (1994-05-01)
DNA fluorochrome staining with Hoechst 33258 bisbenzimide is commonly used for detection of mycoplasma contamination in cell cultures. Photobleaching of Hoechst 33258 is pronounced under the conditions of intense illumination, high magnification and resolution required for detection of mycoplasmas. To...
R Jin et al.
Proceedings of the National Academy of Sciences of the United States of America, 85(23), 8939-8942 (1988-12-01)
We compare the fluorescence properties of bisbenzimide (also known as Hoechst 33258) bound to the minor groove of the poly[d(AT)].poly[d(AT)] duplex with the corresponding fluorescence properties of bisbenzimide dissolved in neat organic solvents and mixed organic/aqueous solvents. Based on these...
Improved microfluorometric DNA determination in biological material using 33258 Hoechst.
C F Cesarone et al.
Analytical biochemistry, 100(1), 188-197 (1979-11-15)
M Tarshis et al.
Journal of immunological methods, 168(2), 245-252 (1994-02-10)
Mycoplasma infection can substantially affect the biological properties of cells in vitro. We have devised a method for the selective killing of mycoplasmas, e.g., A. laidlawii, M. fermentans, M. hyorhinis and M. arginini, from experimentally infected cell cultures. This approach...

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