Bisbenzimide, formerly known as Pibenzimol, Hoechst 33258, is a cell-permeable, benzimidazole dye that binds to the minor groove of double stranded DNA with preference for adenine and thymine-rich sequences.

Physicochemical Properties

Molecular Formula	C25H27CL3N6O
Molecular Weight	533.882
Exact Mass	532.131
Elemental Analysis	C, 56.24; H, 5.10; Cl, 19.92; N, 15.74; O, 3.00
CAS #	23491-45-4
Related CAS #	Hoechst 33258;23491-44-3; Hoechst 33258 trihydrochloride;23491-45-4;Hoechst 33258 analog;258843-62-8;Hoechst 33258 analog 2;23491-54-5;Hoechst 33258 analog 3;23554-98-5;Hoechst 33258 analog 5;23491-55-6;Hoechst 33258 analog 6;129244-66-2
PubChem CID	31953
Appearance	Yellow to green solid powder
Boiling Point	643.1ºC at 760mmHg
Melting Point	314 °C
Flash Point	342.7ºC
LogP	6.639
Hydrogen Bond Donor Count	6
Hydrogen Bond Acceptor Count	5
Rotatable Bond Count	3
Heavy Atom Count	35
Complexity	634
Defined Atom Stereocenter Count	0
InChi Key	4-[6-(4-methyl-1-piperazinyl)[2,6'-bi-1H-benzimidazol]-2'-yl]-phenol, trihydrochloride
InChi Code	SMNPLAKEGAEPJD-UHFFFAOYSA-N
Chemical Name	Bisbenzimide Bisbenzimide trihydrochloride Pibenzimol Pibenzimol HCl Pibenzimol trihydrochloride. H 33258 HOE 33258 Hoechst 33258 NSC 322921.
Synonyms	HOECHST 33258; HOE 33258; Bisbenzimidazole trihydrochloride; Pibenzimol hydrochloride; Hoechst Dye 33258; Hoechst 33258 Trihydrochloride; bisBenzimide H 33258;
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Targets	IC50 = 51 μM (HeLa cells), 32 μM (HL60 cells), and 15 μM (U937 cells)
ln Vitro	1.1 Hoechst working solution preparation: 10 milligrams should dissolve in 5 mL DMSO. It is advised to store the stock solution in the dark at 4°C or -20°C to prevent recurrent freezing and thawing. 1.2 Hoechst stock solution preparation A Hoechst working solution with a final concentration of 10 μg/mL is created by dissolving the stock solution in either PBS or serum-free cell culture medium. Note: Please modify the Hoechst working fluid concentration based on the current circumstances. 1. Staining of cells 2.1 Suspension plates with six wells a. Discard the supernatant after centrifuging at 1000g for three to five minutes at 4°C. Use PBS to wash twice, for five minutes each time. The density of cells is 1×106/mL. c. After adding 1 mL of the working solution, wait three to ten minutes. c. Centrifuge for 3–4 minutes at 400 g and 4°C, discarding the supernatant. d. Use PBS to wash twice, for five minutes each time. e. Re-suspend the cells in PBS or serum-free culture media. observation using a flow cytometer or a fluorescence microscope 2.2 Cells that adhere On sterile coverslips, cultivate adherent cells (a). b. Aspirate extra cells and remove the coverslip from the culture medium. b. Pour in 100 μL of working solution, then give it a little shake to fully cover. Prelude: 3–10 minutes for cells. d. Wash your heart twice, for five minutes each time. Cursor matter or observation using a flow cytometer. Note 1: Please modify the Hoechst working fluid concentration based on the current circumstances. 2. This product is intended only for use in research and development; it cannot be used for home, medical, or other applications. 3. Please wear a lab coat and immediate gloves when operating for your own health and safety.
Enzyme Assay	Pibenzimol/Hoechst 33258 is a fluorescent dye of benzimidazole derivative. Pibenzimol binds to AT-specific sites in the minor groove of duplex DNA and inhibits topoisomerase I, and DNA polymerase, thereby preventing DNA replication. This agent prolongs the G2 phase of the cell cycle and initiates apoptosis in tumor cells. A benzimidazole antifilarial agent; it is fluorescent when it binds to certain nucleotides in DNA, thus providing a tool for the study of DNA replication; it also interferes with mitosis.
Cell Assay	Staining examples: Example 1: Hoechst 33258 may be utilized for the detection of nuclei. Method: For cell staining. 1. Fix, penetrate and seal cells first. 2. Incubate cells with Hoechst 33258 (1:1000; 30 min; dark). 3. Wash cells with PBS for five times and use a fluorescence microscope for image. Example 2: Hoechst 33258 may be utilized for the detection of nuclei and staining nuclei of live and dead cells (apoptotic/necrotic). Method: For cell staining. 1. Incubate cultured cells with 1 mL of the solution prepared with Hoechst 33258 (7.5 μg/mL; 37℃; 5% CO2; 30 min; 1×PBS). 2. Use a Evos FLc Fluorescent Inverted Microscope for image. Example 3: Hoechst 33258 may be utilized for the detection of nuclei, as well as in apoptosis detection assays. Method: For cell staining. 1. Fix cells in paraformaldehyde after transfection and wash cells with PBS. 2. Stain cells with Hoechst 33258 (30 min; 20℃; dark). 3. Use a fluorescence microscope for image.
Toxicity/Toxicokinetics	rat LD50 intravenous 32200 ug/kg BEHAVIORAL: SOMNOLENCE (GENERAL DEPRESSED ACTIVITY); LUNGS, THORAX, OR RESPIRATION: DYSPNEA; SKIN AND APPENDAGES (SKIN): HAIR: OTHER National Technical Information Service., PB84-171206 mouse LD50 intravenous 36900 ug/kg BEHAVIORAL: SOMNOLENCE (GENERAL DEPRESSED ACTIVITY); LUNGS, THORAX, OR RESPIRATION: DYSPNEA; SKIN AND APPENDAGES (SKIN): HAIR: OTHER National Technical Information Service., PB84-171206 dog LD intravenous >22400 ug/kg SENSE ORGANS AND SPECIAL SENSES: LACRIMATION: EYE; LUNGS, THORAX, OR RESPIRATION: DYSPNEA; GASTROINTESTINAL: CHANGES IN STRUCTURE OR FUNCTION OF SALIVARY GLANDS National Technical Information Service., PB84-171206
References	[1]. Newly synthesized bis-benzimidazole derivatives exerting anti-tumor activity through inductionof apoptosis and autophagy. Bioorg Med Chem Lett. 2012 Oct 1;22(19):6297-300. [2]. Synthesis, DNA/RNA affinity and antitumour activity of new aromatic diamidines linked by 3,4-ethylenedioxythiophene. Eur J Med Chem. 2011 Feb;46(2):743-55.
Additional Infomation	A benzimidazole antifilarial agent; it is fluorescent when it binds to certain nucleotides in DNA, thus providing a tool for the study of DNA replication; it also interferes with mitosis. See also: Bisbenzimidazole (annotation moved to).

Solubility Data

Solubility (In Vitro)	DMSO : ~100 mg/mL (~187.31 mM) H2O : ~12.5 mg/mL (~23.41 mM)
Solubility (In Vivo)	Solubility in Formulation 1: 9.09 mg/mL (17.03 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication (<60°C).  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	1.8731 mL	9.3654 mL	18.7308 mL
	5 mM	0.3746 mL	1.8731 mL	3.7462 mL
	10 mM	0.1873 mL	0.9365 mL	1.8731 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.