Hoechst 33258 trihydrochloride (Synonyms: Bisbenzimide, HOE 33258, NSC 322921, Pibenzimol) |
| Catalog No.GC12187 |
Hoechst 33258 trihydrochloride is an analog of the nucleic acid stain Hoechst 33258. It emits blue fluorescence after binding to dsDNA, with a maximum excitation/emission light of 350/461nm.
Products are for research use only. Not for human use. We do not sell to patients.
Cas No.: 23491-45-4
Sample solution is provided at 25 µL, 10mM.
The nucleic acid stain Hoechst 33258 trihydrochloride (Ex/Em: 350/461nm) is frequently utilized as a cell-permeable nuclear counterstain that emits a blue fluorescence upon binding to dsDNA. Hoechst 33258 trihydrochloride is commonly employed in various studies related to cell counting, cell cycle analysis, and cell replication. It is particularly useful in identifying condensed nuclei in apoptotic cells, as well as in combination with BrdU staining for cell-cycle studies. Hoechst 33258 works similarly to Hoechst 33342 but is less cell permeable.
Hoechst dyes are also useful for monitoring cell viability by tracking changes in their emission spectra. As minor groove-binding DNA stains with AT selectivity, the Hoechst dyes are able to bind to all nucleic acids, but they show a greater fluorescence enhancement for AT-rich double-stranded DNA strands compared to GC-rich strands [1]. This property has been exploited to identify Q-bands in chromosomes, which are regions rich in AT base pairs that fluoresce brightly when stained with the quinacrine dye [2].
Fig. Fluorescence excitation and emission spectra of Hoechst 33258 bound to DNA
References:
[1]. Portugal J, Waring MJ. Assignment of DNA binding sites for 4′, 6-diamidine-2-phenylindole and bisbenzimide (Hoechst 33258). A comparative footprinting study. Biochimica et Biophysica Acta (BBA)-Gene Structure and Expression. 1988 Feb 28;949(2):158-68.
[2]. Weisblum B, Haenssler E. Fluorometric properties of the bibenzimidazole derivative Hoechst 33258, a fluorescent probe specific for AT concentration in chromosomal DNA. Chromosoma. 1974 Sep;46(3):255-60.
This plan only provides a guide, please modify it to meet your specific needs.
1. Prepare Hoechst staining solution
(1) Prepare Hoechst dye stock solution: Use DMSO to dissolve solid and prepare a 10 mg/mL Hoechst dye stock solution.
Note: Hoechst stock solution is recommended to be aliquoted and stored in the dark at -4°C or -20°C to avoid repeated freezing and thawing.
(2) Working solution preparation: Use preheated serum-free medium or buffer (such as HBSS or PBS) to dilute the stock solution and prepare a Hoechst working solution with a concentration of 10 μg/mL.
Note: Please adjust the concentration of Hoechst working fluid according to the actual situation and prepare it immediately.
2. Cell staining
2.1 Suspension cells (taking 6-well plate as an example)
(1) Centrifuge suspended cells at 1000g for 3-5 minutes. Discard the supernatant and wash twice with PBS for 5 minutes each time.
(2) Add 1 mL of Hoechst dye working solution and incubate at room temperature in the dark for 5-10 minutes.
(3) After the incubation, centrifuge at 1000g for 5 minutes, remove the supernatant, add PBS and wash 2-3 times, 5 minutes each time.
(4) Use serum-free cell culture medium or PBS to resuspend the cells and observe them through fluorescence microscopy or flow cytometry.
2.2 Adherent cells
(1) Culture adherent cells on sterile coverslips.
(2) Remove the coverslip from the culture medium, suck out the excess culture medium, and place the coverslip in a humid environment.
(3) Add 100μL of Hoechst dye working solution from one corner of the coverslip, shake gently to evenly cover all cells with the dye, and incubate at room temperature in the dark for 5-15 minutes.
(4) Aspirate away the dye working solution, wash the coverslip 2 to 3 times with culture solution, and observe with a fluorescence microscope.
3. Microscope detection: The excitation/emission light of Hoechst 33258 is 365⁄458nm respectively.
Precautions:
① For staining of fixed cells or tissue samples, the fixative needs to be removed by rinsing after fixation;
② Hoechst 33258 staining is usually performed after other staining. If no other staining is required, Hoechst 33258 staining is performed directly;
③ To slow down fluorescence quenching, it is recommended to use anti-fluorescence quenching mounting agent;
④ Fluorescent dyes all have quenching problems, so please try to avoid light;
⑤ Hoechst 33258 is irritating to the human body. For your safety and health, please wear a lab coat and disposable gloves when operating.
References:
[1]. K D Harshman, P B Dervan. Molecular recognition of B-DNA by Hoechst 33258. 1985 Jul 11;13(13):4825-35. doi: 10.1093/nar/13.13.4825.
| Cas No. | 23491-45-4 | SDF | |
| Synonyms | Bisbenzimide, HOE 33258, NSC 322921, Pibenzimol | ||
| Chemical Name | 4-(6-(4-methylpiperazin-1-yl)-1H,1'H-[2,5'-bibenzo[d]imidazol]-2'(3'H)-ylidene)cyclohexa-2,5-dienone trihydrochloride | ||
| Canonical SMILES | CN1CCN(CC1)C2=CC3=C(C=C2)N=C(N3)C4=CC5=C(C=C4)N/C(N5)=C6C=CC(C=C/6)=O.Cl.Cl.Cl | ||
| Formula | C25H27Cl3N6O | M.Wt | 533.88 |
| Solubility | Water : ≥ 72.85 mg/mL (136.45 mM), DMSO : ≥100 mg/mL | Storage | 4°C, protect from light |
| General tips | Please select the appropriate solvent to prepare the stock solution according to the
solubility of the product in different solvents; once the solution is prepared, please store it in
separate packages to avoid product failure caused by repeated freezing and thawing.Storage method
and period of the stock solution: When stored at -80°C, please use it within 6 months; when stored
at -20°C, please use it within 1 month. To increase solubility, heat the tube to 37°C and then oscillate in an ultrasonic bath for some time. |
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| Shipping Condition | Evaluation sample solution: shipped with blue ice. All other sizes available: with RT, or with Blue Ice upon request. | ||
| Prepare stock solution | |||
|
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 |
Step 1: Enter information below (Recommended: An additional animal making an allowance for loss during the experiment)
g
μL
Step 2: Enter the in vivo formulation (This is only the calculator, not formulation. Please contact us first if there is no in vivo formulation at the solubility Section.)
Calculation results:
Working concentration: mg/ml;
Method for preparing DMSO master liquid: mg drug pre-dissolved in μL DMSO ( Master liquid concentration mg/mL, Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug. )
Method for preparing in vivo formulation: Take μL DMSO master liquid, next addμL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O, mix and clarify.
Method for preparing in vivo formulation: Take μL DMSO master liquid, next add μL Corn oil, mix and clarify.
Note: 1. Please make sure the liquid is clear before adding the next solvent.
2. Be sure to add the solvent(s) in order. You must ensure that the solution obtained, in the previous addition, is a clear solution before proceeding to add the next solvent. Physical methods such as vortex, ultrasound or hot water bath can be used to aid dissolving.
3. All of the above co-solvents are available for purchase on the GlpBio website.
Quality Control & SDS
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- Purity: >98.00%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
Average Rating: 5 (Based on Reviews and 30 reference(s) in Google Scholar.)
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