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| Targets |
Nuclear marker dye
The primary "target" of Hoechst 33258 analog is DNA, specifically the minor groove of double-stranded DNA. Hoechst dyes bind non-covalently to the minor groove of DNA, with a preference for A/T-rich sequences. The binding is primarily driven by electrostatic interactions and hydrogen bonding between the dye and the DNA bases. Upon binding to DNA, the dye's fluorescence is significantly enhanced, making it a useful tool for visualizing and quantifying DNA in cells. The Hoechst 33258 analog offers improved brightness and binding affinity compared to the parent compound. |
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| ln Vitro |
Preparation of Hoechst working slution:
1.1 Preparation of stock solution Prepare 1 mg/mL Hoechst stock solution with DMSO. Note: Hoechst stock solution is recommended to be aliquoted and stored in the dark at -4 ℃ or -20 ℃. 1.2 Preparation of working slution Dilute the stock solution with preheated serum-free cell culture medium or PBS to a final concentration of 10 μ g/mL Hoechst working solution. Note: Please adjust the concentration of Hoechst working solution according to your specific needs, and use freshly prepared solutions. 2. Cell staining (suspended cells) 2.1 Centrifuge and collect cells, wash twice with PBS for 5 minutes each time. Cell density is 1 × 10~6/mL 2.2 Add 1 mL of Hoechst working solution and incubate at room temperature for 3-10 minutes. 2.3 400 g, centrifuge for 3-4 minutes, discard the supernatant. 2.4 Wash the cells twice with PBS, each time for 5 minutes. After resuspending cells in 1 mL serum-free medium or PBS, observe them using a fluorescence microscope or flow cytometer. 3. Cell staining (adherent cells) 3.1 Cultivate adherent cells on sterile coverslips. 3.2 Remove the cover glass from the culture medium and aspirate excess culture medium. 3.3 Add 100 μ L of dye working solution, gently shake to completely cover the cells, and incubate for 3-10 minutes. 3.4 Remove the dye working solution, wash 2-3 times with culture medium for 5 minutes each time, and observe using a fluorescence microscope or flow cytometer. In vitro, Hoechst 33258 analog is a fluorescent dye used for staining DNA in live and fixed cells. The dye binds to the minor groove of double-stranded DNA, emitting blue fluorescence with excitation/emission maxima of 365/458 nm. It is commonly used for nuclear counterstaining in fluorescence microscopy, for cell cycle analysis by flow cytometry, and for DNA quantification. The analog offers improved brightness and binding affinity compared to the parent compound, making it a preferred choice for applications requiring high sensitivity. The dye is cell-permeable and can be used to stain live cells without significant toxicity at working concentrations. |
| ln Vivo |
Hoechst dye can bind to DNA in both fixed and live cells and is cell permeable [1].
In vivo, Hoechst 33258 analog is primarily used for labeling and tracking cells in animal models. The dye can be injected into animals to label cells or tissues for subsequent analysis. For example, it can be used to label tumor cells for tracking their dissemination and metastasis in vivo. The dye's blue fluorescence allows for detection using fluorescence microscopy or flow cytometry. However, its use in vivo is limited by its rapid clearance and potential toxicity at high doses. The compound is primarily used for in vitro and ex vivo applications. |
| Enzyme Assay |
DNA binding sites for the minor groove-binding ligands DAPI (4',6-diamidine-2-phenylindole) and Hoechst 33258 (bisbenzimide) have been analysed using DNAase I and micrococcal nuclease footprinting techniques. Both drugs appear to bind to AT-rich regions containing at least four such basepairs. Hoechst 33258 seems to bind relatively poorly to nucleotide sequences containing the alternating step TpA. However, in contrast to DAPI, it can more readily accommodate the presence of guanosine residues at the end of the binding site. We compare the DNA binding sites for DAPI and Hoechst 33258 with those determined for the related minor groove-binding ligands, berenil, netropsin and distamycin A, under comparable conditions, and discuss the importance of using different footprinting probes when analysing drug-DNA interactions[2].
Cell-free assays for Hoechst 33258 analog typically involve assessing its DNA binding properties. A typical protocol involves incubating the dye with calf thymus DNA or a synthetic A/T-rich DNA oligonucleotide in a buffer (e.g., PBS, pH 7.4) at various concentrations. The fluorescence is measured using a fluorescence spectrophotometer with excitation at 350-365 nm and emission at 450-460 nm. The binding affinity (Kd) is determined by fitting the fluorescence enhancement data to a binding isotherm. The dye's selectivity for A/T-rich DNA can be assessed by comparing its binding to different DNA sequences. |
| Cell Assay |
For in vitro cellular experiments, cells are cultured in appropriate media and incubated with Hoechst 33258 analog at concentrations typically ranging from 0.1-10 ug/mL. Cells are incubated for 15-30 minutes at 37degC, washed to remove unbound dye, and then analyzed by fluorescence microscopy or flow cytometry with excitation at 350-365 nm and emission at 450-460 nm. The dye can be used for nuclear counterstaining, cell cycle analysis (by measuring DNA content), or apoptosis detection (by assessing nuclear morphology). Live cells can be stained without significant toxicity at working concentrations.
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| Animal Protocol |
In vivo animal experiments with Hoechst 33258 analog typically involve intravenous or intraperitoneal injection of the dye in mice or rats. A common protocol involves injecting the dye at a dose of 1-10 mg/kg and allowing it to circulate for 1-24 hours. Animals are then euthanized, and tissues are collected for fluorescence microscopy or flow cytometry analysis to identify labeled cells. The dye can be used to track the distribution of labeled cells (e.g., tumor cells, stem cells) in vivo. However, its use is limited by its rapid clearance and potential toxicity at high doses.
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| ADME/Pharmacokinetics |
Hoechst 33258 analog has a molecular weight of 510.58 g/mol and a molecular formula of C29H30N6O3. The dye is soluble in DMSO and should be stored at -20degC, protected from light. As a fluorescent dye, its pharmacokinetic properties are not typically characterized, as it is not used as a therapeutic agent. When used in vivo, the dye is rapidly cleared from the circulation and distributed to tissues. Its fluorescence is stable and photostable, allowing for long-term imaging applications.
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| Toxicity/Toxicokinetics |
The toxicity profile of Hoechst 33258 analog has not been extensively characterized, but Hoechst dyes are generally considered to have low toxicity at working concentrations used for staining. At higher concentrations, the dye may cause DNA damage or other cellular effects due to its intercalation into DNA. The compound should be handled with standard laboratory precautions, including the use of personal protective equipment, and should be protected from light. The compound is intended for research use only.
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| References | |
| Additional Infomation |
This article describes a variety of applications of fluorescence microscopy for detecting deoxyribonucleic acid synthesis. These applications include: (a) analyzing the exchange and separation of sister chromatids during mitosis; (b) locating deoxyribonucleic acid regions on chromosomes containing thymine residues asymmetrically distributed among polynucleotide chains; and (c) detecting late replication regions in metaphase chromosomes. In both fixed cytological specimens and unfixed cultured cells, it was confirmed that the biosynthesis of 5-bromodeoxyuridine incorporated into the interphase nucleus could inhibit Hoechst 33258 fluorescence. Many of the cytological observations described in this article may lay the foundation for future biochemical research. [1]
Hoechst 33258 analog (CAS 258843-62-8) is a modified Hoechst dye that offers improved brightness and binding affinity for DNA compared to the parent compound. It binds to the minor groove of double-stranded DNA, with a preference for A/T-rich sequences. The dye emits blue fluorescence with excitation/emission maxima of 365/458 nm. It is used for nuclear counterstaining, DNA quantification, and cell cycle analysis in fluorescence microscopy and flow cytometry. Hoechst 33258 analog is available as a research compound and is not approved for clinical use. |
| Molecular Formula |
C29H30N6O3
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|---|---|
| Molecular Weight |
510.5869
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| Exact Mass |
510.238
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| CAS # |
258843-62-8
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| Related CAS # |
Hoechst 33258;23491-44-3;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
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| PubChem CID |
12116202
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| Appearance |
Light yellow to yellow solid powder
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| LogP |
4.771
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
8
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| Heavy Atom Count |
38
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| Complexity |
794
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
QXQZJBBLMSBZHP-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C29H30N6O3/c1-34-11-13-35(14-12-34)21-8-10-24-26(18-21)33-29(31-24)20-7-9-23-25(17-20)32-28(30-23)19-4-2-5-22(16-19)38-15-3-6-27(36)37/h2,4-5,7-10,16-18H,3,6,11-15H2,1H3,(H,30,32)(H,31,33)(H,36,37)
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| Chemical Name |
4-[3-[6-[6-(4-methylpiperazin-1-yl)-1H-benzimidazol-2-yl]-1H-benzimidazol-2-yl]phenoxy]butanoic acid
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| Synonyms |
-[3-[6-[6-(4-methylpiperazin-1-yl)-1H-benzimidazol-2-yl]-1H-benzimidazol-2-yl]phenoxy]butanoic acid; SCHEMBL14091141; SCHEMBL16552182; SCHEMBL23191099; DTXSID50477829;
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: This product requires protection from light (avoid light exposure) during transportation and storage. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ≥ 51 mg/mL (~99.88 mM)
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.9585 mL | 9.7926 mL | 19.5852 mL | |
| 5 mM | 0.3917 mL | 1.9585 mL | 3.9170 mL | |
| 10 mM | 0.1959 mL | 0.9793 mL | 1.9585 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.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution 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 stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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