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| Targets |
Marker dye
ortho-iodoHoechst 33258 targets DNA, specifically binding to the minor groove of the double helix. Its binding is non-covalent and driven by a combination of hydrogen bonding and hydrophobic interactions, with a strong preference for A/T-rich sequences containing at least four such base pairs. Studies using DNAase I footprinting have shown that while it binds well to AT-rich regions, it binds relatively poorly to sequences containing the alternating step TpA. However, unlike the related dye DAPI, it can more readily accommodate guanosine residues at the end of its binding site. This specific interaction with DNA is the basis for its function as a fluorescent stain, as the binding event significantly enhances its quantum yield. |
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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. ortho-iodoHoechst 33258 itself is a labeling reagent and does not exhibit classical pharmacological activity like enzyme inhibition. Its primary in vitro function is to serve as a fluorescent probe for DNA. Upon binding to DNA, its fluorescence increases dramatically, enabling the visualization of nuclear material in fixed or living cells. It is used to stain DNA in a variety of in vitro applications, such as quantifying DNA content, assessing cell cycle status via flow cytometry, and identifying nuclei in fluorescence microscopy experiments. The dye's specificity for DNA and its enhanced fluorescence upon binding make it a highly sensitive and reliable tool for these purposes. |
| ln Vivo |
ortho-iodoHoechst 33258 is not administered as a therapeutic agent in vivo. However, it can be used in research settings for in vivo cell tracking or to stain cells for transplantation studies. In such applications, cells are stained with the dye ex vivo and then introduced into an animal model, where they can be subsequently tracked by their fluorescence. Its primary utility, however, remains in vitro and ex vivo for cellular and molecular biology research.
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| 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[3].
In vitro assays for ortho-iodoHoechst 33258 are centered on its use as a DNA stain. A standard protocol involves preparing a 1 mg/mL stock solution in DMSO, which is then diluted with preheated serum-free cell culture medium or PBS to a working concentration, typically around 10 μg/mL. For staining suspended cells, the cells are collected, washed with PBS, and then incubated with the working solution for 3-10 minutes at room temperature. After incubation, the cells are centrifuged, washed, and resuspended for observation under a fluorescence microscope or analysis by flow cytometry. A similar protocol is used for adherent cells grown on coverslips. The staining process is rapid and straightforward, making it a popular choice for routine nuclear staining. |
| Cell Assay |
Cellular assays using ortho-iodoHoechst 33258 typically involve incubating live or fixed cells with the dye. For live-cell imaging, the dye is added directly to the culture medium and incubated for a short period, after which the cells are washed and imaged. The dye's cell permeability allows it to enter live cells without the need for fixation or permeabilization. In fixed-cell applications, the staining protocol is similar, and the dye can be used as a convenient alternative to DAPI. After staining, the cells are analyzed using a fluorescence microscope with UV excitation or a flow cytometer to measure DNA content or assess cell cycle distribution.
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| Animal Protocol |
ortho-iodoHoechst 33258 is not typically administered to animals as a therapeutic or diagnostic agent. Its use in vivo is limited to research applications where cells are labeled ex vivo and then introduced into the animal for tracking purposes. The labeled cells can then be detected by their fluorescence in tissue sections or by imaging the whole animal. This approach is used to study cell migration, homing, and engraftment in various disease models.
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| ADME/Pharmacokinetics |
ortho-iodoHoechst 33258 has a molecular weight of 534.39 g/mol and a chemical formula of C25H23IN6. It is a small, cell-permeable molecule. Its fluorescence properties, with excitation and emission maxima around 350 nm and 460 nm respectively, make it compatible with standard UV/blue light sources. As a fluorescent dye, its pharmacokinetics are not a primary concern for its typical in vitro applications. When used for in vivo tracking, its distribution and clearance would depend on the labeled cells or particles.
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| Toxicity/Toxicokinetics |
ortho-iodoHoechst 33258 is generally considered non-toxic at the low concentrations used for DNA staining. It is a well-established dye that has been widely used in cell biology for decades. As a chemical reagent, standard laboratory safety precautions should be followed, including the use of appropriate personal protective equipment. It is not intended for human therapeutic or diagnostic use and is strictly for research purposes.
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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. [2]
ortho-iodoHoechst 33258 is a member of the Hoechst family of dyes and a derivative of Hoechst 33258. It is used for DNA staining and quantitation in a wide range of biological applications. Its preference for A/T-rich regions of DNA and its enhanced fluorescence upon binding make it a valuable tool for cell cycle analysis, apoptosis detection, and fluorescence microscopy. The compound is a standard reagent in many molecular biology and cell biology laboratories. |
| Molecular Formula |
C25H23IN6
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| Molecular Weight |
534.39432
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| Exact Mass |
534.103
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| CAS # |
158013-41-3
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| PubChem CID |
10324821
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| Appearance |
Pink to red solid powder
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| LogP |
5.132
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
32
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| Complexity |
642
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
UOHARKYRPCGBEI-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C25H23IN6/c1-31-10-12-32(13-11-31)17-7-9-21-23(15-17)29-24(27-21)16-6-8-20-22(14-16)30-25(28-20)18-4-2-3-5-19(18)26/h2-9,14-15H,10-13H2,1H3,(H,27,29)(H,28,30)
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| Chemical Name |
2-(2-iodophenyl)-6-[6-(4-methylpiperazin-1-yl)-1H-benzimidazol-2-yl]-1H-benzimidazole
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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 : ~50 mg/mL (~93.56 mM)
H2O : ~0.67 mg/mL (~1.25 mM) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (4.68 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 + to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.8713 mL | 9.3565 mL | 18.7129 mL | |
| 5 mM | 0.3743 mL | 1.8713 mL | 3.7426 mL | |
| 10 mM | 0.1871 mL | 0.9356 mL | 1.8713 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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