HOE-32020 targets DNA by binding to the minor groove of the double helix. As a member of the Hoechst family of bis-benzimidazole compounds, it exhibits a strong affinity for A/T-rich sequences of DNA. The binding is primarily mediated through hydrogen bonding and hydrophobic interactions with the DNA minor groove, which results in a significant enhancement of fluorescence upon binding. This DNA-binding property makes HOE-32020 a valuable tool for nucleic acid detection and quantification in various research applications.

Hoechst working solution preparation 1.1 Hoechst stock solution preparation Make a 1 mg/mL Hoechst stock solution using DMSO. Note: After filling, Hoechst storage solution should be kept in the dark at -4°C or -20°C. 1.2 Replacing the working solution: Before using the storage solution, add PBS or a high-quality serum-free cell culture medium. Ten μg/mL of Hoechst working solution is the final concentration. Note: Before using, please make sure that the Hoechst working fluid concentration is appropriate for the current circumstances. 2. Suspended cell staining (2.1): Centrifuge cells, add PBS, then wash twice for five minutes each time. Add 1 mL of Hoechst working solution and let it settle for 3–10 minutes, or until the cell density reaches 1×106/mL 2.2. 2.3 Centrifuge for 3–4 minutes at 400 g, then discard. 2.4 Wash the cells twice with PBS, giving them five minutes each time. 2.5 Re-suspend the cells in 1 milliliter of PBS or serum-free water, and use a flow cytometer or fluorescence microscope to observe. 3. Adhesion-based cell staining 3.1 Grow adherent cells on sterile coverslips. 3.2 Aspirate extra cells and remove the coverslip from the culture medium. Step 3: Aspirate the dye working solution, wash the cells 2-3 times with culture media for 5 minutes each time, and use a fluorescence microscope or flow cytometer to monitor. Step 3.3 Add 100 μL of dye working solution and shake gently to completely cover the cells. Observations 1. Kindly modify the Hoechst working fluid concentration based on the current circumstances. Ten minutes. 2. This product may not be used for clinical diagnosis or treatment, nor may it be included into food or medication. It is intended solely for professional use in scientific study. 3. When working, please wear gloves and a lab coat for your own health and safety.
HOE-32020 is not typically evaluated in traditional pharmacological activity assays. Its in vitro activity is assessed based on its fluorescent properties and DNA-binding affinity. The compound exhibits strong fluorescence enhancement upon binding to DNA, with an emission maximum of 495 nm when excited at 350 nm. It stains the nuclei of live cells effectively, allowing for visualization of nuclear morphology and DNA content. The compound is cell-permeable, enabling its use in live cell imaging applications.

HOE-32020 is used for in vivo imaging applications where DNA staining and nuclear visualization are required. It is commonly employed in live cell nuclear labeling studies. However, as a fluorescent dye, it does not exert pharmacological effects in the traditional sense. Its utility lies in its ability to stain DNA in living organisms for research purposes, such as tracking cell division, assessing cell viability, and monitoring nuclear morphology in various biological models.

HOE-32020 does not have a conventional enzyme/receptor binding assay. Its activity is characterized by spectrophotometric and fluorometric measurements. The compound's absorption and emission spectra are recorded to determine its fluorescent properties. DNA-binding assays are performed by titrating the dye with increasing concentrations of DNA and measuring the fluorescence enhancement. The binding affinity and stoichiometry can be determined from these titration curves.

In vitro cellular experiments with HOE-32020 involve incubating live or fixed cells with the dye at concentrations typically ranging from 0.5 to 10 μM. Cells are incubated with the dye for 15-30 minutes at 37°C, followed by washing to remove unbound dye. Stained cells are then visualized using fluorescence microscopy with appropriate excitation and emission filters (typically DAPI or Hoechst filter sets). Flow cytometry can also be used to analyze DNA content and cell cycle distribution.

In vivo animal experiments with HOE-32020 are typically limited to imaging applications. The dye can be administered to animals via intravenous injection or direct application to tissues of interest. Following administration, tissues are harvested and examined under fluorescence microscopy to assess nuclear staining patterns. The compound may also be used in whole-mount staining of embryos or other small organisms for developmental biology studies.

HOE-32020 has a molecular weight of 442.94 g/mol and a molecular formula of C25H23ClN6. It is supplied as a powder or as a 10 mM solution in DMSO. The compound should be protected from light and stored at -80°C for long-term stability. It is soluble in DMSO and aqueous buffers. The dye exhibits excellent photostability and bright fluorescence when bound to DNA.

HOE-32020 is for research use only and is not intended for human or veterinary use. The compound is generally considered to have low toxicity at the concentrations used for staining applications. However, as with all DNA-binding dyes, it should be handled with care due to its potential mutagenic properties. Standard safety precautions, including the use of gloves and eye protection, should be followed when handling this compound.

[1]. Latt SA, Stetten G, Juergens LA, Recent developments in the detection of deoxyribonucleic acid synthesis by 33258 Hoechst fluorescence. The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society 23 (7): 493-505.

[2]. a b c "Hoechst Stains". Invitrogren (Molecular Probes).

[3]. 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 949 (2): 158-68.

HOE-32020 is a member of the Hoechst family of fluorescent DNA probes, which also includes Hoechst 33258 and Hoechst 33342. These dyes are among the most commonly used nuclear stains in biological research. HOE-32020 is distinguished by its specific spectral properties and its ability to stain live cells. The compound is available from various chemical suppliers for research purposes and is used in applications ranging from cell biology to molecular diagnostics.

C25H23CLN6

442.951

442.167

23554-99-6

71576674

Light green to green solid powder

5.181

2

4

3

32

634

0

QXGLWDFFMBHOFL-UHFFFAOYSA-N

InChI=1S/C25H23ClN6/c1-31-10-12-32(13-11-31)19-7-9-21-23(15-19)30-25(28-21)17-4-8-20-22(14-17)29-24(27-20)16-2-5-18(26)6-3-16/h2-9,14-15H,10-13H2,1H3,(H,27,29)(H,28,30)

2-(4-chlorophenyl)-6-[6-(4-methylpiperazin-1-yl)-1H-benzimidazol-2-yl]-1H-benzimidazole

HOE32020; HOE 32020; HOE-32020

2934.99.9001

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.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ≥ 69 mg/mL (~155.78 mM)

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.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*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).

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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)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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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).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)