Hoechst 34580 targets amyloid beta (Aβ) aggregation process (IC50 = 2.3 μM, ThT fluorescence assay) [1]

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.

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1. Aβ aggregation inhibition:
- Hoechst 34580 inhibits Aβ1-42 aggregation in a concentration-dependent manner, with an IC50 of 2.3 μM (50% inhibition) as determined by ThT fluorescence assay [1]
- At 10 μM, it achieves 82% inhibition of Aβ1-42 aggregation compared to the vehicle control [1]
- It shows no significant effect on Aβ1-40 aggregation even at 20 μM, indicating selectivity for Aβ1-42 [1]
2. Aβ fibril formation suppression:
- Transmission Electron Microscopy (TEM) analysis reveals that Hoechst 34580 (10 μM) drastically reduces the number of intact Aβ1-42 fibrils after 72-hour incubation [1]
- The remaining Aβ species appear as amorphous aggregates instead of the typical long, unbranched fibrils observed in the control group [1]
3. Aβ secondary structure modulation:
- Circular Dichroism (CD) spectroscopy shows that Hoechst 34580 (10 μM) reduces the β-sheet content of Aβ1-42 from 45% (control) to 18% [1]
- Corresponding increases in α-helix (from 12% to 29%) and random coil (from 43% to 53%) contents are observed, indicating disruption of fibril-forming structure [1]
4. Protection against Aβ-induced cytotoxicity:
- Hoechst 34580 (1-20 μM) protects SH-SY5Y neuroblastoma cells from Aβ1-42-induced cytotoxicity in a concentration-dependent manner [1]
- Aβ1-42 (20 μM) reduces cell viability to 45% of the control, while co-treatment with 10 μM Hoechst 34580 restores viability to 78% [1]

1. ThT fluorescence assay for Aβ aggregation inhibition:
- Aβ1-42 peptide is dissolved in appropriate buffer to a final concentration of 20 μM [1]
- Hoechst 34580 is serially diluted (0.1 μM to 20 μM) and mixed with Aβ1-42 solution, followed by incubation at 37°C for 72 hours [1]
- Thioflavin T (ThT) reagent is added to the mixture at a final concentration of 20 μM, and fluorescence intensity is measured at an excitation wavelength of 440 nm and emission wavelength of 485 nm [1]
- Inhibition rate is calculated by comparing fluorescence intensity of drug-treated groups to the control, and IC50 is derived from the dose-response curve [1]
2. Circular Dichroism (CD) spectroscopy for Aβ secondary structure analysis:
- Aβ1-42 peptide (20 μM) is incubated with Hoechst 34580 (10 μM) or vehicle at 37°C for 72 hours [1]
- CD spectra are recorded using a CD spectrometer over a wavelength range of 190-260 nm, with a path length of 0.1 cm [1]
- Secondary structure fractions (β-sheet, α-helix, random coil) are calculated using spectral deconvolution software [1]
3. Transmission Electron Microscopy (TEM) for fibril morphology observation:
- Aβ1-42 (20 μM) is incubated with or without Hoechst 34580 (10 μM) at 37°C for 72 hours [1]
- Samples are applied to carbon-coated copper grids and negatively stained with uranyl acetate solution [1]
- Grids are air-dried, and images are acquired using a TEM at an accelerating voltage of 80 kV to observe fibril morphology and quantity [1]

1. Aβ-induced cytotoxicity protection assay (MTT):
- SH-SY5Y cells are seeded in 96-well plates at a density of 5×10^3 cells per well and cultured overnight in DMEM/F12 medium supplemented with fetal bovine serum and antibiotics [1]
- Cells are pre-treated with Hoechst 34580 (1, 5, 10, 20 μM) for 2 hours, then exposed to Aβ1-42 (20 μM) for 48 hours [1]
- MTT reagent is added to each well and incubated for 4 hours at 37°C, after which the supernatant is removed and formazan crystals are dissolved in DMSO [1]
- Absorbance at 570 nm is measured using a microplate reader, and cell viability is calculated relative to the untreated control group [1]

[1]. Discovery of DNA dyes Hoechst 34580 and 33342 as good candidates for inhibiting amyloid beta formation: in silico and in vitro study. J Comput Aided Mol Des. 2016 Aug;30(8):639-50.

Hoechst 34580 is a well-known DNA-binding dye that has been identified as a potential Aβ aggregation inhibitor by computer screening (molecular docking)[1]. Molecular docking studies have shown that it binds to the hydrophobic core region (residues 17-21) of Aβ1-42 and forms hydrogen bonds and hydrophobic interactions with key residues (Phe19, Val24)[1]. It can disrupt the β-sheet formation and fiber aggregation of Aβ1-42 and has a neuroprotective effect against Aβ-induced cytotoxicity, which suggests that it has potential application value in Alzheimer's disease research[1]. Unlike traditional Aβ inhibitors, it has the dual characteristics of a DNA dye and an Aβ regulator, enabling it to be used in biological experiments for multiple purposes[1].

C27H29N7

451.56606

451.248

23555-00-2

Hoechst 34580 tetrahydrochloride;2310135-08-9

448202

Off-white to pink solid powder

5.867

2

5

4

34

672

0

VMCOQLKKSNQANE-UHFFFAOYSA-N

InChI=1S/C27H29N7/c1-32(2)20-7-4-18(5-8-20)26-28-22-10-6-19(16-24(22)30-26)27-29-23-11-9-21(17-25(23)31-27)34-14-12-33(3)13-15-34/h4-11,16-17H,12-15H2,1-3H3,(H,28,30)(H,29,31)

N,N-dimethyl-4-[6-[6-(4-methylpiperazin-1-yl)-1H-benzimidazol-2-yl]-1H-benzimidazol-2-yl]aniline

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 : ≥ 47 mg/mL (~104.08 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.)