Hoechst 33258 trihydrochloride
Based on 23 publication(s) in Google Scholar
Hoechst 33258 trihydrochloride is a marker dye in Hoechst series. Hoechst is A live nuclear marker dye. Hoechst binds to the grooves in the DNA double strand, which tends to be A/ T-rich DNA strand. Although it binds to all nucleic acids, the A/ T-rich double strand DNA significantly enhances fluorescence intensity Therefore,Hoechst dye can be used for living cell labeling. The fluorescence intensity of Hoechst dye increases with the increase of pH of solution.
For research use only. We do not sell to patients.
- Purity: 99.51%
- CAS No.: 23491-45-4
- Formula: C25H27Cl3N6O
- Molecular Weight:533.88
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Storage:
4°C, sealed storage, away from moisture and light
* In solvent : -80°C, 6 months; -20°C, 1 month (sealed storage, away from moisture and light)
To place orders, for customer services and technical support, please contact: MedChemExpress USA
Tel: 609-228-6898 E-mail: [email protected] [email protected]
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Biological Activity
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Chemical Information
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Solvent & Solubility
- Protocol
- Purity & Documentation
- References
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Help & FAQs
Publications Citing Use of MedChemExpress (MCE) Hoechst 33258 trihydrochloride
More- Nat Commun. 2020 Feb 5;11(1):719. [Abstract]
- Chem Eng J. 2026 Jan 19;529:173215.
- Chem Eng J. 365 (2019) 270-281.
- Phytomedicine. 2025 Jul:142:156632. [Abstract]
- Int J Biol Macromol. 2019 Nov 1:140:709-718. [Abstract]
- Virulence. 2022 Dec;13(1):444-457. [Abstract]
- Food Biosci. 11 December 2021, 101501.
- Int Immunopharmacol. 2023 Jun:119:110136. [Abstract]
- Neurochem Res. 2026 Feb 7;51(1):66. [Abstract]
- Acta Biochim Biophys Sin (Shanghai). 2019 Aug 5;51(8):814-825. [Abstract]
- Med Chem Res. 2025 Dec 5.
- Int J Biochem Cell Biol. 2017 Mar:84:75-88. [Abstract]
- J Cell Biochem. 2019 Jun;120(6):9181-9192. [Abstract]
- Discov Oncol. 2025 Feb 18;16(1):199. [Abstract]
- Cancer Manag Res. 2020 Sep 28:12:9197-9209. [Abstract]
- Cancer Manag Res. 2020 Sep 25:12:9097-9111. [Abstract]
- Biochem Biophys Res Commun. 2025 May 5:762:151769. [Abstract]
- Biochem Biophys Res Commun. 2020 Jun 30;527(3):805-810. [Abstract]
- Biochem Cell Biol. 2021 Aug;99(4):403-413. [Abstract]
- Authorea. 2025 Sep 20.
- Linkoping University. 2025.
- Research Square Preprint. 2020 Jun.
- BIOTECHNOLOGY BULLETIN 2018, 34(12):172-178
Customer Validation & Images
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Cell Imaging/Staining
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Cell Imaging/Staining
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IF
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Cell Imaging/Staining
Biological Activity
IC50: 51.31±4.56 μM (HeLa cell), 32.43±3.27 μM (HL60 cell), 15.42±2.16 μM (U937 cell)[1]
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Cell Line
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Type | Value | Description | References |
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| DU-145 | IC50 |
>10 μM
Compound: Hoechst 33258
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Cytotoxicity against human DU145 cells after 48 hrs by SRB assay
Cytotoxicity against human DU145 cells after 48 hrs by SRB assay
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[PMID: 28513176] |
| HeLa | IC50 |
23.52 μM
Compound: Hoechst 33258
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Antiproliferative activity against human HeLa cells after 48 hrs by MTT assay
Antiproliferative activity against human HeLa cells after 48 hrs by MTT assay
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[PMID: 30679134] |
| KG-1 | IC50 |
42 μM
Compound: Hoechst 33258
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Antiproliferative activity against human KG-1 cells assessed as inhibition of cell growth measured after 48 hrs by MTT assay
Antiproliferative activity against human KG-1 cells assessed as inhibition of cell growth measured after 48 hrs by MTT assay
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[PMID: 35152694] |
| MCF7 | IC50 |
48.1 μM
Compound: Hoechst 33258
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Antiproliferative activity against human MCF7 cells after 48 hrs by MTT assay
Antiproliferative activity against human MCF7 cells after 48 hrs by MTT assay
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[PMID: 30679134] |
Guide (The following is our recommended protocol. This protocol is only a guide and should be modified according to your specific needs).
Preparation of Hoechst working solution
1.1 Preparation of stock solution
°Cake 10 mg and dissolve in 5 mL DMSO.
Note: It is recommended that the stock solution be stored at 4°C or - Store at 20°C in the dark and avoid repeated freezing and thawing.
1.2 Preparation of Hoechst working solution
The diluted stock solution was dissolved in serum-free cell culture medium or PBS to obtain Hoechst working solution with a final concentration of 10 μg/mL.
Note: Please adjust the concentration of Hoechst working solution according to the actual situation.
1. Cell staining
2.1 Suspension cells (6-well plate)
a. Centrifuge at 1000 g for 3-5 min at 4°C and discard the supernatant. Wash 2 times with PBS, 5 minutes each. The cell density was 1×106/mL.
b. Add 1 mL of working solution and incubate at room temperature for 3-10 minutes.
c. Centrifuge at 400 g for 3-4 minutes at 4°C and discard the supernatant.
d. Wash 2 times with PBS, 5 min each.
e. Resuspend cells in serum-free cell culture medium or PBS. Fluorescence microscopy or flow cytometry observation.
2.2 Adherent cells
a. Adherent cells were cultured on sterile coverslips.
b. Remove the coverslip from the medium and aspirate excess medium. c. Add 100 μL of working solution, shake gently to completely cover the cells, and incubate at room temperature for 3-10 minutes.
d. Wash 2 times with medium, 5 min each. Fluorescence microscope or flow cytometry observation.
Store
4°C, 1 year. Avoid light
Precautions
1. Please adjust the concentration of Hoechst working solution according to the actual situation.
2. This product is for R&D use only, not for medicine, household or other purposes.
3. For your safety and health, please wear a lab coat and dis
MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.
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Chemical Information
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CAS No. 23491-45-4
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Appearance Solid
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Molecular Weight 533.88
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Formula C25H27Cl3N6O
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Color Yellow to green
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SMILES
OC1=CC=C(C2=NC3=CC=C(C4=NC5=CC=C(N6CCN(C)CC6)C=C5N4)C=C3N2)C=C1.[H]Cl.[H]Cl.[H]Cl
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Synonyms
bisBenzimide H 33258 trihydrochloride; H 33258 trihydrochloride
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Shipping
Room temperature in continental US; may vary elsewhere.
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Storage
4°C, sealed storage, away from moisture and light
* In solvent : -80°C, 6 months; -20°C, 1 month (sealed storage, away from moisture and light)
Publications (23)
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Journal Impact Factor
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Most Recent
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Nat Commun
Liver governs adipose remodelling via extracellular vesicles in response to lipid overload. [Abstract]2020 Feb 5;11(1):719. PMID: 32024826 -
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Phytomedicine
Curcuminoids WM03 inhibits ovarian cancer cisplatin-resistant cells proliferation and reverses cisplatin resistance by targeting DYRK2. [Abstract]2025 Jul:142:156632. PMID: 40315643 -
Int J Biol Macromol
Protein corona of metal-organic framework nanoparticals: Study on the adsorption behavior of protein and cell interaction. [Abstract]2019 Nov 1:140:709-718. PMID: 31445155 -
Virulence
LncRNA ZEB1-AS1/miR-1224-5p / MAP4K4 axis regulates mitochondria-mediated HeLa cell apoptosis in persistent Chlamydia trachomatis infection. [Abstract]2022 Dec;13(1):444-457. PMID: 35266440
Hoechst 33258 trihydrochloride purchased from MedChemExpress. Usage Cited in: Virulence. 2022 Dec;13(1):444-457. [Abstract]
The apoptotic rate was detected by Hoechst staining (400×), HeLa cells were treated with siRNA for 24 h and followed by persistent infection.
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Int Immunopharmacol
2023 Jun:119:110136. PMID: 37075668 -
Neurochem Res
4-Phenylbutyrate Induces Functional Elongation of the Microglial Process Through Activation of Akt. [Abstract]2026 Feb 7;51(1):66. PMID: 41653373 -
Acta Biochim Biophys Sin (Shanghai)
Downregulation of lncRNA AFAP1-AS1 by oridonin inhibits the epithelial-to-mesenchymal transition and proliferation of pancreatic cancer cells. [Abstract]2019 Aug 5;51(8):814-825. PMID: 31314060 -
Hoechst 33258 trihydrochloride purchased from MedChemExpress. Usage Cited in: Med Chem Res. 2025 Dec 5.
The A549 cells were exposed to coumarin (8 μM) or 6e (0, 4, 8 μM), then stained with 50 mM Hoechst 33258, and examined using fluorescence microscopy.
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Int J Biochem Cell Biol
Methylene blue increases the amount of HSF1 through promotion of PKA-mediated increase in HSF1-p300 interaction. [Abstract]2017 Mar:84:75-88. PMID: 28104499 -
J Cell Biochem
Notoginsenoside R1 protects oxygen and glucose deprivation-induced injury by upregulation of miR-21 in cardiomyocytes. [Abstract]2019 Jun;120(6):9181-9192. PMID: 30552708 -
Discov Oncol
Viral IL-10 can promote the proliferation, migration and invasion of nasopharyngeal carcinoma cells and inhibit their apoptosis. [Abstract]2025 Feb 18;16(1):199. PMID: 39964638
Hoechst 33258 trihydrochloride purchased from MedChemExpress. Usage Cited in: Discov Oncol. 2025 Feb 18;16(1):199. [Abstract]
Hoechst 33,258 staining results showing the effect of vIL-10 on the apoptosis of NPC cells.
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Cancer Manag Res
microRNA-139-3p Inhibits Malignant Behaviors of Laryngeal Cancer Cells via the KDM5B/SOX2 Axis and the Wnt/β-Catenin Pathway. [Abstract]2020 Sep 28:12:9197-9209. PMID: 33061611 -
Cancer Manag Res
CREB1 Suppresses Transcription of microRNA-186 to Promote Growth, Invasion and Epithelial-Mesenchymal Transition of Gastric Cancer Cells Through the KRT8/HIF-1α Axis. [Abstract]2020 Sep 25:12:9097-9111. PMID: 33061604
Hoechst 33258 trihydrochloride purchased from MedChemExpress. Usage Cited in: Cancer Manag Res. 2020 Sep 25:12:9097-9111. [Abstract]
The apoptosis rate of GC cells was assessed by Hoechst 33258 staining (5 μg/mL, 30 min).
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Biochem Biophys Res Commun
Tangeretin protects against Aβ1-42-induced toxicity and exploring mitochondria-lysosome interactions in HT22 cells. [Abstract]2025 May 5:762:151769. PMID: 40220719 -
Biochem Biophys Res Commun
Chlamydia trachomatis plasmid-encoded protein pORF5 activates unfolded protein response to induce autophagy via MAPK/ERK signaling pathway. [Abstract]2020 Jun 30;527(3):805-810. PMID: 32446560 -
Biochem Cell Biol
TFAP2A inhibits microRNA-126 expression at the transcriptional level and aggravates ischemic neuronal injury. [Abstract]2021 Aug;99(4):403-413. PMID: 33264079 -
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BIOTECHNOLOGY BULLETIN 2018, 34(12):172-178
Solvent & Solubility
DMSO : 100 mg/mL (187.31 mM; Need ultrasonic; Hygroscopic DMSO has a significant impact on the solubility of product, please use newly opened DMSO)
H2O : 12.5 mg/mL (23.41 mM; ultrasonic and warming and heat to 60°C)
Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month (sealed storage, away from moisture and light). When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.
* Note: If you choose water as the stock solution, please dilute it to the working solution, then filter and sterilize it with a 0.22 μm filter before use.
Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month (sealed storage, away from moisture and light). When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.
* Note: If you choose water as the stock solution, please dilute it to the working solution, then filter and sterilize it with a 0.22 μm filter before use.
Concentration (start) × Volume (start) = Concentration (final) × Volume (final)
Protocol
Hoechst 33258 is prepared as stock solutions in highly pure water. Working solutions in a concentration range of 10-3-10-6 mol/dm3 are prepared prior to testing. Cytotoxic effects of Hoechst 33258 on tested cell lines are determined by the MTT assay. Cells are seeded in 96 micro well flat bottom plates at a concentration of 2×104 cells/mL and left overnight in the CO2 incubator allowing them to attach to the plate surface. Growing medium is replaced with compound supplemented or control medium and incubated for 72 h. Fresh medium with 5 mg/mL of MTT is added onto cells and incubated for 4 h at 37°C. Upon media removal, water insoluble MTT-formazan crystals formed inside the living cells are dissolved in DMSO and the absorbance at 570 nm proportional to the number of living cells is measured on an Elisa Microplate Reader. All experiments are performed at least three times in triplicates.The GI50 value, defined as the compound concentration (μM) leading to cellular growth inhibition by 50%, is calculated and used as a parameter to compare cytotoxicity among the compounds[2].
MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.
Purity & Documentation
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Data Sheet (279 KB)
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SDS (394 KB)
- English - EN (394 KB)
- Français - FR (394 KB)
- Deutsch - DE (394 KB)
- Norwegian - NO (394 KB)
- Español - ES (394 KB)
- Swedish - SV (394 KB)
- Italian - IT (394 KB)
- Korean - KR (394 KB)
- Portuguese - PT (394 KB)
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Handling Instructions (2659 KB)
References
[1]. Wang XJ, et al. Newly synthesized bis-benzimidazole derivatives exerting anti-tumor activity through inductionof apoptosis and autophagy. Bioorg Med Chem Lett. 2012 Oct 1;22(19):6297-300. [Content Brief]
[2]. Stolić I, et al. Synthesis, DNA/RNA affinity and antitumour activity of new aromatic diamidines linked by 3,4-ethylenedioxythiophene. Eur J Med Chem. 2011 Feb;46(2):743-55. [Content Brief]
Complete Stock Solution Preparation Table
Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month (sealed storage, away from moisture and light). When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.
| Optional Solvent | Concentration Solvent Mass | 1 mg | 5 mg | 10 mg | 25 mg |
|---|---|---|---|---|---|
| H2O / DMSO | 1 mM | 1.8731 mL | 9.3654 mL | 18.7308 mL | 46.8270 mL |
| 5 mM | 0.3746 mL | 1.8731 mL | 3.7462 mL | 9.3654 mL | |
| 10 mM | 0.1873 mL | 0.9365 mL | 1.8731 mL | 4.6827 mL | |
| 15 mM | 0.1249 mL | 0.6244 mL | 1.2487 mL | 3.1218 mL | |
| 20 mM | 0.0937 mL | 0.4683 mL | 0.9365 mL | 2.3414 mL | |
| DMSO | 25 mM | 0.0749 mL | 0.3746 mL | 0.7492 mL | 1.8731 mL |
| 30 mM | 0.0624 mL | 0.3122 mL | 0.6244 mL | 1.5609 mL | |
| 40 mM | 0.0468 mL | 0.2341 mL | 0.4683 mL | 1.1707 mL | |
| 50 mM | 0.0375 mL | 0.1873 mL | 0.3746 mL | 0.9365 mL | |
| 60 mM | 0.0312 mL | 0.1561 mL | 0.3122 mL | 0.7805 mL | |
| 80 mM | 0.0234 mL | 0.1171 mL | 0.2341 mL | 0.5853 mL | |
| 100 mM | 0.0187 mL | 0.0937 mL | 0.1873 mL | 0.4683 mL |
* Note: If you choose water as the stock solution, please dilute it to the working solution, then filter and sterilize it with a 0.22 μm filter before use.
Hoechst 33258 trihydrochloride Related Classifications
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