Hoechst 33342 *Ultrapure Grade*
Ordering information
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Additional ordering information
Telephone | 1-800-990-8053 |
Fax | 1-800-609-2943 |
sales@aatbio.com | |
Quotation | Request |
International | See distributors |
Shipping | Standard overnight for United States, inquire for international |
Physical properties
Molecular weight | 561.93 |
Solvent | Water |
Spectral properties
Excitation (nm) | 352 |
Emission (nm) | 454 |
Storage, safety and handling
Certificate of Origin | Download PDF |
H-phrase | H303, H313, H340 |
Hazard symbol | T |
Intended use | Research Use Only (RUO) |
R-phrase | R20, R21, R68 |
Storage | Freeze (< -15 °C); Minimize light exposure |
UNSPSC | 41116134 |
Alternative formats
Hoechst 33342 *20 mM solution in water* |
Overview | ![]() ![]() |
See also: Antibodies and Proteomics, Antibody and Protein Labeling, Bioconjugation, Cell Cycle Assays, Cell Structures and Organelles, Nucleus, Cellular Processes, DNA and RNA Quantitation, DNA Methylation, Flow Cytometry Reagents, Fluorescence Activated Cell Sorting (FACS), Hoechst DNA Stains for Live and Fixed Cells, Immunohistochemistry (IHC), Physiological Probes
CAS 875756-97-1 | Molecular weight 561.93 | Excitation (nm) 352 | Emission (nm) 454 |
The Hoechst stains are a family of fluorescent stains for labeling DNA in fluorescence microscopy. Because these fluorescent stains label DNA, they are also commonly used to visualize nuclei and mitochondria. Two of these closely related bis-benzimides are commonly used: Hoechst 33258 and Hoechst 33342. Both dyes are excited by ultraviolet light at around 350 nm, and both emit blue/cyan fluorescence light around an emission maximum at 461 nm. The Hoechst stains may be used on live or fixed cells, and are often used as a substitute for another nucleic acid stain, DAPI. The key difference between them is that the additional ethyl group of Hoechst 33342 renders it more lipophilic, and thus more able to cross intact cell membranes. In some applications, Hoechst 33258 is significantly less permeant. These dyes can also be used to detect the contents of a sample DNA by plotting a standard emission-to-content curve.
Platform
Fluorescence microscope
Excitation | 350 nm |
Emission | 461 nm |
Recommended plate | Black wall/clear bottom |
Calculators
Common stock solution preparation
Table 1. Volume of Water needed to reconstitute specific mass of Hoechst 33342 *Ultrapure Grade* to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.
0.1 mg | 0.5 mg | 1 mg | 5 mg | 10 mg | |
1 mM | 177.958 µL | 889.791 µL | 1.78 mL | 8.898 mL | 17.796 mL |
5 mM | 35.592 µL | 177.958 µL | 355.916 µL | 1.78 mL | 3.559 mL |
10 mM | 17.796 µL | 88.979 µL | 177.958 µL | 889.791 µL | 1.78 mL |
Molarity calculator
Enter any two values (mass, volume, concentration) to calculate the third.
Mass (Calculate) | Molecular weight | Volume (Calculate) | Concentration (Calculate) | Moles | ||||
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Product Family
Name | Excitation (nm) | Emission (nm) | Extinction coefficient (cm -1 M -1) | Quantum yield |
Hoechst 33258 *CAS 23491-45-4* | 352 | 454 | 460001 | 0.03401 |
Hoechst 33258 *20 mM solution in water* | 352 | 454 | 460001 | 0.03401 |
Hoechst 34580 *CAS 911004-45-0* | 371 | 438 | - | - |
Hoechst 34580 *20 mM solution in water* | 371 | 438 | - | - |
Images

Figure 1. HeLa cells were incubated in 1X HBSS buffer with 5% serum to induce starvation. Following starvation, cells were treated with Autophagy Green™ (Cat No. 23002) working solution for 20 minutes in a 37°C, 5% CO2 incubator and then washed 3 times. Nuclei were labeled with Hoechst 33342 (Cat No. 17530). Lysosomes were labeled with LysoBrite™ Orange (Cat No. 22657).

Figure 2. HeLa cells were seeded in 96-well microplates and incubated at 37 °C, 5% CO2 for 24 hours. Cells were then stained with 5 or 10 µM Hoechst 33342 for 30 minutes at 37 °C, washed, and imaged on a Keyence BZ-X microscope. Afterward, cells were fixed with 4% formaldehyde for 20 minutes at RT, washed and imaged.

Figure 3. Workflow for the “three-in-one” cell death screening assay. HUVECs growing in the 96-well plate for 48 hours are exposed to NPs for 24 hours. Three types of cell death are evaluated simultaneously. A) Cell necrosis is measured spectrophotometrically after mixing an aliquot of cell supernatant with LDH substrate. B) Cell viability is assessed by adding WST-8 substrate to the cells. After three hours of incubation, aliquots of the reaction mixture are transferred into the new plate and measured spectrophotometrically. C) Cell apoptosis is detected after incubating the cells with Hoechst 33342 and fixing them with paraformaldehyde. Images captured under the inverted fluorescence microscope are computationally processed with the specially designed ImageJ macro. Source: An effective “three-in-one” screening assay for testing drug and nanoparticle toxicity in human endothelial cells by Marcela Filipova et al., PLOS, Oct. 2018.

Figure 4. Time-response toxicity of different NPs towards HUVECs as measured by CDS assay. The HUVECs in the 96-well plate were treated with 100 μg/ml of SPION, SiNP and CNTCOOH NPs for 0–24 h. The cell viability was measured by WST-8 assay (A), the cell necrosis was determined by LDH assay (B), and the number of intact cell nuclei (C) and number of apoptotic bodies (D) were counted by ImageJ software after the cells were stained with Hoechst 33342. Each treatment was performed in 6-plicate and the results (n = 3) are expressed as the means ± SEM as tested by one-way ANOVA followed by Dunnett’s test. ***P<0.001, **P<0.01, and *P<0.05, versus the time point 0 hours. Source: An effective “three-in-one” screening assay for testing drug and nanoparticle toxicity in human endothelial cells by Marcela Filipova et al., PLOS, Oct. 2018.

Figure 5. Evaluation of CDS assay performance.
The HUVECs in the 96-well plate were treated with different concentrations of camptothecin, staurosporine or H2O2 for 24 hours (A–D), or with 5 μM camptothecin, 100 nM staurosporine or 2 mM H2O2 for 0, 3, 6, 12 and 24 hours (E–H). Cell viability was measured by WST-8 assay (A, E), and cell necrosis was evaluated by LDH assay (B, F). A count of the intact cell nuclei (C, G) and apoptotic bodies (D, H) was evaluated by ImageJ software after the cells were stained with Hoechst 33342. The WST-8 data and number of cell nuclei and apoptotic bodies were processed with 3h and 3.5h time difference, respectively. The data represent three independent experiments performed in 6-plicates. The bar graphs show the means ± SEM. Repeated measures were statistically tested by one-way ANOVA followed by Dunnett’s post-test. ***P<0.001, **P<0.01, and *P<0.05, versus the negative control. Source: An effective “three-in-one” screening assay for testing drug and nanoparticle toxicity in human endothelial cells by Marcela Filipova et al., PLOS, Oct. 2018.
The HUVECs in the 96-well plate were treated with different concentrations of camptothecin, staurosporine or H2O2 for 24 hours (A–D), or with 5 μM camptothecin, 100 nM staurosporine or 2 mM H2O2 for 0, 3, 6, 12 and 24 hours (E–H). Cell viability was measured by WST-8 assay (A, E), and cell necrosis was evaluated by LDH assay (B, F). A count of the intact cell nuclei (C, G) and apoptotic bodies (D, H) was evaluated by ImageJ software after the cells were stained with Hoechst 33342. The WST-8 data and number of cell nuclei and apoptotic bodies were processed with 3h and 3.5h time difference, respectively. The data represent three independent experiments performed in 6-plicates. The bar graphs show the means ± SEM. Repeated measures were statistically tested by one-way ANOVA followed by Dunnett’s post-test. ***P<0.001, **P<0.01, and *P<0.05, versus the negative control. Source: An effective “three-in-one” screening assay for testing drug and nanoparticle toxicity in human endothelial cells by Marcela Filipova et al., PLOS, Oct. 2018.

Figure 7. Dose-dependent toxicity of diverse NPs towards HUVECs as measured by CDS assay. The HUVECs in the 96-well plate were treated with 0–100 μg/ml of SiNP, SPION or CNTCOOH NPs for 24 hours. The cell viability was measured by WST-8 assay (A), the cell necrosis was gauged by LDH assay (B), and the number of intact cell nuclei (C) and number of apoptotic bodies (D) were counted by ImageJ software after staining the cells with Hoechst 33342. Each measurement was performed in 6-plicate, and the results (n = 3) are expressed as the means ± SEM as tested by one-way ANOVA followed by Dunnett’s test. ***P<0.001, **P<0.01, and *P<0.05, versus the negative control specific for each measurement. Source: An effective “three-in-one” screening assay for testing drug and nanoparticle toxicity in human endothelial cells by Marcela Filipova et al., PLOS, Oct. 2018.
Citations
View all 30 citations: Citation Explorer
Repair Kinetics of DSB-Foci Induced by Proton and $\alpha$-Particle Microbeams of Different Energies
Authors: Belchior, Ana and Canhoto, Jo{\~a}o F and Giesen, Ulrich and Langner, Frank and Rabus, Hans and Schulte, Reinhard
Journal: Life (2022): 2040
Authors: Belchior, Ana and Canhoto, Jo{\~a}o F and Giesen, Ulrich and Langner, Frank and Rabus, Hans and Schulte, Reinhard
Journal: Life (2022): 2040
Repair kinetics of DSB-foci induced by proton and helium ion microbeams of different energies
Authors: Belchior, Ana and Canhoto, Jo{\~a}o F and Giesen, Ulrich and Langner, Frank and Rabus, Hans and Schulte, Reinhard
Journal: arXiv preprint arXiv:2206.08981 (2022)
Authors: Belchior, Ana and Canhoto, Jo{\~a}o F and Giesen, Ulrich and Langner, Frank and Rabus, Hans and Schulte, Reinhard
Journal: arXiv preprint arXiv:2206.08981 (2022)
NAMPT Inhibitor and P73 Activator Represses P53 R175H Mutated HNSCC Cell Proliferation in a Synergistic Manner
Authors: Cai, Bi-He and Bai, Zhi-Yu and Lien, Ching-Feng and Yu, Si-Jie and Lu, Rui-Yu and Wu, Ming-Han and Wu, Wei-Chen and Chen, Chia-Chi and Hsu, Yi-Chiang
Journal: Biomolecules (2022): 438
Authors: Cai, Bi-He and Bai, Zhi-Yu and Lien, Ching-Feng and Yu, Si-Jie and Lu, Rui-Yu and Wu, Ming-Han and Wu, Wei-Chen and Chen, Chia-Chi and Hsu, Yi-Chiang
Journal: Biomolecules (2022): 438
pH-responsive biopolymer-based supramolecular architectures as biodegradable carriers for 1 DOX delivery 2
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Journal: (2021)
Authors: Bkiri, Dounia and Semmate, Noha and Boumart, Zineb and Safini, Najete and Fakri, Fatima Zohra and Bamouh, Zahra and Tadlaoui, Khalid Omari and Fellahi, Siham and Tligui, Noursaid and Fihri, Ouafaa Fassi and others,
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Stimulation of $\alpha$7-nAChRs coordinates autophagy and apoptosis signaling in experimental knee osteoarthritis
Authors: Liu, Yuan and Xu, Shi and Zhang, Haijun and Qian, Kaoliang and Huang, Jiachen and Gu, Xianger and Li, Yan and Fan, Yi and Hu, Jun
Journal: Cell Death \& Disease (2021): 1--12
Authors: Liu, Yuan and Xu, Shi and Zhang, Haijun and Qian, Kaoliang and Huang, Jiachen and Gu, Xianger and Li, Yan and Fan, Yi and Hu, Jun
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Microencapsulated Multi-functionalized Graphene Oxide Equipped With Chloroquine for Efficient and Sustained Sirna Delivery
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Involvement of cancer-derived EMT cells in the accumulation of 18 F-fluorodeoxyglucose in the hypoxic cancer microenvironment
Authors: Sugita, Sachi and Yamato, Masanori and Hatabu, Toshimitsu and Kataoka, Yosky
Journal: Scientific reports (2021): 1--11
Authors: Sugita, Sachi and Yamato, Masanori and Hatabu, Toshimitsu and Kataoka, Yosky
Journal: Scientific reports (2021): 1--11
Studium bun{\v{e}}{\v{c}}n{\'e} toxicity vybran{\`y}ch nano{\v{c}}{\'a}stic v tk{\'a}{\v{n}}ov{\`y}ch kultur{\'a}ch.
Authors: Filipov{\'a}, Marcela
Journal: (2020)
Authors: Filipov{\'a}, Marcela
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Development of in vitro models to investigate the role of decidualisation, PDGF, and ho-1 on stromal--trophoblast interaction
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References
View all 42 references: Citation Explorer
Usefulness of a triple fluorochrome combination Merocyanine 540/Yo-Pro 1/Hoechst 33342 in assessing membrane stability of viable frozen-thawed spermatozoa from Estonian Holstein AI bulls
Authors: Hallap T, Nagy S, Jaakma U, Johannisson A, Rodriguez-Martinez H.
Journal: Theriogenology (2006): 1122
Authors: Hallap T, Nagy S, Jaakma U, Johannisson A, Rodriguez-Martinez H.
Journal: Theriogenology (2006): 1122
Fatty acid synthase and its mRNA concentrations are decreased at different times following Hoechst 33342-induced apoptosis in BC3H-1 myocytes
Authors: Zhang X, Kiechle FL.
Journal: Ann Clin Lab Sci (2006): 185
Authors: Zhang X, Kiechle FL.
Journal: Ann Clin Lab Sci (2006): 185
The DNA minor groove binding agents Hoechst 33258 and 33342 enhance recombinant adeno-associated virus (rAAV) transgene expression
Authors: Li L, Yang L, Kotin RM.
Journal: J Gene Med (2005): 420
Authors: Li L, Yang L, Kotin RM.
Journal: J Gene Med (2005): 420
Resistance mechanism development to the topoisomerase-I inhibitor Hoechst 33342 by Leishmania donovani
Authors: Marquis JF, Hardy I, Olivier M.
Journal: Parasitology (2005): 197
Authors: Marquis JF, Hardy I, Olivier M.
Journal: Parasitology (2005): 197
Acid-base and electronic structure-dependent properties of Hoechst 33342
Authors: Aleman C, Namba AM, Casanovas J.
Journal: J Biomol Struct Dyn (2005): 29
Authors: Aleman C, Namba AM, Casanovas J.
Journal: J Biomol Struct Dyn (2005): 29
Single UV excitation of Hoechst 33342 and propidium iodide for viability assessment of rhesus monkey spermatozoa using flow cytometry
Authors: Cai K, Yang J, Guan M, Ji W, Li Y, Rens W.
Journal: Arch Androl (2005): 371
Authors: Cai K, Yang J, Guan M, Ji W, Li Y, Rens W.
Journal: Arch Androl (2005): 371
Evidence for a qualitative hierarchy within the Hoechst-33342 'side population' (SP) of murine bone marrow cells
Authors: Robinson SN, Seina SM, Gohr JC, Kuszynski CA, Sharp JG.
Journal: Bone Marrow Transplant (2005): 807
Authors: Robinson SN, Seina SM, Gohr JC, Kuszynski CA, Sharp JG.
Journal: Bone Marrow Transplant (2005): 807
Flow cytometric characterization of viable meiotic and postmeiotic cells by Hoechst 33342 in mouse spermatogenesis
Authors: Bastos H, Lassalle B, Chicheportiche A, Riou L, Testart J, Allem and I, Fouchet P.
Journal: Cytometry A (2005): 40
Authors: Bastos H, Lassalle B, Chicheportiche A, Riou L, Testart J, Allem and I, Fouchet P.
Journal: Cytometry A (2005): 40
An in vitro study of Hoechst 33342 redistribution and its effects on cell viability
Authors: Mohorko N, Kregar-Velikonja N, Repovs G, Gorensek M, Bresjanac M.
Journal: Hum Exp Toxicol (2005): 573
Authors: Mohorko N, Kregar-Velikonja N, Repovs G, Gorensek M, Bresjanac M.
Journal: Hum Exp Toxicol (2005): 573
Role of topoisomerases in cytotoxicity induced by DNA ligand Hoechst-33342 and UV-C in a glioma cell line
Authors: Singh S, Dwarakanath BS, Lazar Mathew T.
Journal: Indian J Exp Biol (2005): 313
Authors: Singh S, Dwarakanath BS, Lazar Mathew T.
Journal: Indian J Exp Biol (2005): 313
Application notes
FAQ
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