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Hoechst 33342 *Ultrapure Grade*

HeLa cells were incubated in 1X HBSS buffer with 5% serum to induce starvation. Following starvation, cells were treated with Autophagy Green&trade; (Cat No. 23002) working solution for 20 minutes in a 37&deg;C, 5% CO<sub>2</sub>&nbsp;incubator and then washed 3 times. Nuclei were labeled with Hoechst 33342 (Cat No. 17530). Lysosomes were labeled with LysoBrite&trade; Orange (Cat No. 22657).
HeLa cells were incubated in 1X HBSS buffer with 5% serum to induce starvation. Following starvation, cells were treated with Autophagy Green&trade; (Cat No. 23002) working solution for 20 minutes in a 37&deg;C, 5% CO<sub>2</sub>&nbsp;incubator and then washed 3 times. Nuclei were labeled with Hoechst 33342 (Cat No. 17530). Lysosomes were labeled with LysoBrite&trade; Orange (Cat No. 22657).
HeLa cells were incubated in 1X HBSS buffer with 5% serum to induce starvation. Following starvation, cells were treated with Autophagy Green&trade; (Cat No. 23002) working solution for 20 minutes in a 37&deg;C, 5% CO<sub>2</sub>&nbsp;incubator and then washed 3 times. Nuclei were labeled with Hoechst 33342 (Cat No. 17530). Lysosomes were labeled with LysoBrite&trade; Orange (Cat No. 22657).
HeLa cells were seeded in 96-well microplates and incubated at 37 °C, 5% CO<sub>2</sub> 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.
<strong>Workflow for the &ldquo;three-in-one&rdquo; cell death screening assay.&nbsp;</strong>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:&nbsp;<strong>An effective &ldquo;three-in-one&rdquo; screening assay for testing drug and nanoparticle toxicity in human endothelial cells</strong> by Marcela Filipova et al., <em>PLOS</em>, Oct. 2018.
<strong>Time-response toxicity of different NPs towards HUVECs as measured by CDS assay.&nbsp;</strong>The HUVECs in the 96-well plate were treated with 100 &mu;g/ml of SPION, SiNP and CNTCOOH NPs for 0&ndash;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 &plusmn; SEM as tested by one-way ANOVA followed by Dunnett&rsquo;s test. ***P&lt;0.001, **P&lt;0.01, and *P&lt;0.05, versus the time point 0 hours.&nbsp;Source:&nbsp;<strong>An effective &ldquo;three-in-one&rdquo; screening assay for testing drug and nanoparticle toxicity in human endothelial cells</strong>&nbsp;by Marcela Filipova et al.,&nbsp;<em>PLOS</em>, Oct. 2018.
<strong>Evaluation of CDS assay performance.</strong><br />The HUVECs in the 96-well plate were treated with different concentrations of camptothecin, staurosporine or H<sub>2</sub>O<sub>2</sub> for 24 hours (A&ndash;D), or with 5 &mu;M camptothecin, 100 nM staurosporine or 2 mM H<sub>2</sub>O<sub>2</sub> for 0, 3, 6, 12 and 24 hours (E&ndash;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 &plusmn; SEM. Repeated measures were statistically tested by one-way ANOVA followed by Dunnett&rsquo;s post-test. ***P&lt;0.001, **P&lt;0.01, and *P&lt;0.05, versus the negative control.&nbsp;Source:&nbsp;<strong>An effective &ldquo;three-in-one&rdquo; screening assay for testing drug and nanoparticle toxicity in human endothelial cells</strong>&nbsp;by Marcela Filipova et al.,&nbsp;<em>PLOS</em>, Oct. 2018.
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Physical properties
Molecular weight561.93
SolventWater
Spectral properties
Excitation (nm)352
Emission (nm)454
Storage, safety and handling
Certificate of OriginDownload PDF
H-phraseH303, H313, H340
Hazard symbolT
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R68
StorageFreeze (< -15 °C); Minimize light exposure
UNSPSC41116134
Alternative formats
Hoechst 33342 *20 mM solution in water*

OverviewpdfSDSpdfProtocol


See also: Cell Cycle Assays, Nucleus, DNA Methylation, Fluorescence Activated Cell Sorting (FACS), Hoechst DNA Stains for Live and Fixed Cells, Immunohistochemistry (IHC)
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

Excitation350 nm
Emission461 nm
Recommended plateBlack 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 mg0.5 mg1 mg5 mg10 mg
1 mM177.958 µL889.791 µL1.78 mL8.898 mL17.796 mL
5 mM35.592 µL177.958 µL355.916 µL1.78 mL3.559 mL
10 mM17.796 µL88.979 µL177.958 µL889.791 µL1.78 mL

Molarity calculator

Enter any two values (mass, volume, concentration) to calculate the third.

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles
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Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Excitation (nm)352
Emission (nm)454

Product Family


NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yield
Hoechst 33258 *CAS 23491-45-4*3524544600010.03401
Hoechst 33258 *20 mM solution in water*3524544600010.03401
Hoechst 34580 *CAS 911004-45-0*371438--
Hoechst 34580 *20 mM solution in water*371438--

Images


HeLa cells were incubated in 1X HBSS buffer with 5% serum to induce starvation. Following starvation, cells were treated with Autophagy Green&trade; (Cat No. 23002) working solution for 20 minutes in a 37&deg;C, 5% CO<sub>2</sub>&nbsp;incubator and then washed 3 times. Nuclei were labeled with Hoechst 33342 (Cat No. 17530). Lysosomes were labeled with LysoBrite&trade; Orange (Cat No. 22657).
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).
HeLa cells were seeded in 96-well microplates and incubated at 37 °C, 5% CO<sub>2</sub> 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 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.
<strong>Workflow for the &ldquo;three-in-one&rdquo; cell death screening assay.&nbsp;</strong>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:&nbsp;<strong>An effective &ldquo;three-in-one&rdquo; screening assay for testing drug and nanoparticle toxicity in human endothelial cells</strong> by Marcela Filipova et al., <em>PLOS</em>, Oct. 2018.
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.
<strong>Time-response toxicity of different NPs towards HUVECs as measured by CDS assay.&nbsp;</strong>The HUVECs in the 96-well plate were treated with 100 &mu;g/ml of SPION, SiNP and CNTCOOH NPs for 0&ndash;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 &plusmn; SEM as tested by one-way ANOVA followed by Dunnett&rsquo;s test. ***P&lt;0.001, **P&lt;0.01, and *P&lt;0.05, versus the time point 0 hours.&nbsp;Source:&nbsp;<strong>An effective &ldquo;three-in-one&rdquo; screening assay for testing drug and nanoparticle toxicity in human endothelial cells</strong>&nbsp;by Marcela Filipova et al.,&nbsp;<em>PLOS</em>, 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.
<strong>Evaluation of CDS assay performance.</strong><br />The HUVECs in the 96-well plate were treated with different concentrations of camptothecin, staurosporine or H<sub>2</sub>O<sub>2</sub> for 24 hours (A&ndash;D), or with 5 &mu;M camptothecin, 100 nM staurosporine or 2 mM H<sub>2</sub>O<sub>2</sub> for 0, 3, 6, 12 and 24 hours (E&ndash;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 &plusmn; SEM. Repeated measures were statistically tested by one-way ANOVA followed by Dunnett&rsquo;s post-test. ***P&lt;0.001, **P&lt;0.01, and *P&lt;0.05, versus the negative control.&nbsp;Source:&nbsp;<strong>An effective &ldquo;three-in-one&rdquo; screening assay for testing drug and nanoparticle toxicity in human endothelial cells</strong>&nbsp;by Marcela Filipova et al.,&nbsp;<em>PLOS</em>, 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.
Gallery Image 6
Figure 6. Chemical structure for Hoechst 33342 *Ultrapure Grade* *CAS 23491-52-3*
<strong>Dose-dependent toxicity of diverse NPs towards HUVECs as measured by CDS assay.&nbsp;</strong>The HUVECs in the 96-well plate were treated with 0&ndash;100 &mu;g/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 &plusmn; SEM as tested by one-way ANOVA followed by Dunnett&rsquo;s test. ***P&lt;0.001, **P&lt;0.01, and *P&lt;0.05, versus the negative control specific for each measurement.&nbsp;Source:<strong>&nbsp;An effective &ldquo;three-in-one&rdquo; screening assay for testing drug and nanoparticle toxicity in human endothelial cells&nbsp;</strong>by Marcela Filipova et al.,&nbsp;<em>PLOS</em>, Oct. 2018<strong>.</strong>
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 33 citations: Citation Explorer
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Journal: Microbial Biotechnology (2023)
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Journal: American Journal of Cancer Research (2023): 2998
TLR7 neo-functionalizes to sense dsRNA and trigger antiviral and antibacterial immunity in non-tetrapod vertebrates
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Journal: iScience (2023): 108315
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
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
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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
Authors: Nia, Marzieh Heidari and Ashkar, Said and Munguia-Lopez, Jose G and Kinsella, Joseph M and van de Ven, Theo GM
Journal: (2021)
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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,
Journal: (2021)
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
Microencapsulated Multi-functionalized Graphene Oxide Equipped With Chloroquine for Efficient and Sustained Sirna Delivery
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Journal: (2021)

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
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
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
Resistance mechanism development to the topoisomerase-I inhibitor Hoechst 33342 by Leishmania donovani
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
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
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
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
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
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

Documents


Certificate of origin
Safety data sheet
Product protocol
Certificate of analysis

Application notes


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FAQ


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