Hoechst 33342 20 mM solution in water

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% CO<sub>2</sub> 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.

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Physical properties

Molecular weight	561.93
Solvent	Water

Spectral properties

Excitation (nm)	352
Emission (nm)	454

Storage, safety and handling

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 *Ultrapure Grade*

Overview SDS Protocol

CAS

23491-52-3

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 *20 mM solution in water* 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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Spectrum

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Spectral properties

Excitation (nm)	352
Emission (nm)	454

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. Chemical structure for Hoechst 33342 *20 mM solution in water*

Citations

View all 14 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

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)

Aptamerized silica/gold nanocapsules for stimulated release of doxorubicin through remote two-photon excitation
Authors: Tew, Lih Shin and Lee, Tsung-Hsi and Lo, Leu-Wei and Khung, Yit Lung and Chen, Nai-Tzu
Journal: International Journal of Smart and Nano Materials (2022): 79--99

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

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)

Thoroughly remold the localization and signaling pathway of TLR22
Authors: Ji, Jianfei and Liao, Zhiwei and Rao, Youliang and Li, Wenqian and Yang, Chunrong and Yuan, Gailing and Feng, Hao and Xu, Zhen and Shao, Jianzhong and Su, Jianguo
Journal: Frontiers in immunology (2020): 3003

Plant-derived alkaloid sinomenine potentiates glucocorticoid pharmacodynamics in mitogen-activated human peripheral blood mononuclear cells by regulating the translocation of glucocorticoid receptor
Authors: Xu, Wencheng and Wang, Xiaoqin and Tu, Yuanchao and Masaki, Hiroshi and Tanaka, Sachiko and Onda, Kenji and Sugiyama, Kentaro and Yamada, Haruki and Hirano, Toshihiko
Journal: Phytotherapy research (2019): 187--196

Tetrandrine and cepharanthine induce apoptosis through caspase cascade regulation, cell cycle arrest, MAPK activation and PI3K/Akt/mTOR signal modification in glucocorticoid resistant human leukemia Jurkat T cells
Authors: Xu, Wencheng and Wang, Xiaoqin and Tu, Yuanchao and Masaki, Hiroshi and Tanaka, Sachiko and Onda, Kenji and Sugiyama, Kentaro and Yamada, Haruki and Hirano, Toshihiko
Journal: Chemico-Biological Interactions (2019): 108726

Helicobacter pylori cholesterol glucosylation modulates autophagy for increasing intracellular survival in macrophages
Authors: Lai, Chih-Ho and Huang, Ju-Chun and Cheng, Hsin-Hung and Wu, Meng-Chen and Huang, Mei-Zi and Hsu, Hui-Ying and Chen, Yu-An and Hsu, Chung-Yao and Pan, Yi-Jiun and Chu, Yen-Ting and others, undefined
Journal: Cellular microbiology (2018): e12947

DOTA Functionalized Cross-Linked Small-Molecule Micelles for Theranostics Combining Magnetic Resonance Imaging and Chemotherapy
Authors: Feng, Jianyue and Luo, Qiang and Chen, Yun and Li, Bing and Luo, Kui and Lan, Jingbo and Yu, Yunlong and Zhang, Shiyong
Journal: Bioconjugate chemistry (2018)

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