Hoechst 33342

Ultrapure Grade

Hoechst 33342 Ultrapure Grade is a cell-permeable blue fluorescent dye that binds to AT-rich regions in the minor groove of DNA, enabling live cell nuclear staining, cell cycle analysis, and identification of side population stem cells.

Optimal UV Brightness: Maximum fluorescence under UV or near-UV excitation for clear nuclear visualization
Low Background Noise: Minimal cytoplasmic staining ensures clean nuclear segmentation in imaging applications
Live-Cell Compatible: Cell-permeable design allows real-time nuclear morphology and cell cycle studies
Sigma/Invitrogen Equivalent: Industry-standard performance with enhanced purity and batch-to-batch consistency

Hoechst 33342 spectrum. Hoechst 33342 is a fluorescent compound with an excitation peak at 352 nm and an emission peak at 454 nm. Other spectra of interest include: Hoechst 33258, DAPI (4,6-Diamidino-2-phenylindole), and Hoechst 34580. Hoechst 33342 belongs to the following categories: Cell Cycle Assays, Nucleus, Fluorescence Activated Cell Sorting (FACS), Hoechst DNA Stains for Live and Fixed Cells, Immunohistochemistry (IHC), and Nucleic Acid Building Blocks.

Protocol

Catalog	Size	Price	Quantity
17530	100 mg	Price
17533	1 g	Price

Overview

Hoechst 33342 is a cell membrane-permeant, fluorescent DNA stain acclaimed for its strong binding affinity to A–T-rich regions in the minor groove of double-stranded DNA. This dye surpasses many nuclear stains due to its additional ethyl substituent, which boosts membrane permeability—thus seamlessly crossing the plasma membrane of live cells. As a result, Hoechst 33342 is widely utilized in diverse fields such as cell biology, immunology, neuroscience, and high-throughput drug discovery.

At AAT Bioquest, we deliver easy-to-use Hoechst 33342 solutions (equivalent to formulations from Sigma or Invitrogen). Built on decades of fluorescence chemistry expertise, our brand guarantees:

Optimal brightness under UV or near-UV excitation
Low background noise for clean nuclear segmentation
High consistency across batches due to rigorous QC and thorough purification

Essential Properties

Chemical Family & Basic Properties

Chemical Family: Bisbenzimide (minor groove-binding)
Molecular Weight: 561.93 g/mol

Note on Membrane Permeability: Structurally, Hoechst 33342 has an extra ethyl group for high membrane permeability (Kirby et al., 2024). Once bound to DNA, it emits intense blue fluorescence—ideal for a wide range of imaging and flow cytometry applications.

Excitation & Emission Characteristics

Excitation Maximum: ~350–355 nm (UV or near-UV lasers)
Emission Maximum: ~461 nm (bright blue region)

Its spectral overlap with DAPI allows reuse of existing filter sets. Flow cytometers with a 355 nm or 405 nm laser readily detect Hoechst 33342.

Working Concentration & Dosing Calculations
Given its 561.93 g/mol molecular weight, a 20 mM stock translates to ~11.2 mg/mL. Accurate calculations ensure reproducible staining intensities across experiments.

Key Advantages

Enhanced Membrane Permeability vs. Hoechst 33258 and DAPI
Bright, Stable Fluorescence in the blue emission channel (~461 nm)
High Compatibility with standard immunofluorescence panels
Reduced Photobleaching relative to older UV-excited dyes
Versatility in Live or Fixed Cells for both real-time and endpoint assays

Hoechst 33342 vs. DAPI

Hoechst 33342: More lipophilic, suitable for real-time studies, relatively low toxicity
DAPI: More toxic for live cells, typically used for fixed tissues or endpoint analysis

Mechanism of Action: Minor Groove Binding

Hoechst 33342 is a non-intercalating dye that binds in the DNA minor groove—particularly in A–T–rich regions. This selective binding confers:

High Signal-to-Noise in nuclear labeling
Reliable DNA Quantitation based on fluorescence intensity
Minimal Off-Target Background for clear imaging and flow cytometry

Why Fluorescence Increases Upon DNA Binding
In aqueous solution, free Hoechst 33342 exhibits a relatively low fluorescence quantum yield. However, once the dye inserts into the narrow A–T–rich minor groove:

Structural Confinement: The dye’s molecular motion is restricted, reducing non-radiative energy loss.
Water Exclusion: Fewer water molecules surround the fluorophore, minimizing quenching pathways.
Hydrophobic Interactions: The ethyl substituent forms stabilizing contacts with consecutive A–T pairs, further enhancing fluorescence.

Together, these factors can boost Hoechst 33342 fluorescence by up to 20–30×, making it exceptionally bright once bound to DNA (Lakowicz, 2006).

Additional Utility and Specificity

Minor Groove Affinity: Favors A–T–rich sequences, aiding specialized genomic or epigenetic studies (e.g., histone modification mapping).
Low Off-Target Staining: Results in clean nuclear definition, particularly beneficial for high-content or multi-channel assays.
Efflux Studies: Serves as a model substrate to investigate bacterial ABC transporters (e.g., BmrA), illuminating mechanisms of multidrug resistance (Di Cesare et al., 2024).

Researchers leverage these properties for a range of applications—from routine DNA quantification to probing DNA curvature and exploring conformational changes in complex assays.

Core Applications

Live-Cell and Fixed-Cell Nuclear Staining

Live-Cell Versatility: Lipophilicity facilitates membrane penetration, enabling real-time monitoring of cell division and apoptosis—even in 3D spheroids or organoids.
Fixed-Cell Complement: Hoechst 33342 provides a bright, stable nuclear counterstain in immunofluorescence protocols, synergizing well with red and green fluorophores.

Cell Cycle Analysis

Flow Cytometry: Readily distinguishes G0/G1, S, and G2/M phases with minimal impact on cell viability.
Sorting Applications: Can help isolate stem-like or progenitor cells based on dye efflux (Side Population analysis).

Apoptosis and Cell Death Detection

Nuclear Morphology: Condensed or fragmented nuclei become distinctly visible, allowing clear discrimination of apoptotic cells.
Viability Assays: Often combined with Annexin V or Propidium Iodide (PI) to differentiate between live, apoptotic, and necrotic populations.

High-Content Screening (HCS) and Automated Imaging

Scalability: Ideal for automated nuclear segmentation at scale in multi-well plate formats.
Low Background: Produces consistent, high-contrast signals, facilitating reliable readouts in drug screening.

Side Population (SP) and Stem Cell Analysis

ABC Transporter Assays: Cells that actively efflux Hoechst 33342 form the "Side Population," aiding in identifying stem or progenitor cell fractions.
Minimal Interference: Concentrations under 30 nM often minimize cytotoxic effects, enabling longer-term studies (Fuchs et al., 2023; Hu et al., 2024).

Additional Routine Applications

Mycoplasma Detection: Reveals contamination as small, bright fluorescent spots (confirm via PCR).
Super-Resolution & Multiplexing: Emits in the blue range, freeing other channels for additional probes.

Emerging Insights and Specialized Applications

Recent Literature Highlights

Quiescent Cell Detection: Pairing Hoechst 33342 with Pyronin Y isolates rare G0 cells in leukemic co-cultures (Parker et al., 2024).
Refined Phototoxicity Control: Reducing UV laser exposures (e.g., once every 30–60 minutes) limits bleaching and cell damage (Fuchs et al., 2023).
Deep Learning Integration: Automated segmentation algorithms lower the need for additional immunofluorescent labels (Cooper, 2022).

Automated cell imaging [using Hoechst 33342] …include the ability to effectively compare adherent and suspension cell lines, reduced time to perform the assay, and environmental control allowing for long-term imaging studies. — Featherston et al., 2024

Experimental “Pearls”

Concentration-Dependent Cytotoxicity: Recommended 5–30 nM range for extended live imaging (Fuchs et al., 2023).
Light Exposure: Minimize high-intensity UV illumination to reduce photobleaching in multi-day protocols.
Dual-/Triple-Staining with Hoechst 33342: Combine with Annexin V-FITC, PI, or TUNEL to distinguish apoptosis vs. necrosis.
Buffer Considerations: pH 7.2–7.4 with ensures consistent fluorescence (Van den Berg van Saparoea et al., 2006).

Contrary to the prevailing assumption, Hoechst 33342 can be used in real-time imaging protocols for multiple days at sub-toxic concentrations, greatly expanding live-cell assay capacities. — Fuchs et al., 2023

Specialized & Emerging Applications

Mitochondrial & Membrane Studies: Hoechst 33342 can be useful as an indicator in nano-thermometry or lipid-partitioning assays (Spicer, 2021; Cordeiro, 2023).
Real-Time Gene Delivery Tracking: Sequential Hoechst additions for repeated viral transduction quantification (Hu et al., 2024).
Microbial & Fungal Assays: Effective for visualizing protoplasts in pathogens like Phytophthora cinnamomi (Kharel et al., 2024).

Considerations for Specialized Applications

pH and Ion Strength: Generally stable in physiological ranges; extreme pH shifts can reduce binding efficiency (Cordeiro, 2023).
FRET/FLIM Potential: Hoechst 33342 can serve as a donor fluorophore in the blue range for advanced imaging techniques.
Multiphoton Excitation: Near-infrared lasers help reduce phototoxicity in thicker samples (e.g., organoids).
Partial DNA Saturation: Sub-saturating conditions allow ratiometric A–T analysis in certain genomic assays.
High Autofluorescence Cell Lines: Titrate dye carefully; use gating strategies in flow cytometry to exclude debris.
RNA/Mitochondrial DNA Labeling: Under specific conditions, Hoechst 33342 may bind RNA or mtDNA; maintaining low concentrations and short incubations usually prevents off-target staining.
Photobleaching Strategies: Lower UV power and reduce exposure times to preserve signal.
RBC Lysis: RBCs (no nuclei) remain unlabeled; remove or lyse RBCs in mixed populations for cleaner data.
Membrane Transporter Assays: Hoechst 33342 often serves as a model substrate for bacterial ABC transporters (Hampton et al., 2024; Di Cesare et al., 2024), revealing real-time efflux and multidrug resistance profiles.
Drug Resistance Reversal: Specialized membrane-fusing vehicles plus Hoechst 33342 help dissect transporter-inhibition strategies in cancer cells (Vahdati and Lamprecht, 2024).

By leveraging these detailed insights on Hoechst 33342—from concentration handling to advanced imaging approaches—researchers can optimize assays across a spectrum of cell biology, microbiology, and therapeutic studies.

Frequently Asked Questions

View All FAQ's

What does Hoechst 33342 stain for?
Hoechst 33342 targets nuclear DNA, binding strongly in A–T–rich minor grooves.
Difference between Hoechst 33342 and DAPI?
Hoechst 33342 is more lipophilic, making it ideal for live-cell imaging, whereas DAPI is typically used on fixed or permeabilized cells.
Does Hoechst 33342 show cell death?
While not a classic viability dye, it does reveal nuclear fragmentation (e.g., in apoptotic cells).
Can I stain live cells with Hoechst 33342?
High membrane permeability makes Hoechst 33342 suited for live-cell applications.
What about cytotoxicity?
Hoechst 33342 can be cytotoxic at higher concentrations or extended exposures. We recommend sub-30 nM for multi-day imaging (Fuchs et al., 2023).
How long do Hoechst 33342-stained samples last?
Stained cells can retain fluorescence for days to weeks if kept protected from light at low temperatures.
Does Hoechst 33342 bind RNA or mitochondrial DNA?
Primarily binds double-stranded DNA. Incidental labeling of RNA or mitochondrial DNA (mtDNA) is minimal at standard concentrations.
Which is better, Hoechst 33342 or 33258?
Hoechst 33342 is often preferred for live cells; Hoechst 33258 typically suits fixed-cell setups.
How do I store Hoechst 33342 stock solution?
Keep at ≤−15 °C, shielded from light to maintain stability.
Does DAPI work for live cells?
DAPI is generally not recommended for live cells due to lower permeability and higher toxicity.
Any special concerns for multi-color imaging with Hoechst 33342?
Use a suitable filter set (~350 nm excitation, ~460 nm emission). Plan carefully if using green or red channels to avoid spectral crosstalk.

Further Reading

View All Citations

Arvidsson, M., et al. “An Annotated High-Content Fluorescence Microscopy Dataset with Hoechst 33342-Stained Nuclei and Manually Labelled Outlines.”; Unpublished Dataset/Study, 2022.
Cooper, J., et al. “Lymphocyte Classification from Hoechst-Stained Slides with Deep Learning.” 2022.
Cordeiro, M. M., et al. “Interaction of Hoechst 33342 with POPC Membranes at Different pH Values.” 2023.
Di Cesare, M., et al. (2024). “The transport activity of the multidrug ABC transporter BmrA does not require a wide separation of the nucleotide-binding domains.” J Biol Chem, vol. 300, no. 1, p. 105546.
Featherston, T., et al. (2024). “Comparing automated cell imaging with conventional methods of measuring cell proliferation and viability.” Toxicol Mech Methods, vol. 34, no. 8, pp. 886–896.
Fuchs, H., et al. “Breaking a Dogma: High-Throughput Live-Cell Imaging in Real-Time with Hoechst 33342.” N.d., 2023. (Additional publication info not provided.)
Gill, M. E., et al. “Isolation of Mouse Germ Cells by FACS Using Hoechst 33342 and SYTO16 Double Staining.” N.d., 2024. (Additional publication info not provided.)
Goodell, M. A., et al. “Stem Cell Identification via Dye Efflux.” 1996, 1997.
Hallap, T., et al. “Triple Fluorochrome Combination for Membrane Stability Testing.” 2006.
Hampton, N., et al. (2024). “Strain-level variations of Dirofilaria immitis microfilariae in two biochemical assays.” PLoS One, vol. 19, no. 7, e0307261.
Hou, Y., et al. “Salidroside Intensifies Mitochondrial Function...” 2023.
Hu, X., et al. (2024). “Long-term in vitro monitoring of AAV-transduction efficiencies in real-time with Hoechst 33342.” PLoS One, vol. 19, no. 3, e0298173.
Kharel, A., et al. (2024). “Viable protoplast isolation, organelle visualization and transformation of the globally distributed plant pathogen Phytophthora cinnamomi.” Protoplasma, vol. 261, no. 5, pp. 1073–1092.
Kirby, J., et al. “The Dynamin Inhibitor...” 2024.
Latt S. A., Wohlleb J. C. “Optical studies of the interaction of 33258 Hoechst with DNA, chromatin, and metaphase chromosomes.” Chromosoma. 1975-11-11; 52(4):297–316. doi: 10.1007/BF00364015. PMID: 1192901.
Li, L., et al. “The DNA Minor-Groove Binding Agents Hoechst 33258 and 33342 Enhance Recombinant Adeno-Associated Virus (rAAV) Transgene Expression.” Journal of Gene Medicine, vol. 7, 2005, p. 420.
Manzini, G., et al. “Nucleic Acids Research.” 1983.
Merolli, A., et al. “Hoechst 33342 as a Marker for Imaging Neurites of Dorsal Root Ganglion in vitro.” N.d., 2022. (Additional journal info not provided.)
Parker, J., et al. (2024). “Protocol for in vitro co-culture, proliferation, and cell-cycle analyses of patient-derived leukemia cells.” STAR Protoc, vol. 5, no. 3, p. 103202.
Rahmé, R. “Assaying Cell-Cycle Status Using Flow Cytometry.” 2021.
Rens, C., et al. “Apoptosis Assessment in High-Content and High-Throughput Screening Assays.” 2021.
Spicer, G., et al. “Harnessing DNA for Nanothermometry.” 2021.
Swain, B. M., et al. “Complexities of a Protonatable Substrate in Measurements of Hoechst 33342 Transport by Multidrug Transporter LmrP.” 2020.
Takaoka, Y., et al. “Hoechst-Tagged Fluorescein Diacetate for the Fluorescence Imaging-Based Assessment of Stomatal Dynamics in Arabidopsis thaliana.” N.d., 2020. (Publication details not provided.)
Vahdati, S., and Lamprecht, A. (2024). “Membrane-Fusing Vehicles for Re-Sensitizing Transporter-Mediated Multiple-Drug Resistance in Cancer.” Pharmaceutics, vol. 16, no. 4, p. 493.
Van den Berg van Saparoea, B., et al. “Proton Motive Force-Dependent Hoechst 33342 Transport by the ABC Transporter LmrA of Lactococcus lactis.” Biochemistry, vol. 44, no. 1, 2006, pp. 1693–1700, https://doi.org/10.1021/bi051497y.
Wang, F., et al. “Effective Detection of Hoechst Side-Population Cells by Flow Cytometry.” Journal of Visualized Experiments (JoVE), no. 210, 2024, e67012.
Zhan, F., et al. “Minocycline Alleviates LPS-Induced Cognitive Dysfunction in Mice by Inhibiting the NLRP3/Caspase-1 Pathway.” Aging (Albany NY), vol. 16, no. 3, 2024, pp. 2989–3006.
Zhang, X., and F. L. Kiechle. Annals of Clinical & Laboratory Science, 2006.
Zheng, D., et al. (2024). “High-content image screening to identify chemical modulators for peroxisome and ferroptosis.” Cell Mol Biol Lett, vol. 29, no. 1, p. 26.
Zhu, L., et al. “Schizandrin A Can Inhibit Non-Small Cell Lung Cancer Cell Proliferation...” 2021.

Citations

View all 35 citations: Citation Explorer

Cigarette smoke compromises macrophage innate sensing in response to pneumococcal infection

Authors:

Liao, Wei-Chih and Chou, Chia-Huei and Ho, Mao-Wang and Chen, Jo-Tsen and Chou, Shu-Ling and Huang, Mei-Zi and Bui, Ngoc-Niem and Wu, Hui-Yu and Lee, Chi-Fan and Huang, Wei-Chien and others,

Journal:

Journal of Microbiology, Immunology and Infection (2024)

LGP2 Facilitates Bacterial Escape through Binding Peptidoglycan via EEK Motif and Suppressing NOD2--RIP2 Axis in Cyprinidae and Xenocyprididae Families

Authors:

Liang, Bo and Li, Wenqian and Yang, Chunrong and Su, Jianguo

Journal:

The Journal of Immunology (2024)

TLR7 neo-functionalizes to sense dsRNA and trigger antiviral and antibacterial immunity in non-tetrapod vertebrates

Authors:

Jiang, Rui and Zhu, Wentao and Liao, Zhiwei and Yang, Chunrong and Su, Jianguo

Journal:

iScience (2023): 108315

CHST11-modified chondroitin 4-sulfate as a potential therapeutic target for glioblastoma

Authors:

Lin, You-Cheng and Chu, Yin-Hung and Liao, Wen-Chieh and Chen, Chia-Hua and Hsiao, Wen-Chuan and Ho, Ying-Jui and Yang, Meng-Yin and Liu, Chiung-Hui

Journal:

American Journal of Cancer Research (2023): 2998

Biofilm formation on the surface of monazite and xenotime during bioleaching

Authors:

van Alin, Arya and Corbett, Melissa K and Fathollahzadeh, Homayoun and Tjiam, M Christian and Rickard, William DA and Sun, Xiao and Putnis, Andrew and Eksteen, Jacques and Kaksonen, Anna H and Watkin, Elizabeth

Journal:

Microbial Biotechnology (2023)

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

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

Authors:

Marquis JF, Hardy I, Olivier M.

Journal:

Parasitology (2005): 197

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

Acid-base and electronic structure-dependent properties of Hoechst 33342

Authors:

Aleman C, Namba AM, Casanovas J.

Journal:

J Biomol Struct Dyn (2005): 29

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
CAS	875756-97-1

Instrument settings

Fluorescence microscope
Excitation	350 nm
Emission	461 nm
Recommended plate	Black wall/clear bottom

Documents

Protocol
Safety Data Sheet (Catalog 17530)
Safety Data Sheet (Catalog 17533)
Certificate of Origin
Certificate of Analysis
Brochure: Fluorescence Cell Imaging Probes & Kits
Brochure: Fluorescence Microscopy: Imaging Organelles
Brochure: Nucleic Acid Detection Probes & Assay Kits

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Page updated on November 4, 2025