Hoechst 33342

Hoechst 33342 is a cell membrane-permeant, fluorescent DNA stain with robust live-cell imaging compatibility, ideal for flow cytometry, side population assays, apoptosis detection, and more.

Hoechst 33342 is 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

Why This Matters
Whether you need robust cell cycle analysis, reliable apoptosis detection, or advanced multi-channel imaging, Hoechst 33342 is the go-to nuclear stain—providing an established standard for both routine staining and cutting-edge research.

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

1. Enhanced Membrane Permeability vs. Hoechst 33258 and DAPI
2. Bright, Stable Fluorescence in the blue emission channel (~461 nm)
3. High Compatibility with standard immunofluorescence panels
4. Reduced Photobleaching relative to older UV-excited dyes
5. 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

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.

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.

1. What does Hoechst 33342 stain for?
   Hoechst 33342 targets nuclear DNA, binding strongly in A–T–rich minor grooves.
2. 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.
3. Does Hoechst 33342 show cell death?
   While not a classic viability dye, it does reveal nuclear fragmentation (e.g., in apoptotic cells).
4. Can I stain live cells with Hoechst 33342?
   High membrane permeability makes Hoechst 33342 suited for live-cell applications.
5. 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).
6. 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.
7. 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.
8. Which is better, Hoechst 33342 or 33258?
   Hoechst 33342 is often preferred for live cells; Hoechst 33258 typically suits fixed-cell setups.
9. How do I store Hoechst 33342 stock solution?
   Keep at ≤−15 °C, shielded from light to maintain stability.
10. Does DAPI work for live cells?
    DAPI is generally not recommended for live cells due to lower permeability and higher toxicity.
11. 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.