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

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.