Hoechst 33342 is a popular cell-permeant nuclear counterstain that emits blue fluorescence when bound to DNA. With NucBlue® Live ReadyProbes™ Reagent we have formulated this classic stain in a room temperature-stable solution that is provided in a convenient-to-use dropper bottle. Just tip and drip two drops per ml to stain your cells.

Features of NucBlue® Live ReadyProbes™ Reagent:

• Ready-to-use liquid formulation of high-purity Hoechst 33342
• No need to dilute, weigh, or pipette
• Convenient dropper bottle—just use two drops per ml
• Stable at room temperature—keep handy at your scope or cell culture area

The Classic Blue Nuclear Counterstain for Cellular Imaging
Hoechst 33342 is perhaps the most ubiquitous nuclear fluorescence stain for live and fixed cells and tissue sections. The cell permeable stain binds preferentially to AT regions of DNA. It is excited by UV light and emits blue fluorescence at 460 nm when bound to DNA (Figure 1).

Packaged for Convenience
Typically Hoechst 33342 is supplied as a solid or highly concentrated solution that requires weighing out and diluting several thousand-fold before use. The recommended storage is generally in a freezer or refrigerator. The NucBlue® Live Cell Stain formulation of this imaging workhorse reagent is ready to use and can be stored right next to your microscope or workstation. A liquid formulation in an ultra-convenient dropper bottle allows you to counter-stain your cells whenever and wherever they are ready. Just add two drops per ml for optimally bright and distinct nuclear counter staining.

Multiple Applications
The spectral properties of Hoechst 33342 (2'-[4-ethoxyphenyl]-5-[4-methyl-1-piperazinyl]-2,5'-bi-1H-benzimidazole), including a large Stokes shift, make it ideal for use with green (Alexa Fluor® 488, FITC, GFP) and red (Alexa Fluor® 594, Texas Red®, rhodamine, mCherry, mKate-2) fluorophores in multicolor experiments. Because of its high affinity to DNA, Hoechst 33342 is also frequently used in cell counting, cell cycle, and cell replication studies to distinguish condensed nuclei in apoptotic cells, for cell-cycle studies in combination with Click-iT® EdU or BrdU staining, as a nuclear segmentation tool in high content imaging analysis, and to sort cells based on their DNA content.

Suggestions for Use

• NucBlue® Live Cell Stain may be added directly to cells in full media, or buffer solutions.
• In most cases 2 drops/ml and an incubation of 15 to 30 minutes will give bright nuclear staining; however, optimization may be needed for some cell types, conditions, and applications. In such cases simply add more, or fewer, drops until the optimal staining intensity is obtained. In most cases, staining intensity increases with time if cells are not washed prior to imaging.
• NucBlue® Live Cell Stain is excited by UV light at 360 nm when bound to DNA, with an emission maximum at 460 nm. It is detected through a blue/cyan filter, such as a DAPI filter, blue GFP filters, or the Semrock BrightLine® Alexa Fluor® 350 Dye filter set.

See other Molecular Probes® imaging tools and reagents.