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Viable Hoechst 33342 / 33258 Stain Introduction The Hoechst stains 33342 and 33258 (closely related bis-benzimides) are fluorescent stains for labelling DNA in fluorescence microscopy or flow cytometry. Because these fluorescent stains label DNA, they are also commonly used to visualize nuclei and mitochondria. 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 an alternative to 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. Because the Hoechst stains bind to DNA, they can disrupt DNA replication during cell division. Consequently they are potentially mutagenic and carcinogenic. Care should be taken in their handling and disposal. The following outlines the procedure for staining and analysis of cells using Hoechst 33342 (H342). Modifications may be required depending on the application and with variable response of individual cell types. Staining procedure Hoechst 33342 is a "vital" DNA stain that binds preferentially to A-T base-pairs. The cells require no permeabilization for labeling, but do require physiologic conditions since the dye internalization is an active transport process. This condition typically varies among cell types, but the following procedure will give good results in most cases. 1) Stock H342 solution: H342 should be originally suspended in dH2O at a 1mM concentration (H342 will precipitate in PBS). For a 200 ml solution combine: • 0.112 g Hoechst 33342* • 200 ml of dH20 • 2 ml 95% ethanol Mix thoroughly. Cover the bottle with aluminum foil to protect from the light. Store at 4°C. 2) Cell suspension: This procedure is very sensitive to cell concentration and pH of the media. 3) Cells should be approximately 1-2x106/ml, in buffered media, pH 7.2. It is also helpful to include 2% fetal calf serum/BSA to maintain the cells. 4) Add H342 dye from the stock solution to cell suspension to a final concentration of 10 µM. 5) Cells are then incubated at 37°C for 1 h. Time is a critical factor due to the transport of the dye. Typically, 30 minutes is a minimum, but it is important to remember that the signal may begin to degrade after ~120 minutes. It is recommended that the staining kinetics be empirically defined. 6) Analyze by flow cytometry immediately after incubation. Washing is not recommended (see step 4). H342 stained cells will be illuminated with an argon laser tuned for UV (353-365nm) and resulting fluorescence will be detected at 480nM. H342 has a broad fluorescence range, allowing for co-staining with non-UV excitable fluorophores and correlated analysis of other extrinsic cellular characteristics. Hoechst 33342 will also work with PFA or EtOH-fixed cells, with modification to the above protocol, usually calling for a reduction of Hoechst 33342 concentration (to ~1μM). Dye incubation time is drastically reduced (to ~15 minutes) since the cells are permeabilized while fixing.
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