Jump to a section:IntroductionHoechst and DAPI are popular blue fluorescent, nuclear-specific dyes that can be used to stain live or fixed cells. The dyes have minimal fluorescence in solution, but become brightly fluorescent upon binding to DNA. Therefore, they can be used to stain cells without a wash step. The staining is very stable and non-toxic to live cells for several days or longer.DAPI and Hoechst are minor-groove binding dyes; DAPI has higher affinity for A/T-rich regions of DNA than G/C-rich DNA. Hoechst 33342 and Hoechst 33258 are structurally similar dyes that perform comparably as nuclear counterstains.
Hoechst 33528 is slightly more water soluble than Hoechst 33342, but both dyes are highly cell membrane permeant and widely used in cell cycle studies and as nuclear counterstains for live or fixed cells. They are typically used for staining at 1 ug/mL. DAPI is somewhat less cell membrane permeant than Hoechst dyes, and must be used at a higher concentration (usually 10 ug/mL) for live cell staining.
It can be used for fixed cell staining at 1 ug/mL. We offer DAPI dilactate, a more soluble DAPI salt, which is useful for making stock solutions for cell staining, as well as ready-to-use stock solutions of DAPI and Hoechst in water (see ).Hoechst and DAPI are extremely stable in water at 10 mg/mL, and can be stored at 4°C for years as long as they are protected from light. It is not recommended to store working solutions of Hoechst dye, because the dye will be lost to precipitation or adsorption to the container over time. DAPI is stable in dilute solutions, and can be added directly to antifade mounting medium for long-term use; we also offer EverBrite™ Mounting Media with DAPI (see ).Staining ProtocolsLive cell stainingBelow we provide two protocols for staining live cells with DAPI or Hoechst. Staining by medium exchange results in uniform exposure of cells to probe. However, for some cell types, morphology or viability may be affected by medium exchange. In addition, floating dead cells may be lost during medium removal, and suspension cells must be collected by centrifugation to exchange the medium.
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Direct addition of 10X probe is a convenient staining method that doesn’t require medium exchange, but care must be taken to mix immediately yet gently to avoid high transient probe concentration or disruption of cells by pipetting. Note that we do not recommend adding highly concentrated dye directly to cells in culture, as this will result in local areas of high dye exposure.Live cell staining by medium exchange. Add the dye to complete culture medium. Use Hoechst dyes at 1 ug/mL or DAPI at 10 ug/mL.Note: DAPI or Hoechst can be combined with other fluorescent probes. Remove culture medium from the cells and replace with medium containing dye. Incubate cells at room temperature or 37°C for 5-15 minutes, then image.Note: Washing is not necessary for specific staining, but nuclear staining is stable after washing.Live cell staining by direct addition of 10X probe.
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Add the dye to complete culture medium at 10 times the final recommended staining concentration. Dilute Hoechst dyes to 10 ug/mL, or dilute DAPI to 100 ug/mL.Note: DAPI or Hoechst can be combined with other fluorescent probes.
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Without removing the medium from the cells, add 1/10 volume of 10X dye directly to the well. Immediate mix thoroughly by gently pipetting the medium up and down.