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Protocol: Staining Cells with Hoechst or DAPI Nuclear Stains

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Introduction
Protocol for staining live cells
Protocol for staining fixed cells or tissue sections
Staining bacteria or yeast
Dye technical information
Ordering information

Introduction

Hoechst 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 ordering information).

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 ordering information).

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Staining Protocols

Live cell staining

Below 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. 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

  1. 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.
  2. Remove culture medium from the cells and replace with medium containing dye.
  3. 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

  1. 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.
  2. Without removing the medium from the cells, add 1/10 volume of 10X dye directly to the well.
  3. Immediate mix thoroughly by gently pipetting the medium up and down. For larger well sizes (e.g., 24-well to 6-well plates), the plate can be gently swirled to mix.
  4. 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.

Staining of fixed cells or tissue sections

  1. Add the dye to PBS at 1 ug/mL.
    Note: DAPI or Hoechst can be included together with antibodies or other probes; the dyes also can be diluted in buffers with detergent or blocking agents if convenient.
  2. Add the PBS with dye to cells or tissue sections and incubate at room temperature for at least 5 minutes.
  3. Image the samples; washing is optional but not required.
    Note: Samples can be stored at 4°C after staining and before imaging.
    Note: DAPI can be included directly in antifade mounting medium for one-step mounting and staining. When using DAPI in mounting medium, longer incubation times may be required for DAPI to completely penetrate the cell nuclei.

Staining bacteria or yeast
Hoechst and DAPI stain bacteria more dimly than mammalian cells. Live or killed bacteria (gram-negative or gram-positive) can be stained with 12-15 ug/mL Hoechst or DAPI in PBS or 150 mM NaCl for 30 minutes at room temperature. Dead cells tend to stain more brightly than live cells.

In S. cerevisiae, DAPI and Hoechst preferentially stain dead cells with nuclear and cytoplasmic localization. In live yeast, Hoechst shows dim nuclear and cytoplasmic staining, while DAPI shows dim mitochondrial staining. The dyes can be used to stain yeast at 12-15 ug/mL in PBS.

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Dye Technical Information

DAPI (4′,6-Diamidino-2-Phenylindole)

  • λExEm (with DNA) = 358/461 nm
  • MW = 457.49 (in H2O); 350.25 (dihydrochloride salt); 457.49 (dilactate salt)
  • Molecular formula: C16H17Cl2N5
  • CAS number: 28718-90-3
  • Recommended staining concentration: 10 ug/mL (live cells) or 1 ug/mL (fixed cells)
DAPI

 

Hoechst 33258

  • λExEm (with DNA) = 352/461 nm
  • C25H27Cl3N6O
  • MW: 533.88 (in H2O); 623.96 (pentahydrate)
  • CAS number: 23491-45-4
  • Recommended staining concentration: 1 ug/mL

 

Hoechst 33258, pentahydrate

 

Hoechst 33342

  • λExEm (with DNA) = 350/461 nm
  • MW: 561.93 (in H2O); 615.98 (trihydrochloride trihydrate)
  • Molecular formula: C27H31Cl3N6O
  • CAS number: 23491-52-3
  • Recommended staining concentration: 1 ug/mL

 

Hoechst 33342, trihydrochloride trihydrate

 

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Ordering Information

ProductUnit SizeCatalog Number
Hoechst 33258, 10 mg/mL in H2O10 mL40044
Hoechst 33258, pentahydrate100 mg40045
Hoechst 33342, 10 mg/mL in H2O10 mL40046
Hoechst 33342, trihydrochloride trihydrate100 mg40047
DAPI in H2O, 10 mg/mL1 mL40043
DAPI, dilactate10 mg40009
DAPI, dihydrochloride10 mg40011
EverBrite™ Mounting Medium with DAPI10 mL23002
Drop-n-Stain EverBrite™ Mounting Medium with DAPI10 mL23009
EverBrite™ Hardset Mounting Medium with DAPI10 mL23004

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