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Protocol: Staining Cells with Hoechst or DAPI Nuclear Stains

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Hoechst vs DAPI–What’s the Difference?
How to Stain Live Cells
How to Stain Fixed Cells or Tissue Sections
How to Stain Bacteria or Yeast
DAPI and Hoechst Technical Information
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Hoechst vs DAPI–What’s the Difference?

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.

Are DAPI stains and Hoechst stains the only DNA dyes? No, there are many types of DNA dyes like green, red, and even far-red, that are used depending on the specifics of what you’re trying to stain. DAPI and Hoechst are widely used in many generic products and that’s why they are more common compared to other dyes. Learn more about the other cellular stains available to you.

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).

DAPI, Hoechst, and Photoconversion

In comparing Hoechst staining to DAPI staining, there are a number of important things you should be aware of. Especially when it comes to photoconversion. DAPI photoconversion has many more problems compared to Hoeschst photoconversion. For example, did you know photoconversion of DAPI can cause nuclear stains to fluoresce?

Here’s our general protocol for IF staining of cells for Microscopy: Protocol: Immunofluorescence Staining of Cells for Microscopy

Learn more tech tips just like this on our Tech Tips page.


Hoechst and DAPI Staining Protocols:

How to Stain Live Cells

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.

Some of the other dyes we offer for live cell staining are RedDot™ dyes. The RedDot™ dyes are used to dye the nuclei of live cells, and they come in RedDot™ 1 and RedDot™ 2 options. They are great for short-term imaging and being thermostable and photostable.

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.

How to Stain 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.

How to Stain 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.


DAPI and Hoechst Technical Information

DAPI (4′,6-Diamidino-2-Phenylindole) Staining Protocol

  • 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, dihydrochloride

Hoechst 33258 Staining Protocol

  • 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 Staining Protocol

  • 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

Interested in Ordering?

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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