Image of Hoechst® 33342 packaging

Nuclear stain for HCA/HCS cell demarcation

Hoechst 33342 nucleic acid stain is a popular cell-permeant nuclear counterstain that emits blue fluorescence when bound to dsDNA. It is also available as a solution.

This protocol can be used for:

  • Nuclear demarcation in high-content analysis/screening (HCA/HCS)

This protocol should not be used for:

  • Flow cytometry

You will need the following for this protocol:


Preparing Hoechst dye stock solution

1. Prepare the Hoechst dye stock solution by dissolving the contents of one vial (100 mg) in 10 mL of deionized water (diH2O) to create a 10 mg/mL (16.23 mM) solution. Note: Hoechst dye has poor solubility in water, so sonicate as necessary to dissolve. The 10 mg/mL Hoechst stock solution may be stored at 2–6°C for up to 6 months or at ≤–20°C for longer periods.

Labeling cells

1. Culture cells in an appropriate medium and vessel for HCA.
2. Optional: Add a test compound or drug to the cells, and incubate as desired.
3. Prepare the Hoechst staining solution by adding 5 μL Hoechst stock solution to 10 mL PBS.
4. Mix well.
5. Remove the medium.
6. Add 100 μL staining solution to each well.
7. Incubate for 5–15 minutes, protected from light.
8. Optional: You may image directly in the staining solution, if you wish.
9. Remove the staining solution.
10. Wash the cells 2–3 times in PBS.
11. Image the cells.
Spectral information and storage
 Hoechst 33342
Excitation/Emission (nm)350/461
Standard filter setDAPI
Storage conditions2–6°C or ≤–20°C



Protocol tips

  • Hoechst dye is a known mutagen and should be handled with care.
  • Dissolving Hoechst dye in PBS is not recommended, but phosphate-containing buffers may be used with dilute solutions of the dye.
  • Unbound Hoechst dye has a maximum emission in the 510–540 nm range; a green haze may be observed if too much dye is applied.
  • The fluorescence signal from Hoechst dye is quenched by BrdU.

Cells stained with Hoechst® 33342
Cells stained with Hoechst 33342 and imaged with the Thermo Scientific CellInsight High-Content System.