Hoechst® 33342 Protocol for HCA Instruments
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:
- Cells growing in culture
- Hoechst® 33342, trihydrochloride, trihydrate (Cat. No. H1399)
- Phosphate-buffered saline (PBS)
- Fluorescence microscope
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 set | DAPI |
| EVOS® Light Cube | DAPI |
| Storage conditions | 2–6°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 and imaged with the Thermo Scientific™ CellInsight™ High-Content System.
For Research Use Only. Not for use in diagnostic procedures.