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
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Labeling cells
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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.