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. DAPI and Hoechst are minor-groove binding dyes; DAPI has higher affinity for A/T-rich regions of DNA than G/C-rich DNA.
What is Hoechst staining used for?
Hoechst 33342 is used for specifically staining the nuclei of living or fixed cells and tissues. This stain is commonly used in combination with 5-bromo-2'-deoxyuridine (BrdU) labeling to distinguish the compact chromatin of apoptotic nuclei, to identify replicating cells and to sort cells based on their DNA content.
What is DAPI and why is it used?
DAPI (4′,6-diamidino-2-phenylindole) is a blue-fluorescent DNA stain that exhibits ~20-fold enhancement of fluorescence upon binding to AT regions of dsDNA. DAPI is generally used to stain fixed cells since the dye is cell impermeant, although the stain will enter live cells when used at higher concentrations.
Will Hoechst stain dead cells?
Yes, Hoechst 33342 can stain dead cells, however Hoechst 33358 is the preferred dye that's used for staining dead or fixed cells. Because of its ability to bind to DNA, Hoechst dyes can be used to observe nuclear condensation, thereby distinguishing apoptotic cells from healthy cells as well as necrotic cells.May 6, 2021
Why does Hoechst stain dead cells?
The DNA becomes condensed during apoptosis but not during necrosis. Because of its ability to bind to DNA, Hoechst dyes can be used to observe nuclear condensation, thereby distinguishing apoptotic cells from healthy cells as well as necrotic cells.May 6, 2021
What is the difference between Hoechst and DAPI?
Hoechst dyes are often used as substitutes for another nucleic acid stain called DAPI. Key differences between Hoechst dyes and DAPI are: Hoechst dyes are less toxic than DAPI, which ensures a higher viability of stained cells. The additional ethyl group of the Hoechst dyes renders them more cell-permeable.
Does Hoechst stain dead cells?
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.
Can Hoechst stain dead cells?
Since Hoechst can readily cross cell membranes to stain DNA of living and dead cells, it was used to label the total number of cells. In contrast, PI only enters cells with compromised plasma membranes, thus selectively labeling dead cells.
How do you use Hoechst?
- Add the dye to complete culture medium. Use Hoechst dyes at 1 ug/mL or DAPI at 10 ug/mL.
- Remove culture medium from the cells and replace with medium containing dye.
- Incubate cells at room temperature or 37°C for 5-15 minutes, then image.
How does Hoechst staining work?
Hoechst 33342 binds preferentially to adenine-thymine (A-T) regions of DNA. This stain binds into the minor groove of DNA and exhibits distinct fluorescence emission spectra that are dependent on dye:base pair ratios.
Does Hoechst 33342 stain dead cells?
Yes, Hoechst 33342 can stain dead cells, however Hoechst 33358 is the preferred dye that's used for staining dead or fixed cells. Hoechst dyes are a fluorescent stains that bind to AT-rich regions of the minor grove in DNA.May 6, 2021