How do you do a Hoechst stain?

How do you do a Hoechst stain?

Hoechst and DAPI Staining Protocols:

1. Add the dye to complete culture medium. Use Hoechst dyes at 1 ug/mL or DAPI at 10 ug/mL.
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.

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 33342 is generally used for staining live cells. Hoechst dyes are a fluorescent stains that bind to AT-rich regions of the minor grove in DNA.

Do you need to permeabilize cells for Hoechst staining?

Hoechst dyes are cell-permeable so there is no need to permeabilize them for Hoechst staining.

What is the difference between Hoechst 33342 and 33258?

The key difference between them is that the additional ethyl group of Hoechst 33342 renders it more lipophilic, and thus more able to cross intact cell membranes. In some applications, Hoechst 33258 is significantly less permeant.

What is the difference between DAPI and Hoechst?

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.

How does Hoechst stain DNA?

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.

Is Hoechst 33342 toxic?

Dyes that bind to DNA, such as Hoechst 33342, are commonly used to visualize chromatin in live cells by fluorescence microscopy. A caveat is that the probes themselves should not perturb cellular responses and under normal conditions the dyes are generally non-toxic.

What does a Hoechst stain show?

Hoechst dyes stain the DNA of both living and fixed cells and are compatible with immunohistochemistry applications.

How do you use Hoechst?

Labeling cells

1. Culture cells in an appropriate medium and vessel for fluorescence microscopy.
2. Prepare the Hoechst staining solution by diluting the Hoechst stock solution 1:2,000 in PBS.
3. Remove the medium.
4. Add sufficient staining solution to cover the cells.
5. Incubate for 5–10 minutes, protected from light.

What is the Hoechst method?

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.

Why is Hoechst better than DAPI?

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. There are nuclei staining dyes that allow for viability of cells after staining.

What is DAPI staining used for?

DAPI staining was used to determine the number of nuclei and to assess gross cell morphology. Following light microscopic analyses, the stained cells were processed for electron microscopy.

What is the protocol for staining cells with Hoechst?

Protocol: Staining Cells with Hoechst or DAPI Nuclear Stains. Jump to a section: 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.

What is Hoechst 33342 and Pi double staining?

The staining pattern resulted from the simultaneous use of these dyes makes it possible to distinguish normal, apoptotic, and dead cell populations by flow cytometer and fluorescence microscopy. Figure 1. Hoechst 33342 and PI double staining in MCF-7 cells.

What is the Hoechst 33342 protocol?

Hoechst 33342 Protocol for Imaging Nuclear counterstain for fluorescence microscopy Invitrogen Hoechst 33342 nucleic acid stain is a popular cell-permeant nuclear counterstain that emits blue fluorescence when bound to dsDNA.

What is 33342 stain used for?

Invitrogen Hoechst 33342 nucleic acid stain is a popular cell-permeant nuclear counterstain that emits blue fluorescence when bound to dsDNA. This dye is often used to distinguish condensed pycnotic nuclei in apoptotic cells and for cell cycle studies in combination with BrdU.