Protocol: Staining Cells with Hoechst or DAPI Nuclear Stains - Biotium (2023)

FAQs

What is the difference between DAPI and Hoechst? ›

DAPI is somewhat less cell membrane permeant and more toxic than Hoechst dyes, and is therefore preferred for fixed cell staining over live cell staining. As a fixed cell stain we recommend a DAPI concentration at 1 ug/mL, though live cell staining with DAPI can be performed at higher concentrations (usually 10 ug/mL).

Cells must be permeabilized and/or fixed for DAPI to enter the cell and to bind DNA. Fluorescence increases approximately 20-fold when DAPI is bound to double-stranded DNA. This protocol describes the use of DAPI to label nuclear DNA of cells grown in culture.

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

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. Cells stained with DAPI showed no ultrastructural changes compared to the appearance of cells not stained with DAPI.

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.

One advantage of Hoechst 33342 is that it is membrane permeant and, thus, can stain live cells. Hoechst 33342 binds to adenine-thymine-rich regions of DNA in the minor groove. On binding to DNA, the fluorescence greatly increases.

DAPI staining is normally performed after all other staining. Note that fixation and permeabilization of the sample are not necessary for counterstaining with DAPI.

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.

DAPI is a fluorescent stain that binds strongly to A-T-rich regions in DNA. DAPI and PI only inefficiently pass through an intact cell membrane and, therefore, preferentially stain dead cells.

How do you prepare DAPI for staining? ›

DAPI Staining Solution (ab228549) is a fluorescent stain for labeling DNA in fluorescence microscopy. Since DAPI passes through an intact cell membrane, it can be used to stain live cells and fixed cells.

Posted May 6, 2021. 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.

DAPI is able to be combined with cellular DNA, permeate through the membrane of cell, rapidly enter the nucleus of living cells and bind with DNA to form a DAPI-DNA complex.

For cases where a nuclear dye is desired, e.g. to monitor nuclear size or staining levels, Live Red Dye can be used in place of DAPI but does not require fixation.

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. It is excited by the violet (405 nm) laser line and is commonly used as a nuclear counterstain in fluorescence microscopy, flow cytometry, and chromosome staining.

DAPI (pronounced 'DAPPY', /ˈdæpiː/), or 4′,6-diamidino-2-phenylindole, is a fluorescent stain that binds strongly to adenine–thymine-rich regions in DNA. It is used extensively in fluorescence microscopy.

DAPI (4',6-diamidino-2-phenylindole) (Figure 1) is a well-characterized blue-emitting fluorescent compound widely utilized for nuclear staining. DAPI permeates cell membranes and binds to the minor groove of A/T-rich dsDNA sequences, thus preferentially staining nuclei.

Dyes that bind to DNA, such as Hoechst 33342, are commonly used to visualize chromatin in live cells by fluorescence microscopy.

Therefore, DAPI is used for evaluation of cell death and apoptosis of unfixed cells in flow cytometry. Additionally, DAPI may be used as a nuclear counterstain of fixed cells in imaging or flow cytometry or for determination of DNA content in cell cycle analysis.

What color does Hoechst stain? ›

Hoechst dyes are cell membrane-permeant, minor groove-binding blue fluorescent DNA stains. These dyes are widely used in cell cycle and apoptosis studies as nuclear counterstains.

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.

Before specific staining can occur, tissue samples must undergo preparation through the following stages: Fixation, processing, embedding, sectioning, and sometimes antigen retrieval. In modern histology laboratories, most of these steps are automated.

DAPI Stock Solution Dissolve DAPI in ultrapure water to 1 mg/ml. Stock solution is stable for several months and repeated use if stored protected from light at -20°C. DAPI Working Solution 0.1 μg/ml DAPI in Assay Buffer.

DAPI binds strongly to A-T rich regions in DNA to form a fluorescent complex. It preferentially stains ds-DNA and has a high quantum yield (φf=0.92) when bound to DNA. DAPI is commonly used as a nuclear and chromosome counterstain. It is preferentially used to stain dead cells.

Posted May 6, 2021. Hoechst is minimally toxic to cells especially when used at very low concentrations. It is the preferred dye for staining live cells because of its lower cytotoxicity as compared to other DNA stains.

Fluorescence microscopy of fixed cells uses a fixative agent that renders the cells dead, but maintains cellular structure, allowing the use of specific antibodies and dyes to investigate cell morphology and structure.

Yes, Hoechst is cell permeable. Both, Hoechst 33258 and Hoechst 33342 are cell permeable, however Hoechst 33342 is considerably more cell permeable because of the addition of a lipophilic ethyl group. Hoechst 33342 is usually the preferred choice for staining living cells.

DAPI stains DNA and polyP while maintaining cell viability.

DAPI can penetrate the nucleus without permeabilization and intercalates into the DNA helix. However, high concentrations of DAPI are required for viable cells compared to dying cells, where DNA binding is much more efficient, making DAPI a suitable viability dye in IF staining.

Does DAPI staining emit blue light? ›

It is a stain used very often in fluorescence microscopy, when excited with ultraviolet light DAPI emits a blue light. This makes the stain very popular in fluorescence microscopy making the nuclei visible in a blue colour.

DAPI is a fluorescent stain that binds strongly to A-T-rich regions in DNA. DAPI and PI only inefficiently pass through an intact cell membrane and, therefore, preferentially stain dead cells.

DAPI is considered to be a cell impermeant, meaning it typically cannot pass through the membrane of live cells. However, in high concentrations, DAPI will also mark live cells. DAPI live-cell staining will also label dead cells unless used with a counterstain.

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. For live-cell staining, Hoechst 33342 dye is a popular cell-permeant nuclear counterstain.

One advantage of Hoechst 33342 is that it is membrane permeant and, thus, can stain live cells. Hoechst 33342 binds to adenine-thymine-rich regions of DNA in the minor groove.

Note: When preparing DAPI stock solution, use dimethyl sulfoxide (DMSO) instead of dimethylformamide (DMF), which has been linked to cancer in humans (listed as possible carcinogen by IARC). All operations involving the use of DAPI should be carried out in a chemical fume hood with the sash in the down position.

DAPI (4', 6-diamido- 2-phenylindole) is a commonly used dye for bacterial enumeration, or cell counting. DAPI is a fluorescent dye that stains nucleic acids and is relatively unreactive with inert, non-biological matter, thus making it useful in differentiating between biotic and abiotic components in a sample.