DAPI (4 ', 6-DIAMIDINO-2-PHENYLINDOLE): CHARACTERISTICS, RATIONALE, USE - CHEMISTRY - 2025

The DAPI (4 ', 6-diamidino-2-phenylindole) is a dye by fluorescent property serves as a marker, widely used in the art of fluorescence microscopy or flow cytometry, among others. The fluorescence it emits is bright blue, its excitation occurs between 455-461 nm (UV light).

DAPI dye can pass through the cell membrane of dead cells with great ease. It can also stain the nuclei of living cells, but in this case, the concentration of this must be higher.

Chemical structure of the fluorescent dye DAPI. Source: Image: File: DAPI.svg is a vector version of this file. It should be used in place of this raster image when not inferior / Richard Wheeler (Zephyris) Edited image

The dye is capable of accessing cellular DNA for which it has a special affinity, binding with great avidity to the nitrogenous bases adenine and thymine. For this reason it is very useful in some molecular biology techniques.

This compound belongs to the group of indole dyes and has been shown to have greater sensitivity to DNA than ethidium bromide and propidium iodide, especially on agarose gels.

The use of this fluorescent dye is very broad, as it is useful for: studying changes in DNA in apoptotic processes (cell death) and therefore detecting cells in this process; for DNA footprinting photo (DNA photo printing); to study bacterial contamination; or to visualize nuclear segmentation.

It has also been used in the study of chromosomal bands, in the detection of Mycoplasmas sp DNA, in the DNA-protein interaction, in the staining and counting of cells by immunofluorescence and even to color mature pollen grains.

characteristics

DAPI is the abbreviation of its chemical name (4 ', 6-diamidino-2-phenylindole). Its molecular formula is C ₁₆ H ₁₅ N _5. It has a molecular weight of 350.3. Near the UV light range (345 to 358 nm) the maximum excitation of the DAPI-DNA complex occurs, while the maximum fluorescence emission occurs between 455-461 nm.

This dye is characterized as a yellow powder, but the structures marked with this fluorophore emit a brilliant blue light.

It is a compound soluble in water, however, to accelerate its dissolution some heat can be applied. It can be diluted with PBS but not directly dissolved in it.

Once the dye is prepared, it must be stored in the dark, that is, protected from light, at a temperature of 2 to 8 ° C (refrigerator). Under these conditions, the dye is stable for more than 3 weeks or months.

If it is protected from light but left at room temperature its stability drops to 2 or 3 weeks, but exposed to direct light the deterioration is very fast. If you want to store for much longer, it can be refrigerated at -20 ° C distributed in aliquots.

Basis

This staining is based on generating a nuclear counterstain in the main molecular biology techniques, such as: flow cytometry, fluorescence microscopy and staining of metaphase chromosomes or interphase nuclei, among others.

This technique is based on the great affinity that the dye has for the nitrogenous bases (adenine and thymine) contained in the genetic material (DNA) in the minor groove. While at the cytoplasmic level it leaves very little background.

When the fluorescent dye binds to the adenine and thymine regions of DNA, the fluorescence increases significantly (20 times more). The color it emits is bright blue. Notably, there is no fluorescence emission when binding to GC (guanine-cytosine) base pairs.

It is important to note that although it also has an affinity for RNA, this does not cause a problem, because the highest degree of energy emission from this molecule occurs at another wavelength (500 nm), unlike DNA, which does so at 460 nm. Furthermore, the increase in fluorescence once bound to RNA is only 20%.

DAPI is used more to stain dead (fixed) cells than live cells, since a much higher concentration of the dye is needed to stain the latter, this is because the cell membrane is much less permeable to DAPI when it is alive.

DAPI dye can be used in combination with red and green fluorophores for a multi-color experience.

Use

DAPI (4 ', 6-diamidino-2-phenylindole) is an excellent fluorophore and is therefore widely used in various techniques and for various purposes. The following explains the use of DAPI in the main techniques.

Flow cytometry

The researchers Gohde, Schumann and Zante in 1978 were the first to use and propose DAPI as a fluorophore in the flow cytometry technique, having great success due to its high sensitivity to DNA and its high intensity in fluorescence emission.

The use of DAPI in this technique allows the study of the cell cycle, the quantification of cells and the staining of living and dead cells.

Although there are other colorants, such as ethidium bromide, Hoechst oxide, acridine orange and propidium iodide, DAPI is one of the most widely used as it is more photostable than those previously mentioned.

For this technique it is required to fix the cells, for this, absolute ethanol or 4% paraformaldehyde can be used. The sample is centrifuged and the supernatant is discarded, subsequently the cells are hydrated by adding 5 ml of PBS buffer for 15 minutes.

While time elapses, prepare the DAPI stain with a staining buffer (FOXP3 from BioLegend) at a concentration of 3 µM.

Centrifuge the sample, discard the supernatant, and then cover with 1 ml of DAPI solution for 15 minutes at room temperature.

Take the sample to the flow cytometer with the appropriate laser.

Flow Microfluorometry

Another technique in which DAPI is used is in flow micro-fluorometry together with another fluorophore called mithramycin. Both are useful for quantifying chloroplast DNA individually, but DAPI is best suited for measuring T4 bacteriophage particles.

Hybridization

This technique basically uses DNA probes labeled with a fluorescent dye that can be DAPI.

The sample requires heat treatment to denature the double-stranded DNA and convert it into two single-stranded strands. It is subsequently hybridized with a DAPI-labeled denatured DNA probe that has a sequence of interest.

Later it is washed to eliminate what was not hybridized, a contrast is used to visualize the DNA. The fluorescence microscope allows the observation of the hybridized probe.

This technique has the purpose of detecting specific sequences in chromosomal DNA, being able to make the diagnosis of certain diseases.

These cyto-molecular techniques have been of great help in determining details in the study of karyotypes. For example, he has shown the base pair rich regions of adenosine and thymine called heterochromatic regions or DAPI bands.

This technique is widely used for the study of chromosomes and chromatin in plants and animals, as well as in the diagnosis of prenatal and hematological pathologies in humans.

In this technique, the recommended DAPI concentration is 150 ng / ml for a time of 15 minutes.

Assembled slides should be stored protected from light at 2-8 ° C.

Immunofluorescence staining

Cells are fixed with 4% paraformaldehyde. If other stains are to be used, DAPI is left at the end as a counterstain and the cells are covered with PBS solution for 15 minutes. While time elapses, prepare the DAPI solution by diluting with PBS, such that the final concentration is 300 µM.

Then the excess PBS is removed and covered with DAPI for 5 minutes. Washes several times. The slide is viewed under a fluorescence microscope under the appropriate filter.

Safety sheet

This compound must be handled with care, because it is a compound that has mutagenic properties. Activated carbon is used to eliminate this compound from aqueous solutions that are to be discarded.

Gloves, gown and safety glasses must be used to avoid accidents with this reagent. If contact with skin or mucosa occurs, the area should be washed with enough water.

You should never pipet this reagent by mouth, use pipettes.

Do not contaminate the reagent with microbial agents as this will lead to erroneous results.

Do not dilute the DAPI stain more than recommended, as it will significantly decrease the quality of the stain.

Do not expose the reagent to direct light, or keep warm as this decreases fluorescence.

References

1. Brammer S, Toniazzo C and Poersch L. Corantes commonly involved in plant cytogenetics. Arq. Inst. Biol. 2015, 82. Available from: scielo.
2. Impath Laboratories. DAPI. Available at: menarinidiagnostics.com/
3. Cytocell Laboratories. 2019. Instructions for use of DAPI. available at cytocell.com
4. Elosegi A, Sabater S. Concepts and techniques in river ecology. (2009). Editorial Rubes, Spain. Available at: books.google.co.ve/
5. Novaes R, Penitente A, Talvani A, Natali A, Neves C, Maldonado I. Use of fluorescence in a modified dissector method to estimate the number of myocytes in cardiac tissue. Arch. Bras. Cardiol. 2012; 98 (3): 252-258. Available from: scielo.
6. Rojas-Martínez R, Zavaleta-Mejía E, Rivas-Valencia P. Presence of phytoplasmas in papaya (Carica papaya) in Mexico. Chapingo Magazine. Horticulture series, 2011; 17 (1), 47-50. Available at: scielo.org.