DAPI

DAPI is a cell impermeable, blue fluorescent dye that stains the nuclei of dead, necrotic or fixed cells in fluorescence microscopy and flow cytometry by binding to A-T rich regions of dsDNA.

Excitation and emission

DAPI, or 4',6-diamidino-2-phenylindole, is a fluorescent compound with an excitation peak at 359 nm and an emission peak at 457 nm. It is well excited by the 405 nm violet laser or the 355 nm UV laser. The emission can be captured by a 450/50 nm bandpass filter or using the DAPI filter set configuration on fluorescence microscopes.

Staining principle and mechanism

DAPI is an intercalating dye, binding to the minor groove of A-T rich regions in double stranded DNA (dsDNA). This preference possibly stems from the more negative electrostatic potential of these areas, which would facilitate the formation of hydrogen bonds and hydrophobic interactions between the dye molecules and DNA base pairs. Upon binding to these regions, the quantum yield of DAPI increases more than 20-fold, resulting in bright blue fluorescence when excited by a violet (405 nm) laser. Intercalation with G-C regions, on the other hand, results in minimal to no fluorescence, while binding to A-U rich regions of RNA produces fluorescence but at a much weaker intensity, with the quantum yield of DAPI increasing only about 5-fold.

Permeability

DAPI is cell impermeable at low concentrations. Therefore, it is often used to stain dead, necrotic or fixed cells, where membrane integrity is compromised. At higher concentrations, it is semi-permeable, with the ability to stain live cells. However, due to its high cytotoxicity, the staining of live cells with DAPI is not recommended. Because DAPI primarily stains cell nuclei, where the concentration of dsDNA is highest, it is often used as a co-stain or counterstain for applications such as cell imaging, cell counting and cell sorting, and it has been used to stain a variety of cell types, such as animal, bacterial and fungal.

Protocol and usage

Typical DAPI staining can be performed in 5 to 15 minutes, depending on the protocol used. The staining may be stable for several hours to days under appropriate conditions, though fluorescence intensity may decrease over time. Samples stained with DAPI should be washed after staining to remove any unbound dye and to reduce background fluorescence. To fix cells after staining, paraformaldehyde or other fixative solutions may be used. The staining of cells with DAPI is irreversible.

DAPI vs Hoechst

Some alternatives to DAPI include the Hoechst family of dyes. While both bind to DNA, Hoechst stains tend to have lower cytotoxicity and greater membrane permeability, allowing for their use in staining live cells. DAPI, having poor membrane permeability and greater toxicity, is more often used to stain dead or fixed cells.

Kapuscinski, J. (1995). DAPI: a DNA-specific fluorescent probe. Biotechnic & histochemistry, 70(5), 220-233.

DAPI has a molecular weight of 350.25 daltons. It has moderate solubility in water (20 mg / mL) and yields a clear yellow solution when dissolved. To make a working solution of DAPI, first add deionized water (diH₂O) or dimethylformamide (DMF) to the powdered form. Sonication may be necessary for contents to fully dissolve. After, dilute DAPI solution with PBS to required concentrations. Typical staining protocols use concentrations between 0.5 µM to 5 µM. Once prepared, the solution is stable for several weeks when stored in a dark location at 2 to 6 °C. For longer storage times, keep at < -15 °C.