DAPI (4'-6-diamidino-2-phenylindole) is a DNA-specific blue fluorescent dye used in many biological disciplines, including biochemistry, molecular biology, immunology, and microbiology. Because of its 20-fold fluorescence increase and high binding affinity for adenine-thymine (AT) rich regions of double-stranded DNA (dsDNA), DAPI is an excellent dye for cell counting and sorting, measuring apoptosis, and as a nuclear counterstain in fluorescence microscopy and flow cytometry. While DAPI is more sensitive for use with dsDNA, it also binds to adenine-uracil (AU) regions of RNA but with less affinity and fluorescence enhancement.

When bound to dsDNA, DAPI has an excitation/emission maxima of 359/457 nm. It is well-excited by the violet (405 nm) laser and detected through the DAPI filter. The high quantum yield (Φ = 0.92) of the DAPI/dsDNA complex enables it to emit bright blue fluorescence making DAPI an excellent counterstain in multicolor fluorescent techniques with other green (e.g., iFluor^® 488, Alexa Fluor^® 488, FITC, GFP) and red (e.g., iFluor^® 647, Alexa Fluor^® 647, Cy5, RFP) fluorophores. DAPI will also bind to RNA but at a lower affinity and emit a significantly weaker fluorescence signal, ∼20% as high as the yield of the DAPI/dsDNA complex. The DAPI/RNA complex also exhibits a longer-wavelength emission maximum at ∼500 nm.

In most applications, cell/tissue fixation and permeabilization are done before DAPI staining since the dye is moderately cell impermeant. Increasing the DAPI concentration or incubation time may be necessary. However, this could lead to decreased cell viability. For live-cell DNA staining, Nuclear Violet™ LCS1 and Hoeschts dyes are popular alternatives. They have absorption and fluorescence emission maxima similar to DAPI and are cell-permeant. Besides labeling cell nuclei, DAPI is also used to detect mycoplasma or virus DNA in cell cultures.

AAT Bioquest's portfolio of DAPI reagents includes ready-to-use formulations and FluoroQuest™ anti-fade mounting mediums fortified with DAPI. FluoroQuest™ anti-fade mountants preserve the fluorescence of tissue and cell smears for long-term storage and analysis by reducing the photobleaching rate of DAPI. FluoroQuest™ anti-fade mountants are used in in situ hybridization techniques or other methods requiring DNA staining with blue fluorescence.

One drawback to DAPI is its broad emission spectrum. The very long tail end in the DAPI emission overlaps considerably with green fluorophores, such as FITC and GFP, and will require spectral unmixing to remove artifacts. To address this concern, we offer a variety of different color Nuclear™ LCS1 live-cell nucleic acid stains that can be used as alternatives for DAPI. Nuclear™ LCS1 dyes readily penetrate live cells with healthy membranes and fluoresce strongly upon binding to DNA. Nuclear Violet™ LCS1 is a direct replacement for DAPI.