HeLa cell nuclei stainied with DAPI
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.
Absorption and emission spectra for DAPI.
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.
Table 1. DAPI Specifications
|CAS # ||28718-90-3
|Ex Max (nm) ||359
|Em Max (nm) ||457
|Extinction Coefficient ||27,000 cm-1M-1
|Quantum Yield ||0.58
|Filter set ||DAPI
|Laser line ||Violet (405 nm)
Fluorescence IHC of formaldehyde-fixed, paraffin-embedded Human lung adenocarcinoma positive tissue. Human lung adenocarcinoma positive tissue sections were stained with rabbit anti-EpCam antibody and then incubated with polyHRP-labeled Goat anti-Rabbit IgG secondary antibody followed by iFluor® 488 Styramide™ (Cat No. 45020) stain, respectively. The tissue was mounted in FluoroQuest™ Anti-fading Mounting Medium with DAPI (Cat No. 20005).
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.
Table 2. Product ordering information for DAPI reagents.
|DAPI [4,6-Diamidino-2-phenylindole, dihydrochloride] *10 mM solution in water*||359||457||2 mL||17507|
|DAPI [4,6-Diamidino-2-phenylindole, dihydrochloride] *CAS 28718-90-3*||359||457||10 mg||17510|
|DAPI [4,6-Diamidino-2-phenylindole, dihydrochloride] *CAS 28718-90-3*||359||457||25 mg||17513|
|DAPI [4,6-Diamidino-2-phenylindole, dihydrochloride] *CAS 28718-90-3*||359||457||100 mg||17511|
|DAPI Dilactate||359||457||25 mg||17509|
|FluoroQuest™ Mounting Medium with DAPI||359||457||50 mL||20004|
|FluoroQuest™ Anti-fading Mounting Medium with DAPI||359||457||20 mL||20005|
Nuclear LCS1™ Dyes - Alternatives to DAPI
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.
Emission spectra of DAPI and FITC illustrating considerable cross-talk between 475 and 600 nm.
Fluorescence images of HeLa cells stained with MitoLite™ Green FM (Cat No. 26695) using fluorescence microscope with a FITC filter set. Live cells were co-stained with nuclei stain Nuclear Red™ LCS1 (Cat No. 17542).
Key Features of Nuclear™ LCS1 Dyes
- No-wash, nuclear-specific DNA dyes
- Low toxicity for live cell imaging
- Membrane-permeant dyes that stains nuclei of live, dead, or fixed cekks
- Nuclear Red™ LCS2 compatible with SIM, STED, or STORM
- Choose from violet, yellow, green, orange, red, or far-red fluorescence
Table 3. Product ordering information for Nuclear™ LCS1 DNA stain.
|Nuclear Violet™ LCS1 *5 mM DMSO Solution*||401||460||0.5 mL||17543|
|Nuclear Yellow [Hoechst S769121] *CAS 74681-68-8*[||372||504||0.5 mL||17539|
|Nuclear Green™ LCS1 *5 mM DMSO Solution*||503||527||0.5 mL||17540|
|Nuclear Orange™ LCS1 *5 mM DMSO Solution*||514||556||0.5 mL||17541|
|Nuclear Red™ LCS1 *5 mM DMSO Solution*||622||645||0.5 mL||17542|
|Nuclear Red™ LCS2 *5 mM DMSO Solution*||651||681||0.5 mL||17545|