A blue nuclear counterstain for fluorescence microscopy and cellular imaging.

Thermo Scientific DAPI reagents are a high-purity forms of DAPI dye for fixed-cell, fluorescent staining of DNA content and nuclei for cellular imaging techniques.

DAPI (diamidino-2-phenylindole) is a blue fluorescent probe that fluoresces brightly upon selectively binding to the minor groove of double stranded DNA, where its fluorescence is approximately 20-fold greater than in the nonbound state. Its selectivity for DNA and high cell permeability allows efficient staining of nuclei with little background from the cytoplasm. DAPI is a classic nuclear counterstain for immunofluorescence microscopy, as well as an important component of high-content screening methods requiring cell-based quantitation of DNA content.

As a counterstain in fluorescence imaging methods, DAPI is compatible with antibodies and other probes labeled with fluorescein and rhodamine dyes, as well as with Thermo Scientific DyLight* Fluors. DAPI has greater photostability than Hoechst dyes, although Hoechst 33342 can be use for live cell imaging while use of DAPI is confined to fixed cells. DAPI is offered in powdered solid and aqueous solution forms.

Properties of DAPI Fluorescent Dye:

Application Data:

DAPI counterstaining alongside a phalloidin probe. NIH 3T3 cells were grown in 96-well plates, fixed with paraformaldehyde, and permeabilized for 15 minutes. Cells were incubated for 45 minutes with 3U/mL of DY554-Phalloidin, then for 5 minutes with 1µg/mL of Thermo Scientific DAPI. Stained cells were imaged with the Zeiss Axio Observer .Z1 Microscope (Obj. 20X/0.4NA). Phalloidin binds F-actin, revealing the distribution of actin filaments. DAPI stains DNA, thereby delineating the nuclei.

Dual imaging with DAPI and antibody probes. DU145 prostate cancer cells DU145 were grown in 96-well plates, fixed with paraformaldehyde and permeabilized for 15 minutes. Cells were incubated for 1 hour at room temperature with a mouse anti-human HMGB antibody, then for 45 minutes with Thermo Scientific DyLight 549 Goat Anti-Mouse Secondary Antibody. Finally, Thermo Scientific DAPI was added at 1µg/mL for 5 minutes. Stained cells were imaged with the Thermo Scientific ArrayScan VTI Instrument (Obj. 20X/0.45NA).

Applications for DAPI stain:

• Assaying DNA in solution (ref.4)
• Diagnosing mycoplasmal infection of cell cultures (ref.5)
• Measuring nuclear content and sorting chromosomes in flow cytometry (ref.6)
• Assessing apoptosis (ref.7)
• Detecting nuclei and organellar DNA in immunofluorescent and in situ hybridization procedures (ref.2,8)
• Replacing ethidium bromide for staining DNA in agarose gels (ref.5,9)
• Counterstaining nuclei in histochemical methods when red-fluorescent antibodies have been used to detect specific targets (ref.8)
• Reports also indicate that DAPI will bind to polyphosphates and other polyanions (ref.10), dextran sulfate (ref.11) and SDS (ref.12).

References:

1. Morikawa, K. and Yanagida, J. (1981). Visualization of individual DNA molecules in solution by light microscopy: DAPI staining method. J. Biochem. (Tokyo) 89:693-6.
2. 2. Lawrence, M.E. and Possingham, J.V. (1986). Direct measurement of femtogram amounts of DNA in cells and chloroplasts by quantitative microspectrofluorometry. J. Histochem. Cytochem. 34:761-8.
3. Kubista, M., et al. (1987). Characterization of interaction between DNA and 4', 6-diamidino-2-phenylindole by optical spectroscopy. Biochemistry 26:4545-53.
4. Brunk, C.F., et al. (1979). Assay for nanogram quantities of DNA in cellular homogenates. Anal. Biochem. 92:497-500.
5. Russell, W.C., et al. (1975). A simple cytochemical technique for demonstration of DNA in cells infected with mycoplasmas and viruses. Nature 253:461-2.
6. Hammarton, T.C., et al. (2003). Stage-specific differences in cell cycle control in Trypanosoma brucei revealed by RNA interference of a mitotic cyclin. J. Biol. Chem. 278(25):22877-86.
7. Lai, J., et al. (2003). Loss of HSulf-1 up-regulates heparin-binding growth factor signaling in cancer. J. Biol. Chem. 278(25):23107-17.
8. Soto, P., et al. (2003). SMAD2 and SMAD7 Involvement in the post-translational regulation of Muc4 via the transforming growth factor-b and interferon-g pathways in rat mammary epithelial cells. J. Biol. Chem. 278(22):20338-44.
9. Nairn, R.S., et al. (1982). Comparison of ethidium bromide and 4', 6'-diamidino-2-phenylindole as quantitative fluorescent stains for DNA in agarose gels. J. Biochem. Biophys. Meth. 6:95-103.
10. Tijssen, J.P.F., et al. (1982). Localization of polyphosphates in Saccharomyces fragilis, as revealed by 4', 6-diamidino-2-phenylindole fluorescence. Biochem. Biophys. Acta 721:394-8.
11. Allan, R.A. and Miller, J.J. (1980). Influence of S-adenosylmethionine on 4', 6-diamidino-2-phenylindole dihydrochloride (DAPI)-induced fluorescence of polyphosphate in the yeast vacuole. Can. J. Micro. 26:912-20.
12. Kapuscinski, J. and Skoczylas, B. (1978). Fluorescent complexes of DNA with DAPI (4',6-diamidine-2-phenyl indole dihydrochloride) or DCI (4',6- dicarboxyamide-2-phenyl indole). Nucl. Acids Res. 5:3775-99.

Related Products and Resources:

DyLight Reactive Fluors
Tech Tip #31 – Calculate dye:protein (F/P) molar ratios
All Fluorescent Labeling Reagents – including AMCA, FITC, and TRITC
Immunohistochemistry Reagents – including ABC and DAB staining kits
Thermo Scientific Cellomics* High-Content Screening (HCS) Reagents

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