DAPI | AAT Bioquest

DAPI

4,6-Diamidino-2-phenylindole, dihydrochloride; CAS 28718-90-3

DAPI (4',6-diamidino-2-phenylindole) is a cell-impermeant blue fluorescent DNA stain that binds to AT-rich regions in the minor groove, universally used for nuclear counterstaining in fixed cells and identifying dead cells in live samples.

AT-Rich Minor Groove Binder: 20-fold fluorescence enhancement upon dsDNA binding (K_d ~100 nM)
UV Excitation/Blue Emission: Ex/Em = 358/461 nm, compatible with DAPI filter sets
Nuclear Counterstain Standard: Cell-impermeant for fixed cell nuclear visualization
Dead Cell Indicator: Membrane impermeability restricts staining to compromised cells in live samples

DAPI spectrum. DAPI is a fluorescent compound with an excitation peak at 359 nm and an emission peak at 457 nm. Other spectra of interest include: Hoechst 33258, Hoechst 33342, and Propidium iodide. DAPI (4,6-Diamidino-2-phenylindole) belongs to the following categories: Cell Cycle Assays, Nucleus, DAPI - Blue Fluorescent DNA Stain, Fluorescence Activated Cell Sorting (FACS), and Immunohistochemistry (IHC).

Protocol

COO

COA

Catalog	Size	Price
17510	10 mg	Price
17511	100 mg	Price
17513	25 mg	Price

Overview

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 working concentration

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.

DAPI flow cytometry protocol

Prepare staining buffer sufficient for samples (1 mL per cell sample). Staining buffer is prepared using the following recipe:
100 mM Tris, pH 7.4
150 mM NaCl
1 mM CaCl₂
0.5 mM MgCl₂
0.1% Nonidet P-40
Use the fixation protocol appropriate for your sample.
Dilute DAPI solution with prepared staining buffer to 3 µM.
Centrifuge cell suspension and discard the supernatant. Tap to loosen the pellet and add 1 mL of DAPI staining solution.
Incubate at room temperature for 15 minutes.
Analyze using flow cytometry with 405 nm excitation laser and 450/50 nm bandpass filter (or equivalent channel).

Calculators

Common stock solution preparation

Table 1. Volume of Water needed to reconstitute specific mass of DAPI [4,6-Diamidino-2-phenylindole, dihydrochloride] *CAS 28718-90-3* to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.

	0.1 mg	0.5 mg	1 mg	5 mg	10 mg
1 mM	285.51 µL	1.428 mL	2.855 mL	14.276 mL	28.551 mL
5 mM	57.102 µL	285.51 µL	571.021 µL	2.855 mL	5.71 mL
10 mM	28.551 µL	142.755 µL	285.51 µL	1.428 mL	2.855 mL

Molarity calculator

Enter any two values (mass, volume, concentration) to calculate the third.

Mass (Calculate)		Molecular weight		Volume (Calculate)		Concentration (Calculate)		Moles
mg	÷	350.25 g/mol	=	mL	x	mM	=	mmol

Citations

View all 90 citations: Citation Explorer

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Li, Mengya and Sun, Jian and Zhao, Dong and Zhang, Wen and Xu, Qingan

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Journal of Materials Science: Materials in Medicine (2024): 56

Temperature switchable linkers suitable for triggered drug release in cancer thermo-chemotherapy

Authors:

Ye, Junxiao and Cui, Hui and Liu, Ergang and Pei, Xing and Chai, Meihong and Sun, Lu and Wang, Dongmei and Yang, Victor C and Yu, Fei

Journal:

International Journal of Pharmaceutics (2024): 124757

Tristetraprolin affects invasion-associated genes expression and cell motility in triple-negative breast cancer model

Authors:

Hubiernatorova, Anastasiia and Novak, Josef and Vaskovicova, Michaela and Sekac, David and Kropyvko, Serhii and Hodny, Zdenek

Journal:

Cytoskeleton (2024)

An NIR-responsive hydrogel loaded with polydeoxyribonucleotide nano-vectors for enhanced chronic wound healing

Authors:

Sun, Yanzhen and Li, Yao and Ding, Xiaokang and Xu, Pan and Jing, Xiaodong and Cong, Hailin and Hu, Hao and Yu, Bing and Xu, Fu-Jian

Journal:

Biomaterials (2024): 122789

Emergence of large-scale cell death through ferroptotic trigger waves

Authors:

Co, Hannah KC and Wu, Chia-Chou and Lee, Yi-Chen and Chen, Sheng-hong

Journal:

Nature (2024): 1--9

References

View all 134 references: Citation Explorer

Reliable DNA ploidy determination in dehydrated tissues of vascular plants by DAPI flow cytometry--new prospects for plant research

Authors:

Suda J, Travnicek P.

Journal:

Cytometry A (2006): 273

Identification of all pachytene bivalents in the common shrew using DAPI-staining of synaptonemal complex spreads

Authors:

Belonogova NM, Karamysheva TV, Biltueva LS, Perepelov EA, Minina JM, Polyakov AV, Zhdanova NS, Rubtsov NB, Searle JB, Borodin PM.

Journal:

Chromosome Res (2006): 673

Physical localization of ribosomal genes and chromosome DAPI banding by in situ hybridization in Medicago sativa L

Authors:

Chen JM, Hong YH, Wang YP, Bowley S, Wan JM.

Journal:

Yi Chuan (2006): 184

DNA staining with the fluorochromes EtBr, DAPI and YOYO-1 in the comet assay with tobacco plants after treatment with ethyl methanesulphonate, hyperthermia and DNase-I

Authors:

Gichner T, Mukherjee A, Veleminsky J.

Journal:

Mutat Res (2006): 17

Investigation of chromosomes in varieties and translocation lines of pea Pisum sativum L. by FISH, Ag-NOR, and differential DAPI staining

Authors:

Samatadze TE, Muravenko OM, Bol'sheva NL, Amosova AB, Gostimsckii SA, Zelenin AV.

Journal:

Genetika (2005): 1665

Physical properties

Molecular weight	350.25
Solvent	Water

Spectral properties

Extinction coefficient (cm ^-1 M ^-1)	27000
Excitation (nm)	359
Emission (nm)	457

Storage, safety and handling

Certificate of Origin	Download PDF
H-phrase	H303, H313, H340
Hazard symbol	T
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R68
Storage	Freeze (< -15 °C); Minimize light exposure
UNSPSC	41116134
CAS	28718-90-3

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Page updated on October 8, 2025