DAPI (hydrochloride)
(Synonyms: 4',6-二脒基-2-苯基吲哚二盐酸盐,4',6-Diamidino-2-phenylindole dihydrochloride) 目录号 : GC43378
A fluorescent DNA probe
Cas No.:28718-90-3
Sample solution is provided at 25 µL, 10mM.
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- Purity: >98.00%
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- Datasheet
| Cell experiment [1]: | |
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Cell lines |
bone marrow-derived adherent cells |
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Preparation Method |
After three passages, bone marrow-derived adherent cells were incubated with 50 μg/ml DAPI (Glpbio) at 37°C and 5% CO2 for 2 h. BM-MSCs were treated with trypsin(Glpbio) for 3 min to generate a single-cell suspension, washed three times with DMEM, resuspended in serum-free DMEM, counted, and stored on ice until transplantation. Fluorescence from DAPI labeling on BM-MSCs and cell viability were evaluated for 1, 2, 3, 4, and 8 weeks after cell labeling. |
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Reaction Conditions |
50 μg/ml; at 37°C and 5% CO2 for 2 h |
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Applications |
After three passages in culture, the isolated cell population became homogeneous, showing a monolayer consisting of adherent cells displaying further traits of MSCs, including a typical fibroblast-like morphology and increased proliferation. |
| Animal experiment [2]: | |
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Animal models |
Brain tumor xenograft in nude mice |
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Preparation Method |
Tissue sections for in vivo analysis were collected from animals 3 days after PBS, PAM-RG4/pJDK, or PAM-RG4/pJDK-apoptin injection and immediately paraffinized before further analysis using the DeadEnd™ Fluorometric TUNEL System. Following the given protocol, samples were analyzed using fluorescence microscopy to detect the localized green fluorescence of apoptotic cells and blue fluorescence (DAPI) of cell nuclei. |
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Dosage form |
3 days after injection; 0.5-10μg/ml |
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Applications |
Apoptosis, chromatin condensation, which is caused by the degradation of nuclear membrane proteins, by using DAPI staining. however, when applied in vivo, the experimental group did not show any signs of side effects, indicating a sufficiently low level of cytotoxicity, although it showed similar DAPI and TUNEL assay results. |
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References: [1].Castanheira P, et al. DAPI diffusion after intravitreal injection of mesenchymal stem cells in the injured retina of rats. Cell Transplant. 2009;18(4):423-31. [2].An S, et al. Nonviral gene therapy in vivo with PAM-RG4/apoptin as a potential brain tumor therapeutic. Int J Nanomedicine. 2013;8:821-34. |
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DAPI, as a DNA-specific probe, can form a fluorescent complex by attaching in the minor grove of A-T rich sequences of DNA and nonfluorescent intercalative complexes with double-stranded nucleic acids. DAPI, as a chromosome and nuclear stain, has maximum excitation ultraviolet (UV) light wavelength with 358 nm and emission in the blue range with 461 nm.[1]
DAPI is usually used for flow cytometry, chromosome staining, DNA visualization and quantitation in histochemistry and biochemistry.[2] DAPI is a good substrate of hOCT1 with a Michaelis constant of 8.94 μM.[6].
In vitro, DAPI (1.43μM) can measure mitochondrial permeability transition and mitochondrial membrane depolarization by combining with Annexin V(FITC)and the potentiometric fluorescent dye, tetramethylrhodamine methyl ester (TMRM).[3] In vitro, to evaluate the DAPI fluorescence, SSC buffer (pH 7.2), containing 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04% SDS or Triton X-100 and 2 μg/mL of DAPI dye, there was found that a linear relationship between DAPI fluorescence and Triton X-100 concentrations ranging from 0.01 to 0.04%.[4] In addition, when the concentration of DAPI was increased from 0.4?μM to 400?μM, the total signal intensity increased, implying an increase in the concentration of DAPI molecules bound to the chromosomes.[5] In vitro experiment it shown that treatment with NR 1 μg/mL, DAPI 5 μg/mL, ORO 0.3% (v/v) and CV 0.5% (v/v) provided optimal linearity and coverage of signals over a range of cell densities (corresponding to 10-100% cell confluence).[7].
References:
[1] Karg TJ, et al. Photoconversion of DAPI and Hoechst dyes to green and red-emitting forms after exposure to UV excitation. Chromosoma. 2018 Jun;127(2):235-245.
[2] Kapuscinski J. DAPI: a DNA-specific fluorescent probe. Biotech Histochem. 1995 Sep;70(5):220-33.
[3] Wallberg F, et al. Analysis of Apoptosis and Necroptosis by Fluorescence-Activated Cell Sorting. Cold Spring Harb Protoc. 2016 Apr 1;2016(4):pdb.prot087387.
[4] ?imoliūnas E, et al. DNA-DAPI Interaction-Based Method for Cell Proliferation Rate Evaluation in 3D Structures. Curr Issues Mol Biol. 2021 May 30;43(1):251-263.
[5] Estandarte AK, et al. The use of DAPI fluorescence lifetime imaging for investigating chromatin condensation in human chromosomes. Sci Rep. 2016 Aug 16;6:31417.
[6] Yasujima T, et al. Characterization of human OCT1-mediated transport of DAPI as a fluorescent probe substrate. J Pharm Sci. 2011 Sep;100(9):4006-12.
[7] Phan MAT, et al. Semi-quantification of lipids in human meibomian gland epithelial cells using dual staining microplate assays. Exp Eye Res. 2021 Sep;210:108719.
DAPI 作为一种 DNA 特异性探针，可以通过附着在富含 A-T 的 DNA 序列和带有双链核酸的非荧光嵌入复合物的小树林中形成荧光复合物。 DAPI 作为染色体和核染色剂，最大激发紫外 (UV) 光波长为 358 nm，发射波长为 461 nm 的蓝色。[1]
DAPI 通常用于组织化学和生物化学中的流式细胞术、染色体染色、DNA 可视化和定量。[2] DAPI 是 hOCT1 的良好底物，Michaelis 常数为 8.94 μM。[6].
在体外，DAPI (1.43μM) 可以通过与 Annexin V(FITC) 和电位荧光染料四甲基罗丹明甲酯 (TMRM) 结合来测量线粒体通透性转变和线粒体膜去极化。[3] 在体外，为了评估 DAPI 荧光，SSC 缓冲液 (pH 7.2)，包含 0.01、0.015、0.02、0.025、0.03、0.035、0.04% SDS 或 Triton X-100 和 2 μg/mL 的 DAPI 染料，发现表明 DAPI 荧光与 Triton X-100 浓度在 0.01 到 0.04% 之间呈线性关系。[4] 此外，当 DAPI 浓度从 0.4μM 增加到 400μM 时，总信号强度增加，意味着与染色体结合的 DAPI 分子浓度增加。[5] 体外实验表明，用 NR 1 μg/mL，DAPI 5 μg/mL，ORO 0.3% 处理(v/v) 和 CV 0.5% (v/v) 在一系列细胞密度（对应于 10-100% 细胞 co影响）。[7]。
| Cas No. | 28718-90-3 | SDF | |
| 别名 | 4',6-二脒基-2-苯基吲哚二盐酸盐,4',6-Diamidino-2-phenylindole dihydrochloride | ||
| Canonical SMILES | NC(C(C=C1)=CC=C1C2=CC3=CC=C(C(N)=N)C=C3N2)=N.Cl.Cl | ||
| 分子式 | C16H15N5•2HCl | 分子量 | 350.3 |
| 溶解度 | Insoluble in EtOH; ≥10 mg/mL in Water; ≥53.3 mg/mL in DMSO | 储存条件 | Store at -20°C, protect from light |
| General tips | 请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。 储备液的保存方式和期限：-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。 为了提高溶解度，请将管子加热至37℃，然后在超声波浴中震荡一段时间。 |
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| Shipping Condition | 评估样品解决方案：配备蓝冰进行发货。所有其他可用尺寸：配备RT，或根据请求配备蓝冰。 | ||
| 制备储备液 | |||
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1 mg | 5 mg | 10 mg |
| 1 mM | 2.8547 mL | 14.2735 mL | 28.547 mL |
| 5 mM | 0.5709 mL | 2.8547 mL | 5.7094 mL |
| 10 mM | 0.2855 mL | 1.4273 mL | 2.8547 mL |
| 第一步：请输入基本实验信息（考虑到实验过程中的损耗，建议多配一只动物的药量） | ||||||||||
| 给药剂量 | mg/kg |  |
动物平均体重 | g | |
每只动物给药体积 | ul | |
动物数量 | 只 |
| 第二步：请输入动物体内配方组成（配方适用于不溶于水的药物；不同批次药物配方比例不同，请联系GLPBIO为您提供正确的澄清溶液配方） | ||||||||||
| % DMSO % % Tween 80 % saline | ||||||||||
| 计算重置 | ||||||||||
计算结果:
工作液浓度： mg/ml；
DMSO母液配制方法： mg 药物溶于 μL DMSO溶液（母液浓度 mg/mL，
体内配方配制方法：取 μL DMSO母液，加入 μL PEG300，混匀澄清后加入μL Tween 80，混匀澄清后加入 μL saline，混匀澄清。
1. 首先保证母液是澄清的；
2.
一定要按照顺序依次将溶剂加入，进行下一步操作之前必须保证上一步操作得到的是澄清的溶液，可采用涡旋、超声或水浴加热等物理方法助溶。
3. 以上所有助溶剂都可在 GlpBio 网站选购。
DAPI: a DNA-specific fluorescent probe
Biotech Histochem 1995 Sep;70(5):220-33.PMID:8580206DOI:10.3109/10520299509108199.
DAPI (4',6-diamidino-2-phenylindole) is a DNA-specific probe which forms a fluorescent complex by attaching in the minor grove of A-T rich sequences of DNA. It also forms nonfluorescent intercalative complexes with double-stranded nucleic acids. The physicochemical properties of the dye and its complexes with nucleic acids and history of the development of this dye as a biological stain are described. The application of DAPI as a DNA-specific probe for flow cytometry, chromosome staining, DNA visualization and quantitation in histochemistry and biochemistry is reviewed. The mechanisms of DAPI-nucleic acid complex formation including minor groove binding, intercalation and condensation are discussed.
Labeling nuclear DNA using DAPI
Cold Spring Harb Protoc 2011 Jan 1;2011(1):pdb.prot5556.PMID:21205856DOI:10.1101/pdb.prot5556.
A number of fluorescent stains are available that label DNA and allow easy visualization of the nucleus in interphase cells and chromosomes in mitotic cells, including Hoechst, 4',6-diamidino-2-phenylindole (DAPI), ethidium bromide, propidium iodide, and acridine orange. Although not as bright as the vital Hoechst stains for DNA, DAPI has greater photostability. It is believed that DAPI associates with the minor groove of double-stranded DNA, with a preference for the adenine-thymine clusters. Cells must be permeabilized and/or fixed for DAPI to enter the cell and to bind DNA. Fluorescence increases approximately 20-fold when DAPI is bound to double-stranded DNA. This protocol describes the use of DAPI to label nuclear DNA of cells grown in culture.
Analyzing Cell Death by Nuclear Staining with Hoechst 33342
Cold Spring Harb Protoc 2016 Sep 1;2016(9).PMID:27587774DOI:10.1101/pdb.prot087205.
The nuclei of healthy cells are generally spherical, and the DNA is evenly distributed. During apoptosis the DNA becomes condensed, but this process does not occur during necrosis. Nuclear condensation can therefore be used to distinguish apoptotic cells from healthy cells or necrotic cells. Dyes that bind to DNA, such as Hoechst 33342 or 4',6-diamidino-2-phenylindole (DAPI), can be used to observe nuclear condensation. These dyes fluoresce at 461 nm when excited by ultraviolet light and can therefore be visualized using conventional fluorescent microscopes equipped with light sources that emit light at ∼350 nm and filter sets that permit the transmission of light at ∼460 nm. This protocol describes staining and visualization of cells stained with Hoechst 33342, but it can be adapted for staining with DAPI or other dyes.
DAPI (4',6-diamidino-2-phenylindole) Stains Compact Amyloid Plaques
J Alzheimers Dis 2022;88(3):949-955.PMID:35754268DOI:10.3233/JAD-220072.
DAPI is conventionally used as a nuclear stain for cells in culture or tissue. Here we demonstrate that it binds specifically to the β-sheet core of amyloid-β plaques but not diffuse amyloid-β at the plaque periphery. The specific DAPI induced blue fluorescence is much stronger than amyloid plaque autofluorescence. DAPI staining of fibrillar amyloid deposit may yield a misleading impression of damaged or dying cells. On the other hand, it provides a handy and low-cost means of staining compact amyloid plaques together with cell nuclei in double or triple immunofluorescent studies.
A cautionary (spectral) tail: red-shifted fluorescence by DAPI-DAPI interactions
Biochem Soc Trans 2016 Feb;44(1):46-9.PMID:26862187DOI:10.1042/BST20150231.
The fluorescent dye DAPI is useful for its association with and consequent amplification of an ∼460 nm emission maximum upon binding to dsDNA. Labelling with higher DAPI concentrations is a technique used to reveal Pi polymers [polyphosphate (polyP)], with a red-shift to ∼520-550 nm fluorescence emission. DAPI-polyP emissions of ∼580 nm are also generated upon 415 nm excitation. Red-shifted DAPI emission has been associated with polyP and RNA and has more recently been reported with polyadenylic acid (polyA), specific inositol phosphates (IPs) and heparin. We find that amorphous calcium phosphate (ACP) also demonstrates red-shifted DAPI emission at high DAPI concentrations. This DAPI spectral shift has been attributed to DAPI-DAPI electrostatic interactions enabled by molecules with high negative charge density that increase the local DAPI concentration and favour DAPI molecular proximity, as observed by increasing the dye/phosphate ratio. Excitation of dry DAPI (∼360 nm) confirmed a red-shifted DAPI emission. Whereas enzymatic approaches to modify substrates can help define the nature of DAPI fluorescence signals, multiple approaches beyond red-shifted DAPI excitation/emission are advised before conclusions are drawn about DAPI substrate identification.