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]。