Di-4-ANEPPS
(Synonyms: 氢氧化4-(2-(6-(二丁基氨基)-2-萘基)乙烯基)-1-(3-硫代丙基)吡啶正离子内盐) 目录号 : GC43435Di-4-ANEPPS是一种快速响应的膜电位染料，用于检测膜电位变化
Cas No.:90134-00-2
Sample solution is provided at 25 µL, 10mM.
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Quality Control & SDS
- View current batch:
- Purity: >98.00%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
本方案仅提供一个指导，应根据您的具体需要进行修改。
1.细胞膜染色液制备
(1)配置DMSO或EtOH 储存液:储存液用DMSO或EtOH配置,浓度1~5mM。
注意:未使用的储存液分装保存在-20℃，避免反复冻融。
(2)工作液制备:用合适的缓冲液(如:无血清培养基，HBSS或PBS)稀释储存液，配制浓度为0.5~10μM的工作液。
注意:工作液的最终浓度是根据不同细胞和实验的经验来配制。可以从推荐浓度的十倍以上寻找最佳条件。
2.悬浮细胞染色
(1)悬浮细胞经4°C、1000-1500rpm离心3-5分钟，弃去上清液。用PBS清洗两次，每次5分钟。
(2)加入1mL的Di-4-ANEPPS工作溶液，室温孵育5-30分钟，不同的细胞最佳培养时间不同。
(3)孵育结束后，经1000-1500rpm离心5分钟，去除上清液，加入PBS清洗2-3次，每次5分钟。
(4)用预温的无血清细胞培养基或PBS重悬细胞。通过荧光显微镜或流式细胞术观察。
3.粘壁细胞染色
(1)在无菌盖玻片上培养贴壁细胞。
(2)从培养基中移走盖玻片，吸出过量的培养基，将盖玻片放在潮湿的环境中。
(3)从盖玻片的一角加入100uL的染料工作液，轻轻晃动使染料均匀覆盖所有细胞。
(4)室温条件下孵育5-30分钟，不同的细胞最佳培养时间不同。
(5)吸弃染料工作液，用培养液洗盖玻片2~3次。
4.显微镜检测：Di-4-ANEPPS的激发/发射光分别为495/705nm。
注意事项：
1）荧光染料均存在淬灭问题，请尽量注意避光，以减缓荧光淬灭。
2）为了您的安全和健康，请穿实验服并戴一次性手套操作。
Di-4-ANEPPS is a fast-response membrane potential dye used to detect changes in membrane potential[1]. Di-4-ANEPPS fluoresces to potential changes through very rapid structural changes, so they are suitable for detecting fast and transient potential changes in cells. Di-4-ANEPPS is rapidly internalized in cells and is therefore mainly used in short-term experiments[2]. Di-4-ANEPPS is a highly water-soluble microinjection, the fluorescence is not affected by cell and tissue types, and can be used to measure membrane potential in various tissues and cell types as well as in artificial membranes. Di-4-ANEPPS is a common tool for mapping cardiac electrical activity[3].
Di-4-ANEPPS是一种快速响应的膜电位染料，用于检测膜电位变化[1]。Di-4-ANEPPS通过非常快速的结构变化对电位变化发出荧光，因此它们适合于检测细胞中快速和瞬态的电位变化。Di-4-ANEPPS在细胞中迅速内化，因此主要用于短期实验[2]。Di-4-ANEPPS是高度水溶性的微量注射剂，荧光不受细胞和组织类型的影响，能够用于测量各种组织和细胞类型以及人造膜中的膜电位。Di-4-ANEPPS是绘制心脏电活动的常用工具[3]。
References:
[1]. Maria Pura Hortigon-Vinagre, et al. The Use of Voltage Sensitive Dye di-4-ANEPPS and Video-Based Contractility Measurements to Assess Drug Effects on Excitation-Contraction Coupling in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes. 2021 Mar 1;77(3):280-290. doi: 10.1097/FJC.0000000000000937.
[2]. LM Loew - Pure and Applied Chemistry. Potentiometric dyes: imaging electrical activity of cell membranes.1996 - degruyter.com
[3]. Marina Ronzhina, et al. Di-4-ANEPPS Modulates Electrical Activity and Progress of Myocardial Ischemia in Rabbit Isolated Heart. 2021 Jun 10;12:667065. doi: 10.3389/fphys.2021.667065. eCollection 2021.
| Cas No. | 90134-00-2 | SDF | |
| 别名 | 氢氧化4-(2-(6-(二丁基氨基)-2-萘基)乙烯基)-1-(3-硫代丙基)吡啶正离子内盐 | ||
| Canonical SMILES | CCCCN(CCCC)C(C=C1)=CC2=C1C=C(/C=C/C3=CC=[N+](CCCS([O-])(=O)=O)C=C3)C=C2 | ||
| 分子式 | C28H36N2O3S | 分子量 | 480.7 |
| 溶解度 | Soluble 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 | |||
 |
1 mg | 5 mg | 10 mg |
| 1 mM | 2.0803 mL | 10.4015 mL | 20.803 mL |
| 5 mM | 0.4161 mL | 2.0803 mL | 4.1606 mL |
| 10 mM | 0.208 mL | 1.0401 mL | 2.0803 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。
Di-4-ANEPPS Modulates Electrical Activity and Progress of Myocardial Ischemia in Rabbit Isolated Heart
Front Physiol 2021 Jun 10;12:667065.PMID:34177617DOI:10.3389/fphys.2021.667065.
Aims: Although voltage-sensitive dye Di-4-ANEPPS is a common tool for mapping cardiac electrical activity, reported effects on electrophysiological parameters are rather. The main goals of the study were to reveal effects of the dye on rabbit isolated heart and to verify, whether rabbit isolated heart stained with Di-4-ANEPPS is a suitable tool for myocardial ischemia investigation. Methods and results: Study involved experiments on stained (n = 9) and non-stained (n = 11) Langendorff perfused rabbit isolated hearts. Electrophysiological effects of the dye were evaluated by analysis of various electrogram (EG) parameters using common paired and unpaired statistical tests. It was shown that staining the hearts with Di-4-ANEPPS leads to only short-term sporadic prolongation of impulse conduction through atria and atrioventricular node. On the other hand, significant irreversible slowing of heart rate and ventricular conduction were found in stained hearts as compared to controls. In patch clamp experiments, significant inhibition of sodium current density was observed in differentiated NG108-15 cells stained by the dye. Although no significant differences in mean number of ventricular premature beats were found between the stained and the non-stained hearts in ischemia as well as in reperfusion, all abovementioned results indicate increased arrhythmogenicity. In isolated hearts during ischemia, prominent ischemic patterns appeared in the stained hearts with 3-4 min delay as compared to the non-stained ones. Moreover, the ischemic changes did not achieve the same magnitude as in controls even after 10 min of ischemia. It resulted in poor performance of ischemia detection by proposed EG parameters, as was quantified by receiver operating characteristics analysis. Conclusion: Our results demonstrate significant direct irreversible effect of Di-4-ANEPPS on spontaneous heart rate and ventricular impulse conduction in rabbit isolated heart model. Particularly, this should be considered when Di-4-ANEPPS is used in ischemia studies in rabbit. Delayed attenuated response of such hearts to ischemia might lead to misinterpretation of obtained results.
Characterization of Di-4-ANEPPS with nano-black lipid membranes
Nanoscale 2018 Jan 18;10(3):1090-1098.PMID:29271448DOI:10.1039/c7nr05863b.
We report a platform based on lateral nano-black lipid membranes (nano-BLMs), where electrical measurements and fluorescence microscopy setup are combined, for the calibration of Di-4-ANEPPS, a common voltage sensitive dye (VSD). The advantage of this setup is (1) its flexibility in the choice of lipids and applied voltages, (2) its high stability that enables a high voltage (500 mV) application and long-time measurements and (3) its fluorescence microscopy readout, which can be directly correlated with other fluorescence microscopy experiments using VSDs (e.g. membrane potential measurements in living cells). Using this setup, we observed that the calibration curve of Di-4-ANEPPS is strongly dependent on the net electric charge of the lipids. The developed setup can be used to calibrate VSDs in different lipid environments in order to better understand their fundamental voltage-sensing mechanism in the future.
Di-4-ANEPPS causes photodynamic damage to isolated cardiomyocytes
Pflugers Arch 1994 Apr;426(6):548-51.PMID:8052525DOI:10.1007/BF00378533.
Action potential recordings from isolated guinea pig ventricular cells in the whole-cell recording mode were used to study the toxic and photodynamic properties of the voltage-sensitive fluorescent dye Di-4-ANEPPS. Staining of the cardiomyocytes with Di-4-ANEPPS (30 or 60 microM; 10 min) did not alter the action potential shape. When the stained cells were illuminated (1W/cm2) severe effects on the action potential were observed. There was a prolongation of the action potential duration, occurrence of early afterdepolarizations, reduction of the membrane resting potential and eventually inexcitability. Addition of the antioxidant catalase (100 IU/ml) to the extracellular solution delayed the onset of these effects, suggesting that reactive-oxygen-intermediates take part in Di-4-ANEPPS induced photodynamic damage. Since Di-4-ANEPPS is a very important tool for optical membrane potential recordings in heart tissue and single cardiomyocytes catalase might be useful in suppressing photodynamic damage during optical potential recordings.
The voltage-sensitive dye Di-4-ANEPPS slows conduction velocity in isolated guinea pig hearts
Heart Rhythm 2012 Sep;9(9):1493-500.PMID:22537886DOI:10.1016/j.hrthm.2012.04.034.
Background: Voltage-sensitive dyes are important tools for mapping electrical activity in the heart. However, little is known about the effects of voltage-sensitive dyes on cardiac electrophysiology. Objective: To test the hypothesis that the voltage-sensitive dye Di-4-ANEPPS modulates cardiac impulse propagation. Methods: Electrical and optical mapping experiments were performed in isolated Langendorff perfused guinea pig hearts. The effect of Di-4-ANEPPS on conduction velocity and anisotropy of propagation was quantified. HeLa cells expressing connexin 43 were used to evaluate the effect of Di-4-ANEPPS on gap junctional conductance. Results: In electrical mapping experiments, Di-4-ANEPPS (7.5 μM) was found to decrease both longitudinal and transverse conduction velocities significantly compared with control. No change in the anisotropy of propagation was observed. Similar results were obtained in optical mapping experiments. In these experiments, the effect of Di-4-ANEPPS was dose dependent. Di-4-ANEPPS had no detectable effect on connexin 43-mediated gap junctional conductance in transfected HeLa cells. Conclusion: Our results demonstrate that the voltage-sensitive dye Di-4-ANEPPS directly and dose-dependently modulates cardiac impulse propagation. The effect is not likely mediated by connexin 43 inhibition. Our results highlight an important caveat that should be taken into account when interpreting data obtained using Di-4-ANEPPS in cardiac preparations.
Plasma Membrane Potential of Candida albicans Measured by Di-4-ANEPPS Fluorescence Depends on Growth Phase and Regulatory Factors
Microorganisms 2019 Apr 24;7(4):110.PMID:31022974DOI:10.3390/microorganisms7040110.
The potential of the plasma membrane (Δѱ) regulates the electrochemical potential between the outer and inner sides of cell membranes. The opportunistic fungal pathogen, Candida albicans, regulates the membrane potential in response to environmental conditions, as well as the physiological state of the cell. Here we demonstrate a new method for detection of cell membrane depolarization/permeabilization in C. albicans using the potentiometric zwitterionic dye Di-4-ANEPPS. Di-4-ANEPPS measures the changes in the cell Δѱ depending on the phases of growth and external factors regulating Δѱ, such as potassium or calcium chlorides, amiodarone or DM-11 (inhibitor of H+-ATPase). We also demonstrated that Di-4-ANEPPS is a good tool for fast measurement of the influence of amphipathic compounds on Δѱ.