Acridine Orange 10-Nonyl Bromide (Nonylacridine orange)
(Synonyms: 10-壬基溴代吖啶橙; Nonylacridine orange) 目录号 : GC30183
Acridine Orange 10-Nonyl Bromide（NAO）是心磷脂的高度特异性探针，常用于标记富含CL的线粒体，最大激发光/发射光为489/525nm。
Cas No.:75168-11-5
Sample solution is provided at 25 µL, 10mM.
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Quality Control & SDS
- View current batch:
- Purity: >99.50%
- 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、制备Acridine Orange 10-Nonyl Bromide染色液
（1）染料储存液:使用DMSO将Acridine Orange 10-Nonyl Bromide（NAO）溶解成1-5mM的储存液。
注意:未使用的储存液建议分装后于-4℃或-20℃避光保存（在氮气下储存），避免反复冻融。
（2）染料工作液: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20nM~1μM的染料工作液。
注意:
①由于NAO的使用浓度比较低，建议使用DMSO稀释储存液，配制一个中间储存浓度(比如:100μM)；
②请根据实际情况调整及优化工作液浓度，现用现配。
2、细胞悬浮染色(以6孔板为例)
（1）悬浮细胞经1000g离心3-5min。弃去上清液，使用PBS清洗两次，每次5分钟。
（2）贴壁细胞使用PBS清洗两次，加入胰酶消化细胞，消化完成后经1000g离心3-5min。
（3）加入1mL经37 °C预热的染料工作液重悬细胞，37 °C避光孵育15-30min分钟，不同细胞最佳培养时间不同。
（4）孵育结束后，经1000g离心5分钟，去除上清液，加入PBS清洗2-3次。
（5）使用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技术进行观察
3、细胞贴壁染色
（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染料工作液，轻轻晃动使染料均匀覆盖所有细胞，37 °C避光孵育15-30min分钟。
（4）吸弃染料工作液，使用培养液清洗盖玻片2~3次，通过荧光显微镜进行观察。
4、显微镜检测：Acridine Orange 10-Nonyl Bromide的最大激发光/发射光为489/525nm。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。
References:
[1]. M H Ratinaud, P Leprat, R Julien. In situ flow cytometric analysis of nonyl acridine orange-stained mitochondria from splenocytes. 1988 May;9(3):206-12. doi: 10.1002/cyto.990090304.
Acridine Orange 10-Nonyl Bromide (NAO), an acridine orange derivative, is a highly specific probe for cardiolipin (CL) and is commonly used to label CL-rich mitochondria[1]. Acridine Orange 10-Nonyl Bromide has been proven to specifically bind CL in vitro and is often used as a live cell stain to measure CL content[2]. In rat cortical astrocytes, neonatal cardiomyocytes, and isolated brain mitochondria, Acridine Orange 10-Nonyl binding to mitochondria was abolished by the mitochondrial uncoupler FCCP, demonstrating labeling of mitochondria by Acridine Orange 10-Nonyl Bromide Staining is highly dependent on membrane potential[2]. Acridine Orange 10-Nonyl has a maximum excitation/emission light of 489/525nm and has been used to quantify cardiolipin content and mitochondrial mass in isolated mitochondria and cellular systems[3].
Acridine Orange 10-Nonyl Bromide（NAO）是一种吖啶橙衍生物，是心磷脂（CL）的高度特异性探针，常用于标记富含CL的线粒体[1]。Acridine Orange 10-Nonyl Bromide已被证明在体外特异性结合CL，并常被用作活细胞染色测定CL含量[2]。在大鼠皮质星形胶质细胞、新生儿心肌细胞和分离的脑线粒体中，Acridine Orange 10-Nonyl与线粒体的结合被线粒体解偶联剂FCCP消除，表明Acridine Orange 10-Nonyl Bromide对线粒体的标记染色对膜电位具有高度依赖性[2]。Acridine Orange 10-Nonyl的最大激发光/发射光为489/525nm，并已用于定量分离的线粒体和细胞系统中的心磷脂含量和线粒体质量[3]。
References:
[1].Myriam E Rodriguez,et.Targeting of mitochondria by 10-N-alkyl acridine orange analogues: role of alkyl chain length in determining cellular uptake and localization. 2008 Jun;8(3):237-46. doi: 10.1016/j.mito.2008.04.003. Epub 2008 Apr 25.
[2].Jake Jacobson, Michael R Duchen, Simon J R Heales. Intracellular distribution of the fluorescent dye nonyl acridine orange responds to the mitochondrial membrane potential: implications for assays of cardiolipin and mitochondrial mass. 2002 Jul;82(2):224-33. doi: 10.1046/j.1471-4159.2002.00945.x.
[3]. M H Ratinaud, P Leprat, R Julien. In situ flow cytometric analysis of nonyl acridine orange-stained mitochondria from splenocytes. 1988 May;9(3):206-12. doi: 10.1002/cyto.990090304.
| Cas No. | 75168-11-5 | SDF | |
| 别名 | 10-壬基溴代吖啶橙; Nonylacridine orange | ||
| Canonical SMILES | CCCCCCCCC[N+]1=C2C=C(N(C)C)C=CC2=CC3=C1C=C(N(C)C)C=C3.[Br-] | ||
| 分子式 | C26H38BrN3 | 分子量 | 472.5 |
| 溶解度 | DMSO : 25 mg/mL (52.91 mM) | 储存条件 | Store at 2-8°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 | 2.1164 mL | 10.582 mL | 21.164 mL |
| 5 mM | 0.4233 mL | 2.1164 mL | 4.2328 mL |
| 10 mM | 0.2116 mL | 1.0582 mL | 2.1164 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。
Fluorescent determination of cardiolipin using 10-N-nonyl acridine orange
Cardiolipin (CL) plays an essential role as a marker for cell apoptosis. Quantitative detection of phospholipids (PLs) by UV absorbance is problematic due to the presence of few double bonds in the structure. Although 10-N-nonyl acridine orange (NAO) has been utilized for fluorescent visualization of liposomes and mitochondria through its interaction with CL, in this work, we have developed a specific fluorescent method for CL in solution using NAO. The interaction of sodium n-dodecyl sulfate (SDS), used to treat cells prior to lipid extraction, and other PLs found in cell membranes such as phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidiylserine (PS), and sphingomyelin (SM) with NAO is investigated. The fluorescence intensity of the 0.5 microM NAO signal is strongly quenched by SDS below 25% methanol in water but with a methanol content above 50%, no quenching of NAO by SDS is observed. No fluorescence quenching of NAO with a 50% methanol/50% water solvent by the previously mentioned PLs or 4-20 microM cholesterol with the exception of PG at above 8 microM is noted. Using this 50% methanol/50% water solvent, the fluorescence signal due to the NAO-CL interaction is quite stable from 3 to at least 15 min. With excitation and emission wavelengths set at 518 and 530 nm, respectively, 20 microM NAO provides an inverse linear fluorescence response at 0.2-10 microM CL with a correlation coefficient of 0.9929. The detection limit is 0.2 microM and the limit of quantification is 0.6 microM. Structurally analogous acridine orange and phenosafranin dyes are less effective as fluorescent probes for CL. The CL in the whole cell and membrane samples is quantitatively determined by standard addition to range from 0.2 to 1.5 microM. The level of CL in cell membrane samples, previously subjected to staurosporine which initiates cell apoptosis, is increased but not significantly through use of the t-test.
Binding of 10-N-nonyl acridine orange to cardiolipin-deficient yeast cells: implications for assay of cardiolipin
Assay for apoptosis using the mitochondrial probes, Rhodamine123 and 10-N-nonyl acridine orange
Apoptosis plays a pivotal role in the regulation of cell turnover, and a defect or an excess of apoptosis has been implicated in several human diseases. Apoptosis is activated from an extracellular death signal, or from an internal pathway starting from the endoplasmatic reticulum or the mitochondria. To investigate the mitochondrial compartment during apoptosis, we have established a protocol using fluorochromes and flow cytometry to probe the structure and function of mitochondria kinetically. The protocol could be applied to whole cells or to isolated mitochondria. In the first case, cells are counterstained with ethidium bromide (EB) to evaluate plasma membrane function. The presence of the electrochemical gradient in the mitochondria is probed with Rhodamine123 (Rh123), whereas the structure and the integrity of mitochondria are assessed using 10-N-nonyl-acridine orange (NAO). Not considering the time requested for cell/mitochondria preparation and the activation of apoptosis, the protocol lasts <1 h.
Inhibition of cytochrome c release by 10-N-nonyl acridine orange, a cardiolipin-specific dye, during myocardial ischemia-reperfusion in the rat
The release of cytochrome c from the mitochondria to the cytosol is a critical step for downstream caspase-mediated apoptotic signal transduction in ischemia-reperfusion (I/R)-induced myocardial tissue injury. 10-N-nonyl acridine orange (NAO), a cardiolipin-specific dye, has been shown to inhibit Bid-mediated cytochrome c release from isolated mitochondria in vitro; however, the possible protective effects of NAO and the mechanisms underlying the protection from myocardial I/R-induced tissue injury in a rat model are unknown. Male Sprague-Dawley rats were subjected to a 30-min coronary arterial occlusion followed by reperfusion. All rats received either vehicle or NAO (100 microg/kg iv) 10 min before the occlusion. The infarct size in the heart at 24 h after reperfusion was significantly reduced in NAO-treated rats compared with vehicle-treated rats. NAO treatment significantly reduced the cytosolic cytochrome c contents and caspase-9 activity in the ischemic region but did not affect caspase-8 activity. Furthermore, NAO treatment markedly suppressed the translocation of truncated Bid, a proapoptotic Bcl-2 family member, to the mitochondrial fraction. NAO also suppressed the mitochondrial swelling and oxygen uptake stimulated by calcium overload. The results suggest that NAO possesses protective effects against myocardial I/R injury, which may be due to the suppression of cytochrome c release through blockade of truncated Bid translocation to mitochondria and inhibition of the opening of mitochondrial permeability transition pores.
Use of the fluorescent dye 10-N-nonyl acridine orange in quantitative and location assays of cardiolipin: a study on different experimental models
The fluorescent dye 10-N-nonyl acridine orange (NAO) is extensively used for location and quantitative assays of cardiolipin in living cells on the assumption of its high specificity for cardiolipin; however, the limits and the mechanism of this specificity are not clear. Moreover, whether factors such as the membrane potential in mitochondria may limit the consistency of the results obtained by this method is open to discussion. The aim of this research was to investigate the effects of some experimental factors on the selective fluorescence of NAO in the presence of cardiolipin in artificial and natural membranes (mitochondria). The results show that the fluorescence of NAO, due to interaction with cardiolipin, is significantly modified by factors that control the spatial arrangement of cardiolipin molecules within the space of the membrane under investigation. Moreover, the present observations suggest that the specific effect of cardiolipin is to facilitate the dimerization of this fluorescent dye, thus confirming that reliable measurements of cardiolipin concentration can be obtained only when the NAO/cardiolipin molar ratio is equal to 2. The finding is also reported that in isolated respiring mitochondria the interaction of NAO with cardiolipin is somewhat related to the respiratory state of mitochondria.