Lucigenin (NSC-151912)
(Synonyms: 光泽精; NSC-151912; L-6868) 目录号 : GC33485
A chemiluminescent probe
Cas No.:2315-97-1
Sample solution is provided at 25 µL, 10mM.
Quality Control & SDS
- View current batch:
- Purity: >98.50%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
本方案仅提供一个指导，请根据您的具体需要进行修改。
1.制备染色液
(1)配置储存液:使用DMSO溶解Lucigenin，配置浓度为5-10mM的储存液。
注意:未使用的储存液分装后在-20℃或-80°C避光保存，避免反复冻融。
(2)配置工作液:使用PBS稀释储存液，配制浓度为5-10μM的工作液。
注意:请根据实际情况调整工作液浓度，现用现配。
2.细胞悬浮染色
(1)悬浮细胞：经4°C、1000g离心3-5分钟，弃去上清液，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Lucigenin工作溶液重悬细胞，室温避光孵育5-30分钟。不同细胞最佳孵育时间不同，请根据具体实验需求自行摸索。
(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，每次5分钟。
(5)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.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100μL的染料工作液，轻轻晃动使染料均匀覆盖所有细胞。
(4) 室温避光孵育5-30分钟。不同细胞最佳孵育时间不同，请根据具体实验需求自行摸索。
(5)孵育结束后吸弃染料工作液，使用预温的培养液清洗盖玻片2~3次。
4.显微镜检测：Lucigenin的最大激发/发射波长为455/505nm。
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。
Lucigenin is a chemiluminescent probe used to detect superoxide production and the presence of chloride.1,2 It can be used to detect superoxide production by enzymatic and cellular sources.1,3,4 It is a sensitive method that has been applied to the monitoring of superoxide production from xanthine oxidase, microsomal NADPH cytochrome reductase, and NADPH oxidases of phagocytes, endothelial cells, fibroblasts, and smooth muscle cells of blood vessel walls.3,5 However, it produces similar chemiluminescence signals in isolated aortic and cardiac tissues from wild-type and Nox1-Nox2-Nox4 triple knockout mice, suggesting that it is not selective for NADPH-based ROS production.6 It also reacts with hydrogen peroxide without generating free radical intermediates and has been used to detect lipid hydroperoxide in oils.7 Lucigenin is also used as a fluorescent chloride-sensitive indicator, with its fluorescence being quenched by chloride (ex/em = 455/505 nm, respectively).2,8,9 Lucigenin fluorescence is insensitive to phosphate, sulfate, and nitrate.2
1.Li, Y., Zhu, H., Kuppusamy, P., et al.Validation of lucigenin (Bis-N-methylacridinium) as a chemilumigenic probe for detecting superoxide anion radical production by enzymatic and cellular systemsJ. Biol. Chem.273(4)2015-2023(1998) 2.Biwersi, J., Tulk, B., and Werkman, A.S.Long-wavelength chloride-sensitive fluorescent indicatorsAnal. Biochem.219(1)139-143(1994) 3.Vladimirov, Y.A., and Proskurnina, E.V.Free radicals and cell chemiluminescenceBiochemistry (Mosc.)74(13)1545-1566(2009) 4.Skatchkov, M.P., Sperling, D., Hink, U., et al.Validation of lucigenin as a chemiluminescent probe to monitor vascular superoxide as well as basal vascular nitric oxide productionBiochem. Biophys. Res. Commun.254(2)319-324(1999) 5.Cai, H., Dikalov, S., Griendling, K.K., et al.Detection of reactive oxygen species and nitric oxide in vascular cells and tissues: Comparison of sensitivity and specificityMethods Mol. Med.139293-311(2007) 6.Rezende, F., L?we, O., Helfinger, V., et al.Unchanged NADPH oxidase activity in Nox1-Nox2-Nox4 triple knockout mice – What do NADPH-stimulated chemiluminescence assays really detect?Antioxid. Redox Signal.24(7)392-399(2016) 7.Bunting, J.P., and Gray, D.A.Development of a flow injection chemiluminescent assay for the quantification of lipid hydroperoxidesJ. Am. Oil Chem. Soc.80(10)951-955(2003) 8.Ferdani, R., Li, R., Pajewski, R., et al.Transport of chloride and carboxyfluorescein through phospholipid vesicle membranes by heptapeptide amphiphilesOrg. Biomol. Chem.5(15)2423-2432(2007) 9.Ruedas-Rama, M.J., Orte, A., Hall, E.A.H., et al.A chloride ion nanosensor for time-resolved fluorimetry and fluorescence lifetime imagingAnalyst137(6)1500-1508(2012)
| Cas No. | 2315-97-1 | SDF | |
| 别名 | 光泽精; NSC-151912; L-6868 | ||
| Canonical SMILES | C[N+]1=C2C=CC=CC2=C(C3=C4C=CC=CC4=[N+](C)C5=C3C=CC=C5)C6=C1C=CC=C6.O=N([O-])=O.O=N([O-])=O | ||
| 分子式 | C28H22N4O6 | 分子量 | 510.5 |
| 溶解度 | DMSO : ≥ 25 mg/mL (48.97 mM) | 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 | 1.9589 mL | 9.7943 mL | 19.5886 mL |
| 5 mM | 0.3918 mL | 1.9589 mL | 3.9177 mL |
| 10 mM | 0.1959 mL | 0.9794 mL | 1.9589 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。
Dithiothreitol-Lucigenin Chemiluminescent System for Ultrasensitive Dithiothreitol and Superoxide Dismutase Detection
Anal Chem 2022 Aug 9;94(31):11023-11029.PMID:35878317DOI:10.1021/acs.analchem.2c01690.
1,4-Dithiothreitol (DTT), a highly water-soluble and well-known reducing agent for preservation and regeneration of sulfhydryl groups in biomedical applications, has been developed as an efficient and stable coreactant of Lucigenin for the first time. DTT efficiently reacts with Lucigenin to generate intense chemiluminescence (CL), eliminating the need for external catalysts to facilitate the Lucigenin CL. The DTT-lucigenin CL is approximately 15-fold more intense when compared with the lucigenin-H2O2 classical system. Superoxide dismutase (SOD) remarkably quenches the DTT-lucigenin CL. Based on this phenomenon, a newly developed CL approach for the determination of SOD was proposed with a linear range of 0.01-1.5 μg/mL and a limit of detection of 2.2 ng/mL. Various factors affecting the CL emission of the DTT-lucigenin probe were studied and optimized. Plausible mechanistic pathways for the CL coreaction of Lucigenin with DTT were proposed and fully discussed. Our proposed method not only has the merit of being selective toward the target analytes but also eliminates the need for the complex synthesis of luminescent probes and facilitates the sensitive detection of SOD in human serum and cosmetics SOD raw material with satisfactory recoveries.
Luminol and Lucigenin as detectors for O2.-
Free Radic Biol Med 1993 Oct;15(4):447-51.PMID:8225026DOI:10.1016/0891-5849(93)90044-u.
Univalent oxidation of luminol and univalent reduction of Lucigenin must precede reaction with O2.- if that reaction is to lead to luminescence. The assumption that luminol or Lucigenin, per se, reacts with O2.- in a way leading to luminescence is incorrect, and leads to misinterpretation of results. The chemical reactions leading to the O2(.-)-dependent luminescences of luminol and of Lucigenin are discussed.
Free radicals and cell chemiluminescence
Biochemistry (Mosc) 2009 Dec;74(13):1545-66.PMID:20210708DOI:10.1134/s0006297909130082.
Application of chemiluminescence (CL) for study of free-radical reactions in human and animal cells and tissues is considered in this review. Historically, the study of intrinsic (ultraweak) luminescence gave place to the measurement of CL in the presence of chemical activators (CL probes) and physical activators (sensitizers) of luminescence, which made the method much more sensitive and specific. The methods of CL and EPR are direct methods of radical investigation, though the advantage of the CL method consists in the fact that CL intensity is directly proportional to a steady-state concentration of the radicals responsible for luminescence (first of all, lipid and oxygen radicals) irrespective the activity of these radicals. The mechanisms of CL reactions in the absence of activators and in the presence of luminol and Lucigenin are considered. Examples of various applications of the CL method in medical, biological, and clinical investigations are given including those for estimation of the phagocytic activity of cells, antioxidant activity, determination of toxicity, and other purposes.
[Chemiluminescent assay for biological substances using Lucigenin]
Yakugaku Zasshi 1997 Nov;117(10-11):864-74.PMID:9414596DOI:10.1248/yakushi1947.117.10-11_864.
The chemiluminescent reaction of Lucigenin with various biological substances has been studied. Chemiluminescence of Lucigenin is produced by the addition of either hydrogen peroxide or organic reducing compounds to Lucigenin in an alkaline solution. On the basis of these reaction, we have developed highly sensitive chemiluminescent methods for the detection of enzyme immunoassay, especially using alkaline phosphatase as a label enzyme, and also for HPLC of corticosteroids or p-nitrophenacyl esters of carboxylic acids. The detection limits of enzymes were 10(-19)-10(-20) mol per assay, corticosteroids and p-nitrophenacyl esters were 500 fmol per injection.
Development of lucigenin-N-hydroxyphthalimide chemiluminescence system and its application to sensitive detection of Co2
Spectrochim Acta A Mol Biomol Spectrosc 2022 Oct 15;279:121459.PMID:35700613DOI:10.1016/j.saa.2022.121459.
N-hydroxyphthalimide (NHPI) is an efficient organic catalyst and an important chemical raw material which can be used as an intermediate in organic synthesis of drugs and pesticides. In this study, NHPI has been used as a coreactant of Lucigenin chemiluminescence (CL) for the first time. The CL of the developed system is significantly enhanced in the presence of Co2+. Therefore, we developed a novel lucigenin-NHPI CL method coupled with flow injection analysis for the sensitive, precise, and selective determination of Co2+. The linear range of this method is 1-1000 nM, and the detection limit is 67 pM (S/N = 3). In addition, this method has a good selectivity for Co2+. It has been applied to the detection of Co2+ in lake water, and the standard recovery rate is 95.9-103.2%, indicating that the method is feasible.