Filipin Complex
(Synonyms: 菲律宾菌素复合体) 目录号 : GC18406
A neutral polyene with antifungal activity
Cas No.:11078-21-0
Sample solution is provided at 25 µL, 10mM.
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Quality Control & SDS
- View current batch:
- Purity: >97.50%
- 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)储存液:使用DMSO或无水乙醇溶解Filipin，配置浓度为1-5mM的储存液。
注意:未使用的储存液分装后在-20℃或-80℃避光保存，避免反复冻融。
(2)工作液:使用合适的缓冲液(如：无血清培养基或PBS)稀释储存液，配制浓度为1-250μM的染料工作液。
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：收集细胞，经1000g离心3-5分钟，弃去上清液，用PBS清洗两次，每次约5分钟。
(2)贴壁细胞：使用PBS清洗细胞两次，加入胰酶消化细胞，消化完成后经1000g离心3-5min。
(3)加入1mL的染料工作溶液重悬约5-10x105个细胞，室温避光孵育15-30分钟。
注意：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15-30分钟。
注意：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PBS清洗盖玻片2~3次。
4.显微镜检测：Filipin的最大激发光/发射光波长为380/430nm。
注意事项：
1． Filipin染料染色后，样品的光漂白非常快，建议染色后立即上机检测。
2． 荧光染料均有淬灭的风险，使用过程中请注意避光。
3． 为了您的安全和健康，请穿实验服并戴一次性手套操作。
Filipin complex is a neutral polyene,it has antifungal activity.As a fluorescent compound, Filipin complex can be used for sterol imaging in labeled biological structures,Excitation/emission values of the complexes were 338/480 nm[1，4] . Filipin III is the main component of Filipin complex which is composed of four parts[3] . The interaction of Filipin Complex with steroids was found to be related both to the functional group at the 3-position and the aliphatic chain of the steroid[7] .
Cells were pretreated with 1 and 5 g/mL of Filipin Complex for 1 h and infected with IPNV at a MOI of 1 for 1 h. After 12 h of incubation in the presence of the drug, IPNV VP2/VP3 expression was revealed by immunofluorescence. It shows the effect of Filipin Complex on IPNV infection. No effect on viral infection was observed[2] . The Filipin Complex complex can bind various sterols in aqueous solutions and fungal cell membranes, especially 24α-methyl cholesterol, 24α-ethyl cholesterol, and cholesterol, inducing membrane pit formation and leakage of cell contents[5] . The Filipin Complex complex can inhibit cell growth and mitochondrial terminal electron transport in Saccharomyces cerevisiae[6] .
菲律宾配合物是一种中性多烯，具有抗真菌活性。作为一种荧光化合物，菲律宾配合物可用于标记生物结构中的甾醇成像，配合物的激发/发射值为338/480 nm[1 ，4] . Filipin III是Filipin复合体的主要成分，由四部分组成[3]。发现Filipin Complex与类固醇的相互作用与类固醇3-位官能团和脂肪链有关[7]。
细胞用 1 和 5 g/mL 的菲律宾复合物预处理 1 小时，然后用 MOI 为 1 的 IPNV 感染 1 小时。在药物存在下孵育 12 小时后，通过免疫荧光显示 IPNV VP2/VP3 表达。它显示了 Filipin Complex 对 IPNV 感染的影响。未观察到对病毒感染的影响[2]。 Filipin Complex复合物可结合水溶液和真菌细胞膜中的多种甾醇，尤其是24α-甲基胆固醇、24α-乙基胆固醇和胆固醇，诱导膜凹坑形成和细胞内容物渗漏[5]。 Filipin Complex复合物可抑制酿酒酵母细胞生长和线粒体末端电子传递[6]。
References:
[1]. Castanho MA, Coutinho A, et,al.Absorption and fluorescence spectra of polyene antibiotics in the presence of cholesterol. J Biol Chem. 1992 Jan 5;267(1):204-9. PMID: 1730589
[2]. Levican J, Miranda-Cárdenas C, et,al. Infectious pancreatic necrosis virus enters CHSE-214 cells via macropinocytosis. Sci Rep. 2017 Jun 8;7(1):3068. doi: 10.1038/s41598-017-03036-w. PMID: 28596575; PMCID: PMC5465193.
[3]. Payero TD, Vicente CM, et,al. Functional analysis of filipin tailoring genes from Streptomyces filipinensis reveals alternative routes in filipin III biosynthesis and yields bioactive derivatives. Microb Cell Fact. 2015 Aug 7;14:114. doi: 10.1186/s12934-015-0307-4. PMID: 26246267; PMCID: PMC4527110.
[4]. Kühnl A, Musiol A,et,al.Late Endosomal/Lysosomal Cholesterol Accumulation Is a Host Cell-Protective Mechanism Inhibiting Endosomal Escape of Influenza A Virus. mBio. 2018 Jul 24;9(4):e01345-18. doi: 10.1128/mBio.01345-18. PMID: 30042202; PMCID: PMC6058292.
[5]. Kitajima Y, Sekiya T,et,al. Freeze-fracture ultrastructural alterations induced by filipin, pimaricin, nystatin and amphotericin B in the plasmia membranes of Epidermophyton, Saccharomyces and red complex-induced membrane lesions. Biochim Biophys Acta. 1976 Dec 2;455(2):452-65. doi: 10.1016/0005-2736(76)90317-5. PMID: 793632.
[6]. SHAW PD, ALLAM AM,et,al. EFFECT OF FILIPIN ON THE TERMINAL ELECTRON TRANSPORT SYSTEM OF SACCHAROMYCES CEREVISIAE. Biochim Biophys Acta. 1964 Jul 8;89:33-41. doi: 10.1016/0926-6569(64)90098-7. PMID: 14213010.
[7]. Kleinschmidt MG, Chough KS. Effect of filipin on liposomes prepared with different types of steroids. Plant Physiol. 1972 May;49(5):852-6. doi: 10.1104/pp.49.5.852. PMID: 16658060; PMCID: PMC366064.
| Cas No. | 11078-21-0 | SDF | |
| 别名 | 菲律宾菌素复合体 | ||
| Canonical SMILES | N/A | ||
| 分子式 | C35H58O11 (for Filipin III) | 分子量 | 654.8 |
| 溶解度 | DMF: Soluble,DMSO: Soluble,Ethanol: Soluble,Methanol: Soluble | 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 | 1.5272 mL | 7.6359 mL | 15.2718 mL |
| 5 mM | 0.3054 mL | 1.5272 mL | 3.0544 mL |
| 10 mM | 0.1527 mL | 0.7636 mL | 1.5272 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。
The filipin complex
Separation of the filipin complex by gradient-elution high performance liquid chromatography
A method has been developed for the separation of the filipin complex components by gradient-elution high performance liquid chromatography (HPLC). The elution order for the major filipin components (filipins I approximately IV) was established by first isolating the component fractions by thin-layer chromatography. Each component fraction was then subjected to gradient HPLC. The order of elution for the major filipin components was from first to last: III, IV, II and I. The unexpected reversal in the elution order for filipins III and IV may be evidence that the two filipins are stereoisomeric at the C-1' position. Finally, gradient elution HPLC was used to compare various preparations of filipin. In addition, the technique has been applied to other preparations of polyene antibiotics which have structures similar to that of filipin.
Cholesterol reporter molecules
Cholesterol is a major constituent of the membranes in most eukaryotic cells where it fulfills multiple functions. Cholesterol regulates the physical state of the phospholipid bilayer, affects the activity of several membrane proteins, and is the precursor for steroid hormones and bile acids. Cholesterol plays a crucial role in the formation of membrane microdomains such as "lipid rafts" and caveolae. However, our current understanding on the membrane organization, intracellular distribution and trafficking of cholesterol is rather poor. This is mainly due to inherent difficulties to label and track this small lipid. In this review, we describe different approaches to detect cholesterol in vitro and in vivo. Cholesterol reporter molecules can be classified in two groups: cholesterol binding molecules and cholesterol analogues. The enzyme cholesterol oxidase is used for the determination of cholesterol in serum and food. Susceptibility to cholesterol oxidase can provide information about localization, transfer kinetics, or transbilayer distribution of cholesterol in membranes and cells. The polyene filipin forms a fluorescent complex with cholesterol and is commonly used to visualize the cellular distribution of free cholesterol. Perfringolysin O, a cholesterol binding cytolysin, selectively recognizes cholesterol-rich structures. Photoreactive cholesterol probes are appropriate tools to analyze or to identify cholesterol binding proteins. Among the fluorescent cholesterol analogues one can distinguish probes with intrinsic fluorescence (e.g., dehydroergosterol) from those possessing an attached fluorophore group. We summarize and critically discuss the features of the different cholesterol reporter molecules with a special focus on recent imaging approaches.
Fluorometric evidence for the binding of cholesterol to the filipin complex
Reorganization of membrane cholesterol during membrane fusion in myogenesis in vitro: a study using the filipin-cholesterol complex
Using filipin and freeze-fracture electron microscopy, we examined the distribution of membrane cholesterol during the fusion of myoblasts in vitro. The early stages of fusion were characterized by the depletion of cholesterol from the membrane apposition sites, at which the plasma membranes of two adjacent cells were in close contact. At first, filipin-cholesterol complexes were absent from the plasma membrane of one cell only and were distributed homogeneously on the membrane of the other cell. Eventually, both of the closely apposed membranes became almost completely free the filipin-cholesterol complexes. Membrane fusion took place at several points within the filipin-cholesterol complex-free areas. In later stages, the cytoplasms of the fusing cells became confluent by fenestration of the plasma membranes formed with the filipin-cholesterol complex-free regions. Our observations suggest that membrane cholesterol is reorganized at these fusion sites and that fusion initiated by the juxtaposition of the cholesterol-free areas of each plasma membrane of the adjacent cells.