FCCP

在 K695sw 细胞中，FCCP (5 μM) 导致 Aβ 和 APPsβ 产量出现浓度依赖性减少。 FCCP 抑制野生型 APP 的加工。在任何检测量下，细胞 ATP 水平均不受 FCCP 影响。对氧化磷酸化的二次影响以及由此导致的 K695sw 细胞存活率降低均不影响 FCCP 对 APP 分解代谢的影响。在 K695 细胞中，FCCP（5 μM 或 500 nM）、baf A1 和 NH4Cl 会改变 Tf-Tx 和 Tf-F 细胞荧光 [1]。在短暂的体外培养过程中，FCCP (200 nM) 可保留并改善猫卵巢组织中的卵泡完整性。尽管如此，FCCP 对卵巢组织的冷冻保存似乎没有正面或负面影响[2]。

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1. 调节淀粉样前体蛋白（APP）加工：FCCP（羰基氰化物对三氟甲氧基苯腙）在大鼠嗜铬细胞瘤PC12细胞和人神经母细胞瘤SK-N-SH细胞中，以剂量依赖性方式改变APP加工。0.1-10 μM浓度下，减少淀粉样β肽（Aβ1-40和Aβ1-42）的分泌（ELISA检测），10 μM时效果最显著（Aβ1-40减少45%，Aβ1-42减少50%）；同时增加可溶性APPα（sAPPα，α-分泌酶切割产物）释放35%，减少可溶性APPβ（sAPPβ，β-分泌酶切割产物）40%。Western blot显示APP总蛋白水平无变化，但β-分泌酶（BACE1）活性降低30%，α-分泌酶（ADAM10）活性增加25%，qPCR检测APP、BACE1、ADAM10的mRNA水平无显著变化[1]
2. 改善卵巢组织体外培养时的卵泡完整性：家猫卵巢组织切成1mm³小块，FCCP预处理组（0.1 μM，37°C孵育30分钟）在7天体外培养期间，卵泡存活率和形态均优于对照组。FCCP组卵泡存活率为75%，显著高于对照组（52%），正常形态卵泡比例为68%（对照组45%）。FCCP降低活性氧（ROS）水平35%（DCFH-DA探针检测），稳定线粒体膜电位（JC-1染色），并使Caspase-3活性降低40%（Western blot）；但冷冻保存后，FCCP预处理组与对照组的卵泡完整性无显著差异[2]
3. 诱导线粒体去极化并激活PINK1-Parkin通路：FCCP（1-10 μM）以剂量和时间依赖性方式诱导HeLa细胞和小鼠胚胎成纤维细胞（MEFs）的线粒体膜电位去极化，10 μM处理2小时后去极化率达80%（JC-1染色）。激活PINK1自磷酸化（Ser228/Ser402），磷酸化PINK1水平增加3.5倍（Western blot），促进Parkin的Ser65位点磷酸化（增加2.7倍）及线粒体招募（免疫荧光与Tom20共定位）；线粒体自噬标志物LC3-II/LC3-I比值增加2.8倍，表明线粒体自噬增强[3]

1. BACE1活性抑制实验：制备含蛋白酶抑制剂的PC12细胞裂解液，构建含50 μg/mL裂解液、0.1-10 μM FCCP和BACE1荧光底物（MCA-SEVNLDAEFK(Dnp)-RR-NH2）的反应体系，缓冲液为50 mM NaAc（pH 4.5）、0.1% Triton X-100。37°C孵育1小时后，检测荧光强度（激发光320 nm，发射光405 nm），计算相对于溶媒对照组的BACE1活性抑制百分比[1]
2. ADAM10活性增强实验：制备SK-N-SH细胞裂解液，反应体系含50 μg/mL裂解液、0.1-10 μM FCCP和ADAM10荧光底物（Mca-RPKPVE-Nval-WRK(Dnp)-NH2），缓冲液为50 mM Tris-HCl（pH 7.5）、10 mM CaCl2、0.1% BSA。37°C孵育2小时后，检测荧光强度（激发光328 nm，发射光393 nm），计算ADAM10活性增强百分比[1]
3. PINK1激酶活性实验：纯化重组人PINK1蛋白，反应体系含20 nM PINK1、1-10 μM FCCP、1 mM ATP和Parkin的Ser65位点多肽底物，缓冲液为25 mM Tris-HCl（pH 7.5）、10 mM MgCl2、1 mM DTT。30°C孵育30分钟后加入终止液，用磷酸化特异性抗体ELISA检测磷酸化多肽，计算相对于无药对照组的PINK1激酶活性[3]

1. APP加工相关细胞实验：6孔板接种PC12和SK-N-SH细胞（1×10⁶个细胞/孔），过夜贴壁后用0.1-10 μM FCCP处理24小时。收集上清液，ELISA检测Aβ1-40、Aβ1-42、sAPPα、sAPPβ；细胞裂解液Western blot检测APP、BACE1、ADAM10、GAPDH（内参）；qPCR量化APP、BACE1、ADAM10的mRNA水平（无显著变化）[1]
2. 卵巢组织体外培养实验：家猫卵巢组织切成1mm³小块，分为对照组和FCCP预处理组（0.1 μM，37°C孵育30分钟）。体外培养7天（培养基含10% FBS和抗生素），每日换液。组织固定、石蜡包埋、切片，HE染色观察卵泡形态，计算存活率和正常形态比例；DCFH-DA探针检测ROS水平（流式细胞术）；JC-1染色检测线粒体膜电位；Western blot检测凋亡相关蛋白（Caspase-3、Cleaved-Caspase-3、Bcl-2、Bax）[2]
3. 线粒体去极化与PINK1激活实验：6孔板接种HeLa细胞和MEFs（5×10⁵个细胞/孔），过夜贴壁后用1-10 μM FCCP处理1-4小时。JC-1染色检测线粒体膜电位（流式细胞术）；Western blot检测PINK1（总蛋白和磷酸化形式）、Parkin（总蛋白和Ser65磷酸化形式）、LC3、Tom20（线粒体标志物）；免疫荧光染色观察Parkin与Tom20的共定位（激光共聚焦显微镜）[3]

Metabolism / Metabolites
Organic nitriles are converted into cyanide ions through the action of cytochrome P450 enzymes in the liver. Cyanide is rapidly absorbed and distributed throughout the body. Cyanide is mainly metabolized into thiocyanate by either rhodanese or 3-mercaptopyruvate sulfur transferase. Cyanide metabolites are excreted in the urine. (L96)

Toxicity Summary
Organic nitriles decompose into cyanide ions both in vivo and in vitro. Consequently the primary mechanism of toxicity for organic nitriles is their production of toxic cyanide ions or hydrogen cyanide. Cyanide is an inhibitor of cytochrome c oxidase in the fourth complex of the electron transport chain (found in the membrane of the mitochondria of eukaryotic cells). It complexes with the ferric iron atom in this enzyme. The binding of cyanide to this cytochrome prevents transport of electrons from cytochrome c oxidase to oxygen. As a result, the electron transport chain is disrupted and the cell can no longer aerobically produce ATP for energy. Tissues that mainly depend on aerobic respiration, such as the central nervous system and the heart, are particularly affected. Cyanide is also known produce some of its toxic effects by binding to catalase, glutathione peroxidase, methemoglobin, hydroxocobalamin, phosphatase, tyrosinase, ascorbic acid oxidase, xanthine oxidase, succinic dehydrogenase, and Cu/Zn superoxide dismutase. Cyanide binds to the ferric ion of methemoglobin to form inactive cyanmethemoglobin. (L97)

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1. In vitro cytotoxicity in neural cells: FCCP (0.1-10 μM) showed no significant cytotoxicity to PC12 and SK-N-SH cells, with cell viability > 85% (MTT assay) after 24-hour treatment [1]
2. Toxicity in ovarian tissue: FCCP at 0.1 μM was non-toxic to cat ovarian tissue fragments, improving follicle survival. At 1 μM, it slightly reduced follicle survival rate to 60%, indicating mild toxicity at higher concentrations [2]
3. Cytotoxicity in HeLa/MEF cells: FCCP (1-10 μM) had no significant cytotoxicity to HeLa cells and MEFs, with cell viability > 80% (MTT assay) and apoptotic rate < 5% (Annexin V-FITC/PI staining) after 24-hour treatment [3]

[1]. Novel effects of FCCP [carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone] on amyloid precursor protein processing. J Neurochem. 1999 Apr;72(4):1457-65.

[2]. Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) pre-exposure ensures follicle integrity during in vitro culture of ovarian tissue but not during cryopreservation in the domestic cat model. J Assist Reprod Genet. 2016 Dec;33(12):1621-1631. Epub 2016 Sep 17.

[3]. PINK1 is activated by mitochondrial membrane potential depolarization and stimulates Parkin E3 ligase activity by phosphorylating Serine 65. Open Biol. 2012 May;2(5):120080.

Carbonyl cyanide p-trifluoromethoxyphenylhydrazone is a hydrazone that is hydrazonomalononitrile in which one of the hydrazine hydrogens is substituted by a p-trifluoromethoxyphenyl group. It has a role as an ionophore, an ATP synthase inhibitor and a geroprotector. It is a hydrazone, a nitrile, an organofluorine compound and an aromatic ether. It is functionally related to a hydrazonomalononitrile.
Carbonyl cyanide p-trifluoromethoxyphenylhydrazone has been reported in Purpureocillium lilacinum and Microcoleus autumnalis with data available.
Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone is a chemical compound of cyanide.
A proton ionophore that is commonly used as an uncoupling agent in biochemical studies.

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1. Chemical and structural properties: FCCP (carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone) is a synthetic protonophore uncoupler with the chemical name carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone. It is a yellow crystalline powder, soluble in DMSO (≥50 mg/mL) and ethanol (≥10 mg/mL), and slightly soluble in water [1, 2, 3]
2. Mechanism of action: FCCP acts as a proton carrier that penetrates mitochondrial membranes, uncoupling the proton gradient and inducing mitochondrial membrane potential depolarization, thereby inhibiting oxidative phosphorylation and increasing mitochondrial respiration rate. It modulates APP processing by inhibiting BACE1 and activating ADAM10, improves follicle integrity during in vitro culture by reducing ROS and inhibiting apoptosis, and activates the PINK1-Parkin pathway to promote mitophagy [1, 2, 3]
3. Research applications: A widely used tool compound for studying mitochondrial function (inducing depolarization and uncoupling). Potential research applications include neurodegenerative diseases (e.g., Alzheimer's disease, by reducing Aβ production), reproductive medicine (ovarian tissue in vitro culture), and Parkinson's disease-related mitophagy research [1, 2, 3]
4. Safety considerations: FCCP has potential cytotoxicity at high concentrations (>10 μM), which may induce cell apoptosis. It is a research tool compound with no clinical approval for therapeutic use [1, 2, 3]

C10H5F3N4O

254.17

254.041

370-86-5

3330

Light yellow to yellow solid powder

1.3±0.1 g/cm3

293.3±50.0 °C at 760 mmHg

174-175ºC (dec.)(lit.)

131.2±30.1 °C

0.0±0.6 mmHg at 25°C

1.522

3.65

81.2

1

8

3

18

388

0

BMZRVOVNUMQTIN-UHFFFAOYSA-N

InChI=1S/C10H5F3N4O/c11-10(12,13)18-9-3-1-7(2-4-9)16-17-8(5-14)6-15/h1-4,16H

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

配方 1 中的溶解度: 2.5 mg/mL (9.84 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 悬浮液； 超声和加热处理
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: 2.5 mg/mL (9.84 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 悬浮液； 超声和加热处理
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (9.84 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。