線粒體膜電位（Mitochondrial Membrane Potential,MMP）是判定細胞健康程度、線粒體膜通透性和細胞凋亡的一個重要指標，MMP的喪失通常與細胞凋亡的早期階段有關(guān)。評估線粒體功能狀態(tài)的基于細胞的檢測方法正在成為闡明線粒體活動在藥物誘導毒性、細胞凋亡級聯(lián)以及其他細胞和生化過程中的作用的有用工具。

Enzo Life Sciences的MITO-ID^??Membrane potential detection kit包含一種雙發(fā)射陽離子染料，用于檢測活細胞中的線粒體膜電位（MMP）。在有能量或活躍的細胞中，MITO-ID^??膜電位試劑因其相對負電荷而在線粒體中迅速聚集成發(fā)橙色熒光的聚合體，而在細胞質(zhì)中則以發(fā)綠色熒光的單體存在。然而，在MMP受損的細胞中，MITO-ID?膜電位試劑主要以綠色熒光單體存在于整個細胞質(zhì)中，在線粒體中不再表現(xiàn)出橙色熒光。

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作用機制

該染料的基本化學結(jié)構(gòu)由高度共軛的部分組成，使正電荷廣泛離域。該染料能夠選擇性地進入線粒體，當膜電位增加時，它的顏色會從綠色可逆地變?yōu)槌壬p發(fā)射電位探針）。這種光物理特性是由于在膜極化時可逆地形成J-聚集體，導致在490nm處激發(fā)時，發(fā)射光從～530nm轉(zhuǎn)移到590nm。因此，具有低膜電位的線粒體將積累低濃度的染料，并表現(xiàn)出綠色熒光，而更高極化的線粒體將表現(xiàn)出橙色的熒光。

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?MITO-ID^??Membrane potential detection kit線粒體膜電位檢測試劑盒

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產(chǎn)品特點

●?耐光雙發(fā)射染料，能夠根據(jù)線粒體膜電位狀態(tài)發(fā)出綠色或橙色熒光

●?比JC-1熒光染料靈敏10倍，并具有卓越的水溶性

●?無需洗滌步驟

●?適用于化學/環(huán)境毒性篩選

●?適用于高通量應(yīng)用

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實驗示例

圖1.?用MITO-ID^??Membrane Potential reagent對HeLa細胞的線粒體進行染色，并通過熒光顯微鏡進行觀察。橙色熒光聚集體定位于線粒體（橙色通道），而綠色熒光單體主要定位于細胞質(zhì)（FITC通道）。

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圖2. 對照組和實驗組細胞的流式細胞儀分析。未經(jīng)處理的Jurkat細胞（左）與用1μM CCCP處理15分鐘的Juekat細胞（右），用MITO-ID^??Membrane Potential reagent對細胞進行染色，并使用流式細胞儀檢測。

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產(chǎn)品信息

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部分產(chǎn)品引用文獻

1. Discovery of the 3-Amino-1,2,4-triazine-Based Library as Selective PDK1 Inhibitors with Therapeutic Potential in Highly Aggressive Pancreatic Ductal Adenocarcinoma: D. Carbone, et al.; Int. J. Mol. Sci.?24, 3679 (2023)?

2. Gene signature predicting recurrence in oral squamous cell carcinoma is characterized by increased oxidative phosphorylation: J.K. Noh, et al.; Mol. Oncol.?17, 134 (2023)?

3. Cu(I) and Cu(II) Complexes Based on Lonidamine-Conjugated Ligands Designed to Promote Synergistic Antitumor Effects: F.D. Bello, et al.; Inorg. Chem.?61, 4919 (2022)?

4. Mitochondrial membrane potential-enriched CHO host: a novel and powerful tool for improving biomanufacturing capability: L. Chakrabarti, et al.; MAbs?14, 2020081 (2022)

5. New glycoconjugation strategies for Ruthenium(II) arene complexes via phosphane ligands and assessment of their antiproliferative activity: D. Iacopini, et al.; Bioorg. Chem.?126, 105901 (2022)

6. New tricyclic systems as photosensitizers towards triple negative breast cancer cell: M. Barreca, et al.; Arch. Pharm. Res.?45, 806 (2022)?

7. Role of caspase-8 and/or-9 as biomarkers that can distinguish the potential to cause toxic-and immune related-adverse event, for the progress of acetaminophen-induced liver injury: T. Noda, et al.; Life Sci.?294, 120351 (2022),?Application(s):?Microplate reader?

8. Unveiling the Potential of Innovative Gold(I) and Silver(I) Selenourea Complexes as Anticancer Agents Targeting TrxR and Cellular Redox Homeostasis: M.D. Franco, et al.; Chemistry?28, e202201898 (2022)?

9. Altered inflammatory response in FMRP-deficient microglia: J.M. Parrott, et al.; iScience?24, 103293 (2021),?Application(s):?Fluorescence microscopy?

10. ApoE4 impairs neuron-astrocyte coupling of fatty acid metabolism: G. Qi, et al.; Cell. Rep.?34, 108572 (2021),?Application(s):?Microplate reader?

11. Botrytis cinerea methyl isocitrate lyase mediates oxidative stress tolerance and programmed cell death by modulating cellular succinate levels: L. Oren-Young, et al.; Fungal Genet. Biol.?146, 103484 (2021),?Application(s):?Conidia (fungus spore); microscopy?

12. Copper (II) complexes containing natural flavonoid pomiferin show considerable in vitro cytotoxicity and anti-inflammatory effects: J. Vanco, et al.; Int. J. Mol. Sci.?22, 7626 (2021),?Application(s):?Flow cytometry

13. Depletion of mitochondrial components from extracellular vesicles secreted from astrocytes in a mouse model of fragile X syndrome: B.G. Ha, et al.; Int. J. Mol. Sci.?22, 410 (2021),?Application(s):?Fluorescence microscopy

14. Inhibiting autophagy targets human leukemic stem cells and hypoxic AML blasts by disrupting mitochondrial homeostasis: K.M. Dykstra, et al.; Blood Adv.?5, 2087 (2021)

15. Selective striatal cell loss is ameliorated by regulated autophagy of the cortex: K. Cho & G.W. Kim; Life Sci.?282, 119822 (2021),?Application(s):?Flow cytometry

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MITO-ID^??Membrane potential cytotoxicity kit線粒體膜電位細胞毒性檢測試劑盒

Enzo Life Sciences的MITO-ID^??Membrane potential cytotoxicity kit利用陽離子雙發(fā)射染料檢測線粒體膜電位（MMP）的波動，該染料在細胞質(zhì)中以綠色熒光單體的形式存在，在線粒體中以橙色熒光J聚集體的形式聚集。具有低膜電位的線粒體將積累低濃度的染料并呈現(xiàn)綠色熒光，而更高度極化的線粒體將呈現(xiàn)橙紅色熒光。隨著線粒體功能受損加劇，細胞表現(xiàn)出從橙色熒光到綠色熒光的轉(zhuǎn)變。該試劑盒是一種獨特的HTS測定法，無需洗滌或去除培養(yǎng)基即可實時監(jiān)測線粒體膜電位。

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產(chǎn)品特點

●?靈敏度是JC-1熒光染料的10倍，并具有卓越的水溶性

●?具備光穩(wěn)定性的雙發(fā)射染料

●?無需漂洗/換液步驟

●?單獨的MITO-ID?檢測可用于檢測線粒體質(zhì)量

●?可在較低的藥物/劑量濃度下檢測細胞毒性

●?沒有使用JC-1染料時出現(xiàn)的溶劑偽影

●?適用于高通量應(yīng)用

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實驗示例

圖1. 檢測線粒體紊亂的靈敏度是JC-1的10倍。使用MITO-ID^?膜電位染料（紅色）或JC-1（藍色）在經(jīng)CCCP處理的HeLa細胞中評估線粒體膜電位（MMP）。使用傳統(tǒng)的熒光酶標儀檢測，結(jié)果顯示MMP 隨著CCCP濃度的增加而減少，橙色熒光減少。染料的水溶性優(yōu)化和無需洗滌的實驗方案最大限度減小了可變性，因此Z因子（>0.9）高于使用JC-1.

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圖2. 在藥物篩選中實時檢測有絲分裂毒性。使用BioTek Synergy? Mx熒光酶標儀對線粒體膜電位變化的時間進程研究。HeLa細胞與MITO-ID^?膜電位染料在室溫下孵育30分鐘（不去除血清或培養(yǎng)基）。添加魚藤酮分別達到1μM、3μM和9μM的濃度。橙色信號的減少證明染料對魚藤酮有反應(yīng)。

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產(chǎn)品信息

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部分產(chǎn)品引用文獻

1. Novel Silver Complexes Based on Phosphanes and Ester Derivatives of Bis(pyrazol-1-yl)acetate Ligands Targeting TrxR: New Promising Chemotherapeutic Tools Relevant to SCLC Management: M. Pellei, et al.; Int. J. Mol. Sci.?24, 4091 (2023)?

2. FUNDC1 regulates receptor-mediated mitophagy independently of the PINK1/Parkin-dependent pathway in rotenone-treated SH-SY5Y cells: S.Y. Park, et al.; Food Chem. Toxicol.?137, 111163 (2020),?Application(s):?Fluorescence microscopy and microplate reader

3. Inhibitory role of TRIP-Br1/XIAP in necroptosis under nutrient/serum starvation: Z. Sandag, et al.; Mol. Cells?43, 236 (2020)?

4. NAD hydrolysis by the tuberculosis necrotizing toxin induces lethal oxidative stress in macrophages: D. Pajuelo, et al.; Cell. Microbiol.?22, e13115 (2020),?Application(s):?THP-1 macrophages; microplate reader

5. Impaired autophagic and mitochondrial functions are partially restored by ERT in Gaucher and Fabry diseases: M.M. Ivanova, et al.; PLoS One?14, e0210617 (2019),?Application(s):?Fluorescence microscopy?

6. Inhibitory role of AMPactivated protein kinase in necroptosis of HCT116 colon cancer cells with p53 null mutation under nutrient starvation: D.T. Le, et al.; Int. J. Oncol.?54, 702 (2019)?

7. ROS as a novel indicator to predict anticancer drug efficacy: T. Zaidieh, et al.; BMC Cancer?19, 1224 (2019)?

8. TREM1/3 deficiency impairs tissue repair after acute kidney injury and mitochondrial metabolic flexibility in tubular epithelial cells: A. Tammaro, et al.; Front. Immunol.?10, 1469 (2019),?Application(s):?Microplate reader

9. Anti-cancerous effect of cis-khellactone from Angelica amurensis through the induction of three programmed cell deaths: S. Jung, et al.; Oncotarget?9, 16744 (2018),?Application(s):?Microplate reader?

10. Blue light phototoxicity toward human corneal and conjunctival epithelial cells in basal and hyperosmolar conditions: V. Marek, et al.; Free Radic. Biol. Med.?126, 27 (2018),?Application(s):?Microplate reader

11. cGAS drives noncanonical-inflammasome activation in age-related macular degeneration: N. Kerur, et al.; Nat. Med.?24, 50 (2018)

12. Cytotoxicity of propofol in human induced pluripotent stem cell-derived cardiomyocytes: K. Kido, et al.; J. Anesth.?32, 120 (2018),?Application(s):?Microplate reader

13. Development of novel amino-quinoline-5, 8-dione derivatives as NAD (P) H: quinone oxidoreductase 1 (NQO1) inhibitors with potent antiproliferative activities: Y. Ling, et al.; Eur. J. Med. Chem.?154, 199 (2018)

14. Inhibition of apoptosis using exosomes in Chinese hamster ovary cell culture: S. Han, et al.; Biotechnol. Bioeng.?115, 1331 (2018)

15. Light action spectrum on oxidative stress and mitochondrial damage in A2E-loaded retinal pigment epithelium cells: M. Marie, et al.; Cell Death Disc.?9, 287 (2018)

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