货号：MD01
线粒体自噬检测试剂盒
Mitophagy Detection Kit
储存条件：0-5度保存
运输条件：室温
特点：
只需添加小分子量荧光试剂即可轻松检测线粒体
可以使用荧光显微镜进行活细胞成像
可以与附着的溶酶体染色剂同时染色
选择规格：1set
凑单关联产品TOP5
NO.1. Cell Counting Kit-8 细胞增殖毒性检测
NO.2. FerroOrange 细胞亚铁离子检测
NO.3. Cellular Senescence Detection Kit 细胞衰老检测
NO.4. Annexin V, FITC Apoptosis Detection Kit 细胞凋亡检测
NO.5. DAPGreen – Autophagy Detection 细胞自噬检测
试剂盒内含
| 1 set | .Mtphagy Dye . Lyso Dye |
×1管 ×1管 |
产品概述
线粒体 (Mitochondria) 是细胞中重要的细胞器之一，可以为细胞活力提供能量 。近年有报道去极化线粒体的积累引起的阿尔茨海默病 (Alzheimer’s Disease) 与帕金森病(Parkinson’s Disease)，可能与线粒体自噬有关。线粒体自噬是一种清除机制，可以通过自噬，将氧化应激、DNA损伤因素导致功能失调的线粒体隔离包裹成自噬体(Autophagosome)，再与溶酶体 (Lysosome) 融合后降解。本试剂盒内含Mtphagy Dye (用于检测线粒体自噬) 和Lyso Dye (溶酶体染料)。Mtphagy Dye通过化学结合，固定在细胞内的线粒体上，会发出较弱的荧光。当线粒体发生自噬，损伤的线粒体会与溶酶体融合，pH会下降，变成酸性，此时Mtphagy Dye会产生较强的荧光。如想直观观察Mtphagy Dye标记的线粒体和溶酶体的结合，可联合应用试剂盒中的Lyso Dye (标记溶酶体) 进行双染。
特点：
1）只需添加小分子量荧光试剂即可轻松检测线粒体
2）可以使用荧光显微镜进行活细胞成像
3）可以与附着的溶酶体染色剂同时染色
原理
记载了本产品的检测原理和实验例的论文请看MD01论文实验例中第四篇：Live Cell Imaging of Mitochondrial Autophagy with a Novel Fluorescent Small Molecule
实验例
1.用羰基氰化物间氯苯腙 (CCCP，一种线粒体解偶联剂) 诱导Parkin表达的HeLa细胞线粒体自噬，并通过荧光显微镜进行检测。另外，通过与线粒体染色试剂（MitoBright Deep Red:MT08）一同染色，能够区分出已发生自噬的的线粒体（白色）和未发生自噬的线粒体（紫色）（照片：右侧）。
波长：
Mtphagy Dye:561 nm (Ex)、650 LP nm (Em)
Lyso Dye:488 nm (Ex)、502-554 nm (Em)
MitoBright Deep Red:640 nm (Ex)、656-700 nm (Em)
2.荧光显微镜观察
HeLa细胞用CCCP处理，并与线粒体检测试剂（Mtphagy Dye）和线粒体染色试剂（MitoBright LT Green）共同染色，并经过一段时间（6小时）后进行检测。
<检测条件>
设备：LSM-700 Laser scanning confocal microscope (LSCM)
(Carl Zeiss, Oberkochen, Germany)
激发波长：
MitoBright LT Green 488 nm
Mtphagy Dye 555 nm
物镜：63x
拍摄时间：6小时
拍摄间隔：15秒
3.自噬诱导和线粒体膜电位变化关系的检测
用羰基氰化物间氯苯腙(CCCP，一种线粒体解偶联剂)诱导Parkin表达的HeLa细胞线粒体自噬，并使用线粒体自噬检测试剂盒（Mitophagy Detection Kit：MD01）和线粒体膜电位检测试剂盒（JC-1 MitoMP Detection Kit：MT09）观察荧光结果。
结果证实在未经CCCP处理的细胞中几乎未检测到线粒体自噬的发生，并且线粒体膜电位正常维持。 另一方面，在添加了CCCP的细胞中，证实了线粒体膜电位的降低（JC-1的红色荧光降低）和线粒体自噬的发生（Mtphagy染料的荧光增强）。
<实验条件>
■将Parkin质粒导入HeLa细胞
使用HilyMax（货号：H357）将Parkin质粒引入HeLa细胞中（Parkin质粒/HilyMax试剂：0.1 μg/0.2 μl）
过夜培养后进行检测。
■线粒体自噬检测
向表达Parkin的HeLa细胞中添加0.1 μmol/l Mtphagy工作溶液，并在37°C下孵育30分钟。然后将细胞用HBSS洗涤，加入10 μg/ml CCCP/MEM溶液，并在37℃下孵育2小时。在荧光显微镜下观察细胞。
■线粒体膜电位检测
将10 μg/ml的CCCP/MEM溶液添加至表达Parkin的HeLa细胞中，并在37℃下孵育1.5小时。加入4 μmol/l的JC-1工作液使其终浓度达到2 μmol/l，并在37℃下孵育30分钟。孵育后，将细胞用HBSS洗涤，加入成像缓冲液，并在荧光显微镜下观察细胞。
<检测条件>
■线粒体自噬检测
Ex：561 nm，Em：570-700 nm
■线粒体膜电位检测
绿色Ex：488 nm，Em：500-550 nm
红色Ex：561 nm，Em：560-610 nm
荧光特性
参考文献
| 序号 | 检测对象 | 使用仪器 | 文献 |
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| 17) | 细胞（BAECs） | 荧光显微镜 | N. Kajihara, D. Kukidome, K. Sada, H. Motoshima, N. Furukawa, T. Matsumura, T. Nishikawa and E. Araki, “Low glucose induces mitochondrial reactive oxygen species via fatty acid oxidation in bovine aortic endothelial cells”, J Diabetes Investig, 2017, 8, (6), 750. |
| 18) | 细胞（HT22） | 荧光显微镜 | M. Jin, H. Ni and L. Li, “Leptin Maintained Zinc Homeostasis Against Glutamate-Induced Excitotoxicity by Preventing Mitophagy-Mediated Mitochondrial Activation in HT22 Hippocampal Neuronal Cells.”, Front Neurol, 2018, 9, (9), 332. |
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