Mitotracker 染色操作流程曹海燕.docx

Mitotracker染色实验操作流程1.将细胞种植在confocal 专用DISH里，密度为2104；37培养，24-36h等细胞完全贴壁后染色。2. 准备好无血清培养基、移液器、枪头、避光盒等需要仪器；3. 将mitotracker染色试剂用无血清培养基按1：5000的比例稀释，DAPI试剂（罗氏公司）用无血清培养基按1:1000比例稀释，然后放37孵箱里预热10min。4. 移去DISH里的培养基，加少量无血清培养基冲洗三次，去除残余含血清的培养基；5. 每个DISH钟加入500ul预热的染色液，放37孵箱里孵育20-25min（避光操作）。6. 加入DAPI染色液，500ul/dish，室温避光孵育10min。7. 移去染色液，加入少量无血清培养基冲洗三次，最后加入500-1000ul无血清培养基孵育细胞，准备用激光共聚焦显微镜拍照。注：整个染色过程都要注意避光，避免荧光淬灭，影响染色效果。Mito-TrackerGreen(线粒体绿色荧光探针)产品简介Mito-TrackerGreen是一种线粒体(mitochondria)绿色荧光探针，可以用于活细胞线粒体特异性荧光染色。Mito-TrackerGreen为采用MolecularProbes公司的carbocyanine进行了荧光标记的一种Mito-Tracker，分子量为671.88，可以用作线粒体特异性的荧光探针。和rhodamine123或JC-1相比，Mito-TrackerGreen对于线粒体的染色不依赖于线粒体膜电位。Mito-TrackerGreen可以用于对活细胞的染色，但染色后如果固定会导致染色消退。Mito-TrackerGreen呈绿色荧光，检测时的最大激发波长为490nm，最大发射波长为516nm。按最终工作浓度为20-200nM计算，可以配制约370-3700mlMito-TrackerGreen工作液。 Theory：MitoTracker DyesFixable Probes Although conventional fluorescent stains for mitochondria, such as tetramethylrosamineand rhodamine 123, are readily sequestered by functioning mitochondria, these stains are easily washed out of cells once the mitochondria experience a loss in membrane potential. This characteristic limits the use of such conventional stains in experiments that require cells to be treated with aldehyde fixatives or with other agents that affect the energetic state of the mitochondria. To overcome this limitation, Molecular Probes offers the MitoTracker probesa series of patented mitochondrion-selective stains that are concentrated by active mitochondria and well retained during cell fixation.1 The cell-permeant MitoTracker probes contain a mildly thiol-reactive chloromethyl moiety that appears to be responsible for keeping the dye associated with the mitochondria after fixation. To label mitochondria, cells are simply incubated in submicromolar concentrations of a MitoTracker probe, which passively diffuses across the plasma membrane and accumulates in active mitochondria. Once their mitochondria are labeled, the cells can be treated with an aldehyde-based fixative to allow further processing of the sample. Because most of the MitoTracker probes are also retained after permeabilization with detergents or organic solvents, the sample continues to exhibit the fluorescent staining pattern characteristic of live cells during subsequent processing steps (e.g., immunocytochemistry, in situhybridization, or electron microscopy). In addition, MitoTracker probes eliminate some of the difficulties of working with pathogenic cells because, once the mitochondria are stained, the cells can be treated with fixatives before the sample is analyzed.The MitoTracker probes differ in spectral characteristics and fixability (Table 2). MitoTracker probes are provided in specially packaged sets of 20 vials, each containing 50 g for reconstitution as required. Green-Fluorescent MitoTracker Green FM. Mitochondria in cells stained with nanomolar concentrations of MitoTracker Green FM dye (M7514) exhibit bright green, fluorescein-like fluorescence.2 MitoTracker Green FM dye has the added advantage that it is essentially nonfluorescent in aqueous solutions, only becoming fluorescent once it accumulates in the lipid environment of mitochondria. Hence, background fluorescence is negligible, enabling researchers to clearly visualize mitochondria in live cells immediately following addition of the stain, and without a wash step. Furthermore, MitoTracker Green FM dye is substantiall more photostable than the widely used fluorescent dye rhodamine 123 and produces a brighter, more mitochondrion-selective signal at lower concentrations. Because its emission maxima is blue-shifted approximately 10 nm relative to the emission maximum of rhodamine 123, MitoTracker Green FM dye produces a fluorescent staining pattern that should be better resolved from that of red-fluorescent probes in double-labeling experiments. Although MitoTracker Green FM dye can selectively stain mitochondria both in live cells and in cells that have been fixed, the dye is not well retained after cell permeabilization.原理： MITOTRACKER染料 - 固定式探头虽然传统的荧光染色的线粒体，如tetramethylrosamine 和若丹明123，是由线粒体功能，这些污渍很容易被洗出的细胞一旦遇到线粒体膜电位丧失易于隔离。这一特性限制了在需要的细胞也可以与醛固定剂或与影响线粒体的能量状态的其他药物治疗实验中使用这种常规的污渍。为了克服这个限制，Molecular Probes公司提供MITOTRACKER探针，一系列专利线粒体选择性污渍被浓缩活性线粒体和孔细胞中保留fixation.1细胞穿透物MITOTRACKER的探针含有轻度硫醇反应性氯基部分这似乎是一个负责保持与固定后的线粒体有关的染料。以标记线粒体，细胞被简单地温育在亚微摩尔浓度的一个MITOTRACKER探针，其被动扩散穿过细胞膜并蓄积在线粒体活性的。一旦他们的线粒体被标记，细胞可以与醛系固定剂进行处理，以使样品的进一步处理。因为大多数MITOTRACKER探针也透用洗涤剂或有机溶剂后保留，样品继续表现出的活细胞在随后的加工步骤中的荧光染色模式特性（例如，免疫细胞化学，原位杂交中，或电子显微镜）。此外，MITOTRACKER探针消除一些与致病细胞工作的困难，因为，一旦线粒体染色，细胞可以用固定剂处理前，样品是analyzed.The MITOTRACKER探针中的光谱特性和定影性差异（表2 ）。 MITOTRACKER探针在特殊包装套20的小瓶，每个包含如需要50微克用于重构提供。绿色荧光MITOTRACKER绿色调频。线粒体在细胞沾满MITOTRACKER的纳摩尔浓度绿色调频染料（M7514）表现出明亮的绿色，荧光素样fluorescence.2 MITOTRACKER绿色调频染料具有额外的好处，它是在水溶液中基本上无荧光，只有成为荧光一旦积累在线粒体中的脂质环境。因此，背景荧光是微不