Elajaili Hanan B, Hernandez-Lagunas Laura, Ranguelova Kalina, Dikalov Sergey, Nozik-Grayck Eva
Cardiovascular Pulmonary Research Laboratories and Pediatric Critical Care Medicine, Department of Pediatrics, University of Colorado Anschutz Medical Campus.
Bruker BioSpin Corp.
J Vis Exp. 2019 Jan 11(143). doi: 10.3791/58461.
The accurate and specific detection of reactive oxygen species (ROS) in different cellular and tissue compartments is essential to the study of redox-regulated signaling in biological settings. Electron paramagnetic resonance spectroscopy (EPR) is the only direct method to assess free radicals unambiguously. Its advantage is that it detects physiologic levels of specific species with a high specificity, but it does require specialized technology, careful sample preparation, and appropriate controls to ensure accurate interpretation of the data. Cyclic hydroxylamine spin probes react selectively with superoxide or other radicals to generate a nitroxide signal that can be quantified by EPR spectroscopy. Cell-permeable spin probes and spin probes designed to accumulate rapidly in the mitochondria allow for the determination of superoxide concentration in different cellular compartments. In cultured cells, the use of cell permeable 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH) along with and without cell-impermeable superoxide dismutase (SOD) pretreatment, or use of cell-permeable PEG-SOD, allows for the differentiation of extracellular from cytosolic superoxide. The mitochondrial 1-hydroxy-4-[2-triphenylphosphonio)-acetamido]-2,2,6,6-tetramethyl-piperidine,1-hydroxy-2,2,6,6-tetramethyl-4-[2-(triphenylphosphonio)acetamido] piperidinium dichloride (mito-TEMPO-H) allows for measurement of mitochondrial ROS (predominantly superoxide). Spin probes and EPR spectroscopy can also be applied to in vivo models. Superoxide can be detected in extracellular fluids such as blood and alveolar fluid, as well as tissues such as lung tissue. Several methods are presented to process and store tissue for EPR measurements and deliver intravenous 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine (CPH) spin probe in vivo. While measurements can be performed at room temperature, samples obtained from in vitro and in vivo models can also be stored at -80 °C and analyzed by EPR at 77 K. The samples can be stored in specialized tubing stable at -80 °C and run at 77 K to enable a practical, efficient, and reproducible method that facilitates storing and transferring samples.
准确、特异性地检测不同细胞和组织区室中的活性氧（ROS）对于研究生物环境中氧化还原调节的信号传导至关重要。电子顺磁共振波谱（EPR）是唯一能明确评估自由基的直接方法。其优点是能以高特异性检测特定种类的生理水平，但它确实需要专门技术、仔细的样品制备和适当的对照，以确保对数据的准确解读。环状羟胺自旋探针与超氧化物或其他自由基选择性反应，产生可通过EPR波谱定量的氮氧化物信号。细胞可渗透的自旋探针和设计用于在线粒体中快速积累的自旋探针可用于测定不同细胞区室中的超氧化物浓度。在培养细胞中，使用细胞可渗透的1-羟基-3-甲氧基羰基-2,2,5,5-四甲基吡咯烷（CMH），并进行或不进行细胞不可渗透的超氧化物歧化酶（SOD）预处理，或使用细胞可渗透的聚乙二醇-SOD，可区分细胞外和胞质超氧化物。线粒体1-羟基-4-[2-（三苯基膦基）-乙酰胺基]-2,2,6,6-四甲基哌啶、1-羟基-2,2,6,6-四甲基-4-[2-（三苯基膦基）乙酰胺基]哌啶二氯化物（mito-TEMPO-H）可用于测量线粒体ROS（主要是超氧化物）。自旋探针和EPR波谱也可应用于体内模型。超氧化物可在细胞外液如血液和肺泡液以及组织如肺组织中检测到。介绍了几种用于处理和储存组织以进行EPR测量以及在体内递送静脉注射1-羟基-3-羧基-2,2,5,5-四甲基吡咯烷（CPH）自旋探针的方法。虽然测量可在室温下进行，但从体外和体内模型获得的样品也可储存在-80°C，并在77 K下通过EPR分析。样品可储存在-80°C稳定的专用管中，并在77 K下运行，以实现一种实用、高效且可重复的方法，便于样品的储存和转移。
