Khoo Nicholas K H, Cantu-Medellin Nadiezhda, St Croix Claudette, Kelley Eric E
Departments of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, Pennsylvania.
Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, Pennsylvania.
Curr Protoc Cytom. 2015 Oct 1;74:12.42.1-12.42.11. doi: 10.1002/0471142956.cy1242s74.
A plethora of disease processes are associated with elevated reactive species formation and allied reactions with biomolecules that alter cell signaling, induce overt damage, and promote dysfunction of tissues. Unfortunately, effective detection of reactive species in tissues is wrought with issues that significantly limit capacity for validating species identity, establishing accurate concentrations, and identifying anatomic sites of production. These shortcomings reveal the pressing need for new approaches to more precisely assess reactive species generation in vivo. Herein, we describe an in vivo immuno-spin trapping method for indirectly assessing oxidant levels by detecting free radicals resulting from reaction of oxidants with biomolecules to form stable, immunologically detectable nitrone-biomolecular adducts. This process couples the reactivity and sensitivity of an electron paramagnetic resonance spin trap with the resolution of confocal imaging to visualize the extent of cell and tissue oxidation and anatomic sites of production by detecting resultant free radical formation.
大量疾病过程与活性物质生成增加以及与生物分子的相关反应有关，这些反应会改变细胞信号传导、导致明显损伤并促进组织功能障碍。不幸的是，在组织中有效检测活性物质存在诸多问题，这些问题严重限制了验证物质身份、确定准确浓度以及识别产生部位的能力。这些缺点表明迫切需要新的方法来更精确地评估体内活性物质的产生。在此，我们描述了一种体内免疫自旋捕获方法，通过检测氧化剂与生物分子反应形成稳定的、可免疫检测的硝酮 - 生物分子加合物所产生的自由基，间接评估氧化剂水平。该过程将电子顺磁共振自旋捕获的反应性和灵敏度与共聚焦成像的分辨率相结合，通过检测产生的自由基形成来可视化细胞和组织氧化的程度以及产生部位。
