Department of Chemistry and Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, CA, USA.
Nat Protoc. 2013 Jun;8(6):1249-59. doi: 10.1038/nprot.2013.064. Epub 2013 May 30.
Mitochondria peroxy yellow 1 (MitoPY1) is a small-molecule fluorescent probe that selectively tracks to the mitochondria of live biological specimens and responds to local fluxes of hydrogen peroxide (H(2)O(2)) by a turn-on fluorescence enhancement. This bifunctional dye uses a triphenylphosphonium targeting group and a boronate-based molecular switch to selectively respond to H(2)O(2) over competing reactive oxygen species (ROS) within the mitochondria. MitoPY1 can be used to measure mitochondrial H(2)O(2) levels in both cell culture and tissue models. In this protocol, we describe the synthesis of MitoPY1 and how to use this chemical tool to visualize mitochondrial H(2)O(2) in live cells. The preparation of MitoPY1 is anticipated to take 7-10 d, and assays involving microscopy of cultured mammalian cells can be performed in 1-2 d.
线粒体过氧黄 1（MitoPY1）是一种小分子荧光探针，它选择性地追踪活生物标本的线粒体，并对局部过氧化氢（H2O2）通量做出反应，荧光增强。这种双功能染料使用三苯基膦靶向基团和硼酸盐基分子开关，选择性地响应线粒体中 H2O2 而不是竞争活性氧物种（ROS）。MitoPY1 可用于测量细胞培养物和组织模型中的线粒体 H2O2 水平。在本方案中，我们描述了 MitoPY1 的合成，以及如何使用这种化学工具在活细胞中可视化线粒体 H2O2。MitoPY1 的制备预计需要 7-10 天，涉及培养的哺乳动物细胞显微镜检测的实验可在 1-2 天内完成。
