Preparation and use of MitoPY1 for imaging hydrogen peroxide in mitochondria of live cells.
Preparation and use of MitoPY1 for imaging hydrogen peroxide in mitochondria of live cells.
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DOI：
10.1038/nprot.2013.064
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发表时间：
2013-06
期刊：
影响因子：
14.8
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中科院分区：
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线粒体过氧黄1（MitoPY 1）是一种小分子荧光探针，其选择性地追踪活生物样品的线粒体，并通过开启荧光增强来响应过氧化氢（H2 O2）的局部通量。这种双功能染料使用三苯基磷靶向基团和基于硼酸盐的分子开关，以选择性地响应H2 O2而不是线粒体内的竞争性活性氧（ROS）。MitoPY 1可用于测量细胞培养和组织模型中的线粒体H2 O2水平。在本协议中，我们描述了MitoPY 1的合成以及如何使用这种化学工具来可视化活细胞中的线粒体H2 O2。MitoPY 1的制备预计需要7-10天，而涉及培养的哺乳动物细胞的显微镜检查的测定可以在1-2天内进行。
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 (H2O2) by a turn-on fluorescence enhancement. This bifunctional dye uses a triphenylphosphonium targeting group and a boronate-based molecular switch to selectively respond to H2O2 over competing reactive oxygen species (ROS) within the mitochondria. MitoPY1 can be used to measure mitochondrial H2O2 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 H2O2 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.