使用 TMRM 和 Mitotracker Green 共染对原代人皮肤成纤维细胞进行线粒体形态功能分析。
Mitochondrial Morphofunctional Profiling in Primary Human Skin Fibroblasts Using TMRM and Mitotracker Green Co-staining.
机构信息
Department of Pediatrics, Amalia Children's Hospital, Radboud Center for Mitochondrial Medicine, Radboud University Medical Center, Nijmegen, The Netherlands.
Department of Medical BioSciences, Radboud Center for Mitochondrial Medicine, Radboud University Medical Center, Nijmegen, The Netherlands.
出版信息
Methods Mol Biol. 2025;2878:223-232. doi: 10.1007/978-1-0716-4264-1_12.
Mitochondrial morphology and membrane potential (Δψ) are important readouts of mitochondrial function. Integrated analysis of these parameters in living cells can be performed using fluorescent lipophilic cations, which enter cells and accumulate in the mitochondrial matrix in a Δψ-dependent manner. Here, we describe the use of tetramethylrhodamine methyl ester (TMRM) and Mitotracker Green FM (MG) for mitochondrial morphology and semiquantitative Δψ analysis in living primary human skin fibroblasts (PHSFs). Practically, we present an integrated protocol to quantify mitochondrial morphology parameters and signal intensity using epifluorescence microscopy of PHSFs co-stained with TMRM and MG. This approach performs best using large flat cells like PHSFs, which display a high mitochondria-specific fluorescence signal and are imaged at a relatively high (x40) magnification.
线粒体形态和膜电位（Δψ）是线粒体功能的重要指标。使用荧光亲脂性阳离子可以在活细胞中对这些参数进行综合分析，这些阳离子以依赖于Δψ 的方式进入细胞并在线粒体基质中积累。在这里，我们描述了使用四甲基罗丹明甲酯（TMRM）和 Mitotracker Green FM（MG）对活的原代人皮肤成纤维细胞（PHSF）进行线粒体形态和半定量Δψ 分析的方法。实际上，我们提出了一种综合方案，使用 TMRM 和 MG 共染色的 PHSF 的荧光显微镜对线粒体形态参数和信号强度进行定量。这种方法在使用具有高线粒体特异性荧光信号且以相对较高（x40）放大倍数成像的大平面细胞（如 PHSF）时效果最佳。
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