Live-cell Microscopy and Fluorescence-based Measurement of Luminal pH in Intracellular Organelles.
Live-cell Microscopy and Fluorescence-based Measurement of Luminal pH in Intracellular Organelles.
复制标题
细胞显微镜和基于荧光的细胞内细胞器中的腔pH测量。
DOI：
10.3389/fcell.2017.00071
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发表时间：
2017
影响因子：
5.5
通讯作者：
Morrow EM
中科院分区：
文献类型：
--
作者：
Ma L;Ouyang Q;Werthmann GC;Thompson HM;Morrow EM
内腔pH是细胞内细胞器的重要功能特征。细胞器（如内体、溶酶体和高尔基体）内腔的酸化在基本细胞过程中起着关键作用。因此，测量这些细胞器的腔pH值与基础研究和转化研究都相关。与此同时，准确测量活细胞中的细胞器内pH值可能具有挑战性，并且可能是某些领域研究的限制性障碍。在这里，我们描述了三个强大的方法来严格测量不同的细胞内细胞器的腔pH值，专注于内体，溶酶体和高尔基体。所述方法基于pH敏感性荧光探针的实时成像，并且包括：（1）基于转铁蛋白的pH敏感性和pH不敏感性荧光缀合物的内吞作用的定量比率测量的方案;（2）使用用pH敏感性固有荧光蛋白pHluorin的比率测量变体标记的蛋白质的方案;和（3）使用荧光染料LysoSensor™的方案。我们描述了必要的试剂，关键步骤，以及数据采集和分析的方法和设备。提供了用于衍生自癌细胞系的培养细胞和用于小鼠海马神经元的原代培养物的方案的实施的实例。此外，我们提出了不同的描述intraorganellar pH测量方法的优点和缺点。这些方案可能对许多研究人员有益，从基础科学家到那些专注于患者源性细胞疾病的转化研究人员。
Luminal pH is an important functional feature of intracellular organelles. Acidification of the lumen of organelles such as endosomes, lysosomes, and the Golgi apparatus plays a critical role in fundamental cellular processes. As such, measurement of the luminal pH of these organelles has relevance to both basic research and translational research. At the same time, accurate measurement of intraorganellar pH in living cells can be challenging and may be a limiting hurdle for research in some areas. Here, we describe three powerful methods to measure rigorously the luminal pH of different intracellular organelles, focusing on endosomes, lysosomes, and the Golgi apparatus. The described methods are based on live imaging of pH-sensitive fluorescent probes and include: (1) A protocol based on quantitative, ratiometric measurement of endocytosis of pH-sensitive and pH-insensitive fluorescent conjugates of transferrin; (2) A protocol for the use of proteins tagged with a ratiometric variant of the pH-sensitive intrinsically fluorescent protein pHluorin; and (3) A protocol using the fluorescent dye LysoSensor™. We describe necessary reagents, key procedures, and methods and equipment for data acquisition and analysis. Examples of implementation of the protocols are provided for cultured cells derived from a cancer cell line and for primary cultures of mouse hippocampal neurons. In addition, we present strengths and weaknesses of the different described intraorganellar pH measurement methods. These protocols are likely to be of benefit to many researchers, from basic scientists to those conducting translational research with a focus on diseases in patient-derived cells.