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首页> 外文期刊>Applied Microbiology >Noninvasive High-Throughput Single-Cell Analysis of the Intracellular pH of Saccharomyces cerevisiae by Ratiometric Flow Cytometry
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Noninvasive High-Throughput Single-Cell Analysis of the Intracellular pH of Saccharomyces cerevisiae by Ratiometric Flow Cytometry

机译：比例流式细胞术对酿酒酵母细胞内pH值的无创高通量单细胞分析

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The ability of cells to maintain pH homeostasis in response to environmental changes has elicited interest in basic and applied research and has prompted the development of methods for intracellular pH measurements. Many traditional methods provide information at population level and thus the average values of the studied cell physiological phenomena, excluding the fact that cell cultures are very heterogeneous. Single-cell analysis, on the other hand, offers more detailed insight into population variability, thereby facilitating a considerably deeper understanding of cell physiology. Although microscopy methods can address this issue, they suffer from limitations in terms of the small number of individual cells that can be studied and complicated image processing. We developed a noninvasive high-throughput method that employs flow cytometry to analyze large populations of cells that express pHluorin, a genetically encoded ratiometric fluorescent probe that is sensitive to pH. The method described here enables measurement of the intracellular pH of single cells with high sensitivity and speed, which is a clear improvement compared to previously published methods that either require pretreatment of the cells, measure cell populations, or require complex data analysis. The ratios of fluorescence intensities, which correlate to the intracellular pH, are independent of the expression levels of the pH probe, making the use of transiently or extrachromosomally expressed probes possible. We conducted an experiment on the kinetics of the pH homeostasis of Saccharomyces cerevisiae cultures grown to a stationary phase after ethanol or glucose addition and after exposure to weak acid stress and glucose pulse. Minor populations with pH homeostasis behaving differently upon treatments were identified.
机译：细胞响应环境变化而保持pH稳态的能力引起了基础研究和应用研究的兴趣，并促使细胞内pH测量方法的发展。许多传统方法提供了种群水平的信息，因此也提供了所研究的细胞生理现象的平均值，但不包括细胞培养物非常异质的事实。另一方面，单细胞分析可提供有关种群变异性的更详细的信息，从而有助于对细胞生理学的更深层次的理解。尽管显微镜方法可以解决此问题，但它们在可研究的单个细胞数量少和图像处理复杂方面受到限制。我们开发了一种非侵入性的高通量方法，该方法采用流式细胞仪分析大量表达pHluorin的细胞，pHluorin是一种对pH敏感的遗传编码比例荧光探针。此处描述的方法能够以高灵敏度和速度测量单个细胞的细胞内pH，与以前发布的需要预处理细胞，测量细胞数量或需要复杂数据分析的方法相比，这是一个明显的改进。与细胞内pH相关的荧光强度比与pH探针的表达水平无关，从而可以使用瞬时或染色体外表达的探针。我们进行了一项实验，在添加乙醇或葡萄糖后以及暴露于弱酸胁迫和葡萄糖脉冲后，酿酒酵母培养物的pH稳态趋于稳定。确定了具有不同pH稳态的少数群体，这些群体的行为在治疗后有所不同。

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