High-throughput microfluidics to control and measure signaling dynamics in single yeast cells.
High-throughput microfluidics to control and measure signaling dynamics in single yeast cells.
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DOI：
10.1038/nprot.2015.079
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发表时间：
2015-08
期刊：
影响因子：
14.8
通讯作者：
O'Shea EK
中科院分区：
文献类型：
--
作者：
Hansen AS;Hao N;O'Shea EK
微流体与定量延时荧光显微镜的结合正在改变我们控制、测量和理解单个活细胞中信号动力学的能力。在这里，我们描述了一种管道，它结合了多路微流控细胞培养、用于细胞扰动的自动化可编程流体处理、定量延时显微镜和延时电影的计算分析。我们说明了如何使用这种设置来控制萌芽酵母转录因子MSN2的核定位。使用该方案，我们产生了MSN2定位的振荡，并测量了单个细胞中单个基因的动态基因表达响应。该协议允许一名研究人员在一天内进行多达20项不同的实验，同时收集数千个单细胞的数据。与其他协议相比，本协议相对容易采用，并且具有较高的吞吐量。该方法可广泛用于控制和监测微生物中其他信号转导系统中的单细胞信号动力学。
Microfluidics coupled to quantitative time-lapse fluorescence microscopy is transforming our ability to control, measure, and understand signaling dynamics in single living cells. Here we describe a pipeline that incorporates multiplexed microfluidic cell culture, automated programmable fluid handling for cell perturbation, quantitative time-lapse microscopy, and computational analysis of time-lapse movies. We illustrate how this setup can be used to control the nuclear localization of the budding yeast transcription factor Msn2. Using this protocol, we generate oscillations of Msn2 localization and measure the dynamic gene expression response of individual genes in single cells. The protocol allows a single researcher to perform up to 20 different experiments in a single day, whilst collecting data for thousands of single cells. Compared to other protocols, the present protocol is relatively easy to adopt and higher-throughput. The protocol can be widely used to control and monitor single-cell signaling dynamics in other signal transduction systems in microorganisms.