High-throughput microfluidic single-cell analysis pipeline for studies of signaling dynamics
High-throughput microfluidic single-cell analysis pipeline for studies of signaling dynamics
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DOI：
10.1038/nprot.2014.120
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发表时间：
2014-07-01
期刊：
影响因子：
14.8
通讯作者：
Tay, Savas
中科院分区：
文献类型：
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作者：
Kellogg, Ryan A.;Gomez-Sjoeberg, Rafael;Tay, Savas
单细胞动态过程的时变分析是信号网络定量研究的一项革命性技术。在这里，我们描述了一个实验管道和相关的协议，将微流控细胞培养，精确刺激细胞信号分子或药物，活细胞显微镜，计算机化的细胞跟踪，芯片上的关键蛋白质染色和随后检索的细胞高通量基因表达分析，使用微流控定量PCR（qPCR）。与传统的培养皿方法相比，该管道将实验精度和通量提高了几个数量级，并在细胞和流体处理方面引入了急需的新功能，从而代表了动态单细胞分析的重要一步。微流控膜阀，自动化和简化的协议相结合，现在使一个研究人员能够在1周内生成100万个关于单细胞蛋白定位的数据点，在各种细胞类型和密度下，在48个预先设计的实验条件下，从不同的信号分子或药物，它们的剂量，时间和组合中选择。
Time-dependent analysis of dynamic processes in single live cells is a revolutionary technique for the quantitative studies of signaling networks. Here we describe an experimental pipeline and associated protocol that incorporate microfluidic cell culture, precise stimulation of cells with signaling molecules or drugs, live-cell microscopy, computerized cell tracking, on-chip staining of key proteins and subsequent retrieval of cells for high-throughput gene expression analysis using microfluidic quantitative PCR (qPCR). Compared with traditional culture dish approaches, this pipeline enhances experimental precision and throughput by orders of magnitude and introduces much-desired new capabilities in cell and fluid handling, thus representing a major step forward in dynamic single-cell analysis. A combination of microfluidic membrane valves, automation and a streamlined protocol now enables a single researcher to generate 1 million data points on single-cell protein localization within 1 week, in various cell types and densities, under 48 predesigned experimental conditions selected from different signaling molecules or drugs, their doses, timings and combinations.