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High-throughput microfluidic single-cell analysis pipeline for studies of signaling dynamics

Nature Protocols volume 9, pages 1713–1726 (2014)Cite this article

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Abstract

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.

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Figure 1: Single-cell live-imaging and gene expression analysis pipeline.
Figure 2: Photos of the microfluidic cell culture system setup.
Figure 3: Cell culture chip layout.
Figure 4: Cell culture chip control GUI.
Figure 5: Anticipated results.

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Acknowledgements

We acknowledge S. Quake for supervising earlier stages of this work. We thank M. Covert and his group for useful discussions and for making an earlier version of the image analysis software available, as well as for the gift of 3T3 cells with the p65-DsRed fusion protein. This work was supported by a European Research Council (ERC) starting grant and a Swiss National Science Foundation grant to S. Tay.

Author information

Author notes

  1. Rafael Gómez-Sjöberg & Anne A Leyrat

    Present address: Present addresses: Quanticel Pharmaceuticals, San Francisco, California, USA (R.G.-S.); Fluidigm Corporation, South San Francisco, California, USA (A.A.L.).,

Authors and Affiliations

  1. Department of Biosystems Science and Engineering,

    Ryan A Kellogg & Savaş Tay

  2. ETH Zürich, Zurich, Switzerland

    Ryan A Kellogg & Savaş Tay

  3. Department of Bioengineering, Stanford University, Stanford, California, USA

    Rafael Gómez-Sjöberg

  4. Department of Bioengineering, Stanford University, Stanford, California, USA

    Anne A Leyrat

Authors
  1. Ryan A Kellogg
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  4. Savaş Tay
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Contributions

R.A.K. optimized the protocol and developed methods for cell retrieval and gene expression analysis, and wrote the manuscript. R.G-.S. and A.A.L. are the original developers of the microfluidic cell culture chip and software. S.T. optimized the protocol for cell signaling studies and supervised the signaling project. All authors edited the manuscript.

Corresponding author

Correspondence to Savaş Tay.

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Competing interests

A.A.L. is an employee of Fluidigm Corporation. The remaining authors declare no competing financial interests.

Supplementary information

Supplementary Data

AutoCad DXF chip design file for cell culture chip molds (flow and control layers) (TXT 25643 kb)

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Kellogg, R., Gómez-Sjöberg, R., Leyrat, A. et al. High-throughput microfluidic single-cell analysis pipeline for studies of signaling dynamics. Nat Protoc 9, 1713–1726 (2014). https://doi.org/10.1038/nprot.2014.120

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