Protocol | Published:

High-throughput microfluidics to control and measure signaling dynamics in single yeast cells

Nature Protocols volume 10, pages 11811197 (2015) | Download Citation

Abstract

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. By 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, while collecting data for thousands of single cells. Compared with other protocols, the present protocol is relatively easy to adopt and of higher throughput. The protocol can be widely used to control and monitor single-cell signaling dynamics in other signal transduction systems in microorganisms.

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Acknowledgements

We thank M. McClean and S. Ramanathan for their help with setting up the original Y-channel microfluidic device. We thank D. MacLaurin and E. Zwiebach-Cohen for discussions. We thank the O'Shea laboratory for discussions and comments on the manuscript. This work was performed in part at the Center for Nanoscale Systems at Harvard University, a member of the National Nanotechnology Infrastructure Network, which is supported by the National Science Foundation under NSF award no. ECS-0335765. This work was supported by the Howard Hughes Medical Institute (to E.K.O'S.) and the US National Institutes of Health grant R01 GM111458 (to N.H.).

Author information

Affiliations

  1. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA.

    • Anders S Hansen
    •  & Erin K O'Shea
  2. Howard Hughes Medical Institute, Harvard University, Northwest Laboratory, Cambridge, Massachusetts, USA.

    • Anders S Hansen
    •  & Erin K O'Shea
  3. Faculty of Arts and Sciences Center for Systems Biology, Harvard University, Northwest Laboratory, Cambridge, Massachusetts, USA.

    • Anders S Hansen
    •  & Erin K O'Shea
  4. Section of Molecular Biology, Division of Biological Sciences, University of California San Diego, La Jolla, California, USA.

    • Nan Hao
  5. Department of Molecular and Cellular Biology, Harvard University, Northwest Laboratory, Cambridge, Massachusetts, USA.

    • Erin K O'Shea

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Contributions

A.S.H. developed the multiplexed microfluidic device, the automated fluid control system, developed MATLAB code and wrote the protocol. N.H. developed the original method of using microfluidics to control analog-sensitive kinases and Msn2 localization. E.K.O'S. supervised the projects. A.S.H., N.H. and E.K.O'S. wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Erin K O'Shea.

Integrated supplementary information

Supplementary figures

  1. 1.

    Control experiment for Figure 2.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figure 1, Supplementary Tutorials 1 and 2

  2. 2.

    Supplementary Data 4: Microscope holder

    Microscope holder sketch in ‘PDF’ format.

Zip files

  1. 1.

    Supplementary Data 1: Transparency mask

    Raw transparency mask file in ‘.eps’ format.

  2. 2.

    Supplementary Data 2: Valve control scripts

    A zip-compressed folder containing MATLAB scripts for controlling and interfacing with solenoid electrovalves as described in Box 1 and Supplementary Tutorial 1.

  3. 3.

    Supplementary Data 3: Image analysis scripts

    A zip-compressed folder containing MATLAB scripts for image analysis as described in Supplementary Tutorial 2.

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DOI

https://doi.org/10.1038/nprot.2015.079

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