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Single-cell analysis and sorting using droplet-based microfluidics

Nature Protocols volume 8, pages 870–891 (2013)Cite this article

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Abstract

We present a droplet-based microfluidics protocol for high-throughput analysis and sorting of single cells. Compartmentalization of single cells in droplets enables the analysis of proteins released from or secreted by cells, thereby overcoming one of the major limitations of traditional flow cytometry and fluorescence-activated cell sorting. As an example of this approach, we detail a binding assay for detecting antibodies secreted from single mouse hybridoma cells. Secreted antibodies are detected after only 15 min by co-compartmentalizing single mouse hybridoma cells, a fluorescent probe and single beads coated with anti-mouse IgG antibodies in 50-pl droplets. The beads capture the secreted antibodies and, when the captured antibodies bind to the probe, the fluorescence becomes localized on the beads, generating a clearly distinguishable fluorescence signal that enables droplet sorting at ∼200 Hz as well as cell enrichment. The microfluidic system described is easily adapted for screening other intracellular, cell-surface or secreted proteins and for quantifying catalytic or regulatory activities. In order to screen ∼1 million cells, the microfluidic operations require 2–6 h; the entire process, including preparation of microfluidic devices and mammalian cells, requires 5–7 d.

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Figure 1
Figure 2: Design of microfluidic devices.
Figure 3
Figure 4: Principle of experimental design.
Figure 5: Fluorescence analysis and sorting of droplets.
Figure 6: Schematic of the optical setup.
Figure 7: Compartmentalized single hybridoma cells before and after microfluidic sorting.

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Acknowledgements

We are grateful to R. Sperling for the kind gift of surfactant and D. Aubrecht for his help in developing droplet detection and sorting methods. This work was supported by the National Science Foundation (NSF) (DMR-1006546), the National Institutes of Health (NIH) (P01GM096971 and 5R01EB014703-02), the Harvard Materials Science Research and Engineering Center (DMR-0820484) and the Lithuanian Research Council (MIP-048/2012). W.L.U. acknowledges support from a Canadian National Sciences and Engineering Research Council (NSERC) Postgraduate Scholarship (PGS D). J.A.H. and J.G. were supported by NIH SBIR grant no. 1R43AI082861-01. This work was performed in part at the Center for Nanoscale Systems (CNS), a member of the National Nanotechnology Infrastructure Network (NNIN), which is supported by the NSF under award no. ECS-0335765. CNS is part of Harvard University.

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Authors and Affiliations

  1. School of Engineering and Applied Sciences (SEAS), Harvard University, Cambridge, Massachusetts, USA

    Linas Mazutis, W Lloyd Ung, David A Weitz & John A Heyman

  2. Institute of Biotechnology, Vilnius University, Vilnius, Lithuania

    Linas Mazutis

  3. HabSel Inc.,, Cambridge, Massachusetts, USA

    John Gilbert & John A Heyman

  4. École Supérieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI ParisTech), Centre National de la Recherche Scientifique (CNRS) UMR 7084, Paris, France.,

    Andrew D Griffiths

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Contributions

L.M. and J.A.H. performed the experiments described in this protocol, W.L.U. provided the LabVIEW software, L.M., J.A.H. and A.D.G. analyzed the data; J.G. set up the detection system, all authors edited and proofread the paper.

Corresponding authors

Correspondence to Andrew D Griffiths or John A Heyman.

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Competing interests

The authors are inventors on a patent application (PCT/US2008/008563) including some of the ideas described in this manuscript.

Supplementary information

Supplementary Video 1

Cell and bead co-encapsulation (MOV 353 kb)

Supplementary Video 2

Droplet containing cells and beads reinjection and spacing (MOV 1756 kb)

Supplementary Video 3

Droplet sorting (MOV 495 kb)

Supplementary Data (*dwg format, to be viewed with AutoCAD software)

AutoCad designs of the microfluidic chips (ZIP 1189 kb)

Supplementary Figure 1

Droplet volume as a function of flow rate. Microfluidic channels were 25 μm deep and the design is provided in Supplementary Material and indicated in Figure 2a. The flow rate for the aqueous phase was kept at 180 μl/h, while the flow rate for the continuous phase was varied from 160 μl/h to 300 μl/h (PDF 285 kb)

Supplementary Note

Plate-based sandwich assay to measure secreted antibody (PDF 258 kb)

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Mazutis, L., Gilbert, J., Ung, W. et al. Single-cell analysis and sorting using droplet-based microfluidics. Nat Protoc 8, 870–891 (2013). https://doi.org/10.1038/nprot.2013.046

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