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Single-cell analysis and sorting using droplet-based microfluidics
Open accessAbstract
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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Citations
Cites
High-throughput droplet digital PCR system for absolute quantitation of DNA copy number
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Microdroplet-based PCR enrichment for large-scale targeted sequencing
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Droplet-based single cell RNAseq tools: a practical guide
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“Microfluidic droplet emulsion technology [7, 8] is emerging as a powerful facilitator for cellular and metabolic engineering, as various devices enable precise single-cell compartmentalization [9], culturing [10], phenotyping [11], and sorting [12].”
The Chromatin Regulator HMGA1a Undergoes Phase Separation in the Nucleus**
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Metadata
- Authors
- Mazutis, L., Gilbert, J., Ung, W. L., Weitz, D. A., Griffiths, A. D., & Heyman, J. A.
- Venue
- Nature Protocols
- Published
- 2013-04-04
- Volume
- 8
- Issue
- 5
- Pages
- 870-891
- ISSN
- 1750-2799 (ISSN-L)
- 1754-2189
- OpenAlex
- W2015554529
- Cited by
- 1,355
- References
- 2
- FWCI
- 41.98
- Type
- journal article
- Language
- en
- Open access
- green
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