Single-cell analysis and sorting using droplet-based microfluidics

By L. Mazutis, J. Gilbert, W. L. Ung, David A. Weitz, A. Griffiths, J. Heyman. Nature Protocols, 2013

1,241 citations recorded by the source corpus.

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.

Subfields: Biomedical Engineering, Electrical and Electronic Engineering

View Paper: https://www.semanticscholar.org/paper/31e3213b9e3f5dc91a3134e306a9ef50fb81ecb8

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In Biomedical Engineering

In Electrical and Electronic Engineering

David A. Weitz

Citations in the corpus

Exponential scaling of single-cell RNA-seq in the past decade (2017, methodology): “ number of IFC’s to create big data sets (Shalek et al., 2013; Zeisel et al., 2015). More recently, methods emerged to randomly capture and manipulate individual cells in nanoliter droplet emulsions (Mazutis et al., 2013). The inDrop and Drop-seq protocols both present strategies to isolate cells in”
The technology and biology of single-cell RNA sequencing. (2015, background): “Additional promising technologies on the horizon are microdroplet-based microfluidics methods, which have the potential to capture thousands of cells in a single experiment (Mazutis et al., 2013), and microwell technologies such as CytoSeq (Fan et al., 2015).”
Droplet microfluidics: A tool for biology, chemistry and nanotechnology (2016, neutral): “Here, the probability of finding a certain number of cells inside a droplet follows the Poisson distribution [28].”
Droplet barcoding for single cell transcriptomics applied to embryonic stem cells (2015, positive): “Cells can be compartmentalized into droplets and assayed for different bio-molecules (Mazutis et al. 2013), their genes amplified (Eastburn et al. 2013) and droplets sorted at high-throughput rates (Agresti et al. 2010).”