Format

Send to

Choose Destination
Nat Protoc. 2013 May;8(5):870-91. doi: 10.1038/nprot.2013.046. Epub 2013 Apr 4.

Single-cell analysis and sorting using droplet-based microfluidics.

Author information

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

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.

PMID:
23558786
PMCID:
PMC4128248
DOI:
10.1038/nprot.2013.046
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Nature Publishing Group Icon for PubMed Central
Loading ...
Support Center