Optimized double emulsion flow cytometry with high-throughput single droplet isolation

• Published in: bioRxiv
• Main Authors: Brower, Kara K, Carswell-Crumpton, Catherine, Klemm, Sandy, Cruz, Bianca, Kim, Gaeun, Calhoun, Suzanne G K, Nichols, Lisa, dyce, Polly M
• Format: Paper
• Language: English
• Published: Cold Spring Harbor Cold Spring Harbor Laboratory Press 08.11.2019
• Subjects: Flow cytometry; Microfluidics; Phenotyping; Statistical analysis
• DOI: 10.1101/803460

Droplet microfluidics has made large impacts in diverse areas such as enzyme evolution, chemical product screening, polymer engineering, and single-cell analysis. However, while droplet reactions have become increasingly sophisticated, phenotyping droplets by a fluorescent signal and sorting them to isolate variants-of-interest remains a field-wide bottleneck. Here, we present an optimized double emulsion workflow, sdDE-FACS, that enables high-throughput phenotyping, selection, and sorting of droplets using standard flow cytometers. Using a 130 μm nozzle, we demonstrate robust post-sort recovery of intact droplets, with little to no shear-induced droplet breakage, at high sort frequency (12-14 kHz) across two instruments. We report the first quantitative plate statistics for droplet double emulsion isolation and demonstrate single droplet recovery with >70% efficiency. In addition, we establish complete downstream recovery of nucleic acids from single, sorted double emulsion droplets, comparable to the capabilities of single-cell FACS. This work resolves several hurdles in the field of high-throughput droplet analysis and paves the way for a variety of new droplet assays, including rare variant isolation and multiparameter single-cell analysis, marrying the full power of flow cytometry with droplet microfluidics.