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

2019 ◽  
Author(s):  
Kara K. Brower ◽  
Catherine Carswell-Crumpton ◽  
Sandy Klemm ◽  
Bianca Cruz ◽  
Gaeun Kim ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Single Cell ◽  
High Throughput ◽  
Single Cell Analysis ◽  
Double Emulsion ◽  
Droplet Microfluidics ◽  
Cell Analysis ◽  
Single Droplet ◽  
Emulsion Droplets ◽  
Droplet Sorting

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 industry-standard FACS instruments. We report the first quantitative plate statistics for double emulsion droplet 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, an advance in droplet sorting comparable with 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.

scholarly journals Double emulsion flow cytometry with high-throughput single droplet isolation and nucleic acid recovery

Lab on a Chip ◽  
2020 ◽  
Vol 20 (12) ◽  
pp. 2062-2074 ◽  
Author(s):  
Kara K. Brower ◽  
Catherine Carswell-Crumpton ◽  
Sandy Klemm ◽  
Bianca Cruz ◽  
Gaeun Kim ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Nucleic Acid ◽  
High Throughput ◽  
Double Emulsion ◽  
Single Droplet ◽  
Emulsion Droplets ◽  
Acid Recovery ◽  
Emulsion Flow ◽  
Double Emulsion Droplets

We have developed a novel workflow (sdDE-FACS, s̲ingle d̲roplet D̲ouble E̲mulsion FACS) that allows robust production, screening, and sorting of single double emulsion droplets with complete nucleic acid recovery.

scholarly journals Development of Droplet Microfluidics Enabling High-Throughput Single-Cell Analysis

Molecules ◽  
2016 ◽  
Vol 21 (7) ◽  
pp. 881 ◽  
Author(s):  
Na Wen ◽  
Zhan Zhao ◽  
Beiyuan Fan ◽  
Deyong Chen ◽  
Dong Men ◽  
...  
Keyword(s):  
Single Cell ◽  
High Throughput ◽  
Single Cell Analysis ◽  
Droplet Microfluidics ◽  
Cell Analysis

scholarly journals Droplet flow cytometry for single-cell analysis

RSC Advances ◽  
2021 ◽  
Vol 11 (34) ◽  
pp. 20944-20960
Author(s):  
Ming Li ◽  
Hangrui Liu ◽  
Siyuan Zhuang ◽  
Keisuke Goda
Keyword(s):  
Flow Cytometry ◽  
Single Cell ◽  
High Throughput ◽  
Single Cell Analysis ◽  
Cell Analysis ◽  
Droplet Flow ◽  
Recent Advances

This work reviews recent advances in the integration of emulsion microdroplets and flow cytometry technologies, so-called droplet flow cytometry (DFC), for high-throughput single-cell analysis.

scholarly journals Large-scale label-free single-cell analysis of paramylon in Euglena gracilis by high-throughput broadband Raman flow cytometry

2020 ◽  
Vol 11 (4) ◽  
pp. 1752 ◽  
Author(s):  
Kotaro Hiramatsu ◽  
Koji Yamada ◽  
Matthew Lindley ◽  
Kengo Suzuki ◽  
Keisuke Goda
Keyword(s):  
Flow Cytometry ◽  
Single Cell ◽  
High Throughput ◽  
Euglena Gracilis ◽  
Large Scale ◽  
Single Cell Analysis ◽  
Cell Analysis ◽  
Label Free

scholarly journals Noninvasive High-Throughput Single-Cell Analysis of HIV Protease Activity Using Ratiometric Flow Cytometry

Sensors ◽  
2013 ◽  
Vol 13 (12) ◽  
pp. 16330-16346 ◽  
Author(s):  
Rok Gaber ◽  
Andreja Majerle ◽  
Roman Jerala ◽  
Mojca Benčina
Keyword(s):  
Flow Cytometry ◽  
Single Cell ◽  
High Throughput ◽  
Protease Activity ◽  
Single Cell Analysis ◽  
Hiv Protease ◽  
Cell Analysis

scholarly journals Noninvasive High-Throughput Single-Cell Analysis of the Intracellular pH of Saccharomyces cerevisiae by Ratiometric Flow Cytometry

2013 ◽  
Vol 79 (23) ◽  
pp. 7179-7187 ◽  
Author(s):  
Mari Valkonen ◽  
Dominik Mojzita ◽  
Merja Penttilä ◽  
Mojca Benčina
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Flow Cytometry ◽  
Single Cell ◽  
Intracellular Ph ◽  
High Throughput ◽  
Single Cell Analysis ◽  
Cell Physiology ◽  
Complex Data ◽  
Cell Analysis ◽  
Ph Homeostasis

ABSTRACTThe ability of cells to maintain pH homeostasis in response to environmental changes has elicited interest in basic and applied research and has prompted the development of methods for intracellular pH measurements. Many traditional methods provide information at population level and thus the average values of the studied cell physiological phenomena, excluding the fact that cell cultures are very heterogeneous. Single-cell analysis, on the other hand, offers more detailed insight into population variability, thereby facilitating a considerably deeper understanding of cell physiology. Although microscopy methods can address this issue, they suffer from limitations in terms of the small number of individual cells that can be studied and complicated image processing. We developed a noninvasive high-throughput method that employs flow cytometry to analyze large populations of cells that express pHluorin, a genetically encoded ratiometric fluorescent probe that is sensitive to pH. The method described here enables measurement of the intracellular pH of single cells with high sensitivity and speed, which is a clear improvement compared to previously published methods that either require pretreatment of the cells, measure cell populations, or require complex data analysis. The ratios of fluorescence intensities, which correlate to the intracellular pH, are independent of the expression levels of the pH probe, making the use of transiently or extrachromosomally expressed probes possible. We conducted an experiment on the kinetics of the pH homeostasis ofSaccharomyces cerevisiaecultures grown to a stationary phase after ethanol or glucose addition and after exposure to weak acid stress and glucose pulse. Minor populations with pH homeostasis behaving differently upon treatments were identified.

scholarly journals Sequentially addressable dielectrophoretic array for high-throughput sorting of large-volume biological compartments

2020 ◽  
Vol 6 (22) ◽  
pp. eaba6712 ◽  
Author(s):  
A. Isozaki ◽  
Y. Nakagawa ◽  
M. H. Loo ◽  
Y. Shibata ◽  
N. Tanaka ◽  
...  
Keyword(s):  
Single Cell ◽  
High Speed ◽  
Single Cell Analysis ◽  
Droplet Microfluidics ◽  
Cell Analysis ◽  
Green Biotechnology ◽  
Speed Flow ◽  
Droplet Sorting ◽  
On Chip

Droplet microfluidics has become a powerful tool in precision medicine, green biotechnology, and cell therapy for single-cell analysis and selection by virtue of its ability to effectively confine cells. However, there remains a fundamental trade-off between droplet volume and sorting throughput, limiting the advantages of droplet microfluidics to small droplets (<10 pl) that are incompatible with long-term maintenance and growth of most cells. We present a sequentially addressable dielectrophoretic array (SADA) sorter to overcome this problem. The SADA sorter uses an on-chip array of electrodes activated and deactivated in a sequence synchronized to the speed and position of a passing target droplet to deliver an accumulated dielectrophoretic force and gently pull it in the direction of sorting in a high-speed flow. We use it to demonstrate large-droplet sorting with ~20-fold higher throughputs than conventional techniques and apply it to long-term single-cell analysis of Saccharomyces cerevisiae based on their growth rate.

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