scholarly journals Separations-encoded microparticles for single-cell western blotting

2019 ◽  
Author(s):  
Burcu Gumuscu ◽  
Amy Elizabeth Herr
Keyword(s):  
Single Cell ◽  
Single Cells ◽  
Thin Sheet ◽  
Protein Electrophoresis ◽  
Microfluidic Channels ◽  
Fluorescence Signal ◽  
Analytical Sensitivity ◽  
Planar Array ◽  
Single Cell Isolation ◽  
Mechanical Release

Direct measurement of proteins from single cells has been realized at the microscale using microfluidic channels, capillaries, and semi-enclosed microwell arrays. Although powerful, these formats are constrained, with the enclosed geometries proving cumbersome for multistage assays, including electrophoresis followed by immunoprobing. We introduce a hybrid microfluidic format that toggles between a planar microwell array and a suspension of microparticles. The planar array is stippled in a thin sheet of polyacrylamide gel, for efficient single-cell isolation and protein electrophoresis of hundreds-to-thousands of cells. Upon mechanical release, array elements become a suspension of separations-encoded microparticles for more efficient immunoprobing due to enhanced mass transfer. Dehydrating microparticles offer improved analytical sensitivity owing to in-gel concentration of fluorescence signal for high-throughput single-cell targeted proteomics.

scholarly journals Isolating and culturing of single microbial cells by laser ejection sorting technology

2021 ◽  
Author(s):  
Peng Liang ◽  
Bo Liu ◽  
Yun Wang ◽  
Kunxiang Liu ◽  
Yinping Zhao ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Single Cell ◽  
Single Cells ◽  
Lactobacillus Rhamnosus ◽  
Fluorescence Signal ◽  
Microbial Cells ◽  
E Coli ◽  
Average Survival ◽  
Forward Transfer ◽  
Single Cell Isolation

Single cell isolation and cultivation play an important role in studying physiology, gene expression and functions of microorganisms. A series of single-cell isolation technologies have been developed, among which single-cell ejection technology is one of the most promising. Single cell ejection technology has applied Laser Induced Forward Transfer Technique (LIFT) to isolate bacteria but the viability (or recovery rate) of cells after sorting has not been clarified in the current research progress. In this work, to keep the cells alive as much as possible, we propose a three-layer LIFT system (top layer: 25-nm aluminum film; second layer: 3 μm agar media; third layer: liquid containing bacterial) for the isolation and cultivation of single Gram-negative ( E. coli ), Gram-positive ( Lactobacillus rhamnosus GG, LGG), and eukaryotic microorganisms ( Saccharomyces cerevisiae ). The experiment results showed that the average survival rates for ejected pure single cells were 63% for Saccharomyces cerevisiae , 22% for E. coli DH5α, and 74% for LGG. In addition, we successfully isolated and cultured the GFP expressing E. coli JM109 from the mixture containing complex communities of soil bacteria by fluorescence signal. The average survival rate of E. coli JM109 was demonstrated to be 25.3%. In this study, the isolated and cultured single colonies were further confirmed by colony PCR and sequencing. Such precise sorting and cultivation technique of live single microbial cells could be coupled with other microscopic approaches to isolate single microorganisms with specific functions, revealing their roles in the natural community. Importance We developed a laser induced forward transfer (LIFT) technology to accurately isolate single live microbial cells. The cultivation recovery rates of the ejected single cells were 63% for Saccharomyces cerevisiae , 22% for E. coli DH5α, and 74% for Lactobacillus rhamnosus GG (LGG). Coupled LIFT with fluorescent microscope, we demonstrated that single cells of GFP expressing E. coli JM109 were sorted according to fluorescence signal from a complex community of soil bacteria, and subsequently cultured with 25% cultivation recovery rate. This single cell live sorting technology could isolate single microbes with specific functions, revealing their roles in the natural community.

A microfluidic device enabling deterministic single cell trapping and release

Lab on a Chip ◽  
2021 ◽  
Author(s):  
Huichao Chai ◽  
Yongxiang Feng ◽  
Fei Liang ◽  
Wenhui Wang
Keyword(s):  
Chemical Analysis ◽  
Single Cell ◽  
Microfluidic Device ◽  
Single Cells ◽  
Cell Isolation ◽  
Cell Trapping ◽  
Analysis Techniques ◽  
Single Cell Trapping ◽  
Single Cell Isolation ◽  
Made In

Successful single-cell isolation is a pivotal technique for subsequent biological and chemical analysis of single cells. Although significant advances have been made in single-cell isolation and analysis techniques, most passive...

scholarly journals High-Throughput Single-Cell Cultivation on Microfluidic Streak Plates

2016 ◽  
Vol 82 (7) ◽  
pp. 2210-2218 ◽  
Author(s):  
Cheng-Ying Jiang ◽  
Libing Dong ◽  
Jian-Kang Zhao ◽  
Xiaofang Hu ◽  
Chaohua Shen ◽  
...  
Keyword(s):  
Single Cell ◽  
High Throughput ◽  
Single Cells ◽  
Response Analysis ◽  
Bacterial Strains ◽  
Content Type ◽  
Chemical Gradients ◽  
Droplet Array ◽  
Polycyclic Aromatic ◽  
Single Cell Isolation

ABSTRACTThis paper describes the microfluidic streak plate (MSP), a facile method for high-throughput microbial cell separation and cultivation in nanoliter sessile droplets. The MSP method builds upon the conventional streak plate technique by using microfluidic devices to generate nanoliter droplets that can be streaked manually or robotically onto petri dishes prefilled with carrier oil for cultivation of single cells. In addition, chemical gradients could be encoded in the droplet array for comprehensive dose-response analysis. The MSP method was validated by using single-cell isolation ofEscherichia coliand antimicrobial susceptibility testing ofPseudomonas aeruginosaPAO1. The robustness of the MSP work flow was demonstrated by cultivating a soil community that degrades polycyclic aromatic hydrocarbons. Cultivation in droplets enabled detection of the richest species diversity with better coverage of rare species. Moreover, isolation and cultivation of bacterial strains by MSP led to the discovery of several species with high degradation efficiency, including fourMycobacteriumisolates and a previously unknown fluoranthene-degradingBlastococcusspecies.

scholarly journals A practical solution for preserving single cells for RNA sequencing

2017 ◽  
Author(s):  
Moustafa Attar ◽  
Eshita Sharma ◽  
Shuqiang Li ◽  
Claire Bryer ◽  
Laura Cubitt ◽  
...  
Keyword(s):  
Single Cell ◽  
Single Cells ◽  
Rna Seq ◽  
Rna Integrity ◽  
Tissue Integrity ◽  
Free Amine ◽  
Gene Level ◽  
Single Cell Isolation ◽  
Amine Groups ◽  
Cell Preservation

AbstractThe design and implementation of single-cell experiments is often limited by their requirement for fresh starting material. We have adapted a method for histological tissue fixation using dithio-bis(succinimidyl propionate) (DSP), or Lomant’s Reagent, to stabilise cell samples for single-cell transcriptomic applications. DSP is a reversible cross-linker of free amine groups that has previously been shown to preserve tissue integrity for histology while maintaining RNA integrity and yield in bulk RNA extractions. Although RNA-seq data from DSP-fixed single cells appears to be prone to characteristic artefacts, such as slightly reduced yield of cDNA and a detectable 3’ bias in comparison with fresh cells, cell preservation using DSP does not appear to substantially reduce RNA complexity at the gene level. In addition, there is evidence that instantaneous fixation of cells can reduce inter-cell technical variability. The ability of DSP-fixed cells to retain commonly used dyes, such as propidium iodide, enables the tracking of experimental sub-populations and the recording of cell viability at the point of fixation. Preserving cells using DSP will remove several barriers in the staging of single-cell experiments, including the transport of samples and the scheduling of shared equipment for downstream single-cell isolation and processing.

scholarly journals Laser induced isolation and cultivation of single microbial cells

2020 ◽  
Author(s):  
Peng Liang ◽  
Huan Wang ◽  
Yun Wang ◽  
Yinping Zhao ◽  
Wei E. Huang ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Single Cell ◽  
Cell Sorting ◽  
Single Cells ◽  
Lactobacillus Rhamnosus ◽  
Microbial Cells ◽  
E Coli ◽  
Eukaryotic Microorganisms ◽  
Cultivation Technique ◽  
Single Cell Isolation

AbstractSingle cell isolation and cultivation play an important role in studying physiology, gene expression and functions of microorganisms. Laser Induced Forward Transfer Technique (LIFT) has been applied to isolate single cells but the cell viability after sorting is unclear. We demonstrate that a three-layer LIFT system could be applied to isolate single cells of Gram-negative (E. coli), Gram-positive (Lactobacillus rhamnosus GG, LGG), and eukaryotic microorganisms (Saccharomyces cerevisiae) and the sorted single cells were able to be cultured. The experiment results showed that the average cultivation recovery rate of the ejected single cells were 58% for Saccharomyces cerevisiae, 22% for E. coli, and 74% for Lactobacillus rhamnosus GG (LGG). The identities of the cultured cells from single cell sorting were confirmed by using colony PCR with 16S-rRNA for bacteria and large unit rRNA for yeast and subsequent sequencing. This precise sorting and cultivation technique of live single microbial cells can be coupled with other microscopic approaches (e.g. fluorescent and Raman microscopy) to culture single microorganisms with specific functions, revealing their roles in the natural community.ImportanceSingle cell isolation and cultivation are crucial to recover microorganisms for the study of physiology, gene expression and functions. We developed a laser induced cell sorting technology to precisely isolate single microbial cells from a microscopic slide. More importantly, the isolated single microbial cells are still viable for cultivation. We demonstrate to apply the live sorting method to isolate and cultivate single cells of Gram-negative (E. coli), Gram-positive (Lactobacillus rhamnosus GG, LGG), and eukaryotic microorganisms (Saccharomyces cerevisiae). This precise sorting and cultivation technique can be coupled with other microscopic approaches (e.g. fluorescent and Raman microscopy) to culture specifically targeted single microorganisms from microbial community.Abstract Graphic

Characterization of the Effect of Geometry on Single Cell Adhesion Strength Using a Microfluidic Device

2007 ◽  
Author(s):  
Kevin V. Christ ◽  
Kyle B. Williamson ◽  
Kristyn S. Masters ◽  
Kevin T. Turner
Keyword(s):  
Cell Adhesion ◽  
Single Cell ◽  
Adhesion Strength ◽  
Single Cells ◽  
Microfluidic Channel ◽  
Reynolds Numbers ◽  
Microfluidic Channels ◽  
Low Reynolds Numbers ◽  
Physiological Processes ◽  
Micropipette Manipulation

Cell adhesion plays a crucial role in a number of fundamental physiological processes and is important in the development of implantable biomaterials. Cell adhesion strength has previously been measured using a range of techniques, including population assays (e.g., centrifugation [1], hydrodynamic flow [2]) and single-cell methods (e.g., AFM [3], micropipette manipulation [4]). Population studies are unable to provide detailed information about individual cell behavior, while the single-cell methods are often time-consuming and difficult to perform. Microfluidic channels present a way to generate well-defined stress fields on cells [5]. The small dimensions of these channels result in low Reynolds numbers that allow for the generation of sufficiently large stresses to detach well-spread cells under laminar flow conditions. In the present work, a microfluidic channel was used to controllably load adhered single-cells to detachment and measure the adhesion strength. Using this assay, the effect of cell geometry on adhesion strength was investigated.

scholarly journals Microfluidics 3D gel-island chip for single cell isolation and lineage-dependent drug responses study

Lab on a Chip ◽  
2016 ◽  
Vol 16 (13) ◽  
pp. 2504-2512 ◽  
Author(s):  
Zhixiong Zhang ◽  
Yu-Chih Chen ◽  
Yu-Heng Cheng ◽  
Yi Luan ◽  
Euisik Yoon
Keyword(s):  
Single Cell ◽  
Drug Response ◽  
Cell Tracking ◽  
Single Cells ◽  
Cell Isolation ◽  
Microfluidic Platform ◽  
Drug Responses ◽  
3D Microenvironment ◽  
Single Cell Isolation

This paper reports a novel gel-island microfluidic platform enabling single-cell tracking in biomimetic 3D microenvironment for investigating heterogeneous drug response of single cells.

Impedance-Based Single-Cell Pipetting

2020 ◽  
Vol 25 (3) ◽  
pp. 222-233
Author(s):  
David Bonzon ◽  
Georges Muller ◽  
Jean-Baptiste Bureau ◽  
Nicolas Uffer ◽  
Nicolas Beuchat ◽  
...  
Keyword(s):  
Cell Viability ◽  
Single Cell ◽  
Gold Standard ◽  
Single Cells ◽  
Cell Isolation ◽  
Design Parameters ◽  
Cell Line Development ◽  
A Cell ◽  
Single Cell Isolation ◽  
Biological Methods

Many biological methods are based on single-cell isolation. In single-cell line development, the gold standard involves the dilution of cells by means of a pipet. This process is time-consuming as it is repeated over several weeks to ensure clonality. Here, we report the modeling, designing, and testing of a disposable pipet tip integrating a cell sensor based on the Coulter principle. We investigate, test, and discuss the effects of design parameters on the sensor performances with an analytical model. We also describe a system that enables the dispensing of single cells using an instrumented pipet coupled with the sensing tip. Most importantly, this system allows the recording of an impedance trace to be used as proof of single-cell isolation. We assess the performances of the system with beads and cells. Finally, we show that the electrical detection has no effect on cell viability.

scholarly journals Massively parallel single-cell genome sequencing enables high-resolution analysis of soil and marine microbiome

2020 ◽  
Author(s):  
Yohei Nishikawa ◽  
Masato Kogawa ◽  
Masahito Hosokawa ◽  
Katsuhiko Mineta ◽  
Kai Takahashi ◽  
...  
Keyword(s):  
Single Cell ◽  
Genome Sequencing ◽  
Single Cells ◽  
Gene Clusters ◽  
Sequencing Platform ◽  
High Resolution Analysis ◽  
Resolution Analysis ◽  
Single Cell Isolation ◽  
Cell Genome ◽  
Single Cell Genome

AbstractTo improve our understanding of the environmental microbiome, we developed a single-cell genome sequencing platform, named SAG-gel, which utilizes gel beads for single-cell isolation, cell lysis, and whole genome amplification (WGA) for sequencing. SAG-gel enables serial, parallel and independent reactions of > 100,000 single cells in a single tube, delivering high-quality genome recovery with storable randomized single-cell genome libraries. From soil and marine environmental sources, we acquired 734 partial genomes that are recapitulated in 231 species, 35% of which were assigned as high-to-medium qualities. We found that each genome to be almost unique and 98.7% of them were newly identified, implying the complex genetic diversities across 44 phyla. The various metabolic capabilities including virulence factors and biosynthetic gene clusters were found across the lineages at single-cell resolution. This technology will accelerate the accumulation of reference genomes of uncharacterized environmental microbes and provide us new insights for their roles.

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