scholarly journals Simultaneous detection of vesicular content and exocytotic release with two electrodes in and at a single cell

2021 ◽  
Author(s):  
Chaoyi Gu ◽  
Andrew Ewing
Keyword(s):  
Single Cell ◽  
Transmitter Release ◽  
Simultaneous Detection

We developed a technique employing two electrodes to simultaneously and dynamically monitor vesicular neurotransmitter storage and vesicular transmitter release in and at the same cell. To do this, two electrochemical...

scholarly journals SUGAR-seq enables simultaneous detection of glycans, epitopes, and the transcriptome in single cells

2021 ◽  
Vol 7 (8) ◽  
pp. eabe3610
Author(s):  
Conor J. Kearney ◽  
Stephin J. Vervoort ◽  
Kelly M. Ramsbottom ◽  
Izabela Todorovski ◽  
Emily J. Lelliott ◽  
...  
Keyword(s):  
Single Cell ◽  
Posttranslational Modification ◽  
Cellular Differentiation ◽  
Single Cells ◽  
Simultaneous Detection ◽  
Surface Protein ◽  
Rna Seq ◽  
Cell Level ◽  
Simultaneous Integration ◽  
Unique Surface

Multimodal single-cell RNA sequencing enables the precise mapping of transcriptional and phenotypic features of cellular differentiation states but does not allow for simultaneous integration of critical posttranslational modification data. Here, we describe SUrface-protein Glycan And RNA-seq (SUGAR-seq), a method that enables detection and analysis of N-linked glycosylation, extracellular epitopes, and the transcriptome at the single-cell level. Integrated SUGAR-seq and glycoproteome analysis identified tumor-infiltrating T cells with unique surface glycan properties that report their epigenetic and functional state.

scholarly journals Development of microfluidic acoustic flow cytometry based on simultaneous ultrasound backscatter and photoacoustics for micron and sub-micron size objects

2021 ◽  
Author(s):  
Vaskar Gnyawali
Keyword(s):  
Single Cell ◽  
Simultaneous Detection ◽  
Flow Cytometer ◽  
Center Frequency ◽  
Nanosecond Laser ◽  
Sufficient Information ◽  
Cell Analysis ◽  
Acoustic Flow ◽  
Ultrasound Backscatter ◽  
Focal Zone

I developed a flow cytometer based on simultaneous detection of ultra-high frequency ultrasound backscatter and photoacoustic waves from individual micron scale objects, such as, cells, microparticles, and microbubbles owing in a microuidic channel. Individual micron scale objects are ow focused through a focal zone, where both ultrasound and laser pulses focus, in a microchannel of a polydimethylsiloxane (PDMS) based microuidic device. At the focal zone, the objects are simultaneously insonified by ultrasound (center frequency 375 MHz) and irradiated by nanosecond laser (532 nm wavelength) pulses. The interactions generate ultrasound backscatter and photoacoustic signals from the individual objects, which are strongly dependent on their size, morphology, and biomechanical properties, such as the Young's modulus, and optical absorption properties. These parameters can be extracted by analyzing the unique spectral features of the detected signals. At frequencies less than 100 MHz, the signals from the micron scale objects do not contain these unique spectral signatures, thus higher frequencies are required. Cell analysis is the main application of interest using the acoustic flow cytometer. Combining ultrasound backscatter and photoacoustics results in sufficient information about a single cell that can be used for single cell analysis and for diagnostics applications. However, the usage of this system is not limited to biological cells. This system can also be used for analyzing individual microbubbles, which are used as ultrasound contrast agents. During my research, a novel microuidic technique is developed to generate microbubbles of desired sizes by shrinking microbubbles from O(100) _m by applying a suitable vacuum pressure. These shrunken bubbles of different sizes can be used as samples to validate the acoustic ow system for microbubble analysis.

SIFTER: Electrophoretic complex fractionation protocol

2021 ◽  
Author(s):  
Julea Vlassakis ◽  
Louise L Hansen ◽  
Amy E Herr
Keyword(s):  
Single Cell ◽  
Protein Interaction ◽  
Gel Electrophoresis ◽  
Protein Complexes ◽  
Single Cells ◽  
Polyacrylamide Gel Electrophoresis ◽  
Simultaneous Detection ◽  
Single Cell Protein ◽  
Cell Protein ◽  
A Cell

Abstract We introduce micro-arrayed, differential detergent fractionation for the simultaneous detection of protein complexes in 100s of individual cells with SIFTER (Single-cell protein Interaction Fractionation Through Electrophoresis and immunoassay Readout). Size-based fractionation of protein complexes is accomplished with five assay steps. First, a cell suspension generated by trypsinization is introduced onto a microwell array, and single cells are settled into the microwells by gravity. Cells are lysed in F-actin stabilization buffer that is delivered by a hydrogel lid. Second, the protein complexes are fractionated from the smaller monomers by polyacrylamide gel electrophoresis. Monomers are electrophoresed into the gel and are immobilized using a UV-induced covalent reaction to benzophenone. Third, a protein-complex depolymerization buffer is introduced by another hydrogel lid. Fourth, the recently depolymerized complexes are electrophoresed into a region of the gel separate from the immobilized monomers, where the complex fraction are in turn immobilized. Fifth, in-gel immunoprobing detects the immobilized populations of monomer and depolymerized complexes. These general steps are built on previously published protocols for bulk actin studies, single-cell western blotting, and bidirectional separations1-4.

Distinct Pathogenesis of Clonal Hematopoiesis Revealed By Single Cell RNA Sequencing Integrated with Highly Sensitive Genotyping Method

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 34-34
Author(s):  
Masahiro Marshall Nakagawa ◽  
Ryosaku Inagaki ◽  
Yutaka Kuroda ◽  
Yasuhito Nannya ◽  
Lanying Zhao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Single Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Simultaneous Detection ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Single Cell Sequencing ◽  
Clonal Hematopoiesis

Background Recent evidence suggests that age-related clonal hematopoiesis (CH) might represent the earliest precursor of myeloid neoplasms. Although the exact mechanism of clonal selection that shapes CH is still to be elucidated, both cell intrinsic and non-cell intrinsic effects of mutations, including the interplay between mutated cells and the bone marrow environment, are thought to play important roles, which are best studied using single-cell sequencing analysis of both mutations and gene expression. Methods We performed single-cell sequencing of hematopoietic stem and progenitors (HSPCs) from BM of the 16 patients with CH along with 16 control patients without CH identified by screening otherwise healthy individuals who received hip joint replacement, using a novel platform that enables simultaneous detection of gene mutations and expression based on the Fluidigm C1-HT system. Sequence data were analyzed with Seurat (Stuart et al Cell 2019) with integration of genotyping information. Cells were clustered and each cluster was assigned by marker-gene expressions for major cell-types in HSPCs, including hematopoietic stem cell (HSC)-like and erythroid progenitors. Cells were grouped by their genotypes and pathway analysis were performed. Results In total, we identified 35 subjects who had CH-related mutations, including those affecting DNMT3A, TET2, ASXL1, SF3B1, PPM1D, IDH1, GNB1 and TP53, of which 11 had more than one CH-related mutation. Most of these mutations showed a low variant allele frequency (VAF) ≤ 0.05. However, clones having double mutations of DNMT3A/TET2 or those having biallelic TET2 mutations tended to show a higher VAF as high as 0.4, suggesting an enhanced clonal advantage for clones having multiple mutations. Using our novel single-cell platform, we analyzed 3,767 cells from control patients without CH and 1,474 mutated cells and 7,234 wild-type (WT) cells from patients with CH. By targeting both genomic DNA and RNA, we successfully obtained a sufficient number of single-cell reads for genes whose expression was too low to evaluate by only targeting RNA, such as TET2 and DNMT3A. Although some clones having a high-VAF mutation caused a skewed clustering to be detected as a CH clone, many clones with low-VAF mutations did not make distinct clusters, indicating the importance of genotyping at a single cell level to identify and characterize mutated cells. Simultaneous detection of genotype and expression allowed us to see the effect of CH-mutations on cell phenotype and differentiation. For example, cells having compound TET2/DNMT3A mutations were significantly enriched in the erythroid cluster, while another clone with double TET2 mutations were more enriched in the HSC-like cluster, compared to cells from individuals without CH (WTcont). These are in line with the previous findings of TET2/DNMT3A double knockout mice or TET2 knockout mice, respectively. In another case with an IDH1 mutation, IDH1-mutated (MUTIDH1) cells less contributed to the HSC-like fraction, showing an enhancement of cell proliferation-signature, compared to WT (WTIDH1) cells in the same patient. Strikingly, compared to WTcont cells, WTIDH1 cells were significantly enriched in the HSC-like fraction and showed an enhanced expression of cytokine-related pathway genes, which was in line with a finding seen in mouse cells treated with 2-hydroxy-glutalate, an mutant IDH-related oncometabolite. Similarly, when compared to WTcont cells, WT cells from patients with DNMT3A- (WTDNMT3A) or TET2- (WTTET2) mutated CH significantly showed an enhanced cell proliferation. HSC-like WTTET2 cells also showed aberrant IFN-response signatures compared to corresponding WTcont cells, which was confirmed in competitive transplantation of Tet2 heterozygous knockout (hKO) and WT cells in a mouse model; HSPCs of WT competitors transplanted with Tet2-hKO cells showed a significant enhancement of IFN-response signatures compared to those transplanted with WT cells. Intriguingly, monocytes of Tet2-hKO donors showed aberrant expression of S100a8/a9, which might contribute to the non-cell intrinsic effect of Tet2-hKO cells. Conclusions In CH, not only mutated cells but also surrounding WT cells show an aberrant gene expression phenotype, suggesting the presence of non-cell autonomous phenotype or an altered bone marrow environment that favors the positive selection of CH-clones. Disclosures Nakagawa: Sumitomo Dainippon Pharma Co., Ltd.: Research Funding. Inagaki:Sumitomo Dainippon Pharma Co., Ltd.: Current Employment. Ogawa:Eisai Co., Ltd.: Research Funding; KAN Research Institute, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Asahi Genomics Co., Ltd.: Current equity holder in private company; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Chordia Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Ultrasensitive and Simultaneous Detection of Two Cytokines Secreted by Single Cell in Microfluidic Droplets via Magnetic-Field Amplified SERS

2019 ◽  
Vol 91 (3) ◽  
pp. 2551-2558 ◽  
Author(s):  
Dan Sun ◽  
Fanghao Cao ◽  
Weiqing Xu ◽  
Qidan Chen ◽  
Wei Shi ◽  
...  
Keyword(s):  
Magnetic Field ◽  
Single Cell ◽  
Simultaneous Detection ◽  
Microfluidic Droplets

scholarly journals PREIMPLANTATION GENETIC TESTING: Single-cell technologies at the forefront of PGT and embryo research

Reproduction ◽  
2020 ◽  
Vol 160 (5) ◽  
pp. A19-A31
Author(s):  
Olga Tšuiko ◽  
Elia Fernandez Gallardo ◽  
Thierry Voet ◽  
Joris Robert Vermeesch
Keyword(s):  
Single Cell ◽  
Embryo Development ◽  
Single Gene ◽  
Simultaneous Detection ◽  
Embryo Research ◽  
Chromosomal Mosaicism ◽  
Genetic Constitution ◽  
Aneuploidy Screening ◽  
Cell Technologies ◽  
The Impact

While chromosomal mosaicism in the embryo was observed already in the 1990s using both karyotyping and FISH technologies, the full extent of this phenomenon and the overall awareness of the consequences of chromosomal instability on embryo development has only come with the advent of sophisticated single-cell technologies. High-throughput techniques, such as DNA microarrays and massive parallel sequencing, have shifted single-cell genome research from evaluating a few loci at a time to the ability to perform comprehensive screening of all 24 chromosomes. The development of genome-wide single-cell haplotyping methods have also enabled for simultaneous detection of single-gene disorders and aneuploidy using a single universal protocol. Today, three decades later haplotyping-based embryo testing is performed worldwide to reliably detect virtually any Mendelian hereditary disease with a known cause, including autosomal-recessive, autosomal-dominant and X-linked disorders. At the same time, these single-cell assays have also provided unique insight into the complexity of embryo genome dynamics, by elucidating mechanistic origin, nature and developmental fate of embryonic aneuploidy. Understanding the impact of postzygotically acquired genomic aberrations on embryo development is essential to determine the still controversial diagnostic value of aneuploidy screening. For that reason, considerable efforts have been put into linking the genetic constitution of the embryo not only to its morphology and implantation potential, but more importantly to its transcriptome using single-cell RNA sequencing. Collectively, these breakthrough technologies have revolutionized single-cell research and clinical practice in assisted reproduction and led to unique discoveries in early embryogenesis.

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