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1.
Cell Prolif ; 53(4): e12788, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32153074

RESUMO

OBJECTIVES: Terahertz (THz)-based imaging techniques hold great potential for biological and biomedical applications, which nevertheless are hampered by the low spatial resolution of conventional THz imaging systems. In this work, we report a high-performance photoconductive antenna microprobe-based near-field THz time-domain spectroscopy scanning microscope. MATERIALS AND METHODS: A single watermelon pulp cell was prepared on a clean quartz slide and covered by a thin polyethylene film. The high performance near-field THz microscope was developed based on a coherent THz time-domain spectroscopy system coupled with a photoconductive antenna microprobe. The sample was imaged in transmission mode. RESULTS: We demonstrate the direct imaging of the morphology of single watermelon pulp cells in the natural dehydration process with our near-field THz microscope. CONCLUSIONS: Given the label-free and non-destructive nature of THz detection techniques, our near-field microscopy-based single-cell imaging approach sheds new light on studying biological samples with THz.


Assuntos
Microscopia de Varredura por Sonda/instrumentação , Análise de Célula Única/instrumentação , Imagem Terahertz/instrumentação , Citrullus/citologia , Dessecação , Desenho de Equipamento , Humanos
2.
PLoS One ; 15(1): e0227601, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31978064

RESUMO

The diversity of living cells, in both size and internal complexity, calls for imaging methods with adaptable spatial resolution. Soft x-ray tomography (SXT) is a three-dimensional imaging technique ideally suited to visualizing and quantifying the internal organization of single cells of varying sizes in a near-native state. The achievable resolution of the soft x-ray microscope is largely determined by the objective lens, but switching between objectives is extremely time-consuming and typically undertaken only during microscope maintenance procedures. Since the resolution of the optic is inversely proportional to the depth of focus, an optic capable of imaging the thickest cells is routinely selected. This unnecessarily limits the achievable resolution in smaller cells and eliminates the ability to obtain high-resolution images of regions of interest in larger cells. Here, we describe developments to overcome this shortfall and allow selection of microscope optics best suited to the specimen characteristics and data requirements. We demonstrate that switchable objective capability advances the flexibility of SXT to enable imaging cells ranging in size from bacteria to yeast and mammalian cells without physically modifying the microscope, and we demonstrate the use of this technology to image the same specimen with both optics.


Assuntos
Imageamento Tridimensional/métodos , Análise de Célula Única/métodos , Tomografia por Raios X/instrumentação , Tomografia por Raios X/métodos , Linfócitos B/citologia , Desenho de Equipamento , Escherichia coli/citologia , Humanos , Schizosaccharomyces/citologia , Análise de Célula Única/instrumentação
3.
Talanta ; 206: 120174, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31514890

RESUMO

A method of simultaneous cell counting and determination of metals in single cells using time-resolved inductively coupled plasma-mass spectrometry (ICP-MS) was reported. A facile, low cost and highly efficient single-cell introduction system of time-resolved ICP-MS consists of a flow cell, a visual contrast calibration device, a customized nebulizer and a fabricated spray chamber. The flow cell includes a cell sample tube, a sheath liquid tube and a flow chamber. The visual contrast calibration device was composed of a microscope with a 16 × microscope objective (160 × total magnification). The flow chamber was used to combine a flow of red blood cell suspension (0.800 µL/min) and a flow of PBS (4.40 µL/min) into the nebulizer. The intact cells were directly introduced with the single-cell introduction system into the plasma via nebulizing, and then ion plumes corresponding to single cells were individually detected with mass spectrometer. The frequency of the spikes directly reflects the number of cells, and the intensity of spikes is proportional to the concentration of copper within one cell. The single-cell introduction system can be transported into the ICP-MS via a customized transport system with 100% efficiency. A high cell introduction efficiency into the plasma supports for a reduction of cell consumption. The Cu signal frequency was about 120 cell events per minute. This single-cell introduction system simplifies the introduction of individual and intact cells. The copper content in single red blood cell was 0.20-0.40 fg.


Assuntos
Cobre/análise , Eritrócitos/química , Humanos , Limite de Detecção , Espectrometria de Massas/instrumentação , Espectrometria de Massas/métodos , Nebulizadores e Vaporizadores , Análise de Célula Única/instrumentação , Análise de Célula Única/métodos
4.
Nat Commun ; 10(1): 5729, 2019 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-31844066

RESUMO

While Tn-Seq is a powerful tool to determine genome-wide bacterial fitness in high-throughput, culturing transposon-mutant libraries in pools can mask community or other complex single-cell phenotypes. Droplet Tn-Seq (dTn-Seq) solves this problem by microfluidics facilitated encapsulation of individual transposon mutants into growth medium-in-oil droplets, thereby enabling isolated growth, free from the influence of the population. Here we describe and validate microfluidic chip design, production, encapsulation, and dTn-Seq sample preparation. We determine that 1-3% of mutants in Streptococcus pneumoniae have a different fitness when grown in isolation and show how dTn-Seq can help identify leads for gene function, including those involved in hyper-competence, processing of alpha-1-acid glycoprotein, sensitivity against the human leukocyte elastase and microcolony formation. Additionally, we show dTn-Seq compatibility with microscopy, FACS and investigations of bacterial cell-to-cell and bacteria-host cell interactions. dTn-Seq reduces costs and retains the advantages of Tn-Seq, while expanding the method's original applicability.


Assuntos
Elementos de DNA Transponíveis/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Técnicas Analíticas Microfluídicas/métodos , Análise de Sequência de DNA/métodos , Análise de Célula Única/métodos , DNA Bacteriano/genética , Biblioteca Gênica , Genes Bacterianos/genética , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Mutação , Análise de Célula Única/instrumentação , Streptococcus pneumoniae/genética
5.
Nat Commun ; 10(1): 4576, 2019 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-31594952

RESUMO

Single-cell ATAC-seq (scATAC-seq) profiles the chromatin accessibility landscape at single cell level, thus revealing cell-to-cell variability in gene regulation. However, the high dimensionality and sparsity of scATAC-seq data often complicate the analysis. Here, we introduce a method for analyzing scATAC-seq data, called Single-Cell ATAC-seq analysis via Latent feature Extraction (SCALE). SCALE combines a deep generative framework and a probabilistic Gaussian Mixture Model to learn latent features that accurately characterize scATAC-seq data. We validate SCALE on datasets generated on different platforms with different protocols, and having different overall data qualities. SCALE substantially outperforms the other tools in all aspects of scATAC-seq data analysis, including visualization, clustering, and denoising and imputation. Importantly, SCALE also generates interpretable features that directly link to cell populations, and can potentially reveal batch effects in scATAC-seq experiments.


Assuntos
/métodos , Análise de Dados , Modelos Estatísticos , Análise de Célula Única/métodos , Animais , Análise por Conglomerados , Conjuntos de Dados como Assunto , Células HEK293 , Humanos , Leucemia/genética , Neoplasias Mamárias Experimentais/genética , Camundongos , Distribuição Normal , Análise de Célula Única/instrumentação , Células-Tronco
6.
Nat Protoc ; 14(11): 3126-3143, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31554956

RESUMO

Mutations are the driving force of evolution and the source of important pathologies. The characterization of the dynamics and effects of mutations on fitness is therefore central to our understanding of evolution and to human health. This protocol describes how to implement two methods that we recently developed: mutation visualization (MV) and microfluidic mutation accumulation (µMA), which allow the occurrence of mutations created by DNA replication errors (MV) and the evolution of cell fitness during MA (µMA) to be followed directly in individual cells of Escherichia coli. MV provides a quantitative characterization of the dynamics of mutation occurrences, and µMA allows precise estimation of the distribution of fitness effects (DFEs) of mutations. Both methods use microfluidics and time-lapse microscopy, and a fluorescent mismatch repair (MMR) MutL protein is used as a marker for nascent mutations. Here, we present a single protocol describing how to implement the MV and µMA methods, including detailed procedures for microfluidic setup installation, data acquisition and data analysis and interpretation. Using this procedure, the microfluidic setup installation can be completed within 1 d, and automated data acquisition takes 2-4 d.


Assuntos
Escherichia coli/genética , Mutação , Análise de Célula Única/instrumentação , Replicação do DNA , Desenho de Equipamento , Escherichia coli/citologia , Proteínas de Escherichia coli/genética , Viabilidade Microbiana , Técnicas Analíticas Microfluídicas/instrumentação , Microscopia de Fluorescência/instrumentação , Acúmulo de Mutações
7.
Nat Commun ; 10(1): 3544, 2019 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-31391463

RESUMO

Simultaneous measurement of proteins and mRNA in single cells enables quantitative understanding and modeling of cellular functions. Here, we present an automated microfluidic system for multi-parameter and ultra-sensitive protein/mRNA measurements in single cells. Our technology improves the sensitivity of digital proximity ligation assay by up to 55-fold, with a detection limit of 2277 proteins per cell and with detection efficiency of as few as 29 protein molecules. Our measurements using this system reveal higher mRNA/protein correlation in single mammalian cells than previous estimates. Furthermore, time-lapse imaging of herpes simplex virus 1 infected epithelial cells enabled by our device shows that expression of ICP4 -a major transcription factor regulating hundreds of viral genes- is only partially correlated with viral protein counts, suggesting that many cells go through abortive infection. These results highlight the importance of high-sensitivity protein/mRNA quantification for understanding fundamental molecular mechanisms in individual cells.


Assuntos
Proteínas/isolamento & purificação , RNA Mensageiro/isolamento & purificação , Análise de Célula Única/métodos , Animais , Dosagem de Genes , Humanos , Microscopia Intravital/instrumentação , Microscopia Intravital/métodos , Dispositivos Lab-On-A-Chip , Limite de Detecção , Microfluídica/instrumentação , Microfluídica/métodos , Análise de Célula Única/instrumentação , Imagem com Lapso de Tempo/instrumentação , Imagem com Lapso de Tempo/métodos , Células Vero
8.
Anal Chim Acta ; 1076: 118-124, 2019 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-31203955

RESUMO

The quantification of low concentration proteins can facilitate the discovery of some significant biomarkers, and provide us a more profound understanding of cell heterogeneity when applied to single cell analysis. However, most state-of- art single cell protein detection platforms are bulky, expensive and complicated. Here we report a simple and low cost microfluidic dPCR (digital polymerase chain reaction) chip-based proximity ligation assay (PLA) for the quantification of low concentration proteins. First, standard hCSTB (human cystatin B) protein was used to optimize the related experimental conditions. Comparing to ordinary PLA tests, the results showed that our method achieved femtomolar limit of detection (LOD) with a linear dynamic range over three to four orders of magnitude. Then human CD147 protein, a reported biomarker for hepatoma carcinoma, was detected in single HepG2 and L02 cells. The results showed that there were wide disparities in single cell CD147 abundance for both of the two cell lines. And the average CD147 protein content in single HepG2 cells displayed 2-fold increase in comparison to that in single L02 cells. Comparing to the research findings obtained at bulk level, our method can provide more useful information for diagnosis and targeted therapy of tumors.


Assuntos
Basigina/análise , Cistatina B/análise , Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas/métodos , Biomarcadores Tumorais/análise , Linhagem Celular Tumoral , Humanos , Limite de Detecção , Técnicas Analíticas Microfluídicas/instrumentação , Reação em Cadeia da Polimerase/instrumentação , Reação em Cadeia da Polimerase/métodos , Análise de Célula Única/instrumentação , Análise de Célula Única/métodos
9.
IEEE Trans Nanobioscience ; 18(3): 369-372, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31180894

RESUMO

Fine needle aspirate sampling of tumors requires acquisition of sufficient cells to complete a diagnosis. Aspirates through such fine needles are typically composed of small cell clusters in suspension, making them readily amenable to microfluidic analysis. Here we show a microfluidic device with integrated electrodes capable of interrogating and identifying cellular components in a patient-derived sample of dissociated tumor cells using micro-electrical impedance spectroscopy ( µ EIS). We show that the µ EIS system can distinguish dissociated tumor cells in a sample consisting of red blood cell (RBCs) and peripheral blood mononucleated cells (PBMCs). Our µ EIS system can also distinguish dissociated tumor cells from normal cells and we show results for five major cancer types, specifically, lung, thyroid, breast, ovarian, and kidney cancer. Moreover, our µ EIS system can make these distinctions in a label-free manner, thereby opening the possibility of integration into standard clinical workflows at the point of care.


Assuntos
Espectroscopia Dielétrica , Neoplasias/patologia , Análise de Célula Única , Células Tumorais Cultivadas/citologia , Biópsia por Agulha Fina , Espectroscopia Dielétrica/instrumentação , Espectroscopia Dielétrica/métodos , Desenho de Equipamento , Citometria de Fluxo/instrumentação , Humanos , Técnicas Analíticas Microfluídicas/instrumentação , Análise de Célula Única/instrumentação , Análise de Célula Única/métodos
10.
Nat Protoc ; 14(7): 2015-2035, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31168087

RESUMO

Measurements of a single entity underpin knowledge of the heterogeneity and stochastics in the behavior of molecules, nanoparticles, and cells. Electrochemistry provides a direct and fast method to analyze single entities as it probes electron/charge-transfer processes. However, a highly reproducible electrochemical-sensing nanointerface is often hard to fabricate because of a lack of control of the fabrication processes at the nanoscale. In comparison with conventional micro/nanoelectrodes with a metal wire inside, we present a general and easily implemented protocol that describes how to fabricate and use a wireless nanopore electrode (WNE). Nanoscale metal deposition occurs at the tip of the nanopipette, providing an electroactive sensing interface. The WNEs utilize a dynamic ionic flow instead of a metal wire to sense the interfacial redox process. WNEs provide a highly controllable interface with a 30- to 200-nm diameter. This protocol presents the construction and characterization of two types of WNEs-the open-type WNE and closed-type WNE-which can be used to achieve reproducible electrochemical measurements of single entities. Combined with the related signal amplification mechanisms, we also describe how WNEs can be used to detect single redox molecules/ions, analyze the metabolism of single cells, and discriminate single nanoparticles in a mixture. This protocol is broadly applicable to studies of living cells, nanomaterials, and sensors at the single-entity level. The total time required to complete the protocol is ~10-18 h. Each WNE costs ~$1-$3.


Assuntos
Técnicas Eletroquímicas/instrumentação , Eletrodos , Nanoporos , Nanotecnologia/métodos , Técnicas Eletroquímicas/métodos , Desenho de Equipamento , Humanos , Células MCF-7 , Nanopartículas/análise , Oxirredução , Técnicas de Patch-Clamp/instrumentação , Técnicas de Patch-Clamp/métodos , Análise de Célula Única/instrumentação , Análise de Célula Única/métodos , Tecnologia sem Fio
11.
Nat Commun ; 10(1): 2163, 2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-31092822

RESUMO

Molecular analysis of circulating tumor cells (CTCs) at single-cell resolution offers great promise for cancer diagnostics and therapeutics from simple liquid biopsy. Recent development of massively parallel single-cell RNA-sequencing (scRNA-seq) provides a powerful method to resolve the cellular heterogeneity from gene expression and pathway regulation analysis. However, the scarcity of CTCs and the massive contamination of blood cells limit the utility of currently available technologies. Here, we present Hydro-Seq, a scalable hydrodynamic scRNA-seq barcoding technique, for high-throughput CTC analysis. High cell-capture efficiency and contamination removal capability of Hydro-Seq enables successful scRNA-seq of 666 CTCs from 21 breast cancer patient samples at high throughput. We identify breast cancer drug targets for hormone and targeted therapies and tracked individual cells that express markers of cancer stem cells (CSCs) as well as of epithelial/mesenchymal cell state transitions. Transcriptome analysis of these cells provides insights into monitoring target therapeutics and processes underlying tumor metastasis.


Assuntos
Neoplasias da Mama/patologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Células Neoplásicas Circulantes/patologia , Células-Tronco Neoplásicas/patologia , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/isolamento & purificação , Neoplasias da Mama/sangue , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/genética , Linhagem Celular , Transição Epitelial-Mesenquimal , Feminino , Perfilação da Expressão Gênica/instrumentação , Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Ensaios de Triagem em Larga Escala/instrumentação , Ensaios de Triagem em Larga Escala/métodos , Humanos , Biópsia Líquida/instrumentação , Biópsia Líquida/métodos , Camundongos , Análise de Sequência de RNA/instrumentação , Análise de Sequência de RNA/métodos , Análise de Célula Única/instrumentação , Análise de Célula Única/métodos
12.
Proc Inst Mech Eng H ; 233(7): 683-694, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31113284

RESUMO

Single-cell analysis serves as an important approach to study cell functions and interactions. Catering to the demand of Big Data Era, fast reactions for single cells and paralleled high-throughput analysis have become an urgent need. Microdroplet in microfluidics has advantages of modularity and integrity, as well as high throughput and sensitivity, which present great potential in the field of single-cell analysis. This review is carried out on three aspects to introduce microdroplet chips for single-cell analysis: droplet formation, droplet detection and practical functions. Structures of droplet formation are categorized into three types, including T-shaped channel, flow-involved channel and three-dimensional micro-vortice. The detection methods, including fluorescence, Raman spectroscopy, mass spectroscopy and electrochemical detection, are summarized from applications. Both pros and cons for existing techniques are reviewed and discussed. The functions of microdroplets-on-chip cover cell culture, nucleic acid test and cell identification. For each field, principles/mechanisms and/or schematic images are laconically introduced. Microdroplet in microfluidics has become a major research direction in single-cell analysis. With updated methods of droplet formation such as inertial ordering and micro-vortice, microdroplets-based biochips will expect high throughput detection and high-accuracy trace detection for clinical diagnosis in the near future.


Assuntos
Análise de Célula Única/métodos , Análise Serial de Tecidos/métodos , Humanos , Análise de Célula Única/instrumentação , Análise Serial de Tecidos/instrumentação
13.
Lab Chip ; 19(11): 2009-2018, 2019 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-31065640

RESUMO

Cytotoxicity exerted by cytotoxic lymphocytes against cancer cells is an essential cellular function for successful cancer immunotherapy. Standard cytotoxicity assays mostly provide population level information, whereas live cell imaging-based cytotoxicity assays can assess single cell level heterogeneity. However, long term tracking of individual cytotoxic lymphocyte-hematological cancer cell interactions is technically challenging because both cells can float around and form multi-cellular aggregates. To overcome this limitation, single hematological cancer cell arrays with immobilized hematological cancer cells are fabricated using microwell arrays. Using this new platform, single cell level natural killer (NK) cell cytotoxicity against leukemic cells is quantitatively assessed. Depending on microwell surface adhesiveness and inter-microwell distances, distinct modes of NK-leukemic cell interactions that result in different NK cell cytotoxicity are observed. For microwell arrays coated with bovine serum albumin, which prevents cell adhesion, NK cells stably contacted cancer cells with rounded morphologies, whereas for microwell arrays coated with fibronectin (FN), which triggers integrin signals, NK cells contacting cancer cells exhibited dynamic behaviors with elongated morphologies and constantly explored extracellular spaces by membrane extension. In addition, FN on extracellular spaces facilitate NK cell detachment from leukemic cells after killing by providing anchorage for force transmission, and promote cytotoxicity and serial killing. Single hematologic cell arrays are not only an efficient method for lymphocyte cytotoxicity analysis but also a useful tool to study the role of signaling molecules in extracellular spaces on lymphocyte cytotoxicity.


Assuntos
Espaço Extracelular/metabolismo , Neoplasias Hematológicas/imunologia , Neoplasias Hematológicas/patologia , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Análise de Célula Única/instrumentação , Análise Serial de Tecidos/instrumentação , Adesão Celular , Linhagem Celular Tumoral , Humanos
14.
Talanta ; 200: 169-176, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31036170

RESUMO

Circulating tumor cells (CTCs) are rare cancer cells that are shed from the tumors into the peripheral blood and are instrumental in distant metastasis. Early detection of CTCs can therefore improve prognoses and help design patient-specific treatment regimen. However, the current CTC isolation techniques have poor efficacy and selectivity, owing to the rarity and heterogeneity of the CTCs. We designed a microchip for integrated single-cell isolation of CTCs - based on cell size and immuno-phenotype - and analysis. Each isolation unit consisted of a trap channel, a bypass channel, and a release channel. The larger cells were preferentially captured at the trap channels and flushed out selectively via release microvalves according to their immuno-phenotype. The average recovery rate and purity of lung cancer cells isolated from a spiked WBC population were respectively 92.5% and 94% using the microchip, which were significantly higher compared to that obtained using anti-CD45 magnetic beads. In addition, the isolated cancer cells were analyzed on chip for the surface markers of epithelial mesenchymal transition. Taken together, the integrated microchip is a promising tool for the isolation and analysis of CTCs in the clinical setting.


Assuntos
Separação Celular/instrumentação , Separação Celular/métodos , Neoplasias Pulmonares/patologia , Técnicas Analíticas Microfluídicas/instrumentação , Células Neoplásicas Circulantes/patologia , Análise de Célula Única/instrumentação , Análise de Célula Única/métodos , Linhagem Celular Tumoral , Humanos
15.
Nat Commun ; 10(1): 2194, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-31097704

RESUMO

Although the physical properties of chromosomes, including their morphology, mechanics, and dynamics are crucial for their biological function, many basic questions remain unresolved. Here we directly image the circular chromosome in live E. coli with a broadened cell shape. We find that it exhibits a torus topology with, on average, a lower-density origin of replication and an ultrathin flexible string of DNA at the terminus of replication. At the single-cell level, the torus is strikingly heterogeneous, with blob-like Mbp-size domains that undergo major dynamic rearrangements, splitting and merging at a minute timescale. Our data show a domain organization underlying the chromosome structure of E. coli, where MatP proteins induce site-specific persistent domain boundaries at Ori/Ter, while transcription regulators HU and Fis induce weaker transient domain boundaries throughout the genome. These findings provide an architectural basis for the understanding of the dynamic spatial organization of bacterial genomes in live cells.


Assuntos
Cromossomos Bacterianos/química , DNA Bacteriano/química , DNA Circular/química , Escherichia coli/genética , Genoma Bacteriano , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos Bacterianos/metabolismo , Replicação do DNA , DNA Bacteriano/metabolismo , DNA Circular/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Microscopia Intravital/instrumentação , Microscopia Intravital/métodos , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Conformação de Ácido Nucleico , Análise de Célula Única/instrumentação , Análise de Célula Única/métodos
16.
Anal Chim Acta ; 1063: 127-135, 2019 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-30967176

RESUMO

Characterizing cell behavior is important to modern medical diagnoses as the changes of cell behavior are often indicators of huge diseases. In order to gain enough information about cells, developing novel methods of cell sorting and imaging is an important task. With development of micro-fabrication technologies, more advanced miniaturized devices are applied to cell research. Here, a portable and easy-to-use chip with high-density periodic micro-well array is designed and fabricated to capture target cells specifically. Combining with aptamer-silver conjugates and FAM functioned report probes, the sandwich assay was successfully applied for imaging cells. Any well of the chip is carefully designed to provide abundant information on single cells. Since there are 19,200 microwells in a single chip, more information is available. Compared to other cells, such as HEK-293, MCF-7, U2OS and Ramos cells, the sandwich assay shows high specificity towards target cell CCRF-CEM. What's more, the applications of the chip can be further expanded to other cells imaging if suitable aptamers were selected. This high-density micro-well array of aptamer-silver conjugates is hopeful to play an important role in medical diagnosis in the future.


Assuntos
Aptâmeros de Nucleotídeos/química , Separação Celular/instrumentação , Separação Celular/métodos , Citometria de Fluxo/métodos , Imagem Óptica/métodos , Prata/química , Análise de Célula Única/instrumentação , Análise de Célula Única/métodos , Linhagem Celular Tumoral , Células HEK293 , Humanos , Microscopia de Força Atômica , Microscopia Eletrônica de Varredura , Imagem Óptica/instrumentação
17.
Adv Exp Med Biol ; 1129: 63-79, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30968361

RESUMO

In this review, we describe the BD Rhapsody™ Single-Cell Analysis System, a platform that allows high-throughput capture of nucleic acids from single cells using a simple cartridge workflow and a multitier barcoding system. The resulting captured information can be used to generate various types of next-generation sequencing (NGS) libraries, including whole transcriptome analysis for discovery biology and targeted RNA analysis for high sensitivity transcript detection. The BD Rhapsody system can be used with emerging applications, such as BD™ AbSeq assays, to profile gene expression in both mRNA and protein level to provide ultra-high resolution analysis of single cells.


Assuntos
Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , RNA Mensageiro/análise , Análise de Célula Única/instrumentação , Análise de Célula Única/métodos , Análise de Sequência de RNA , Transcriptoma
18.
Analyst ; 144(9): 2942-2953, 2019 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-30939180

RESUMO

Adipocyte hypertrophy and hyperplasia are important parameters in describing abnormalities in adipogenesis that are concomitant to diseases such as obesity, diabetes, anorexia nervosa and osteoporosis. Therefore, technical developments in the detection of adipocytes become an important driving factor in adipogenesis research. Current techniques such as optical microscopy and flow cytometry are available in detection and examination of adipocytes, driving cell- and molecular-based research of adipogenesis. Even though microscopy techniques are common and straightforward, they are restricted in terms of manipulation and separation of the cells. Flow cytometry is an alternative, but mature adipocytes are fragile and cannot withstand the flow process. Other separation methods usually require labeling of the cells or usage of microfluidic platforms that utilize fluids with different densities. Magnetic levitation is a novel label-free technology with the principle of movement of cells towards the lower magnetic field in a paramagnetic medium depending on their individual densities. In this study, we used a magnetic levitation device for density-based single cell detection of differentiated adipogenic cells in heterogeneous populations. Results showed that the magnetic levitation platform was sensitive to changes in the lipid content of mesenchymal stem cells committed to adipogenesis and it could be successfully used to detect the adipogenic differentiation of the cells.


Assuntos
Adipócitos/citologia , Células da Medula Óssea/citologia , Células-Tronco Mesenquimais/citologia , Técnicas Analíticas Microfluídicas/métodos , Análise de Célula Única/métodos , Adipogenia/fisiologia , Animais , Células Cultivadas , Dispositivos Lab-On-A-Chip , Fenômenos Magnéticos , Imãs , Camundongos , Técnicas Analíticas Microfluídicas/instrumentação , Análise de Célula Única/instrumentação
19.
Lab Chip ; 19(10): 1706-1727, 2019 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-30997473

RESUMO

Droplet based scRNA-seq systems such as Drop-seq, inDrop and Chromium 10X have been the catalyst for the wide adoption of high-throughput scRNA-seq technologies in the research laboratory. In order to understand the capabilities of these systems to deeply interrogate biology; here we provide a practical guide through all the steps involved in a typical scRNA-seq experiment. Through comparing and contrasting these three main droplet based systems (and their derivatives), we provide an overview of all critical considerations in obtaining high quality and biologically relevant data. We also discuss the limitations of these systems and how they fit into the emerging field of Genomic Cytometry.


Assuntos
/instrumentação , RNA/genética , Análise de Célula Única/instrumentação , Análise de Célula Única/métodos , Humanos , Tamanho da Partícula , Propriedades de Superfície
20.
Methods Mol Biol ; 1979: 3-8, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31028628

RESUMO

Transcriptomics has been revolutionized by massive throughput RNA-seq. To date, the ongoing decrease in sequencing cost and recent eruption of single-cell related protocols have boosted a demand for single-cell RNA sequencing projects. Although the single-cell RNA-Seq (scRNA-Seq) approach is close to the conventional "bulk" RNA-seq, several features that are unique to scRNA-seq should be taken into consideration in order to obtain high-quality libraries and unbiased sequencing data.In this chapter I give recommendations for setting up the single cell-suitable laboratory environment.


Assuntos
Perfilação da Expressão Gênica/métodos , RNA/genética , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Desenho de Equipamento , Perfilação da Expressão Gênica/instrumentação , Genoma , Humanos , Laboratórios , Controle de Qualidade , Análise de Sequência de RNA/instrumentação , Análise de Célula Única/instrumentação , Manejo de Espécimes/métodos
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