Inhibition of Retinoblastoma Protein Inactivation

DTIC Science & Technology

2017-11-01

SUBJECT TERMS cell cycle, Retinoblastoma protein, E2F transcription factor, high throughput screen, drug discovery, x-ray crystallography 16. SECURITY...screening by x-ray crystallography . 2.0 KEYWORDS Retinoblastoma (Rb) pathway, E2F transcription factor, cancer, cell-cycle inhibition, activation...modulation, inhibition, high throughput screening, fragment-based screening, x-ray crystallography . 3.0 ACCOMPLISHMENTS Summary: We

A three-dimensional in vitro ovarian cancer coculture model using a high-throughput cell patterning platform

PubMed Central

Rizvi, Imran; Moon, Sangjun; Hasan, Tayyaba; Demirci, Utkan

2013-01-01

In vitro 3D cancer models that provide a more accurate representation of disease in vivo are urgently needed to improve our understanding of cancer pathology and to develop better cancer therapies. However, development of 3D models that are based on manual ejection of cells from micropipettes suffer from inherent limitations such as poor control over cell density, limited repeatability, low throughput, and, in the case of coculture models, lack of reproducible control over spatial distance between cell types (e.g., cancer and stromal cells). In this study, we build on a recently introduced 3D model in which human ovarian cancer (OVCAR-5) cells overlaid on Matrigel™ spontaneously form multicellular acini. We introduce a high-throughput automated cell printing system to bioprint a 3D coculture model using cancer cells and normal fibroblasts micropatterned on Matrigel™. Two cell types were patterned within a spatially controlled microenvironment (e.g., cell density, cell-cell distance) in a high-throughput and reproducible manner; both cell types remained viable during printing and continued to proliferate following patterning. This approach enables the miniaturization of an established macro-scale 3D culture model and would allow systematic investigation into the multiple unknown regulatory feedback mechanisms between tumor and stromal cells and provide a tool for high-throughput drug screening. PMID:21298805

An image analysis toolbox for high-throughput C. elegans assays

PubMed Central

Wählby, Carolina; Kamentsky, Lee; Liu, Zihan H.; Riklin-Raviv, Tammy; Conery, Annie L.; O’Rourke, Eyleen J.; Sokolnicki, Katherine L.; Visvikis, Orane; Ljosa, Vebjorn; Irazoqui, Javier E.; Golland, Polina; Ruvkun, Gary; Ausubel, Frederick M.; Carpenter, Anne E.

2012-01-01

We present a toolbox for high-throughput screening of image-based Caenorhabditis elegans phenotypes. The image analysis algorithms measure morphological phenotypes in individual worms and are effective for a variety of assays and imaging systems. This WormToolbox is available via the open-source CellProfiler project and enables objective scoring of whole-animal high-throughput image-based assays of C. elegans for the study of diverse biological pathways relevant to human disease. PMID:22522656

Defining the taxonomic domain of applicability for mammalian-based high-throughput screening assays

EPA Science Inventory

Cell-based high throughput screening (HTS) technologies are becoming mainstream in chemical safety evaluations. The US Environmental Protection Agency (EPA) Toxicity Forecaster (ToxCastTM) and the multi-agency Tox21 Programs have been at the forefront in advancing this science, m...

High throughput system for magnetic manipulation of cells, polymers, and biomaterials

PubMed Central

Spero, Richard Chasen; Vicci, Leandra; Cribb, Jeremy; Bober, David; Swaminathan, Vinay; O’Brien, E. Timothy; Rogers, Stephen L.; Superfine, R.

2008-01-01

In the past decade, high throughput screening (HTS) has changed the way biochemical assays are performed, but manipulation and mechanical measurement of micro- and nanoscale systems have not benefited from this trend. Techniques using microbeads (particles ∼0.1–10 μm) show promise for enabling high throughput mechanical measurements of microscopic systems. We demonstrate instrumentation to magnetically drive microbeads in a biocompatible, multiwell magnetic force system. It is based on commercial HTS standards and is scalable to 96 wells. Cells can be cultured in this magnetic high throughput system (MHTS). The MHTS can apply independently controlled forces to 16 specimen wells. Force calibrations demonstrate forces in excess of 1 nN, predicted force saturation as a function of pole material, and powerlaw dependence of F∼r−2.7±0.1. We employ this system to measure the stiffness of SR2+ Drosophila cells. MHTS technology is a key step toward a high throughput screening system for micro- and nanoscale biophysical experiments. PMID:19044357

High-Throughput Non-Contact Vitrification of Cell-Laden Droplets Based on Cell Printing

NASA Astrophysics Data System (ADS)

Shi, Meng; Ling, Kai; Yong, Kar Wey; Li, Yuhui; Feng, Shangsheng; Zhang, Xiaohui; Pingguan-Murphy, Belinda; Lu, Tian Jian; Xu, Feng

2015-12-01

Cryopreservation is the most promising way for long-term storage of biological samples e.g., single cells and cellular structures. Among various cryopreservation methods, vitrification is advantageous by employing high cooling rate to avoid the formation of harmful ice crystals in cells. Most existing vitrification methods adopt direct contact of cells with liquid nitrogen to obtain high cooling rates, which however causes the potential contamination and difficult cell collection. To address these limitations, we developed a non-contact vitrification device based on an ultra-thin freezing film to achieve high cooling/warming rate and avoid direct contact between cells and liquid nitrogen. A high-throughput cell printer was employed to rapidly generate uniform cell-laden microdroplets into the device, where the microdroplets were hung on one side of the film and then vitrified by pouring the liquid nitrogen onto the other side via boiling heat transfer. Through theoretical and experimental studies on vitrification processes, we demonstrated that our device offers a high cooling/warming rate for vitrification of the NIH 3T3 cells and human adipose-derived stem cells (hASCs) with maintained cell viability and differentiation potential. This non-contact vitrification device provides a novel and effective way to cryopreserve cells at high throughput and avoid the contamination and collection problems.

High-Throughput Non-Contact Vitrification of Cell-Laden Droplets Based on Cell Printing

PubMed Central

Shi, Meng; Ling, Kai; Yong, Kar Wey; Li, Yuhui; Feng, Shangsheng; Zhang, Xiaohui; Pingguan-Murphy, Belinda; Lu, Tian Jian; Xu, Feng

2015-01-01

Cryopreservation is the most promising way for long-term storage of biological samples e.g., single cells and cellular structures. Among various cryopreservation methods, vitrification is advantageous by employing high cooling rate to avoid the formation of harmful ice crystals in cells. Most existing vitrification methods adopt direct contact of cells with liquid nitrogen to obtain high cooling rates, which however causes the potential contamination and difficult cell collection. To address these limitations, we developed a non-contact vitrification device based on an ultra-thin freezing film to achieve high cooling/warming rate and avoid direct contact between cells and liquid nitrogen. A high-throughput cell printer was employed to rapidly generate uniform cell-laden microdroplets into the device, where the microdroplets were hung on one side of the film and then vitrified by pouring the liquid nitrogen onto the other side via boiling heat transfer. Through theoretical and experimental studies on vitrification processes, we demonstrated that our device offers a high cooling/warming rate for vitrification of the NIH 3T3 cells and human adipose-derived stem cells (hASCs) with maintained cell viability and differentiation potential. This non-contact vitrification device provides a novel and effective way to cryopreserve cells at high throughput and avoid the contamination and collection problems. PMID:26655688

Continuous inertial microparticle and blood cell separation in straight channels with local microstructures.

PubMed

Wu, Zhenlong; Chen, Yu; Wang, Moran; Chung, Aram J

2016-02-07

Fluid inertia which has conventionally been neglected in microfluidics has been gaining much attention for particle and cell manipulation because inertia-based methods inherently provide simple, passive, precise and high-throughput characteristics. Particularly, the inertial approach has been applied to blood separation for various biomedical research studies mainly using spiral microchannels. For higher throughput, parallelization is essential; however, it is difficult to realize using spiral channels because of their large two dimensional layouts. In this work, we present a novel inertial platform for continuous sheathless particle and blood cell separation in straight microchannels containing microstructures. Microstructures within straight channels exert secondary flows to manipulate particle positions similar to Dean flow in curved channels but with higher controllability. Through a balance between inertial lift force and microstructure-induced secondary flow, we deterministically position microspheres and cells based on their sizes to be separated downstream. Using our inertial platform, we successfully sorted microparticles and fractionized blood cells with high separation efficiencies, high purities and high throughputs. The inertial separation platform developed here can be operated to process diluted blood with a throughput of 10.8 mL min(-1)via radially arrayed single channels with one inlet and two rings of outlets.

Label-free cancer cell separation from human whole blood using inertial microfluidics at low shear stress.

PubMed

Lee, Myung Gwon; Shin, Joong Ho; Bae, Chae Yun; Choi, Sungyoung; Park, Je-Kyun

2013-07-02

We report a contraction-expansion array (CEA) microchannel device that performs label-free high-throughput separation of cancer cells from whole blood at low Reynolds number (Re). The CEA microfluidic device utilizes hydrodynamic field effect for cancer cell separation, two kinds of inertial effects: (1) inertial lift force and (2) Dean flow, which results in label-free size-based separation with high throughput. To avoid cell damages potentially caused by high shear stress in conventional inertial separation techniques, the CEA microfluidic device isolates the cells with low operational Re, maintaining high-throughput separation, using nondiluted whole blood samples (hematocrit ~45%). We characterized inertial particle migration and investigated the migration of blood cells and various cancer cells (MCF-7, SK-BR-3, and HCC70) in the CEA microchannel. The separation of cancer cells from whole blood was demonstrated with a cancer cell recovery rate of 99.1%, a blood cell rejection ratio of 88.9%, and a throughput of 1.1 × 10(8) cells/min. In addition, the blood cell rejection ratio was further improved to 97.3% by a two-step filtration process with two devices connected in series.

The high throughput biomedicine unit at the institute for molecular medicine Finland: high throughput screening meets precision medicine.

PubMed

Pietiainen, Vilja; Saarela, Jani; von Schantz, Carina; Turunen, Laura; Ostling, Paivi; Wennerberg, Krister

2014-05-01

The High Throughput Biomedicine (HTB) unit at the Institute for Molecular Medicine Finland FIMM was established in 2010 to serve as a national and international academic screening unit providing access to state of the art instrumentation for chemical and RNAi-based high throughput screening. The initial focus of the unit was multiwell plate based chemical screening and high content microarray-based siRNA screening. However, over the first four years of operation, the unit has moved to a more flexible service platform where both chemical and siRNA screening is performed at different scales primarily in multiwell plate-based assays with a wide range of readout possibilities with a focus on ultraminiaturization to allow for affordable screening for the academic users. In addition to high throughput screening, the equipment of the unit is also used to support miniaturized, multiplexed and high throughput applications for other types of research such as genomics, sequencing and biobanking operations. Importantly, with the translational research goals at FIMM, an increasing part of the operations at the HTB unit is being focused on high throughput systems biological platforms for functional profiling of patient cells in personalized and precision medicine projects.

Gold-coated polydimethylsiloxane microwells for high-throughput electrochemiluminescence analysis of intracellular glucose at single cells.

PubMed

Xia, Juan; Zhou, Junyu; Zhang, Ronggui; Jiang, Dechen; Jiang, Depeng

2018-06-04

In this communication, a gold-coated polydimethylsiloxane (PDMS) chip with cell-sized microwells was prepared through a stamping and spraying process that was applied directly for high-throughput electrochemiluminescence (ECL) analysis of intracellular glucose at single cells. As compared with the previous multiple-step fabrication of photoresist-based microwells on the electrode, the preparation process is simple and offers fresh electrode surface for higher luminescence intensity. More luminescence intensity was recorded from cell-retained microwells than that at the planar region among the microwells that was correlated with the content of intracellular glucose. The successful monitoring of intracellular glucose at single cells using this PDMS chip will provide an alternative strategy for high-throughput single-cell analysis. Graphical abstract ᅟ.

Development of a high-throughput microscale cell disruption platform for Pichia pastoris in rapid bioprocess design.

PubMed

Bláha, Benjamin A F; Morris, Stephen A; Ogonah, Olotu W; Maucourant, Sophie; Crescente, Vincenzo; Rosenberg, William; Mukhopadhyay, Tarit K

2018-01-01

The time and cost benefits of miniaturized fermentation platforms can only be gained by employing complementary techniques facilitating high-throughput at small sample volumes. Microbial cell disruption is a major bottleneck in experimental throughput and is often restricted to large processing volumes. Moreover, for rigid yeast species, such as Pichia pastoris, no effective high-throughput disruption methods exist. The development of an automated, miniaturized, high-throughput, noncontact, scalable platform based on adaptive focused acoustics (AFA) to disrupt P. pastoris and recover intracellular heterologous protein is described. Augmented modes of AFA were established by investigating vessel designs and a novel enzymatic pretreatment step. Three different modes of AFA were studied and compared to the performance high-pressure homogenization. For each of these modes of cell disruption, response models were developed to account for five different performance criteria. Using multiple responses not only demonstrated that different operating parameters are required for different response optima, with highest product purity requiring suboptimal values for other criteria, but also allowed for AFA-based methods to mimic large-scale homogenization processes. These results demonstrate that AFA-mediated cell disruption can be used for a wide range of applications including buffer development, strain selection, fermentation process development, and whole bioprocess integration. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:130-140, 2018. © 2017 American Institute of Chemical Engineers.

High-throughput measurements of the optical redox ratio using a commercial microplate reader.

PubMed

Cannon, Taylor M; Shah, Amy T; Walsh, Alex J; Skala, Melissa C

2015-01-01

There is a need for accurate, high-throughput, functional measures to gauge the efficacy of potential drugs in living cells. As an early marker of drug response in cells, cellular metabolism provides an attractive platform for high-throughput drug testing. Optical techniques can noninvasively monitor NADH and FAD, two autofluorescent metabolic coenzymes. The autofluorescent redox ratio, defined as the autofluorescence intensity of NADH divided by that of FAD, quantifies relative rates of cellular glycolysis and oxidative phosphorylation. However, current microscopy methods for redox ratio quantification are time-intensive and low-throughput, limiting their practicality in drug screening. Alternatively, high-throughput commercial microplate readers quickly measure fluorescence intensities for hundreds of wells. This study found that a commercial microplate reader can differentiate the receptor status of breast cancer cell lines (p < 0.05) based on redox ratio measurements without extrinsic contrast agents. Furthermore, microplate reader redox ratio measurements resolve response (p < 0.05) and lack of response (p > 0.05) in cell lines that are responsive and nonresponsive, respectively, to the breast cancer drug trastuzumab. These studies indicate that the microplate readers can be used to measure the redox ratio in a high-throughput manner and are sensitive enough to detect differences in cellular metabolism that are consistent with microscopy results.

The next generation CdTe technology- Substrate foil based solar cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ferekides, Chris

The main objective of this project was the development of one of the most promising Photovoltaic (PV) materials CdTe into a versatile, cost effective, and high throughput technology, by demonstrating substrate devices on foil substrates using high throughput fabrication conditions. The typical CdTe cell is of the superstrate configuration where the solar cell is fabricated on a glass superstrate by the sequential deposition of a TCO, n-type heterojunction partner, p-CdTe absorber, and back contact. Large glass modules are heavy and present significant challenges during manufacturing (uniform heating, etc.). If a substrate CdTe cell could be developed (the main goal ofmore » this project) a roll-to-toll high throughput technology could be developed.« less

Metabolomics Approach for Toxicity Screening of Volatile Substances

EPA Science Inventory

In 2007 the National Research Council envisioned the need for inexpensive, high throughput, cell based toxicity testing methods relevant to human health. High Throughput Screening (HTS) in vitro screening approaches have addressed these problems by using robotics. However, the ch...

Automated Analysis of siRNA Screens of Virus Infected Cells Based on Immunofluorescence Microscopy

NASA Astrophysics Data System (ADS)

Matula, Petr; Kumar, Anil; Wörz, Ilka; Harder, Nathalie; Erfle, Holger; Bartenschlager, Ralf; Eils, Roland; Rohr, Karl

We present an image analysis approach as part of a high-throughput microscopy screening system based on cell arrays for the identification of genes involved in Hepatitis C and Dengue virus replication. Our approach comprises: cell nucleus segmentation, quantification of virus replication level in cells, localization of regions with transfected cells, cell classification by infection status, and quality assessment of an experiment. The approach is fully automatic and has been successfully applied to a large number of cell array images from screening experiments. The experimental results show a good agreement with the expected behavior of positive as well as negative controls and encourage the application to screens from further high-throughput experiments.

Multiplex High-Throughput Targeted Proteomic Assay To Identify Induced Pluripotent Stem Cells.

PubMed

Baud, Anna; Wessely, Frank; Mazzacuva, Francesca; McCormick, James; Camuzeaux, Stephane; Heywood, Wendy E; Little, Daniel; Vowles, Jane; Tuefferd, Marianne; Mosaku, Olukunbi; Lako, Majlinda; Armstrong, Lyle; Webber, Caleb; Cader, M Zameel; Peeters, Pieter; Gissen, Paul; Cowley, Sally A; Mills, Kevin

2017-02-21

Induced pluripotent stem cells have great potential as a human model system in regenerative medicine, disease modeling, and drug screening. However, their use in medical research is hampered by laborious reprogramming procedures that yield low numbers of induced pluripotent stem cells. For further applications in research, only the best, competent clones should be used. The standard assays for pluripotency are based on genomic approaches, which take up to 1 week to perform and incur significant cost. Therefore, there is a need for a rapid and cost-effective assay able to distinguish between pluripotent and nonpluripotent cells. Here, we describe a novel multiplexed, high-throughput, and sensitive peptide-based multiple reaction monitoring mass spectrometry assay, allowing for the identification and absolute quantitation of multiple core transcription factors and pluripotency markers. This assay provides simpler and high-throughput classification into either pluripotent or nonpluripotent cells in 7 min analysis while being more cost-effective than conventional genomic tests.

web cellHTS2: a web-application for the analysis of high-throughput screening data.

PubMed

Pelz, Oliver; Gilsdorf, Moritz; Boutros, Michael

2010-04-12

The analysis of high-throughput screening data sets is an expanding field in bioinformatics. High-throughput screens by RNAi generate large primary data sets which need to be analyzed and annotated to identify relevant phenotypic hits. Large-scale RNAi screens are frequently used to identify novel factors that influence a broad range of cellular processes, including signaling pathway activity, cell proliferation, and host cell infection. Here, we present a web-based application utility for the end-to-end analysis of large cell-based screening experiments by cellHTS2. The software guides the user through the configuration steps that are required for the analysis of single or multi-channel experiments. The web-application provides options for various standardization and normalization methods, annotation of data sets and a comprehensive HTML report of the screening data analysis, including a ranked hit list. Sessions can be saved and restored for later re-analysis. The web frontend for the cellHTS2 R/Bioconductor package interacts with it through an R-server implementation that enables highly parallel analysis of screening data sets. web cellHTS2 further provides a file import and configuration module for common file formats. The implemented web-application facilitates the analysis of high-throughput data sets and provides a user-friendly interface. web cellHTS2 is accessible online at http://web-cellHTS2.dkfz.de. A standalone version as a virtual appliance and source code for platforms supporting Java 1.5.0 can be downloaded from the web cellHTS2 page. web cellHTS2 is freely distributed under GPL.

Real-time Image Processing for Microscopy-based Label-free Imaging Flow Cytometry in a Microfluidic Chip.

PubMed

Heo, Young Jin; Lee, Donghyeon; Kang, Junsu; Lee, Keondo; Chung, Wan Kyun

2017-09-14

Imaging flow cytometry (IFC) is an emerging technology that acquires single-cell images at high-throughput for analysis of a cell population. Rich information that comes from high sensitivity and spatial resolution of a single-cell microscopic image is beneficial for single-cell analysis in various biological applications. In this paper, we present a fast image-processing pipeline (R-MOD: Real-time Moving Object Detector) based on deep learning for high-throughput microscopy-based label-free IFC in a microfluidic chip. The R-MOD pipeline acquires all single-cell images of cells in flow, and identifies the acquired images as a real-time process with minimum hardware that consists of a microscope and a high-speed camera. Experiments show that R-MOD has the fast and reliable accuracy (500 fps and 93.3% mAP), and is expected to be used as a powerful tool for biomedical and clinical applications.

Inertial-ordering-assisted droplet microfluidics for high-throughput single-cell RNA-sequencing.

PubMed

Moon, Hui-Sung; Je, Kwanghwi; Min, Jae-Woong; Park, Donghyun; Han, Kyung-Yeon; Shin, Seung-Ho; Park, Woong-Yang; Yoo, Chang Eun; Kim, Shin-Hyun

2018-02-27

Single-cell RNA-seq reveals the cellular heterogeneity inherent in the population of cells, which is very important in many clinical and research applications. Recent advances in droplet microfluidics have achieved the automatic isolation, lysis, and labeling of single cells in droplet compartments without complex instrumentation. However, barcoding errors occurring in the cell encapsulation process because of the multiple-beads-in-droplet and insufficient throughput because of the low concentration of beads for avoiding multiple-beads-in-a-droplet remain important challenges for precise and efficient expression profiling of single cells. In this study, we developed a new droplet-based microfluidic platform that significantly improved the throughput while reducing barcoding errors through deterministic encapsulation of inertially ordered beads. Highly concentrated beads containing oligonucleotide barcodes were spontaneously ordered in a spiral channel by an inertial effect, which were in turn encapsulated in droplets one-by-one, while cells were simultaneously encapsulated in the droplets. The deterministic encapsulation of beads resulted in a high fraction of single-bead-in-a-droplet and rare multiple-beads-in-a-droplet although the bead concentration increased to 1000 μl -1 , which diminished barcoding errors and enabled accurate high-throughput barcoding. We successfully validated our device with single-cell RNA-seq. In addition, we found that multiple-beads-in-a-droplet, generated using a normal Drop-Seq device with a high concentration of beads, underestimated transcript numbers and overestimated cell numbers. This accurate high-throughput platform can expand the capability and practicality of Drop-Seq in single-cell analysis.

Recent Advances in Nanobiotechnology and High-Throughput Molecular Techniques for Systems Biomedicine

PubMed Central

Kim, Eung-Sam; Ahn, Eun Hyun; Chung, Euiheon; Kim, Deok-Ho

2013-01-01

Nanotechnology-based tools are beginning to emerge as promising platforms for quantitative high-throughput analysis of live cells and tissues. Despite unprecedented progress made over the last decade, a challenge still lies in integrating emerging nanotechnology-based tools into macroscopic biomedical apparatuses for practical purposes in biomedical sciences. In this review, we discuss the recent advances and limitations in the analysis and control of mechanical, biochemical, fluidic, and optical interactions in the interface areas of nanotechnology-based materials and living cells in both in vitro and in vivo settings. PMID:24258011

A simple cell-based high throughput screening (HTS) assay for inhibitors of Salmonella enterica RNA polymerase containing the general stress response regulator RpoS (σS).

PubMed

Campos-Gomez, Javier; Benitez, Jorge A

2018-07-01

RNA polymerase containing the stress response regulator σ S subunit (RpoS) plays a key role in bacterial survival in hostile environments in nature and during infection. Here we devise and validate a simple cell-based high throughput luminescence assay for this holoenzyme suitable for screening large chemical libraries in a robotic platform. Copyright © 2018 Elsevier B.V. All rights reserved.

Inhibition of Retinoblastoma Protein Inactivation

DTIC Science & Technology

2016-09-01

Retinoblastoma protein, E2F transcription factor, high throughput screen, drug discovery, x-ray crystallography 16. SECURITY CLASSIFICATION OF: 17...developed a method to perform fragment based screening by x-ray crystallography . 2.0 KEYWORDS Retinoblastoma (Rb) pathway, E2F transcription factor...cancer, cell-cycle inhibition, activation, modulation, inhibition, high throughput screening, fragment-based screening, x-ray crystallography

High-throughput synchronization of mammalian cell cultures by spiral microfluidics.

PubMed

Lee, Wong Cheng; Bhagat, Ali Asgar S; Lim, Chwee Teck

2014-01-01

The development of mammalian cell cycle synchronization techniques has greatly advanced our understanding of many cellular regulatory events and mechanisms specific to different phases of the cell cycle. In this chapter, we describe a high-throughput microfluidic-based approach for cell cycle synchronization. By exploiting the relationship between cell size and its phase in the cell cycle, large numbers of synchronized cells can be obtained by size fractionation in a spiral microfluidic channel. Protocols for the synchronization of primary cells such as mesenchymal stem cells, and immortal cell lines such as Chinese hamster ovarian cells (CHO-CD36) and HeLa cells are provided as examples.

In Vitro Toxicity Screening Technique for Volatile Substances Using Flow-Through System#

EPA Science Inventory

In 2007 the National Research Council envisioned the need for inexpensive, high throughput, cell based toxicity testing methods relevant to human health. High Throughput Screening (HTS) in vitro screening approaches have addressed these problems by using robotics. However the cha...

RNA isolation from mammalian cells using porous polymer monoliths: an approach for high-throughput automation.

PubMed

Chatterjee, Anirban; Mirer, Paul L; Zaldivar Santamaria, Elvira; Klapperich, Catherine; Sharon, Andre; Sauer-Budge, Alexis F

2010-06-01

The life science and healthcare communities have been redefining the importance of ribonucleic acid (RNA) through the study of small molecule RNA (in RNAi/siRNA technologies), micro RNA (in cancer research and stem cell research), and mRNA (gene expression analysis for biologic drug targets). Research in this field increasingly requires efficient and high-throughput isolation techniques for RNA. Currently, several commercial kits are available for isolating RNA from cells. Although the quality and quantity of RNA yielded from these kits is sufficiently good for many purposes, limitations exist in terms of extraction efficiency from small cell populations and the ability to automate the extraction process. Traditionally, automating a process decreases the cost and personnel time while simultaneously increasing the throughput and reproducibility. As the RNA field matures, new methods for automating its extraction, especially from low cell numbers and in high throughput, are needed to achieve these improvements. The technology presented in this article is a step toward this goal. The method is based on a solid-phase extraction technology using a porous polymer monolith (PPM). A novel cell lysis approach and a larger binding surface throughout the PPM extraction column ensure a high yield from small starting samples, increasing sensitivity and reducing indirect costs in cell culture and sample storage. The method ensures a fast and simple procedure for RNA isolation from eukaryotic cells, with a high yield both in terms of quality and quantity. The technique is amenable to automation and streamlined workflow integration, with possible miniaturization of the sample handling process making it suitable for high-throughput applications.

Optimization and high-throughput screening of antimicrobial peptides.

PubMed

Blondelle, Sylvie E; Lohner, Karl

2010-01-01

While a well-established process for lead compound discovery in for-profit companies, high-throughput screening is becoming more popular in basic and applied research settings in academia. The development of combinatorial libraries combined with easy and less expensive access to new technologies have greatly contributed to the implementation of high-throughput screening in academic laboratories. While such techniques were earlier applied to simple assays involving single targets or based on binding affinity, they have now been extended to more complex systems such as whole cell-based assays. In particular, the urgent need for new antimicrobial compounds that would overcome the rapid rise of drug-resistant microorganisms, where multiple target assays or cell-based assays are often required, has forced scientists to focus onto high-throughput technologies. Based on their existence in natural host defense systems and their different mode of action relative to commercial antibiotics, antimicrobial peptides represent a new hope in discovering novel antibiotics against multi-resistant bacteria. The ease of generating peptide libraries in different formats has allowed a rapid adaptation of high-throughput assays to the search for novel antimicrobial peptides. Similarly, the availability nowadays of high-quantity and high-quality antimicrobial peptide data has permitted the development of predictive algorithms to facilitate the optimization process. This review summarizes the various library formats that lead to de novo antimicrobial peptide sequences as well as the latest structural knowledge and optimization processes aimed at improving the peptides selectivity.

A High Throughput Phenotypic Screening reveals compounds that counteract premature osteogenic differentiation of HGPS iPS-derived mesenchymal stem cells

PubMed Central

Lo Cicero, Alessandra; Jaskowiak, Anne-Laure; Egesipe, Anne-Laure; Tournois, Johana; Brinon, Benjamin; Pitrez, Patricia R.; Ferreira, Lino; de Sandre-Giovannoli, Annachiara; Levy, Nicolas; Nissan, Xavier

2016-01-01

Hutchinson-Gilford progeria syndrome (HGPS) is a rare fatal genetic disorder that causes systemic accelerated aging in children. Thanks to the pluripotency and self-renewal properties of induced pluripotent stem cells (iPSC), HGPS iPSC-based modeling opens up the possibility of access to different relevant cell types for pharmacological approaches. In this study, 2800 small molecules were explored using high-throughput screening, looking for compounds that could potentially reduce the alkaline phosphatase activity of HGPS mesenchymal stem cells (MSCs) committed into osteogenic differentiation. Results revealed seven compounds that normalized the osteogenic differentiation process and, among these, all-trans retinoic acid and 13-cis-retinoic acid, that also decreased progerin expression. This study highlights the potential of high-throughput drug screening using HGPS iPS-derived cells, in order to find therapeutic compounds for HGPS and, potentially, for other aging-related disorders. PMID:27739443

A High Throughput Phenotypic Screening reveals compounds that counteract premature osteogenic differentiation of HGPS iPS-derived mesenchymal stem cells.

PubMed

Lo Cicero, Alessandra; Jaskowiak, Anne-Laure; Egesipe, Anne-Laure; Tournois, Johana; Brinon, Benjamin; Pitrez, Patricia R; Ferreira, Lino; de Sandre-Giovannoli, Annachiara; Levy, Nicolas; Nissan, Xavier

2016-10-14

Hutchinson-Gilford progeria syndrome (HGPS) is a rare fatal genetic disorder that causes systemic accelerated aging in children. Thanks to the pluripotency and self-renewal properties of induced pluripotent stem cells (iPSC), HGPS iPSC-based modeling opens up the possibility of access to different relevant cell types for pharmacological approaches. In this study, 2800 small molecules were explored using high-throughput screening, looking for compounds that could potentially reduce the alkaline phosphatase activity of HGPS mesenchymal stem cells (MSCs) committed into osteogenic differentiation. Results revealed seven compounds that normalized the osteogenic differentiation process and, among these, all-trans retinoic acid and 13-cis-retinoic acid, that also decreased progerin expression. This study highlights the potential of high-throughput drug screening using HGPS iPS-derived cells, in order to find therapeutic compounds for HGPS and, potentially, for other aging-related disorders.

Micro-patterned agarose gel devices for single-cell high-throughput microscopy of E. coli cells.

PubMed

Priest, David G; Tanaka, Nobuyuki; Tanaka, Yo; Taniguchi, Yuichi

2017-12-21

High-throughput microscopy of bacterial cells elucidated fundamental cellular processes including cellular heterogeneity and cell division homeostasis. Polydimethylsiloxane (PDMS)-based microfluidic devices provide advantages including precise positioning of cells and throughput, however device fabrication is time-consuming and requires specialised skills. Agarose pads are a popular alternative, however cells often clump together, which hinders single cell quantitation. Here, we imprint agarose pads with micro-patterned 'capsules', to trap individual cells and 'lines', to direct cellular growth outwards in a straight line. We implement this micro-patterning into multi-pad devices called CapsuleHotel and LineHotel for high-throughput imaging. CapsuleHotel provides ~65,000 capsule structures per mm 2 that isolate individual Escherichia coli cells. In contrast, LineHotel provides ~300 line structures per mm that direct growth of micro-colonies. With CapsuleHotel, a quantitative single cell dataset of ~10,000 cells across 24 samples can be acquired and analysed in under 1 hour. LineHotel allows tracking growth of > 10 micro-colonies across 24 samples simultaneously for up to 4 generations. These easy-to-use devices can be provided in kit format, and will accelerate discoveries in diverse fields ranging from microbiology to systems and synthetic biology.

SeqAPASS to evaluate conservation of high-throughput screening targets across non-mammalian species

EPA Science Inventory

Cell-based high-throughput screening (HTS) and computational technologies are being applied as tools for toxicity testing in the 21st century. The U.S. Environmental Protection Agency (EPA) embraced these technologies and created the ToxCast Program in 2007, which has served as a...

High-throughput screening based on label-free detection of small molecule microarrays

NASA Astrophysics Data System (ADS)

Zhu, Chenggang; Fei, Yiyan; Zhu, Xiangdong

2017-02-01

Based on small-molecule microarrays (SMMs) and oblique-incidence reflectivity difference (OI-RD) scanner, we have developed a novel high-throughput drug preliminary screening platform based on label-free monitoring of direct interactions between target proteins and immobilized small molecules. The screening platform is especially attractive for screening compounds against targets of unknown function and/or structure that are not compatible with functional assay development. In this screening platform, OI-RD scanner serves as a label-free detection instrument which is able to monitor about 15,000 biomolecular interactions in a single experiment without the need to label any biomolecule. Besides, SMMs serves as a novel format for high-throughput screening by immobilization of tens of thousands of different compounds on a single phenyl-isocyanate functionalized glass slide. Based on the high-throughput screening platform, we sequentially screened five target proteins (purified target proteins or cell lysate containing target protein) in high-throughput and label-free mode. We found hits for respective target protein and the inhibition effects for some hits were confirmed by following functional assays. Compared to traditional high-throughput screening assay, the novel high-throughput screening platform has many advantages, including minimal sample consumption, minimal distortion of interactions through label-free detection, multi-target screening analysis, which has a great potential to be a complementary screening platform in the field of drug discovery.

Droplet Array-Based 3D Coculture System for High-Throughput Tumor Angiogenesis Assay.

PubMed

Du, Xiaohui; Li, Wanming; Du, Guansheng; Cho, Hansang; Yu, Min; Fang, Qun; Lee, Luke P; Fang, Jin

2018-03-06

Angiogenesis is critical for tumor progression and metastasis, and it progresses through orchestral multicellular interactions. Thus, there is urgent demand for high-throughput tumor angiogenesis assays for concurrent examination of multiple factors. For investigating tumor angiogenesis, we developed a microfluidic droplet array-based cell-coculture system comprising a two-layer polydimethylsiloxane chip featuring 6 × 9 paired-well arrays and an automated droplet-manipulation device. In each droplet-pair unit, tumor cells were cultured in 3D in one droplet by mixing cell suspensions with Matrigel, and in the other droplet, human umbilical vein endothelial cells (HUVECs) were cultured in 2D. Droplets were fused by a newly developed fusion method, and tumor angiogenesis was assayed by coculturing tumor cells and HUVECs in the fused droplet units. The 3D-cultured tumor cells formed aggregates harboring a hypoxic center-as observed in vivo-and secreted more vascular endothelial growth factor (VEGF) and more strongly induced HUVEC tubule formation than did 2D-cultured tumor cells. Our single array supported 54 assays in parallel. The angiogenic potentials of distinct tumor cells and their differential responses to antiangiogenesis agent, Fingolimod, could be investigated without mutual interference in a single array. Our droplet-based assay is convenient to evaluate multicellular interaction in high throughput in the context of tumor sprouting angiogenesis, and we envision that the assay can be extensively implementable for studying other cell-cell interactions.

Recent advances in nanobiotechnology and high-throughput molecular techniques for systems biomedicine.

PubMed

Kim, Eung-Sam; Ahn, Eun Hyun; Chung, Euiheon; Kim, Deok-Ho

2013-12-01

Nanotechnology-based tools are beginning to emerge as promising platforms for quantitative high-throughput analysis of live cells and tissues. Despite unprecedented progress made over the last decade, a challenge still lies in integrating emerging nanotechnology-based tools into macroscopic biomedical apparatuses for practical purposes in biomedical sciences. In this review, we discuss the recent advances and limitations in the analysis and control of mechanical, biochemical, fluidic, and optical interactions in the interface areas of nanotechnologybased materials and living cells in both in vitro and in vivo settings.

Increasing the coverage area through relay node deployment in long term evolution advanced cellular networks

NASA Astrophysics Data System (ADS)

Aldhaibani, Jaafar A.; Ahmad, R. B.; Yahya, A.; Azeez, Suzan A.

2015-05-01

Wireless multi-hop relay networks have become very important technologies in mobile communications. These networks ensure high throughput and coverage extension with a low cost. The poor capacity at cell edges is not enough to meet with growing demand of high capacity and throughput irrespective of user's placement in the cellular network. In this paper we propose optimal placement of relay node that provides maximum achievable rate at users and enhances the throughput and coverage at cell edge region. The proposed scheme is based on the outage probability at users and taken on account the interference between nodes. Numerical analyses along with simulation results indicated there are an improvement in capacity for users at the cell edge is 40% increment from all cell capacity.

Automated image-based assay for evaluation of HIV neutralization and cell-to-cell fusion inhibition.

PubMed

Sheik-Khalil, Enas; Bray, Mark-Anthony; Özkaya Şahin, Gülsen; Scarlatti, Gabriella; Jansson, Marianne; Carpenter, Anne E; Fenyö, Eva Maria

2014-08-30

Standardized techniques to detect HIV-neutralizing antibody responses are of great importance in the search for an HIV vaccine. Here, we present a high-throughput, high-content automated plaque reduction (APR) assay based on automated microscopy and image analysis that allows evaluation of neutralization and inhibition of cell-cell fusion within the same assay. Neutralization of virus particles is measured as a reduction in the number of fluorescent plaques, and inhibition of cell-cell fusion as a reduction in plaque area. We found neutralization strength to be a significant factor in the ability of virus to form syncytia. Further, we introduce the inhibitory concentration of plaque area reduction (ICpar) as an additional measure of antiviral activity, i.e. fusion inhibition. We present an automated image based high-throughput, high-content HIV plaque reduction assay. This allows, for the first time, simultaneous evaluation of neutralization and inhibition of cell-cell fusion within the same assay, by quantifying the reduction in number of plaques and mean plaque area, respectively. Inhibition of cell-to-cell fusion requires higher quantities of inhibitory reagent than inhibition of virus neutralization.

Microarray platform affords improved product analysis in mammalian cell growth studies

PubMed Central

Li, Lingyun; Migliore, Nicole; Schaefer, Eugene; Sharfstein, Susan T.; Dordick, Jonathan S.; Linhardt, Robert J.

2014-01-01

High throughput (HT) platforms serve as cost-efficient and rapid screening method for evaluating the effect of cell culture conditions and screening of chemicals. The aim of the current study was to develop a high-throughput cell-based microarray platform to assess the effect of culture conditions on Chinese hamster ovary (CHO) cells. Specifically, growth, transgene expression and metabolism of a GS/MSX CHO cell line, which produces a therapeutic monoclonal antibody, was examined using microarray system in conjunction with conventional shake flask platform in a non-proprietary medium. The microarray system consists of 60 nl spots of cells encapsulated in alginate and separated in groups via an 8-well chamber system attached to the chip. Results show the non-proprietary medium developed allows cell growth, production and normal glycosylation of recombinant antibody and metabolism of the recombinant CHO cells in both the microarray and shake flask platforms. In addition, 10.3 mM glutamate addition to the defined base media results in lactate metabolism shift in the recombinant GS/MSX CHO cells in the shake flask platform. Ultimately, the results demonstrate that the high-throughput microarray platform has the potential to be utilized for evaluating the impact of media additives on cellular processes, such as, cell growth, metabolism and productivity. PMID:24227746

Integrated Droplet-Based Microextraction with ESI-MS for Removal of Matrix Interference in Single-Cell Analysis.

PubMed

Zhang, Xiao-Chao; Wei, Zhen-Wei; Gong, Xiao-Yun; Si, Xing-Yu; Zhao, Yao-Yao; Yang, Cheng-Dui; Zhang, Si-Chun; Zhang, Xin-Rong

2016-04-29

Integrating droplet-based microfluidics with mass spectrometry is essential to high-throughput and multiple analysis of single cells. Nevertheless, matrix effects such as the interference of culture medium and intracellular components influence the sensitivity and the accuracy of results in single-cell analysis. To resolve this problem, we developed a method that integrated droplet-based microextraction with single-cell mass spectrometry. Specific extraction solvent was used to selectively obtain intracellular components of interest and remove interference of other components. Using this method, UDP-Glc-NAc, GSH, GSSG, AMP, ADP and ATP were successfully detected in single MCF-7 cells. We also applied the method to study the change of unicellular metabolites in the biological process of dysfunctional oxidative phosphorylation. The method could not only realize matrix-free, selective and sensitive detection of metabolites in single cells, but also have the capability for reliable and high-throughput single-cell analysis.

A novel hanging spherical drop system for the generation of cellular spheroids and high throughput combinatorial drug screening.

PubMed

Neto, A I; Correia, C R; Oliveira, M B; Rial-Hermida, M I; Alvarez-Lorenzo, C; Reis, R L; Mano, J F

2015-04-01

We propose a novel hanging spherical drop system for anchoring arrays of droplets of cell suspension based on the use of biomimetic superhydrophobic flat substrates, with controlled positional adhesion and minimum contact with a solid substrate. By facing down the platform, it was possible to generate independent spheroid bodies in a high throughput manner, in order to mimic in vivo tumour models on the lab-on-chip scale. To validate this system for drug screening purposes, the toxicity of the anti-cancer drug doxorubicin in cell spheroids was tested and compared to cells in 2D culture. The advantages presented by this platform, such as feasibility of the system and the ability to control the size uniformity of the spheroid, emphasize its potential to be used as a new low cost toolbox for high-throughput drug screening and in cell or tissue engineering.

Validation of high-throughput single cell analysis methodology.

PubMed

Devonshire, Alison S; Baradez, Marc-Olivier; Morley, Gary; Marshall, Damian; Foy, Carole A

2014-05-01

High-throughput quantitative polymerase chain reaction (qPCR) approaches enable profiling of multiple genes in single cells, bringing new insights to complex biological processes and offering opportunities for single cell-based monitoring of cancer cells and stem cell-based therapies. However, workflows with well-defined sources of variation are required for clinical diagnostics and testing of tissue-engineered products. In a study of neural stem cell lines, we investigated the performance of lysis, reverse transcription (RT), preamplification (PA), and nanofluidic qPCR steps at the single cell level in terms of efficiency, precision, and limit of detection. We compared protocols using a separate lysis buffer with cell capture directly in RT-PA reagent. The two methods were found to have similar lysis efficiencies, whereas the direct RT-PA approach showed improved precision. Digital PCR was used to relate preamplified template copy numbers to Cq values and reveal where low-quality signals may affect the analysis. We investigated the impact of calibration and data normalization strategies as a means of minimizing the impact of inter-experimental variation on gene expression values and found that both approaches can improve data comparability. This study provides validation and guidance for the application of high-throughput qPCR workflows for gene expression profiling of single cells. Copyright © 2014 Elsevier Inc. All rights reserved.

High-throughput screening with nanoimprinting 3D culture for efficient drug development by mimicking the tumor environment.

PubMed

Yoshii, Yukie; Furukawa, Takako; Waki, Atsuo; Okuyama, Hiroaki; Inoue, Masahiro; Itoh, Manabu; Zhang, Ming-Rong; Wakizaka, Hidekatsu; Sogawa, Chizuru; Kiyono, Yasushi; Yoshii, Hiroshi; Fujibayashi, Yasuhisa; Saga, Tsuneo

2015-05-01

Anti-cancer drug development typically utilizes high-throughput screening with two-dimensional (2D) cell culture. However, 2D culture induces cellular characteristics different from tumors in vivo, resulting in inefficient drug development. Here, we report an innovative high-throughput screening system using nanoimprinting 3D culture to simulate in vivo conditions, thereby facilitating efficient drug development. We demonstrated that cell line-based nanoimprinting 3D screening can more efficiently select drugs that effectively inhibit cancer growth in vivo as compared to 2D culture. Metabolic responses after treatment were assessed using positron emission tomography (PET) probes, and revealed similar characteristics between the 3D spheroids and in vivo tumors. Further, we developed an advanced method to adopt cancer cells from patient tumor tissues for high-throughput drug screening with nanoimprinting 3D culture, which we termed Cancer tissue-Originated Uniformed Spheroid Assay (COUSA). This system identified drugs that were effective in xenografts of the original patient tumors. Nanoimprinting 3D spheroids showed low permeability and formation of hypoxic regions inside, similar to in vivo tumors. Collectively, the nanoimprinting 3D culture provides easy-handling high-throughput drug screening system, which allows for efficient drug development by mimicking the tumor environment. The COUSA system could be a useful platform for drug development with patient cancer cells. Copyright © 2015 Elsevier Ltd. All rights reserved.

SPIM-fluid: open source light-sheet based platform for high-throughput imaging

PubMed Central

Gualda, Emilio J.; Pereira, Hugo; Vale, Tiago; Estrada, Marta Falcão; Brito, Catarina; Moreno, Nuno

2015-01-01

Light sheet fluorescence microscopy has recently emerged as the technique of choice for obtaining high quality 3D images of whole organisms/embryos with low photodamage and fast acquisition rates. Here we present an open source unified implementation based on Arduino and Micromanager, which is capable of operating Light Sheet Microscopes for automatized 3D high-throughput imaging on three-dimensional cell cultures and model organisms like zebrafish, oriented to massive drug screening. PMID:26601007

Process in manufacturing high efficiency AlGaAs/GaAs solar cells by MO-CVD

NASA Technical Reports Server (NTRS)

Yeh, Y. C. M.; Chang, K. I.; Tandon, J.

1984-01-01

Manufacturing technology for mass producing high efficiency GaAs solar cells is discussed. A progress using a high throughput MO-CVD reactor to produce high efficiency GaAs solar cells is discussed. Thickness and doping concentration uniformity of metal oxide chemical vapor deposition (MO-CVD) GaAs and AlGaAs layer growth are discussed. In addition, new tooling designs are given which increase the throughput of solar cell processing. To date, 2cm x 2cm AlGaAs/GaAs solar cells with efficiency up to 16.5% were produced. In order to meet throughput goals for mass producing GaAs solar cells, a large MO-CVD system (Cambridge Instrument Model MR-200) with a susceptor which was initially capable of processing 20 wafers (up to 75 mm diameter) during a single growth run was installed. In the MR-200, the sequencing of the gases and the heating power are controlled by a microprocessor-based programmable control console. Hence, operator errors can be reduced, leading to a more reproducible production sequence.

Identifying genes that extend life span using a high-throughput screening system.

PubMed

Chen, Cuiying; Contreras, Roland

2007-01-01

We developed a high-throughput functional genomic screening system that allows identification of genes prolonging lifespan in the baker's yeast Saccharomyces cerevisiae. The method is based on isolating yeast mother cells with a higher than average number of cell divisions as indicated by the number of bud scars on their surface. Fluorescently labeled wheat germ agglutinin (WGA) was used for specific staining of chitin, a major component of bud scars. The critical new steps in our bud-scar-sorting system are the use of small microbeads, which allows successive rounds of purification and regrowth of the mother cells (M-cell), and utilization of flow cytometry to sort and isolate cells with a longer lifespan based on the number of bud scars specifically labeled with WGA.

Automated Microfluidic Instrument for Label-Free and High-Throughput Cell Separation.

PubMed

Zhang, Xinjie; Zhu, Zhixian; Xiang, Nan; Long, Feifei; Ni, Zhonghua

2018-03-20

Microfluidic technologies for cell separation were reported frequently in recent years. However, a compact microfluidic instrument enabling thoroughly automated cell separation is still rarely reported until today due to the difficult hybrid between the macrosized fluidic control system and the microsized microfluidic device. In this work, we propose a novel and automated microfluidic instrument to realize size-based separation of cancer cells in a label-free and high-throughput manner. Briefly, the instrument is equipped with a fully integrated microfluidic device and a set of robust fluid-driven and control units, and the instrument functions of precise fluid infusion and high-throughput cell separation are guaranteed by a flow regulatory chip and two cell separation chips which are the key components of the microfluidic device. With optimized control programs, the instrument is successfully applied to automatically sort human breast adenocarcinoma cell line MCF-7 from 5 mL of diluted human blood with a high recovery ratio of ∼85% within a rapid processing time of ∼23 min. We envision that our microfluidic instrument will be potentially useful in many biomedical applications, especially cell separation, enrichment, and concentration for the purpose of cell culture and analysis.

High-throughput analysis of yeast replicative aging using a microfluidic system

PubMed Central

Jo, Myeong Chan; Liu, Wei; Gu, Liang; Dang, Weiwei; Qin, Lidong

2015-01-01

Saccharomyces cerevisiae has been an important model for studying the molecular mechanisms of aging in eukaryotic cells. However, the laborious and low-throughput methods of current yeast replicative lifespan assays limit their usefulness as a broad genetic screening platform for research on aging. We address this limitation by developing an efficient, high-throughput microfluidic single-cell analysis chip in combination with high-resolution time-lapse microscopy. This innovative design enables, to our knowledge for the first time, the determination of the yeast replicative lifespan in a high-throughput manner. Morphological and phenotypical changes during aging can also be monitored automatically with a much higher throughput than previous microfluidic designs. We demonstrate highly efficient trapping and retention of mother cells, determination of the replicative lifespan, and tracking of yeast cells throughout their entire lifespan. Using the high-resolution and large-scale data generated from the high-throughput yeast aging analysis (HYAA) chips, we investigated particular longevity-related changes in cell morphology and characteristics, including critical cell size, terminal morphology, and protein subcellular localization. In addition, because of the significantly improved retention rate of yeast mother cell, the HYAA-Chip was capable of demonstrating replicative lifespan extension by calorie restriction. PMID:26170317

Label-free high-throughput detection and quantification of circulating melanoma tumor cell clusters by linear-array-based photoacoustic tomography

NASA Astrophysics Data System (ADS)

Hai, Pengfei; Zhou, Yong; Zhang, Ruiying; Ma, Jun; Li, Yang; Shao, Jin-Yu; Wang, Lihong V.

2017-04-01

Circulating tumor cell (CTC) clusters, arising from multicellular groupings in a primary tumor, greatly elevate the metastatic potential of cancer compared with single CTCs. High-throughput detection and quantification of CTC clusters are important for understanding the tumor metastatic process and improving cancer therapy. Here, we applied a linear-array-based photoacoustic tomography (LA-PAT) system and improved the image reconstruction for label-free high-throughput CTC cluster detection and quantification in vivo. The feasibility was first demonstrated by imaging CTC cluster ex vivo. The relationship between the contrast-to-noise ratios (CNRs) and the number of cells in melanoma tumor cell clusters was investigated and verified. Melanoma CTC clusters with a minimum of four cells could be detected, and the number of cells could be computed from the CNR. Finally, we demonstrated imaging of injected melanoma CTC clusters in rats in vivo. Similarly, the number of cells in the melanoma CTC clusters could be quantified. The data showed that larger CTC clusters had faster clearance rates in the bloodstream, which agreed with the literature. The results demonstrated the capability of LA-PAT to detect and quantify melanoma CTC clusters in vivo and showed its potential for tumor metastasis study and cancer therapy.

Unsupervised Clustering of Subcellular Protein Expression Patterns in High-Throughput Microscopy Images Reveals Protein Complexes and Functional Relationships between Proteins

PubMed Central

Handfield, Louis-François; Chong, Yolanda T.; Simmons, Jibril; Andrews, Brenda J.; Moses, Alan M.

2013-01-01

Protein subcellular localization has been systematically characterized in budding yeast using fluorescently tagged proteins. Based on the fluorescence microscopy images, subcellular localization of many proteins can be classified automatically using supervised machine learning approaches that have been trained to recognize predefined image classes based on statistical features. Here, we present an unsupervised analysis of protein expression patterns in a set of high-resolution, high-throughput microscope images. Our analysis is based on 7 biologically interpretable features which are evaluated on automatically identified cells, and whose cell-stage dependency is captured by a continuous model for cell growth. We show that it is possible to identify most previously identified localization patterns in a cluster analysis based on these features and that similarities between the inferred expression patterns contain more information about protein function than can be explained by a previous manual categorization of subcellular localization. Furthermore, the inferred cell-stage associated to each fluorescence measurement allows us to visualize large groups of proteins entering the bud at specific stages of bud growth. These correspond to proteins localized to organelles, revealing that the organelles must be entering the bud in a stereotypical order. We also identify and organize a smaller group of proteins that show subtle differences in the way they move around the bud during growth. Our results suggest that biologically interpretable features based on explicit models of cell morphology will yield unprecedented power for pattern discovery in high-resolution, high-throughput microscopy images. PMID:23785265

Advantages and application of label-free detection assays in drug screening.

PubMed

Cunningham, Brian T; Laing, Lance G

2008-08-01

Adoption is accelerating for a new family of label-free optical biosensors incorporated into standard format microplates owing to their ability to enable highly sensitive detection of small molecules, proteins and cells for high-throughput drug discovery applications. Label-free approaches are displacing other detection technologies owing to their ability to provide simple assay procedures for hit finding/validation, accessing difficult target classes, screening the interaction of cells with drugs and analyzing the affinity of small molecule inhibitors to target proteins. This review describes several new drug discovery applications that are under development for microplate-based photonic crystal optical biosensors and the key issues that will drive adoption of the technology. Microplate-based optical biosensors are enabling a variety of cell-based assays, inhibition assays, protein-protein binding assays and protein-small molecule binding assays to be performed with high-throughput and high sensitivity.

High-Throughput Phenotyping of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes and Neurons Using Electric Field Stimulation and High-Speed Fluorescence Imaging

PubMed Central

Daily, Neil J.; Du, Zhong-Wei

2017-01-01

Abstract Electrophysiology of excitable cells, including muscle cells and neurons, has been measured by making direct contact with a single cell using a micropipette electrode. To increase the assay throughput, optical devices such as microscopes and microplate readers have been used to analyze electrophysiology of multiple cells. We have established a high-throughput (HTP) analysis of action potentials (APs) in highly enriched motor neurons and cardiomyocytes (CMs) that are differentiated from human induced pluripotent stem cells (iPSCs). A multichannel electric field stimulation (EFS) device enabled the ability to electrically stimulate cells and measure dynamic changes in APs of excitable cells ultra-rapidly (>100 data points per second) by imaging entire 96-well plates. We found that the activities of both neurons and CMs and their response to EFS and chemicals are readily discerned by our fluorescence imaging-based HTP phenotyping assay. The latest generation of calcium (Ca2+) indicator dyes, FLIPR Calcium 6 and Cal-520, with the HTP device enables physiological analysis of human iPSC-derived samples highlighting its potential application for understanding disease mechanisms and discovering new therapeutic treatments. PMID:28525289

Microfluidic Imaging Flow Cytometry by Asymmetric-detection Time-stretch Optical Microscopy (ATOM).

PubMed

Tang, Anson H L; Lai, Queenie T K; Chung, Bob M F; Lee, Kelvin C M; Mok, Aaron T Y; Yip, G K; Shum, Anderson H C; Wong, Kenneth K Y; Tsia, Kevin K

2017-06-28

Scaling the number of measurable parameters, which allows for multidimensional data analysis and thus higher-confidence statistical results, has been the main trend in the advanced development of flow cytometry. Notably, adding high-resolution imaging capabilities allows for the complex morphological analysis of cellular/sub-cellular structures. This is not possible with standard flow cytometers. However, it is valuable for advancing our knowledge of cellular functions and can benefit life science research, clinical diagnostics, and environmental monitoring. Incorporating imaging capabilities into flow cytometry compromises the assay throughput, primarily due to the limitations on speed and sensitivity in the camera technologies. To overcome this speed or throughput challenge facing imaging flow cytometry while preserving the image quality, asymmetric-detection time-stretch optical microscopy (ATOM) has been demonstrated to enable high-contrast, single-cell imaging with sub-cellular resolution, at an imaging throughput as high as 100,000 cells/s. Based on the imaging concept of conventional time-stretch imaging, which relies on all-optical image encoding and retrieval through the use of ultrafast broadband laser pulses, ATOM further advances imaging performance by enhancing the image contrast of unlabeled/unstained cells. This is achieved by accessing the phase-gradient information of the cells, which is spectrally encoded into single-shot broadband pulses. Hence, ATOM is particularly advantageous in high-throughput measurements of single-cell morphology and texture - information indicative of cell types, states, and even functions. Ultimately, this could become a powerful imaging flow cytometry platform for the biophysical phenotyping of cells, complementing the current state-of-the-art biochemical-marker-based cellular assay. This work describes a protocol to establish the key modules of an ATOM system (from optical frontend to data processing and visualization backend), as well as the workflow of imaging flow cytometry based on ATOM, using human cells and micro-algae as the examples.

Ultrafast Microfluidic Cellular Imaging by Optical Time-Stretch.

PubMed

Lau, Andy K S; Wong, Terence T W; Shum, Ho Cheung; Wong, Kenneth K Y; Tsia, Kevin K

2016-01-01

There is an unmet need in biomedicine for measuring a multitude of parameters of individual cells (i.e., high content) in a large population efficiently (i.e., high throughput). This is particularly driven by the emerging interest in bringing Big-Data analysis into this arena, encompassing pathology, drug discovery, rare cancer cell detection, emulsion microdroplet assays, to name a few. This momentum is particularly evident in recent advancements in flow cytometry. They include scaling of the number of measurable colors from the labeled cells and incorporation of imaging capability to access the morphological information of the cells. However, an unspoken predicament appears in the current technologies: higher content comes at the expense of lower throughput, and vice versa. For example, accessing additional spatial information of individual cells, imaging flow cytometers only achieve an imaging throughput ~1000 cells/s, orders of magnitude slower than the non-imaging flow cytometers. In this chapter, we introduce an entirely new imaging platform, namely optical time-stretch microscopy, for ultrahigh speed and high contrast label-free single-cell (in a ultrafast microfluidic flow up to 10 m/s) imaging and analysis with an ultra-fast imaging line-scan rate as high as tens of MHz. Based on this technique, not only morphological information of the individual cells can be obtained in an ultrafast manner, quantitative evaluation of cellular information (e.g., cell volume, mass, refractive index, stiffness, membrane tension) at nanometer scale based on the optical phase is also possible. The technology can also be integrated with conventional fluorescence measurements widely adopted in the non-imaging flow cytometers. Therefore, these two combinatorial and complementary measurement capabilities in long run is an attractive platform for addressing the pressing need for expanding the "parameter space" in high-throughput single-cell analysis. This chapter provides the general guidelines of constructing the optical system for time stretch imaging, fabrication and design of the microfluidic chip for ultrafast fluidic flow, as well as the image acquisition and processing.

Continuous cell introduction and rapid dynamic lysis for high-throughput single-cell analysis on microfludic chips with hydrodynamic focusing.

PubMed

Xu, Chun-Xiu; Yin, Xue-Feng

2011-02-04

A chip-based microfluidic system for high-throughput single-cell analysis is described. The system was integrated with continuous introduction of individual cells, rapid dynamic lysis, capillary electrophoretic (CE) separation and laser induced fluorescence (LIF) detection. A cross microfluidic chip with one sheath-flow channel located on each side of the sampling channel was designed. The labeled cells were hydrodynamically focused by sheath-flow streams and sequentially introduced into the cross section of the microchip under hydrostatic pressure generated by adjusting liquid levels in the reservoirs. Combined with the electric field applied on the separation channel, the aligned cells were driven into the separation channel and rapidly lysed within 33ms at the entry of the separation channel by Triton X-100 added in the sheath-flow solution. The maximum rate for introducing individual cells into the separation channel was about 150cells/min. The introduction of sheath-flow streams also significantly reduced the concentration of phosphate-buffered saline (PBS) injected into the separation channel along with single cells, thus reducing Joule heating during electrophoretic separation. The performance of this microfluidic system was evaluated by analysis of reduced glutathione (GSH) and reactive oxygen species (ROS) in single erythrocytes. A throughput of 38cells/min was obtained. The proposed method is simple and robust for high-throughput single-cell analysis, allowing for analysis of cell population with considerable size to generate results with statistical significance. Copyright © 2010 Elsevier B.V. All rights reserved.

Developing a gene biomarker at the tipping point of adaptive and adverse responses in human bronchial epithelial cells

EPA Science Inventory

Determining mechanism-based biomarkers that distinguish adaptive and adverse cellular processes is critical to understanding the health effects of environmental exposures. Shifting from in vivo, low-throughput toxicity studies to high-throughput screening (HTS) paradigms and risk...

A High-Throughput Assay for Screening of Natural Products that Enhanced Tumoricidal Activity of NK Cells.

PubMed

Gong, Chenyuan; Ni, Zhongya; Yao, Chao; Zhu, Xiaowen; Ni, Lulu; Wang, Lixin; Zhu, Shiguo

2015-01-01

Recently, immunotherapy has shown a lot of promise in cancer treatment and different immune cell types are involved in this endeavor. Among different immune cell populations, NK cells are also an important component in unleashing the therapeutic activity of immune cells. Therefore, in order to enhance the tumoricidal activity of NK cells, identification of new small-molecule natural products is important. Despite the availability of different screening methods for identification of natural products, a simple, economic and high-throughput method is lacking. Hence, in this study, we have developed a high-throughput assay for screening and indentifying natural products that can enhance NK cell-mediated killing of cancer cells. We expanded human NK cell population from human peripheral blood mononuclear cells (PBMCs) by culturing these PBMCs with membrane-bound IL-21 and CD137L engineered K562 cells. Next, expanded NK cells were co-cultured with non-small cell lung cancer (NSCLC) cells with or without natural products and after 24 h of co-culturing, harvested supernatants were analyzed for IFN-γ secretions by ELISA method. We screened 502 natural products and identified that 28 candidates has the potential to induce IFN-γ secretion by NK cells to varying degrees. Among the 28 natural product candidates, we further confirmed and analyzed the potential of one molecule, andrographolide. It actually increased IFN-γ secretion by NK cells and enhanced NK cell-mediated killing of NSCLC cells. Our results demonstrated that this IFN-γ based high-throughput assay for screening of natural products for NK cell tumoricidal activity is a simple, economic and reliable method.

CellCognition: time-resolved phenotype annotation in high-throughput live cell imaging.

PubMed

Held, Michael; Schmitz, Michael H A; Fischer, Bernd; Walter, Thomas; Neumann, Beate; Olma, Michael H; Peter, Matthias; Ellenberg, Jan; Gerlich, Daniel W

2010-09-01

Fluorescence time-lapse imaging has become a powerful tool to investigate complex dynamic processes such as cell division or intracellular trafficking. Automated microscopes generate time-resolved imaging data at high throughput, yet tools for quantification of large-scale movie data are largely missing. Here we present CellCognition, a computational framework to annotate complex cellular dynamics. We developed a machine-learning method that combines state-of-the-art classification with hidden Markov modeling for annotation of the progression through morphologically distinct biological states. Incorporation of time information into the annotation scheme was essential to suppress classification noise at state transitions and confusion between different functional states with similar morphology. We demonstrate generic applicability in different assays and perturbation conditions, including a candidate-based RNA interference screen for regulators of mitotic exit in human cells. CellCognition is published as open source software, enabling live-cell imaging-based screening with assays that directly score cellular dynamics.

High-speed Fourier ptychographic microscopy based on programmable annular illuminations.

PubMed

Sun, Jiasong; Zuo, Chao; Zhang, Jialin; Fan, Yao; Chen, Qian

2018-05-16

High-throughput quantitative phase imaging (QPI) is essential to cellular phenotypes characterization as it allows high-content cell analysis and avoids adverse effects of staining reagents on cellular viability and cell signaling. Among different approaches, Fourier ptychographic microscopy (FPM) is probably the most promising technique to realize high-throughput QPI by synthesizing a wide-field, high-resolution complex image from multiple angle-variably illuminated, low-resolution images. However, the large dataset requirement in conventional FPM significantly limits its imaging speed, resulting in low temporal throughput. Moreover, the underlying theoretical mechanism as well as optimum illumination scheme for high-accuracy phase imaging in FPM remains unclear. Herein, we report a high-speed FPM technique based on programmable annular illuminations (AIFPM). The optical-transfer-function (OTF) analysis of FPM reveals that the low-frequency phase information can only be correctly recovered if the LEDs are precisely located at the edge of the objective numerical aperture (NA) in the frequency space. By using only 4 low-resolution images corresponding to 4 tilted illuminations matching a 10×, 0.4 NA objective, we present the high-speed imaging results of in vitro Hela cells mitosis and apoptosis at a frame rate of 25 Hz with a full-pitch resolution of 655 nm at a wavelength of 525 nm (effective NA = 0.8) across a wide field-of-view (FOV) of 1.77 mm 2 , corresponding to a space-bandwidth-time product of 411 megapixels per second. Our work reveals an important capability of FPM towards high-speed high-throughput imaging of in vitro live cells, achieving video-rate QPI performance across a wide range of scales, both spatial and temporal.

pH measurement and a rational and practical pH control strategy for high throughput cell culture system.

PubMed

Zhou, Haiying; Purdie, Jennifer; Wang, Tongtong; Ouyang, Anli

2010-01-01

The number of therapeutic proteins produced by cell culture in the pharmaceutical industry continues to increase. During the early stages of manufacturing process development, hundreds of clones and various cell culture conditions are evaluated to develop a robust process to identify and select cell lines with high productivity. It is highly desirable to establish a high throughput system to accelerate process development and reduce cost. Multiwell plates and shake flasks are widely used in the industry as the scale down model for large-scale bioreactors. However, one of the limitations of these two systems is the inability to measure and control pH in a high throughput manner. As pH is an important process parameter for cell culture, this could limit the applications of these scale down model vessels. An economical, rapid, and robust pH measurement method was developed at Eli Lilly and Company by employing SNARF-4F 5-(-and 6)-carboxylic acid. The method demonstrated the ability to measure the pH values of cell culture samples in a high throughput manner. Based upon the chemical equilibrium of CO(2), HCO(3)(-), and the buffer system, i.e., HEPES, we established a mathematical model to regulate pH in multiwell plates and shake flasks. The model calculates the required %CO(2) from the incubator and the amount of sodium bicarbonate to be added to adjust pH to a preset value. The model was validated by experimental data, and pH was accurately regulated by this method. The feasibility of studying the pH effect on cell culture in 96-well plates and shake flasks was also demonstrated in this study. This work shed light on mini-bioreactor scale down model construction and paved the way for cell culture process development to improve productivity or product quality using high throughput systems. Copyright 2009 American Institute of Chemical Engineers

A high content, high throughput cellular thermal stability assay for measuring drug-target engagement in living cells.

PubMed

Massey, Andrew J

2018-01-01

Determining and understanding drug target engagement is critical for drug discovery. This can be challenging within living cells as selective readouts are often unavailable. Here we describe a novel method for measuring target engagement in living cells based on the principle of altered protein thermal stabilization / destabilization in response to ligand binding. This assay (HCIF-CETSA) utilizes high content, high throughput single cell immunofluorescent detection to determine target protein levels following heating of adherent cells in a 96 well plate format. We have used target engagement of Chk1 by potent small molecule inhibitors to validate the assay. Target engagement measured by this method was subsequently compared to target engagement measured by two alternative methods (autophosphorylation and CETSA). The HCIF-CETSA method appeared robust and a good correlation in target engagement measured by this method and CETSA for the selective Chk1 inhibitor V158411 was observed. However, these EC50 values were 23- and 12-fold greater than the autophosphorylation IC50. The described method is therefore a valuable advance in the CETSA method allowing the high throughput determination of target engagement in adherent cells.

A radial flow microfluidic device for ultra-high-throughput affinity-based isolation of circulating tumor cells.

PubMed

Murlidhar, Vasudha; Zeinali, Mina; Grabauskiene, Svetlana; Ghannad-Rezaie, Mostafa; Wicha, Max S; Simeone, Diane M; Ramnath, Nithya; Reddy, Rishindra M; Nagrath, Sunitha

2014-12-10

Circulating tumor cells (CTCs) are believed to play an important role in metastasis, a process responsible for the majority of cancer-related deaths. But their rarity in the bloodstream makes microfluidic isolation complex and time-consuming. Additionally the low processing speeds can be a hindrance to obtaining higher yields of CTCs, limiting their potential use as biomarkers for early diagnosis. Here, a high throughput microfluidic technology, the OncoBean Chip, is reported. It employs radial flow that introduces a varying shear profile across the device, enabling efficient cell capture by affinity at high flow rates. The recovery from whole blood is validated with cancer cell lines H1650 and MCF7, achieving a mean efficiency >80% at a throughput of 10 mL h(-1) in contrast to a flow rate of 1 mL h(-1) standardly reported with other microfluidic devices. Cells are recovered with a viability rate of 93% at these high speeds, increasing the ability to use captured CTCs for downstream analysis. Broad clinical application is demonstrated using comparable flow rates from blood specimens obtained from breast, pancreatic, and lung cancer patients. Comparable CTC numbers are recovered in all the samples at the two flow rates, demonstrating the ability of the technology to perform at high throughputs. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Microfluidic Impedance Flow Cytometry Enabling High-Throughput Single-Cell Electrical Property Characterization

PubMed Central

Chen, Jian; Xue, Chengcheng; Zhao, Yang; Chen, Deyong; Wu, Min-Hsien; Wang, Junbo

2015-01-01

This article reviews recent developments in microfluidic impedance flow cytometry for high-throughput electrical property characterization of single cells. Four major perspectives of microfluidic impedance flow cytometry for single-cell characterization are included in this review: (1) early developments of microfluidic impedance flow cytometry for single-cell electrical property characterization; (2) microfluidic impedance flow cytometry with enhanced sensitivity; (3) microfluidic impedance and optical flow cytometry for single-cell analysis and (4) integrated point of care system based on microfluidic impedance flow cytometry. We examine the advantages and limitations of each technique and discuss future research opportunities from the perspectives of both technical innovation and clinical applications. PMID:25938973

Use of early passage fetal intestinal epithelial cells in semi-high-throughput screening assays: an approach to identify new innate immune system adjuvants.

PubMed

Buckner, Diana; Wilson, Suzanne; Kurk, Sandra; Hardy, Michele; Miessner, Nicole; Jutila, Mark A

2006-09-01

Innate immune system stimulants (innate adjuvants) offer complementary approaches to vaccines and antimicrobial compounds to increase host resistance to infection. The authors established fetal bovine intestinal epithelial cell (BIEC) cultures to screen natural product and synthetic compound libraries for novel mucosal adjuvants. They showed that BIECs from fetal intestine maintained an in vivo phenotype as reflected in cytokeratin expression, expression of antigens restricted to intestinal enterocytes, and induced interleukin-8 (IL-8) production. BIECs could be infected by and support replication of bovine rotavirus. A semi-high-throughput enzyme-linked immunosorbent assay-based assay that measured IL-8 production by BIECs was established and used to screen commercially available natural compounds for novel adjuvant activity. Five novel hits were identified, demonstrating the utility of the assay for selecting and screening new epithelial cell adjuvants. Although the identified compounds had not previously been shown to induce IL-8 production in epithelial cells, other known functions for 3 of the 5 were consistent with this activity. Statistical analysis of the throughput data demonstrated that the assay is adaptable to a high-throughput format for screening both synthetic and natural product derived compound libraries.

Identification of Novel Compounds Inhibiting Chikungunya Virus-Induced Cell Death by High Throughput Screening of a Kinase Inhibitor Library

PubMed Central

Gomes, Rafael G. B.; da Silva, Camila T.; Taniguchi, Juliana B.; No, Joo Hwan; Lombardot, Benoit; Schwartz, Olivier; Hansen, Michael A. E.; Freitas-Junior, Lucio H.

2013-01-01

Chikungunya virus (CHIKV) is a mosquito-borne arthrogenic alphavirus that causes acute febrile illness in humans accompanied by joint pains and in many cases, persistent arthralgia lasting weeks to years. The re-emergence of CHIKV has resulted in numerous outbreaks in the eastern hemisphere, and threatens to expand in the foreseeable future. Unfortunately, no effective treatment is currently available. The present study reports the use of resazurin in a cell-based high-throughput assay, and an image-based high-content assay to identify and characterize inhibitors of CHIKV-infection in vitro. CHIKV is a highly cytopathic virus that rapidly kills infected cells. Thus, cell viability of HuH-7 cells infected with CHIKV in the presence of compounds was determined by measuring metabolic reduction of resazurin to identify inhibitors of CHIKV-associated cell death. A kinase inhibitor library of 4,000 compounds was screened against CHIKV infection of HuH-7 cells using the resazurin reduction assay, and the cell toxicity was also measured in non-infected cells. Seventy-two compounds showing ≥50% inhibition property against CHIKV at 10 µM were selected as primary hits. Four compounds having a benzofuran core scaffold (CND0335, CND0364, CND0366 and CND0415), one pyrrolopyridine (CND0545) and one thiazol-carboxamide (CND3514) inhibited CHIKV-associated cell death in a dose-dependent manner, with EC50 values between 2.2 µM and 7.1 µM. Based on image analysis, these 6 hit compounds did not inhibit CHIKV replication in the host cell. However, CHIKV-infected cells manifested less prominent apoptotic blebs typical of CHIKV cytopathic effect compared with the control infection. Moreover, treatment with these compounds reduced viral titers in the medium of CHIKV-infected cells by up to 100-fold. In conclusion, this cell-based high-throughput screening assay using resazurin, combined with the image-based high content assay approach identified compounds against CHIKV having a novel antiviral activity - inhibition of virus-induced CPE - likely by targeting kinases involved in apoptosis. PMID:24205414

High content screening of ToxCast compounds using Vala Sciences’ complex cell culturing systems (SOT)

EPA Science Inventory

US EPA’s ToxCast research program evaluates bioactivity for thousands of chemicals utilizing high-throughput screening assays to inform chemical testing decisions. Vala Sciences provides high content, multiplexed assays that utilize quantitative cell-based digital image analysis....

Vision-based Nano Robotic System for High-throughput Non-embedded Cell Cutting

NASA Astrophysics Data System (ADS)

Shang, Wanfeng; Lu, Haojian; Wan, Wenfeng; Fukuda, Toshio; Shen, Yajing

2016-03-01

Cell cutting is a significant task in biology study, but the highly productive non-embedded cell cutting is still a big challenge for current techniques. This paper proposes a vision-based nano robotic system and then realizes automatic non-embedded cell cutting with this system. First, the nano robotic system is developed and integrated with a nanoknife inside an environmental scanning electron microscopy (ESEM). Then, the positions of the nanoknife and the single cell are recognized, and the distance between them is calculated dynamically based on image processing. To guarantee the positioning accuracy and the working efficiency, we propose a distance-regulated speed adapting strategy, in which the moving speed is adjusted intelligently based on the distance between the nanoknife and the target cell. The results indicate that the automatic non-embedded cutting is able to be achieved within 1-2 mins with low invasion benefiting from the high precise nanorobot system and the sharp edge of nanoknife. This research paves a way for the high-throughput cell cutting at cell’s natural condition, which is expected to make significant impact on the biology studies, especially for the in-situ analysis at cellular and subcellular scale, such as cell interaction investigation, neural signal transduction and low invasive cell surgery.

Cytopathological image analysis using deep-learning networks in microfluidic microscopy.

PubMed

Gopakumar, G; Hari Babu, K; Mishra, Deepak; Gorthi, Sai Siva; Sai Subrahmanyam, Gorthi R K

2017-01-01

Cytopathologic testing is one of the most critical steps in the diagnosis of diseases, including cancer. However, the task is laborious and demands skill. Associated high cost and low throughput drew considerable interest in automating the testing process. Several neural network architectures were designed to provide human expertise to machines. In this paper, we explore and propose the feasibility of using deep-learning networks for cytopathologic analysis by performing the classification of three important unlabeled, unstained leukemia cell lines (K562, MOLT, and HL60). The cell images used in the classification are captured using a low-cost, high-throughput cell imaging technique: microfluidics-based imaging flow cytometry. We demonstrate that without any conventional fine segmentation followed by explicit feature extraction, the proposed deep-learning algorithms effectively classify the coarsely localized cell lines. We show that the designed deep belief network as well as the deeply pretrained convolutional neural network outperform the conventionally used decision systems and are important in the medical domain, where the availability of labeled data is limited for training. We hope that our work enables the development of a clinically significant high-throughput microfluidic microscopy-based tool for disease screening/triaging, especially in resource-limited settings.

Automated analysis of siRNA screens of cells infected by hepatitis C and dengue viruses based on immunofluorescence microscopy images

NASA Astrophysics Data System (ADS)

Matula, Petr; Kumar, Anil; Wörz, Ilka; Harder, Nathalie; Erfle, Holger; Bartenschlager, Ralf; Eils, Roland; Rohr, Karl

2008-03-01

We present an image analysis approach as part of a high-throughput microscopy siRNA-based screening system using cell arrays for the identification of cellular genes involved in hepatitis C and dengue virus replication. Our approach comprises: cell nucleus segmentation, quantification of virus replication level in the neighborhood of segmented cell nuclei, localization of regions with transfected cells, cell classification by infection status, and quality assessment of an experiment and single images. In particular, we propose a novel approach for the localization of regions of transfected cells within cell array images, which combines model-based circle fitting and grid fitting. By this scheme we integrate information from single cell array images and knowledge from the complete cell arrays. The approach is fully automatic and has been successfully applied to a large number of cell array images from screening experiments. The experimental results show a good agreement with the expected behaviour of positive as well as negative controls and encourage the application to screens from further high-throughput experiments.

Towards high-throughput automated targeted femtosecond laser-based transfection of adherent cells

NASA Astrophysics Data System (ADS)

Antkowiak, Maciej; Torres-Mapa, Maria Leilani; Gunn-Moore, Frank; Dholakia, Kishan

2011-03-01

Femtosecond laser induced cell membrane poration has proven to be an attractive alternative to the classical methods of drug and gene delivery. It is a selective, sterile, non-contact technique that offers a highly localized operation, low toxicity and consistent performance. However, its broader application still requires the development of robust, high-throughput and user-friendly systems. We present a system capable of unassisted enhanced targeted optoinjection and phototransfection of adherent mammalian cells with a femtosecond laser. We demonstrate the advantages of a dynamic diffractive optical element, namely a spatial light modulator (SLM) for precise three dimensional positioning of the beam. It enables the implementation of a "point-and-shoot" system in which using the software interface a user simply points at the cell and a predefined sequence of precisely positioned doses can be applied. We show that irradiation in three axial positions alleviates the problem of exact beam positioning on the cell membrane and doubles the number of viably optoinjected cells when compared with a single dose. The presented system enables untargeted raster scan irradiation which provides transfection of adherent cells at the throughput of 1 cell per second.

High throughput ion-channel pharmacology: planar-array-based voltage clamp.

PubMed

Kiss, Laszlo; Bennett, Paul B; Uebele, Victor N; Koblan, Kenneth S; Kane, Stefanie A; Neagle, Brad; Schroeder, Kirk

2003-02-01

Technological advances often drive major breakthroughs in biology. Examples include PCR, automated DNA sequencing, confocal/single photon microscopy, AFM, and voltage/patch-clamp methods. The patch-clamp method, first described nearly 30 years ago, was a major technical achievement that permitted voltage-clamp analysis (membrane potential control) of ion channels in most cells and revealed a role for channels in unimagined areas. Because of the high information content, voltage clamp is the best way to study ion-channel function; however, throughput is too low for drug screening. Here we describe a novel breakthrough planar-array-based HT patch-clamp technology developed by Essen Instruments capable of voltage-clamping thousands of cells per day. This technology provides greater than two orders of magnitude increase in throughput compared with the traditional voltage-clamp techniques. We have applied this method to study the hERG K(+) channel and to determine the pharmacological profile of QT prolonging drugs.

Microscale Laminar Vortices for High-Purity Extraction and Release of Circulating Tumor Cells.

PubMed

Hur, Soojung Claire; Che, James; Di Carlo, Dino

2017-01-01

Circulating tumor cells (CTCs) are disseminated tumor cells that reflect the tumors of origin and can provide a liquid biopsy that would potentially enable noninvasive tumor profiling, treatment monitoring, and identification of targeted treatments. Accurate and rapid purification of CTCs holds great potential to improve cancer care but the task remains technically challenging. Microfluidic isolation of CTCs within microscale vortices enables high-throughput and size-based purification of rare CTCs from bodily fluids. Collected cells are highly pure, viable, and easily accessible, allowing seamless integration with various downstream applications. Here, we describe how to fabricate the High-Throughput Vortex Chip (Vortex-HT) and to process diluted whole blood for CTC collection. Lastly, immunostaining and imaging protocols for CTC classification and corresponding CTC image galleries are reported.

High resolution light-sheet based high-throughput imaging cytometry system enables visualization of intra-cellular organelles

NASA Astrophysics Data System (ADS)

Regmi, Raju; Mohan, Kavya; Mondal, Partha Pratim

2014-09-01

Visualization of intracellular organelles is achieved using a newly developed high throughput imaging cytometry system. This system interrogates the microfluidic channel using a sheet of light rather than the existing point-based scanning techniques. The advantages of the developed system are many, including, single-shot scanning of specimens flowing through the microfluidic channel at flow rate ranging from micro- to nano- lit./min. Moreover, this opens-up in-vivo imaging of sub-cellular structures and simultaneous cell counting in an imaging cytometry system. We recorded a maximum count of 2400 cells/min at a flow-rate of 700 nl/min, and simultaneous visualization of fluorescently-labeled mitochondrial network in HeLa cells during flow. The developed imaging cytometry system may find immediate application in biotechnology, fluorescence microscopy and nano-medicine.

A review of the theory, methods and recent applications of high-throughput single-cell droplet microfluidics

NASA Astrophysics Data System (ADS)

Lagus, Todd P.; Edd, Jon F.

2013-03-01

Most cell biology experiments are performed in bulk cell suspensions where cell secretions become diluted and mixed in a contiguous sample. Confinement of single cells to small, picoliter-sized droplets within a continuous phase of oil provides chemical isolation of each cell, creating individual microreactors where rare cell qualities are highlighted and otherwise undetectable signals can be concentrated to measurable levels. Recent work in microfluidics has yielded methods for the encapsulation of cells in aqueous droplets and hydrogels at kilohertz rates, creating the potential for millions of parallel single-cell experiments. However, commercial applications of high-throughput microdroplet generation and downstream sensing and actuation methods are still emerging for cells. Using fluorescence-activated cell sorting (FACS) as a benchmark for commercially available high-throughput screening, this focused review discusses the fluid physics of droplet formation, methods for cell encapsulation in liquids and hydrogels, sensors and actuators and notable biological applications of high-throughput single-cell droplet microfluidics.

Subnuclear foci quantification using high-throughput 3D image cytometry

NASA Astrophysics Data System (ADS)

Wadduwage, Dushan N.; Parrish, Marcus; Choi, Heejin; Engelward, Bevin P.; Matsudaira, Paul; So, Peter T. C.

2015-07-01

Ionising radiation causes various types of DNA damages including double strand breaks (DSBs). DSBs are often recognized by DNA repair protein ATM which forms gamma-H2AX foci at the site of the DSBs that can be visualized using immunohistochemistry. However most of such experiments are of low throughput in terms of imaging and image analysis techniques. Most of the studies still use manual counting or classification. Hence they are limited to counting a low number of foci per cell (5 foci per nucleus) as the quantification process is extremely labour intensive. Therefore we have developed a high throughput instrumentation and computational pipeline specialized for gamma-H2AX foci quantification. A population of cells with highly clustered foci inside nuclei were imaged, in 3D with submicron resolution, using an in-house developed high throughput image cytometer. Imaging speeds as high as 800 cells/second in 3D were achieved by using HiLo wide-field depth resolved imaging and a remote z-scanning technique. Then the number of foci per cell nucleus were quantified using a 3D extended maxima transform based algorithm. Our results suggests that while most of the other 2D imaging and manual quantification studies can count only up to about 5 foci per nucleus our method is capable of counting more than 100. Moreover we show that 3D analysis is significantly superior compared to the 2D techniques.

20180312 - Application of a Multiplexed High Content Imaging (HCI) Based Cell Viability and Apoptosis Chemical Screening Assay with Results in MCF-7 Cells (SOT)

EPA Science Inventory

The NCCT high throughput transcriptomics (HTTr) screening program uses whole transcriptome profiling assay in human-derived cells to collect concentration-response data for large numbers (100s-1000s) of environmental chemicals. To contextualize HTTr data, chemical effects on cell...

Single-cell genome sequencing at ultra-high-throughput with microfluidic droplet barcoding.

PubMed

Lan, Freeman; Demaree, Benjamin; Ahmed, Noorsher; Abate, Adam R

2017-07-01

The application of single-cell genome sequencing to large cell populations has been hindered by technical challenges in isolating single cells during genome preparation. Here we present single-cell genomic sequencing (SiC-seq), which uses droplet microfluidics to isolate, fragment, and barcode the genomes of single cells, followed by Illumina sequencing of pooled DNA. We demonstrate ultra-high-throughput sequencing of >50,000 cells per run in a synthetic community of Gram-negative and Gram-positive bacteria and fungi. The sequenced genomes can be sorted in silico based on characteristic sequences. We use this approach to analyze the distributions of antibiotic-resistance genes, virulence factors, and phage sequences in microbial communities from an environmental sample. The ability to routinely sequence large populations of single cells will enable the de-convolution of genetic heterogeneity in diverse cell populations.

Development of a high-throughput screening system for identification of novel reagents regulating DNA damage in human dermal fibroblasts.

PubMed

Bae, Seunghee; An, In-Sook; An, Sungkwan

2015-09-01

Ultraviolet (UV) radiation is a major inducer of skin aging and accumulated exposure to UV radiation increases DNA damage in skin cells, including dermal fibroblasts. In the present study, we developed a novel DNA repair regulating material discovery (DREAM) system for the high-throughput screening and identification of putative materials regulating DNA repair in skin cells. First, we established a modified lentivirus expressing the luciferase and hypoxanthine phosphoribosyl transferase (HPRT) genes. Then, human dermal fibroblast WS-1 cells were infected with the modified lentivirus and selected with puromycin to establish cells that stably expressed luciferase and HPRT (DREAM-F cells). The first step in the DREAM protocol was a 96-well-based screening procedure, involving the analysis of cell viability and luciferase activity after pretreatment of DREAM-F cells with reagents of interest and post-treatment with UVB radiation, and vice versa. In the second step, we validated certain effective reagents identified in the first step by analyzing the cell cycle, evaluating cell death, and performing HPRT-DNA sequencing in DREAM-F cells treated with these reagents and UVB. This DREAM system is scalable and forms a time-saving high-throughput screening system for identifying novel anti-photoaging reagents regulating DNA damage in dermal fibroblasts.

Developing a novel fiber optic fluorescence device for multiplexed high-throughput cytotoxic screening.

PubMed

Lee, Dennis; Barnes, Stephen

2010-01-01

The need for new pharmacological agents is unending. Yet the drug discovery process has changed substantially over the past decade and continues to evolve in response to new technologies. There is presently a high demand to reduce discovery time by improving specific lab disciplines and developing new technology platforms in the area of cell-based assay screening. Here we present the developmental concept and early stage testing of the Ab-Sniffer, a novel fiber optic fluorescence device for high-throughput cytotoxicity screening using an immobilized whole cell approach. The fused silica fibers are chemically functionalized with biotin to provide interaction with fluorescently labeled, streptavidin functionalized alginate-chitosan microspheres. The microspheres are also functionalized with Concanavalin A to facilitate binding to living cells. By using lymphoma cells and rituximab in an adaptation of a well-known cytotoxicity protocol we demonstrate the utility of the Ab-Sniffer for functional screening of potential drug compounds rather than indirect, non-functional screening via binding assay. The platform can be extended to any assay capable of being tied to a fluorescence response including multiple target cells in each well of a multi-well plate for high-throughput screening.

Pediatric Glioblastoma Therapies Based on Patient-Derived Stem Cell Resources

DTIC Science & Technology

2014-11-01

genomic DNA and then subjected to Illumina high-throughput sequencing . In this analysis, shRNAs lost in the GSC population represent candidate gene...and genomic DNA and then subjected to Illumina high-throughput sequencing . In this analysis, shRNAs lost in the GSC population represent candidate...PRISM 7900 Sequence Detection System ( Genomics Resource, FHCRC). Relative transcript abundance was analyzed using the 2−ΔΔCt method. TRIzol (Invitrogen

Emory University: High-Throughput Protein-Protein Interaction Dataset for Lung Cancer-Associated Genes | Office of Cancer Genomics

Cancer.gov

To discover novel PPI signaling hubs for lung cancer, CTD2 Center at Emory utilized large-scale genomics datasets and literature to compile a set of lung cancer-associated genes. A library of expression vectors were generated for these genes and utilized for detecting pairwise PPIs with cell lysate-based TR-FRET assays in high-throughput screening format. Read the abstract.

Genome-wide RNAi Screening to Identify Host Factors That Modulate Oncolytic Virus Therapy.

PubMed

Allan, Kristina J; Mahoney, Douglas J; Baird, Stephen D; Lefebvre, Charles A; Stojdl, David F

2018-04-03

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact virus replication. Here we present the application of this technology to uncovering host targets that specifically modulate the replication of Maraba virus, an oncolytic rhabdovirus, and vaccinia virus with the goal of enhancing therapy. While the protocol has been tested for use with oncolytic Maraba virus and oncolytic vaccinia virus, this approach is applicable to other oncolytic viruses and can also be utilized for identifying host targets that modulate virus replication in mammalian cells in general. This protocol describes the development and validation of an assay for high-throughput RNAi screening in mammalian cells, the key considerations and preparation steps important for conducting a primary high-throughput RNAi screen, and a step-by-step guide for conducting a primary high-throughput RNAi screen; in addition, it broadly outlines the methods for conducting secondary screen validation and tertiary validation studies. The benefit of high-throughput RNAi screening is that it allows one to catalogue, in an extensive and unbiased fashion, host factors that modulate any aspect of virus replication for which one can develop an in vitro assay such as infectivity, burst size, and cytotoxicity. It has the power to uncover biotherapeutic targets unforeseen based on current knowledge.

Hydrogel-Based Fluorescent Dual pH and Oxygen Sensors Loaded in 96-Well Plates for High-Throughput Cell Metabolism Studies.

PubMed

Wu, Shanshan; Wu, Siying; Yi, Zheyuan; Zeng, Fei; Wu, Weizhen; Qiao, Yuan; Zhao, Xingzhong; Cheng, Xing; Tian, Yanqing

2018-02-13

In this study, we developed fluorescent dual pH and oxygen sensors loaded in multi-well plates for in-situ and high-throughput monitoring of oxygen respiration and extracellular acidification during microbial cell growth for understanding metabolism. Biocompatible PHEMA-co-PAM materials were used as the hydrogel matrix. A polymerizable oxygen probe (OS2) derived from PtTFPP and a polymerizable pH probe (S2) derived from fluorescein were chemically conjugated into the matrix to solve the problem of the probe leaching from the matrix. Gels were allowed to cure directly on the bottom of 96-well plates at room-temperature via redox polymerization. The influence of matrix's composition on the sensing behaviors was investigated to optimize hydrogels with enough robustness for repeatable use with good sensitivity. Responses of the dual sensing hydrogels to dissolved oxygen (DO) and pH were studied. These dual oxygen-pH sensing plates were successfully used for microbial cell-based screening assays, which are based on the measurement of fluorescence intensity changes induced by cellular oxygen consumption and pH changes during microbial growth. This method may provide a real-time monitoring of cellular respiration, acidification, and a rapid kinetic assessment of multiple samples for cell viability as well as high-throughput drug screening. All of these assays can be carried out by a conventional plate reader.

Crystal Symmetry Algorithms in a High-Throughput Framework for Materials

NASA Astrophysics Data System (ADS)

Taylor, Richard

The high-throughput framework AFLOW that has been developed and used successfully over the last decade is improved to include fully-integrated software for crystallographic symmetry characterization. The standards used in the symmetry algorithms conform with the conventions and prescriptions given in the International Tables of Crystallography (ITC). A standard cell choice with standard origin is selected, and the space group, point group, Bravais lattice, crystal system, lattice system, and representative symmetry operations are determined. Following the conventions of the ITC, the Wyckoff sites are also determined and their labels and site symmetry are provided. The symmetry code makes no assumptions on the input cell orientation, origin, or reduction and has been integrated in the AFLOW high-throughput framework for materials discovery by adding to the existing code base and making use of existing classes and functions. The software is written in object-oriented C++ for flexibility and reuse. A performance analysis and examination of the algorithms scaling with cell size and symmetry is also reported.

Rapid formation of size-controllable multicellular spheroids via 3D acoustic tweezers.

PubMed

Chen, Kejie; Wu, Mengxi; Guo, Feng; Li, Peng; Chan, Chung Yu; Mao, Zhangming; Li, Sixing; Ren, Liqiang; Zhang, Rui; Huang, Tony Jun

2016-07-05

The multicellular spheroid is an important 3D cell culture model for drug screening, tissue engineering, and fundamental biological research. Although several spheroid formation methods have been reported, the field still lacks high-throughput and simple fabrication methods to accelerate its adoption in drug development industry. Surface acoustic wave (SAW) based cell manipulation methods, which are known to be non-invasive, flexible, and high-throughput, have not been successfully developed for fabricating 3D cell assemblies or spheroids, due to the limited understanding on SAW-based vertical levitation. In this work, we demonstrated the capability of fabricating multicellular spheroids in the 3D acoustic tweezers platform. Our method used drag force from microstreaming to levitate cells in the vertical direction, and used radiation force from Gor'kov potential to aggregate cells in the horizontal plane. After optimizing the device geometry and input power, we demonstrated the rapid and high-throughput nature of our method by continuously fabricating more than 150 size-controllable spheroids and transferring them to Petri dishes every 30 minutes. The spheroids fabricated by our 3D acoustic tweezers can be cultured for a week with good cell viability. We further demonstrated that spheroids fabricated by this method could be used for drug testing. Unlike the 2D monolayer model, HepG2 spheroids fabricated by the 3D acoustic tweezers manifested distinct drug resistance, which matched existing reports. The 3D acoustic tweezers based method can serve as a novel bio-manufacturing tool to fabricate complex 3D cell assembles for biological research, tissue engineering, and drug development.

Sense and sensibility: the use of cell death biomarker assays in high-throughput anticancer drug screening and monitoring treatment responses.

PubMed

Shoshan, Maria C; Havelka, Associate Professor Principal Investigator Aleksandra Mandic; Neumann, Frank; Linder, Stig

2006-11-01

Cell-based screening allows identification of biologically active compounds, for example, potential anticancer drugs. In this review, various screening assays are discussed in terms of what they measure and how this affects interpretation and relevance. High-throughput (HT) assays of viability based on the reduction of exogenous substrates do not always reflect viability or cell number levels. Membrane integrity assays can be used for HT quantification of cell death, but are non-specific as to the death mode. Several HT assays monitor end point apoptosis. Screening libraries at a single concentration (micromolar) can prevent detection of potent apoptosis inducers, as high concentrations may induce mainly necrosis. Using monolayer cultures limits the significance of cell-based screening as the properties of monolayer cells differ from tumours in vivo. Spheroid cultures are more physiological, but are impractical for screening by conventional methods. The authors have developed an assay quantifying accumulation of a caspase-cleaved protein specific for epithelial cells. It provides an integrated measure of apoptosis in two- and three-dimensional cultures and can be used as a blood biomarker assay for tumour apoptosis in vivo.

Blood group genotyping: from patient to high-throughput donor screening.

PubMed

Veldhuisen, B; van der Schoot, C E; de Haas, M

2009-10-01

Blood group antigens, present on the cell membrane of red blood cells and platelets, can be defined either serologically or predicted based on the genotypes of genes encoding for blood group antigens. At present, the molecular basis of many antigens of the 30 blood group systems and 17 human platelet antigens is known. In many laboratories, blood group genotyping assays are routinely used for diagnostics in cases where patient red cells cannot be used for serological typing due to the presence of auto-antibodies or after recent transfusions. In addition, DNA genotyping is used to support (un)-expected serological findings. Fetal genotyping is routinely performed when there is a risk of alloimmune-mediated red cell or platelet destruction. In case of patient blood group antigen typing, it is important that a genotyping result is quickly available to support the selection of donor blood, and high-throughput of the genotyping method is not a prerequisite. In addition, genotyping of blood donors will be extremely useful to obtain donor blood with rare phenotypes, for example lacking a high-frequency antigen, and to obtain a fully typed donor database to be used for a better matching between recipient and donor to prevent adverse transfusion reactions. Serological typing of large cohorts of donors is a labour-intensive and expensive exercise and hampered by the lack of sufficient amounts of approved typing reagents for all blood group systems of interest. Currently, high-throughput genotyping based on DNA micro-arrays is a very feasible method to obtain a large pool of well-typed blood donors. Several systems for high-throughput blood group genotyping are developed and will be discussed in this review.

A high-throughput in vitro ring assay for vasoactivity using magnetic 3D bioprinting

PubMed Central

Tseng, Hubert; Gage, Jacob A.; Haisler, William L.; Neeley, Shane K.; Shen, Tsaiwei; Hebel, Chris; Barthlow, Herbert G.; Wagoner, Matthew; Souza, Glauco R.

2016-01-01

Vasoactive liabilities are typically assayed using wire myography, which is limited by its high cost and low throughput. To meet the demand for higher throughput in vitro alternatives, this study introduces a magnetic 3D bioprinting-based vasoactivity assay. The principle behind this assay is the magnetic printing of vascular smooth muscle cells into 3D rings that functionally represent blood vessel segments, whose contraction can be altered by vasodilators and vasoconstrictors. A cost-effective imaging modality employing a mobile device is used to capture contraction with high throughput. The goal of this study was to validate ring contraction as a measure of vasoactivity, using a small panel of known vasoactive drugs. In vitro responses of the rings matched outcomes predicted by in vivo pharmacology, and were supported by immunohistochemistry. Altogether, this ring assay robustly models vasoactivity, which could meet the need for higher throughput in vitro alternatives. PMID:27477945

Novel KCNQ2 channel activators discovered using fluorescence-based and automated patch-clamp-based high-throughput screening techniques

PubMed Central

Yue, Jin-feng; Qiao, Guan-hua; Liu, Ni; Nan, Fa-jun; Gao, Zhao-bing

2016-01-01

Aim: To establish an improved, high-throughput screening techniques for identifying novel KCNQ2 channel activators. Methods: KCNQ2 channels were stably expressed in CHO cells (KCNQ2 cells). Thallium flux assay was used for primary screening, and 384-well automated patch-clamp IonWorks Barracuda was used for hit validation. Two validated activators were characterized using a conventional patch-clamp recording technique. Results: From a collection of 80 000 compounds, the primary screening revealed a total of 565 compounds that potentiated the fluorescence signals in thallium flux assay by more than 150%. When the 565 hits were examined in IonWorks Barracuda, 38 compounds significantly enhanced the outward currents recorded in KCNQ2 cells, and were confirmed as KCNQ2 activators. In the conventional patch-clamp recordings, two validated activators ZG1732 and ZG2083 enhanced KCNQ2 currents with EC50 values of 1.04±0.18 μmol/L and 1.37±0.06 μmol/L, respectively. Conclusion: The combination of thallium flux assay and IonWorks Barracuda assay is an efficient high-throughput screening (HTS) route for discovering KCNQ2 activators. PMID:26725738

Cell-based medicinal chemistry optimization of high-throughput screening (HTS) hits for orally active antimalarials. Part 1: challenges in potency and absorption, distribution, metabolism, excretion/pharmacokinetics (ADME/PK).

PubMed

Chatterjee, Arnab K

2013-10-24

Malaria represents a significant health issue, and novel and effective drugs are needed to address parasite resistance that has emerged to the current drug arsenal. Antimalarial drug discovery has historically benefited from a whole-cell (phenotypic) screening approach to identify lead molecules. This approach has been utilized by several groups to optimize weakly active antimalarial pharmacophores, such as the quinolone scaffold, to yield potent and highly efficacious compounds that are now poised to enter clinical trials. More recently, GNF/Novartis, GSK, and others have employed the same approach in high-throughput screening (HTS) of large compound libraries to find novel scaffolds that have also been optimized to clinical candidates by GNF/Novartis. This perspective outlines some of the inherent challenges in cell-based medicinal chemistry optimization, including optimization of oral exposure and hERG activity.

Cytotoxicity Test Based on Human Cells Labeled with Fluorescent Proteins: Fluorimetry, Photography, and Scanning for High-Throughput Assay.

PubMed

Kalinina, Marina A; Skvortsov, Dmitry A; Rubtsova, Maria P; Komarova, Ekaterina S; Dontsova, Olga A

2018-06-01

High- and medium-throughput assays are now routine methods for drug screening and toxicology investigations on mammalian cells. However, a simple and cost-effective analysis of cytotoxicity that can be carried out with commonly used laboratory equipment is still required. The developed cytotoxicity assays are based on human cell lines stably expressing eGFP, tdTomato, mCherry, or Katushka2S fluorescent proteins. Red fluorescent proteins exhibit a higher signal-to-noise ratio, due to less interference by medium autofluorescence, in comparison to green fluorescent protein. Measurements have been performed on a fluorescence scanner, a plate fluorimeter, and a camera photodocumentation system. For a 96-well plate assay, the sensitivity per well and the measurement duration were 250 cells and 15 min for the scanner, 500 cells and 2 min for the plate fluorimeter, and 1000 cells and less than 1 min for the camera detection. These sensitivities are similar to commonly used MTT (tetrazolium dye) assays. The used scanner and the camera had not been previously applied for cytotoxicity evaluation. An image processing scheme for the high-resolution scanner is proposed that significantly diminishes the number of control wells, even for a library containing fluorescent substances. The suggested cytotoxicity assay has been verified by measurements of the cytotoxicity of several well-known cytotoxic drugs and further applied to test a set of novel bacteriotoxic compounds in a medium-throughput format. The fluorescent signal of living cells is detected without disturbing them and adding any reagents, thus allowing to investigate time-dependent cytotoxicity effects on the same sample of cells. A fast, simple and cost-effective assay is suggested for cytotoxicity evaluation based on mammalian cells expressing fluorescent proteins and commonly used laboratory equipment.

Niche-based screening identifies small-molecule inhibitors of leukemia stem cells.

PubMed

Hartwell, Kimberly A; Miller, Peter G; Mukherjee, Siddhartha; Kahn, Alissa R; Stewart, Alison L; Logan, David J; Negri, Joseph M; Duvet, Mildred; Järås, Marcus; Puram, Rishi; Dancik, Vlado; Al-Shahrour, Fatima; Kindler, Thomas; Tothova, Zuzana; Chattopadhyay, Shrikanta; Hasaka, Thomas; Narayan, Rajiv; Dai, Mingji; Huang, Christina; Shterental, Sebastian; Chu, Lisa P; Haydu, J Erika; Shieh, Jae Hung; Steensma, David P; Munoz, Benito; Bittker, Joshua A; Shamji, Alykhan F; Clemons, Paul A; Tolliday, Nicola J; Carpenter, Anne E; Gilliland, D Gary; Stern, Andrew M; Moore, Malcolm A S; Scadden, David T; Schreiber, Stuart L; Ebert, Benjamin L; Golub, Todd R

2013-12-01

Efforts to develop more effective therapies for acute leukemia may benefit from high-throughput screening systems that reflect the complex physiology of the disease, including leukemia stem cells (LSCs) and supportive interactions with the bone marrow microenvironment. The therapeutic targeting of LSCs is challenging because LSCs are highly similar to normal hematopoietic stem and progenitor cells (HSPCs) and are protected by stromal cells in vivo. We screened 14,718 compounds in a leukemia-stroma co-culture system for inhibition of cobblestone formation, a cellular behavior associated with stem-cell function. Among those compounds that inhibited malignant cells but spared HSPCs was the cholesterol-lowering drug lovastatin. Lovastatin showed anti-LSC activity in vitro and in an in vivo bone marrow transplantation model. Mechanistic studies demonstrated that the effect was on target, via inhibition of HMG-CoA reductase. These results illustrate the power of merging physiologically relevant models with high-throughput screening.

Niche-based screening identifies small-molecule inhibitors of leukemia stem cells

PubMed Central

Mukherjee, Siddhartha; Kahn, Alissa R; Stewart, Alison L; Logan, David J; Negri, Joseph M; Duvet, Mildred; Järås, Marcus; Puram, Rishi; Dancik, Vlado; Al-Shahrour, Fatima; Kindler, Thomas; Tothova, Zuzana; Chattopadhyay, Shrikanta; Hasaka, Thomas; Narayan, Rajiv; Dai, Mingji; Huang, Christina; Shterental, Sebastian; Chu, Lisa P; Haydu, J Erika; Shieh, Jae Hung; Steensma, David P; Munoz, Benito; Bittker, Joshua A; Shamji, Alykhan F; Clemons, Paul A; Tolliday, Nicola J; Carpenter, Anne E; Gilliland, D Gary; Stern, Andrew M; Moore, Malcolm A S; Scadden, David T; Schreiber, Stuart L; Ebert, Benjamin L; Golub, Todd R

2014-01-01

Efforts to develop more effective therapies for acute leukemia may benefit from high-throughput screening systems that reflect the complex physiology of the disease, including leukemia stem cells (LSCs) and supportive interactions with the bone-marrow microenvironment. The therapeutic targeting of LSCs is challenging because LSCs are highly similar to normal hematopoietic stem and progenitor cells (HSPCs) and are protected by stromal cells in vivo. We screened 14,718 compounds in a leukemia-stroma co-culture system for inhibition of cobblestone formation, a cellular behavior associated with stem-cell function. Among those that inhibited malignant cells but spared HSPCs was the cholesterol-lowering drug lovastatin. Lovastatin showed anti-LSC activity in vitro and in an in vivo bone marrow transplantation model. Mechanistic studies demonstrated that the effect was on-target, via inhibition of HMGCoA reductase. These results illustrate the power of merging physiologically-relevant models with high-throughput screening. PMID:24161946

Droplet barcoding for single cell transcriptomics applied to embryonic stem cells

PubMed Central

Klein, Allon M; Mazutis, Linas; Akartuna, Ilke; Tallapragada, Naren; Veres, Adrian; Li, Victor; Peshkin, Leonid; Weitz, David A; Kirschner, Marc W

2015-01-01

Summary It has long been the dream of biologists to map gene expression at the single cell level. With such data one might track heterogeneous cell sub-populations, and infer regulatory relationships between genes and pathways. Recently, RNA sequencing has achieved single cell resolution. What is limiting is an effective way to routinely isolate and process large numbers of individual cells for quantitative in-depth sequencing. We have developed a high-throughput droplet-microfluidic approach for barcoding the RNA from thousands of individual cells for subsequent analysis by next-generation sequencing. The method shows a surprisingly low noise profile and is readily adaptable to other sequencing-based assays. We analyzed mouse embryonic stem cells, revealing in detail the population structure and the heterogeneous onset of differentiation after LIF withdrawal. The reproducibility of these high-throughput single cell data allowed us to deconstruct cell populations and infer gene expression relationships. PMID:26000487

Continuous high throughput molecular adhesion based cell sorting using ridged microchannels

NASA Astrophysics Data System (ADS)

Tasadduq, Bushra; Wang, Gonghao; Alexeev, Alexander; Sarioglu, Ali Fatih; Sulchek, Todd

2016-11-01

Cell molecular interactions govern important physiological processes such as stem cell homing, inflammation and cancer metastasis. But due to a lack of effective separation technologies selective to these interactions it is challenging to specifically sort cells. Other label free separation techniques based on size, stiffness and shape do not provide enough specificity to cell type, and correlation to clinical condition. We propose a novel microfluidic device capable of high throughput molecule dependent separation of cells by flowing them through a microchannel decorated with molecule specific coated ridges. The unique aspect of this sorting design is the use of optimized gap size which is small enough to lightly squeeze the cells while flowing under the ridged part of the channel to increase the surface area for interaction between the ligand on cell surface and coated receptor molecule but large enough so that biomechanical markers, stiffness and viscoelasticity, do not dominate the cell separation mechanism. We are able to separate Jurkat cells based on its expression of PSGL-1ligand using ridged channel coated with P selectin at a flow rate of 0.045ml/min and achieve 2-fold and 5-fold enrichment of PSGL-1 positive and negative Jurkat cells respectively.

Pinched flow coupled shear-modulated inertial microfluidics for high-throughput rare blood cell separation.

PubMed

Bhagat, Ali Asgar S; Hou, Han Wei; Li, Leon D; Lim, Chwee Teck; Han, Jongyoon

2011-06-07

Blood is a highly complex bio-fluid with cellular components making up >40% of the total volume, thus making its analysis challenging and time-consuming. In this work, we introduce a high-throughput size-based separation method for processing diluted blood using inertial microfluidics. The technique takes advantage of the preferential cell focusing in high aspect-ratio microchannels coupled with pinched flow dynamics for isolating low abundance cells from blood. As an application of the developed technique, we demonstrate the isolation of cancer cells (circulating tumor cells (CTCs)) spiked in blood by exploiting the difference in size between CTCs and hematologic cells. The microchannel dimensions and processing parameters were optimized to enable high throughput and high resolution separation, comparable to existing CTC isolation technologies. Results from experiments conducted with MCF-7 cells spiked into whole blood indicate >80% cell recovery with an impressive 3.25 × 10(5) fold enrichment over red blood cells (RBCs) and 1.2 × 10(4) fold enrichment over peripheral blood leukocytes (PBL). In spite of a 20× sample dilution, the fast operating flow rate allows the processing of ∼10(8) cells min(-1) through a single microfluidic device. The device design can be easily customized for isolating other rare cells from blood including peripheral blood leukocytes and fetal nucleated red blood cells by simply varying the 'pinching' width. The advantage of simple label-free separation, combined with the ability to retrieve viable cells post enrichment and minimal sample pre-processing presents numerous applications for use in clinical diagnosis and conducting fundamental studies.

A high throughput respirometric assay for mitochondrial biogenesis and toxicity

PubMed Central

Beeson, Craig C.; Beeson, Gyda C.; Schnellmann, Rick G.

2010-01-01

Mitochondria are a common target of toxicity for drugs and other chemicals, and results in decreased aerobic metabolism and cell death. In contrast, mitochondrial biogenesis restores cell vitality and there is a need for new agents to induce biogenesis. Current cell-based models of mitochondrial biogenesis or toxicity are inadequate because cultured cell lines are highly glycolytic with minimal aerobic metabolism and altered mitochondrial physiology. In addition, there are no high-throughput, real-time assays that assess mitochondrial function. We adapted primary cultures of renal proximal tubular cells (RPTC) that exhibit in vivo levels of aerobic metabolism, are not glycolytic, and retain higher levels of differentiated functions and used the Seahorse Biosciences analyzer to measure mitochondrial function in real time in multi-well plates. Using uncoupled respiration as a marker of electron transport chain (ETC) integrity, the nephrotoxicants cisplatin, HgCl2 and gentamicin exhibited mitochondrial toxicity prior to decreases in basal respiration and cell death. Conversely, using FCCP-uncoupled respiration as a marker of maximal ETC activity, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), SRT1720, resveratrol, daidzein, and metformin produced mitochondrial biogenesis in RPTC. The merger of the RPTC model and multi-well respirometry results in a single high throughput assay to measure mitochondrial biogenesis and toxicity, and nephrotoxic potential. PMID:20465991

Microfluidic cell chips for high-throughput drug screening

PubMed Central

Chi, Chun-Wei; Ahmed, AH Rezwanuddin; Dereli-Korkut, Zeynep; Wang, Sihong

2016-01-01

The current state of screening methods for drug discovery is still riddled with several inefficiencies. Although some widely used high-throughput screening platforms may enhance the drug screening process, their cost and oversimplification of cell–drug interactions pose a translational difficulty. Microfluidic cell-chips resolve many issues found in conventional HTS technology, providing benefits such as reduced sample quantity and integration of 3D cell culture physically more representative of the physiological/pathological microenvironment. In this review, we introduce the advantages of microfluidic devices in drug screening, and outline the critical factors which influence device design, highlighting recent innovations and advances in the field including a summary of commercialization efforts on microfluidic cell chips. Future perspectives of microfluidic cell devices are also provided based on considerations of present technological limitations and translational barriers. PMID:27071838

High-Throughput Cancer Cell Sphere Formation for 3D Cell Culture.

PubMed

Chen, Yu-Chih; Yoon, Euisik

2017-01-01

Three-dimensional (3D) cell culture is critical in studying cancer pathology and drug response. Though 3D cancer sphere culture can be performed in low-adherent dishes or well plates, the unregulated cell aggregation may skew the results. On contrary, microfluidic 3D culture can allow precise control of cell microenvironments, and provide higher throughput by orders of magnitude. In this chapter, we will look into engineering innovations in a microfluidic platform for high-throughput cancer cell sphere formation and review the implementation methods in detail.

Gene cassette knock-in in mammalian cells and zygotes by enhanced MMEJ.

PubMed

Aida, Tomomi; Nakade, Shota; Sakuma, Tetsushi; Izu, Yayoi; Oishi, Ayu; Mochida, Keiji; Ishikubo, Harumi; Usami, Takako; Aizawa, Hidenori; Yamamoto, Takashi; Tanaka, Kohichi

2016-11-28

Although CRISPR/Cas enables one-step gene cassette knock-in, assembling targeting vectors containing long homology arms is a laborious process for high-throughput knock-in. We recently developed the CRISPR/Cas-based precise integration into the target chromosome (PITCh) system for a gene cassette knock-in without long homology arms mediated by microhomology-mediated end-joining. Here, we identified exonuclease 1 (Exo1) as an enhancer for PITCh in human cells. By combining the Exo1 and PITCh-directed donor vectors, we achieved convenient one-step knock-in of gene cassettes and floxed allele both in human cells and mouse zygotes. Our results provide a technical platform for high-throughput knock-in.

Using iterative cluster merging with improved gap statistics to perform online phenotype discovery in the context of high-throughput RNAi screens

PubMed Central

Yin, Zheng; Zhou, Xiaobo; Bakal, Chris; Li, Fuhai; Sun, Youxian; Perrimon, Norbert; Wong, Stephen TC

2008-01-01

Background The recent emergence of high-throughput automated image acquisition technologies has forever changed how cell biologists collect and analyze data. Historically, the interpretation of cellular phenotypes in different experimental conditions has been dependent upon the expert opinions of well-trained biologists. Such qualitative analysis is particularly effective in detecting subtle, but important, deviations in phenotypes. However, while the rapid and continuing development of automated microscope-based technologies now facilitates the acquisition of trillions of cells in thousands of diverse experimental conditions, such as in the context of RNA interference (RNAi) or small-molecule screens, the massive size of these datasets precludes human analysis. Thus, the development of automated methods which aim to identify novel and biological relevant phenotypes online is one of the major challenges in high-throughput image-based screening. Ideally, phenotype discovery methods should be designed to utilize prior/existing information and tackle three challenging tasks, i.e. restoring pre-defined biological meaningful phenotypes, differentiating novel phenotypes from known ones and clarifying novel phenotypes from each other. Arbitrarily extracted information causes biased analysis, while combining the complete existing datasets with each new image is intractable in high-throughput screens. Results Here we present the design and implementation of a novel and robust online phenotype discovery method with broad applicability that can be used in diverse experimental contexts, especially high-throughput RNAi screens. This method features phenotype modelling and iterative cluster merging using improved gap statistics. A Gaussian Mixture Model (GMM) is employed to estimate the distribution of each existing phenotype, and then used as reference distribution in gap statistics. This method is broadly applicable to a number of different types of image-based datasets derived from a wide spectrum of experimental conditions and is suitable to adaptively process new images which are continuously added to existing datasets. Validations were carried out on different dataset, including published RNAi screening using Drosophila embryos [Additional files 1, 2], dataset for cell cycle phase identification using HeLa cells [Additional files 1, 3, 4] and synthetic dataset using polygons, our methods tackled three aforementioned tasks effectively with an accuracy range of 85%–90%. When our method is implemented in the context of a Drosophila genome-scale RNAi image-based screening of cultured cells aimed to identifying the contribution of individual genes towards the regulation of cell-shape, it efficiently discovers meaningful new phenotypes and provides novel biological insight. We also propose a two-step procedure to modify the novelty detection method based on one-class SVM, so that it can be used to online phenotype discovery. In different conditions, we compared the SVM based method with our method using various datasets and our methods consistently outperformed SVM based method in at least two of three tasks by 2% to 5%. These results demonstrate that our methods can be used to better identify novel phenotypes in image-based datasets from a wide range of conditions and organisms. Conclusion We demonstrate that our method can detect various novel phenotypes effectively in complex datasets. Experiment results also validate that our method performs consistently under different order of image input, variation of starting conditions including the number and composition of existing phenotypes, and dataset from different screens. In our findings, the proposed method is suitable for online phenotype discovery in diverse high-throughput image-based genetic and chemical screens. PMID:18534020

DOE Office of Scientific and Technical Information (OSTI.GOV)

Heusinkveld, Harm J.; Westerink, Remco H.S., E-mail: R.Westerink@uu.nl

Calcium plays a crucial role in virtually all cellular processes, including neurotransmission. The intracellular Ca{sup 2+} concentration ([Ca{sup 2+}]{sub i}) is therefore an important readout in neurotoxicological and neuropharmacological studies. Consequently, there is an increasing demand for high-throughput measurements of [Ca{sup 2+}]{sub i}, e.g. using multi-well microplate readers, in hazard characterization, human risk assessment and drug development. However, changes in [Ca{sup 2+}]{sub i} are highly dynamic, thereby creating challenges for high-throughput measurements. Nonetheless, several protocols are now available for real-time kinetic measurement of [Ca{sup 2+}]{sub i} in plate reader systems, though the results of such plate reader-based measurements have beenmore » questioned. In view of the increasing use of plate reader systems for measurements of [Ca{sup 2+}]{sub i} a careful evaluation of current technologies is warranted. We therefore performed an extensive set of experiments, using two cell lines (PC12 and B35) and two fluorescent calcium-sensitive dyes (Fluo-4 and Fura-2), for comparison of a linear plate reader system with single cell fluorescence microscopy. Our data demonstrate that the use of plate reader systems for high-throughput real-time kinetic measurements of [Ca{sup 2+}]{sub i} is associated with many pitfalls and limitations, including erroneous sustained increases in fluorescence, limited sensitivity and lack of single cell resolution. Additionally, our data demonstrate that probenecid, which is often used to prevent dye leakage, effectively inhibits the depolarization-evoked increase in [Ca{sup 2+}]{sub i}. Overall, the data indicate that the use of current plate reader-based strategies for high-throughput real-time kinetic measurements of [Ca{sup 2+}]{sub i} is associated with caveats and limitations that require further investigation. - Research Highlights: > The use of plate readers for high-throughput screening of intracellular Ca{sup 2+} is associated with many pitfalls and limitations. > Single cell fluorescent microscopy is recommended for measurements of intracellular Ca{sup 2+}. > Dual-wavelength dyes (Fura-2) are preferred over single-wavelength dyes (Fluo-4) for measurements of intracellular Ca{sup 2+}. > Probenecid prevents dye leakage but abolishes depolarization-evoked Ca{sup 2+} influx, severely hampering measurements of Ca{sup 2+}. > In general, care should be taken when interpreting data from high-throughput kinetic measurements.« less

Classification of large circulating tumor cells isolated with ultra-high throughput microfluidic Vortex technology.

PubMed

Che, James; Yu, Victor; Dhar, Manjima; Renier, Corinne; Matsumoto, Melissa; Heirich, Kyra; Garon, Edward B; Goldman, Jonathan; Rao, Jianyu; Sledge, George W; Pegram, Mark D; Sheth, Shruti; Jeffrey, Stefanie S; Kulkarni, Rajan P; Sollier, Elodie; Di Carlo, Dino

2016-03-15

Circulating tumor cells (CTCs) are emerging as rare but clinically significant non-invasive cellular biomarkers for cancer patient prognosis, treatment selection, and treatment monitoring. Current CTC isolation approaches, such as immunoaffinity, filtration, or size-based techniques, are often limited by throughput, purity, large output volumes, or inability to obtain viable cells for downstream analysis. For all technologies, traditional immunofluorescent staining alone has been employed to distinguish and confirm the presence of isolated CTCs among contaminating blood cells, although cells isolated by size may express vastly different phenotypes. Consequently, CTC definitions have been non-trivial, researcher-dependent, and evolving. Here we describe a complete set of objective criteria, leveraging well-established cytomorphological features of malignancy, by which we identify large CTCs. We apply the criteria to CTCs enriched from stage IV lung and breast cancer patient blood samples using the High Throughput Vortex Chip (Vortex HT), an improved microfluidic technology for the label-free, size-based enrichment and concentration of rare cells. We achieve improved capture efficiency (up to 83%), high speed of processing (8 mL/min of 10x diluted blood, or 800 μL/min of whole blood), and high purity (avg. background of 28.8±23.6 white blood cells per mL of whole blood). We show markedly improved performance of CTC capture (84% positive test rate) in comparison to previous Vortex designs and the current FDA-approved gold standard CellSearch assay. The results demonstrate the ability to quickly collect viable and pure populations of abnormal large circulating cells unbiased by molecular characteristics, which helps uncover further heterogeneity in these cells.

Classification of large circulating tumor cells isolated with ultra-high throughput microfluidic Vortex technology

PubMed Central

Che, James; Yu, Victor; Dhar, Manjima; Renier, Corinne; Matsumoto, Melissa; Heirich, Kyra; Garon, Edward B.; Goldman, Jonathan; Rao, Jianyu; Sledge, George W.; Pegram, Mark D.; Sheth, Shruti; Jeffrey, Stefanie S.; Kulkarni, Rajan P.; Sollier, Elodie; Di Carlo, Dino

2016-01-01

Circulating tumor cells (CTCs) are emerging as rare but clinically significant non-invasive cellular biomarkers for cancer patient prognosis, treatment selection, and treatment monitoring. Current CTC isolation approaches, such as immunoaffinity, filtration, or size-based techniques, are often limited by throughput, purity, large output volumes, or inability to obtain viable cells for downstream analysis. For all technologies, traditional immunofluorescent staining alone has been employed to distinguish and confirm the presence of isolated CTCs among contaminating blood cells, although cells isolated by size may express vastly different phenotypes. Consequently, CTC definitions have been non-trivial, researcher-dependent, and evolving. Here we describe a complete set of objective criteria, leveraging well-established cytomorphological features of malignancy, by which we identify large CTCs. We apply the criteria to CTCs enriched from stage IV lung and breast cancer patient blood samples using the High Throughput Vortex Chip (Vortex HT), an improved microfluidic technology for the label-free, size-based enrichment and concentration of rare cells. We achieve improved capture efficiency (up to 83%), high speed of processing (8 mL/min of 10x diluted blood, or 800 μL/min of whole blood), and high purity (avg. background of 28.8±23.6 white blood cells per mL of whole blood). We show markedly improved performance of CTC capture (84% positive test rate) in comparison to previous Vortex designs and the current FDA-approved gold standard CellSearch assay. The results demonstrate the ability to quickly collect viable and pure populations of abnormal large circulating cells unbiased by molecular characteristics, which helps uncover further heterogeneity in these cells. PMID:26863573

Comparison of submerged and unsubmerged printing of ovarian cancer cells.

PubMed

Davidoff, Sherry N; Au, David; Smith, Samuel; Brooks, Amanda E; Brooks, Benjamin D

2015-01-01

A high-throughput cell based assay would greatly aid in the development and screening of ovarian cancer drug candidates. Previously, a three-dimensional microfluidic printer that is not only capable of controlling the location of cell deposition, but also of maintaining a liquid, nutrient rich environment to preserve cellular phenotype has been developed (Wasatch Microfluidics). In this study, we investigated the impact (i.e., viability, density, and phenotype) of depositing cells on a surface submerged in cell culture media. It was determined that submersion of the microfluidic print head in cell media did not alter the cell density, viability, or phenotype.. This article describes an in depth study detailing the impact of one of the fundamental components of a 3D microfluidic cell printer designed to mimic the in vivo cell environment. Development of such a tool holds promise as a high-throughput drug-screening platform for new cancer therapeutics.

Microfluidic guillotine for single-cell wound repair studies

NASA Astrophysics Data System (ADS)

Blauch, Lucas R.; Gai, Ya; Khor, Jian Wei; Sood, Pranidhi; Marshall, Wallace F.; Tang, Sindy K. Y.

2017-07-01

Wound repair is a key feature distinguishing living from nonliving matter. Single cells are increasingly recognized to be capable of healing wounds. The lack of reproducible, high-throughput wounding methods has hindered single-cell wound repair studies. This work describes a microfluidic guillotine for bisecting single Stentor coeruleus cells in a continuous-flow manner. Stentor is used as a model due to its robust repair capacity and the ability to perform gene knockdown in a high-throughput manner. Local cutting dynamics reveals two regimes under which cells are bisected, one at low viscous stress where cells are cut with small membrane ruptures and high viability and one at high viscous stress where cells are cut with extended membrane ruptures and decreased viability. A cutting throughput up to 64 cells per minute—more than 200 times faster than current methods—is achieved. The method allows the generation of more than 100 cells in a synchronized stage of their repair process. This capacity, combined with high-throughput gene knockdown in Stentor, enables time-course mechanistic studies impossible with current wounding methods.

Comparison of PC12 and Cerebellar Granule Cell Cultures for Evaluating Neurite Outgrowth Using High Content Screening

EPA Science Inventory

Development of high-throughput assays for chemical screening and hazard identification is a pressing priority worldwide. One approach uses in vitro, cell-based assays which recapitulate biological events observed in vivo. Neurite outgrowth is one such critical cellular process un...

COMPARISON OF NEUROSCREEN-1 AND CEREBELLAR GRANULE CELL CULTURES FOR EVALUATING NEURITE OUTGROWTH USING THE ARRAYSCAN HIGH CONTENT ANALYSIS SYSTEM

EPA Science Inventory

A major challenge facing the Environmental Protection Agency is the development of high-throughput screening assays amendable to resource-efficient developmental neurotoxicity for chemical screening and toxicity prioritization. One approach uses in vitro, cell-based assays which...

Ion channel drug discovery and research: the automated Nano-Patch-Clamp technology.

PubMed

Brueggemann, A; George, M; Klau, M; Beckler, M; Steindl, J; Behrends, J C; Fertig, N

2004-01-01

Unlike the genomics revolution, which was largely enabled by a single technological advance (high throughput sequencing), rapid advancement in proteomics will require a broader effort to increase the throughput of a number of key tools for functional analysis of different types of proteins. In the case of ion channels -a class of (membrane) proteins of great physiological importance and potential as drug targets- the lack of adequate assay technologies is felt particularly strongly. The available, indirect, high throughput screening methods for ion channels clearly generate insufficient information. The best technology to study ion channel function and screen for compound interaction is the patch clamp technique, but patch clamping suffers from low throughput, which is not acceptable for drug screening. A first step towards a solution is presented here. The nano patch clamp technology, which is based on a planar, microstructured glass chip, enables automatic whole cell patch clamp measurements. The Port-a-Patch is an automated electrophysiology workstation, which uses planar patch clamp chips. This approach enables high quality and high content ion channel and compound evaluation on a one-cell-at-a-time basis. The presented automation of the patch process and its scalability to an array format are the prerequisites for any higher throughput electrophysiology instruments.

A high-throughput direct fluorescence resonance energy transfer-based assay for analyzing apoptotic proteases using flow cytometry and fluorescence lifetime measurements.

PubMed

Suzuki, Miho; Sakata, Ichiro; Sakai, Takafumi; Tomioka, Hiroaki; Nishigaki, Koichi; Tramier, Marc; Coppey-Moisan, Maïté

2015-12-15

Cytometry is a versatile and powerful method applicable to different fields, particularly pharmacology and biomedical studies. Based on the data obtained, cytometric studies are classified into high-throughput (HTP) or high-content screening (HCS) groups. However, assays combining the advantages of both are required to facilitate research. In this study, we developed a high-throughput system to profile cellular populations in terms of time- or dose-dependent responses to apoptotic stimulations because apoptotic inducers are potent anticancer drugs. We previously established assay systems involving protease to monitor live cells for apoptosis using tunable fluorescence resonance energy transfer (FRET)-based bioprobes. These assays can be used for microscopic analyses or fluorescence-activated cell sorting. In this study, we developed FRET-based bioprobes to detect the activity of the apoptotic markers caspase-3 and caspase-9 via changes in bioprobe fluorescence lifetimes using a flow cytometer for direct estimation of FRET efficiencies. Different patterns of changes in the fluorescence lifetimes of these markers during apoptosis were observed, indicating a relationship between discrete steps in the apoptosis process. The findings demonstrate the feasibility of evaluating collective cellular dynamics during apoptosis. Copyright © 2015 Elsevier Inc. All rights reserved.

An Image-Based Algorithm for Precise and Accurate High Throughput Assessment of Drug Activity against the Human Parasite Trypanosoma cruzi

PubMed Central

Moraes, Carolina Borsoi; Yang, Gyongseon; Kang, Myungjoo; Freitas-Junior, Lucio H.; Hansen, Michael A. E.

2014-01-01

We present a customized high content (image-based) and high throughput screening algorithm for the quantification of Trypanosoma cruzi infection in host cells. Based solely on DNA staining and single-channel images, the algorithm precisely segments and identifies the nuclei and cytoplasm of mammalian host cells as well as the intracellular parasites infecting the cells. The algorithm outputs statistical parameters including the total number of cells, number of infected cells and the total number of parasites per image, the average number of parasites per infected cell, and the infection ratio (defined as the number of infected cells divided by the total number of cells). Accurate and precise estimation of these parameters allow for both quantification of compound activity against parasites, as well as the compound cytotoxicity, thus eliminating the need for an additional toxicity-assay, hereby reducing screening costs significantly. We validate the performance of the algorithm using two known drugs against T.cruzi: Benznidazole and Nifurtimox. Also, we have checked the performance of the cell detection with manual inspection of the images. Finally, from the titration of the two compounds, we confirm that the algorithm provides the expected half maximal effective concentration (EC50) of the anti-T. cruzi activity. PMID:24503652

High-Throughput Mechanobiology Screening Platform Using Micro- and Nanotopography.

PubMed

Hu, Junqiang; Gondarenko, Alexander A; Dang, Alex P; Bashour, Keenan T; O'Connor, Roddy S; Lee, Sunwoo; Liapis, Anastasia; Ghassemi, Saba; Milone, Michael C; Sheetz, Michael P; Dustin, Michael L; Kam, Lance C; Hone, James C

2016-04-13

We herein demonstrate the first 96-well plate platform to screen effects of micro- and nanotopographies on cell growth and proliferation. Existing high-throughput platforms test a limited number of factors and are not fully compatible with multiple types of testing and assays. This platform is compatible with high-throughput liquid handling, high-resolution imaging, and all multiwell plate-based instrumentation. We use the platform to screen for topographies and drug-topography combinations that have short- and long-term effects on T cell activation and proliferation. We coated nanofabricated "trench-grid" surfaces with anti-CD3 and anti-CD28 antibodies to activate T cells and assayed for interleukin 2 (IL-2) cytokine production. IL-2 secretion was enhanced at 200 nm trench width and >2.3 μm grating pitch; however, the secretion was suppressed at 100 nm width and <0.5 μm pitch. The enhancement on 200 nm grid trench was further amplified with the addition of blebbistatin to reduce contractility. The 200 nm grid pattern was found to triple the number of T cells in long-term expansion, a result with direct clinical applicability in adoptive immunotherapy.

Automatic Segmentation of High-Throughput RNAi Fluorescent Cellular Images

PubMed Central

Yan, Pingkum; Zhou, Xiaobo; Shah, Mubarak; Wong, Stephen T. C.

2010-01-01

High-throughput genome-wide RNA interference (RNAi) screening is emerging as an essential tool to assist biologists in understanding complex cellular processes. The large number of images produced in each study make manual analysis intractable; hence, automatic cellular image analysis becomes an urgent need, where segmentation is the first and one of the most important steps. In this paper, a fully automatic method for segmentation of cells from genome-wide RNAi screening images is proposed. Nuclei are first extracted from the DNA channel by using a modified watershed algorithm. Cells are then extracted by modeling the interaction between them as well as combining both gradient and region information in the Actin and Rac channels. A new energy functional is formulated based on a novel interaction model for segmenting tightly clustered cells with significant intensity variance and specific phenotypes. The energy functional is minimized by using a multiphase level set method, which leads to a highly effective cell segmentation method. Promising experimental results demonstrate that automatic segmentation of high-throughput genome-wide multichannel screening can be achieved by using the proposed method, which may also be extended to other multichannel image segmentation problems. PMID:18270043

High-throughput microfluidic single-cell digital polymerase chain reaction.

PubMed

White, A K; Heyries, K A; Doolin, C; Vaninsberghe, M; Hansen, C L

2013-08-06

Here we present an integrated microfluidic device for the high-throughput digital polymerase chain reaction (dPCR) analysis of single cells. This device allows for the parallel processing of single cells and executes all steps of analysis, including cell capture, washing, lysis, reverse transcription, and dPCR analysis. The cDNA from each single cell is distributed into a dedicated dPCR array consisting of 1020 chambers, each having a volume of 25 pL, using surface-tension-based sample partitioning. The high density of this dPCR format (118,900 chambers/cm(2)) allows the analysis of 200 single cells per run, for a total of 204,000 PCR reactions using a device footprint of 10 cm(2). Experiments using RNA dilutions show this device achieves shot-noise-limited performance in quantifying single molecules, with a dynamic range of 10(4). We performed over 1200 single-cell measurements, demonstrating the use of this platform in the absolute quantification of both high- and low-abundance mRNA transcripts, as well as micro-RNAs that are not easily measured using alternative hybridization methods. We further apply the specificity and sensitivity of single-cell dPCR to performing measurements of RNA editing events in single cells. High-throughput dPCR provides a new tool in the arsenal of single-cell analysis methods, with a unique combination of speed, precision, sensitivity, and specificity. We anticipate this approach will enable new studies where high-performance single-cell measurements are essential, including the analysis of transcriptional noise, allelic imbalance, and RNA processing.

In Vitro Toxicity Screening Technique for Volatile Substances ...

EPA Pesticide Factsheets

In 2007 the National Research Council envisioned the need for inexpensive, high throughput, cell based toxicity testing methods relevant to human health. High Throughput Screening (HTS) in vitro screening approaches have addressed these problems by using robotics. However the challenge is that many of these chemicals are volatile and not amenable to HTS robotic liquid handling applications. We assembled an in vitro cell culture apparatus capable of screening volatile chemicals for toxicity with potential for miniaturization for high throughput. BEAS-2B lung cells were grown in an enclosed culture apparatus under air-liquid interface (ALI) conditions, and exposed to an array of xenobiotics in 5% CO2. Use of ALI conditions allows direct contact of cells with a gas xenobiotic, as well as release of endogenous gaseous molecules without interference by medium on the apical surface. To identify potential xenobiotic-induced perturbations in cell homeostasis, we monitored for alterations of endogenously-produced gaseous molecules in air directly above the cells, termed “headspace”. Alterations in specific endogenously-produced gaseous molecules (e.g., signaling molecules nitric oxide (NO) and carbon monoxide (CO) in headspace is indicative of xenobiotic-induced perturbations of specific cellular processes. Additionally, endogenously produced volatile organic compounds (VOCs) may be monitored in a nonspecific, discovery manner to determine whether cell processes are

Rapid high-throughput cloning and stable expression of antibodies in HEK293 cells.

PubMed

Spidel, Jared L; Vaessen, Benjamin; Chan, Yin Yin; Grasso, Luigi; Kline, J Bradford

2016-12-01

Single-cell based amplification of immunoglobulin variable regions is a rapid and powerful technique for cloning antigen-specific monoclonal antibodies (mAbs) for purposes ranging from general laboratory reagents to therapeutic drugs. From the initial screening process involving small quantities of hundreds or thousands of mAbs through in vitro characterization and subsequent in vivo experiments requiring large quantities of only a few, having a robust system for generating mAbs from cloning through stable cell line generation is essential. A protocol was developed to decrease the time, cost, and effort required by traditional cloning and expression methods by eliminating bottlenecks in these processes. Removing the clonal selection steps from the cloning process using a highly efficient ligation-independent protocol and from the stable cell line process by utilizing bicistronic plasmids to generate stable semi-clonal cell pools facilitated an increased throughput of the entire process from plasmid assembly through transient transfections and selection of stable semi-clonal cell pools. Furthermore, the time required by a single individual to clone, express, and select stable cell pools in a high-throughput format was reduced from 4 to 6months to only 4 to 6weeks. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Development of Droplet Microfluidics Enabling High-Throughput Single-Cell Analysis.

PubMed

Wen, Na; Zhao, Zhan; Fan, Beiyuan; Chen, Deyong; Men, Dong; Wang, Junbo; Chen, Jian

2016-07-05

This article reviews recent developments in droplet microfluidics enabling high-throughput single-cell analysis. Five key aspects in this field are included in this review: (1) prototype demonstration of single-cell encapsulation in microfluidic droplets; (2) technical improvements of single-cell encapsulation in microfluidic droplets; (3) microfluidic droplets enabling single-cell proteomic analysis; (4) microfluidic droplets enabling single-cell genomic analysis; and (5) integrated microfluidic droplet systems enabling single-cell screening. We examine the advantages and limitations of each technique and discuss future research opportunities by focusing on key performances of throughput, multifunctionality, and absolute quantification.

High-Throughput Single-Cell RNA Sequencing and Data Analysis.

PubMed

Sagar; Herman, Josip Stefan; Pospisilik, John Andrew; Grün, Dominic

2018-01-01

Understanding biological systems at a single cell resolution may reveal several novel insights which remain masked by the conventional population-based techniques providing an average readout of the behavior of cells. Single-cell transcriptome sequencing holds the potential to identify novel cell types and characterize the cellular composition of any organ or tissue in health and disease. Here, we describe a customized high-throughput protocol for single-cell RNA-sequencing (scRNA-seq) combining flow cytometry and a nanoliter-scale robotic system. Since scRNA-seq requires amplification of a low amount of endogenous cellular RNA, leading to substantial technical noise in the dataset, downstream data filtering and analysis require special care. Therefore, we also briefly describe in-house state-of-the-art data analysis algorithms developed to identify cellular subpopulations including rare cell types as well as to derive lineage trees by ordering the identified subpopulations of cells along the inferred differentiation trajectories.

High-Throughput Screening Using a Whole-Cell Virus Replication Reporter Gene Assay to Identify Inhibitory Compounds against Rift Valley Fever Virus Infection.

PubMed

Islam, Md Koushikul; Baudin, Maria; Eriksson, Jonas; Öberg, Christopher; Habjan, Matthias; Weber, Friedemann; Överby, Anna K; Ahlm, Clas; Evander, Magnus

2016-04-01

Rift Valley fever virus (RVFV) is an emerging virus that causes serious illness in humans and livestock. There are no approved vaccines or treatments for humans. The purpose of the study was to identify inhibitory compounds of RVFV infection without any preconceived idea of the mechanism of action. A whole-cell-based high-throughput drug screening assay was developed to screen 28,437 small chemical compounds targeting RVFV infection. To accomplish both speed and robustness, a replication-competent NSs-deleted RVFV expressing a fluorescent reporter gene was developed. Inhibition of fluorescence intensity was quantified by spectrophotometry and related to virus infection in human lung epithelial cells (A549). Cell toxicity was assessed by the Resazurin cell viability assay. After primary screening, 641 compounds were identified that inhibited RVFV infection by ≥80%, with ≥50% cell viability at 50 µM concentration. These compounds were subjected to a second screening regarding dose-response profiles, and 63 compounds with ≥60% inhibition of RVFV infection at 3.12 µM compound concentration and ≥50% cell viability at 25 µM were considered hits. Of these, six compounds with high inhibitory activity were identified. In conclusion, the high-throughput assay could efficiently and safely identify several promising compounds that inhibited RVFV infection. © 2016 Society for Laboratory Automation and Screening.

CyTOF workflow: differential discovery in high-throughput high-dimensional cytometry datasets

PubMed Central

Nowicka, Malgorzata; Krieg, Carsten; Weber, Lukas M.; Hartmann, Felix J.; Guglietta, Silvia; Becher, Burkhard; Levesque, Mitchell P.; Robinson, Mark D.

2017-01-01

High dimensional mass and flow cytometry (HDCyto) experiments have become a method of choice for high throughput interrogation and characterization of cell populations.Here, we present an R-based pipeline for differential analyses of HDCyto data, largely based on Bioconductor packages. We computationally define cell populations using FlowSOM clustering, and facilitate an optional but reproducible strategy for manual merging of algorithm-generated clusters. Our workflow offers different analysis paths, including association of cell type abundance with a phenotype or changes in signaling markers within specific subpopulations, or differential analyses of aggregated signals. Importantly, the differential analyses we show are based on regression frameworks where the HDCyto data is the response; thus, we are able to model arbitrary experimental designs, such as those with batch effects, paired designs and so on. In particular, we apply generalized linear mixed models to analyses of cell population abundance or cell-population-specific analyses of signaling markers, allowing overdispersion in cell count or aggregated signals across samples to be appropriately modeled. To support the formal statistical analyses, we encourage exploratory data analysis at every step, including quality control (e.g. multi-dimensional scaling plots), reporting of clustering results (dimensionality reduction, heatmaps with dendrograms) and differential analyses (e.g. plots of aggregated signals). PMID:28663787

CellSegm - a MATLAB toolbox for high-throughput 3D cell segmentation

PubMed Central

2013-01-01

The application of fluorescence microscopy in cell biology often generates a huge amount of imaging data. Automated whole cell segmentation of such data enables the detection and analysis of individual cells, where a manual delineation is often time consuming, or practically not feasible. Furthermore, compared to manual analysis, automation normally has a higher degree of reproducibility. CellSegm, the software presented in this work, is a Matlab based command line software toolbox providing an automated whole cell segmentation of images showing surface stained cells, acquired by fluorescence microscopy. It has options for both fully automated and semi-automated cell segmentation. Major algorithmic steps are: (i) smoothing, (ii) Hessian-based ridge enhancement, (iii) marker-controlled watershed segmentation, and (iv) feature-based classfication of cell candidates. Using a wide selection of image recordings and code snippets, we demonstrate that CellSegm has the ability to detect various types of surface stained cells in 3D. After detection and outlining of individual cells, the cell candidates can be subject to software based analysis, specified and programmed by the end-user, or they can be analyzed by other software tools. A segmentation of tissue samples with appropriate characteristics is also shown to be resolvable in CellSegm. The command-line interface of CellSegm facilitates scripting of the separate tools, all implemented in Matlab, offering a high degree of flexibility and tailored workflows for the end-user. The modularity and scripting capabilities of CellSegm enable automated workflows and quantitative analysis of microscopic data, suited for high-throughput image based screening. PMID:23938087

CellSegm - a MATLAB toolbox for high-throughput 3D cell segmentation.

PubMed

Hodneland, Erlend; Kögel, Tanja; Frei, Dominik Michael; Gerdes, Hans-Hermann; Lundervold, Arvid

2013-08-09

: The application of fluorescence microscopy in cell biology often generates a huge amount of imaging data. Automated whole cell segmentation of such data enables the detection and analysis of individual cells, where a manual delineation is often time consuming, or practically not feasible. Furthermore, compared to manual analysis, automation normally has a higher degree of reproducibility. CellSegm, the software presented in this work, is a Matlab based command line software toolbox providing an automated whole cell segmentation of images showing surface stained cells, acquired by fluorescence microscopy. It has options for both fully automated and semi-automated cell segmentation. Major algorithmic steps are: (i) smoothing, (ii) Hessian-based ridge enhancement, (iii) marker-controlled watershed segmentation, and (iv) feature-based classfication of cell candidates. Using a wide selection of image recordings and code snippets, we demonstrate that CellSegm has the ability to detect various types of surface stained cells in 3D. After detection and outlining of individual cells, the cell candidates can be subject to software based analysis, specified and programmed by the end-user, or they can be analyzed by other software tools. A segmentation of tissue samples with appropriate characteristics is also shown to be resolvable in CellSegm. The command-line interface of CellSegm facilitates scripting of the separate tools, all implemented in Matlab, offering a high degree of flexibility and tailored workflows for the end-user. The modularity and scripting capabilities of CellSegm enable automated workflows and quantitative analysis of microscopic data, suited for high-throughput image based screening.

Laser-Induced Fluorescence Detection in High-Throughput Screening of Heterogeneous Catalysts and Single Cells Analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Su, Hui

2001-01-01

Laser-induced fluorescence detection is one of the most sensitive detection techniques and it has found enormous applications in various areas. The purpose of this research was to develop detection approaches based on laser-induced fluorescence detection in two different areas, heterogeneous catalysts screening and single cell study. First, we introduced laser-induced imaging (LIFI) as a high-throughput screening technique for heterogeneous catalysts to explore the use of this high-throughput screening technique in discovery and study of various heterogeneous catalyst systems. This scheme is based on the fact that the creation or the destruction of chemical bonds alters the fluorescence properties of suitablymore » designed molecules. By irradiating the region immediately above the catalytic surface with a laser, the fluorescence intensity of a selected product or reactant can be imaged by a charge-coupled device (CCD) camera to follow the catalytic activity as a function of time and space. By screening the catalytic activity of vanadium pentoxide catalysts in oxidation of naphthalene, we demonstrated LIFI has good detection performance and the spatial and temporal resolution needed for high-throughput screening of heterogeneous catalysts. The sample packing density can reach up to 250 x 250 subunits/cm 2 for 40-μm wells. This experimental set-up also can screen solid catalysts via near infrared thermography detection.« less

Droplet Microarray Based on Superhydrophobic-Superhydrophilic Patterns for Single Cell Analysis.

PubMed

Jogia, Gabriella E; Tronser, Tina; Popova, Anna A; Levkin, Pavel A

2016-12-09

Single-cell analysis provides fundamental information on individual cell response to different environmental cues and is a growing interest in cancer and stem cell research. However, current existing methods are still facing challenges in performing such analysis in a high-throughput manner whilst being cost-effective. Here we established the Droplet Microarray (DMA) as a miniaturized screening platform for high-throughput single-cell analysis. Using the method of limited dilution and varying cell density and seeding time, we optimized the distribution of single cells on the DMA. We established culturing conditions for single cells in individual droplets on DMA obtaining the survival of nearly 100% of single cells and doubling time of single cells comparable with that of cells cultured in bulk cell population using conventional methods. Our results demonstrate that the DMA is a suitable platform for single-cell analysis, which carries a number of advantages compared with existing technologies allowing for treatment, staining and spot-to-spot analysis of single cells over time using conventional analysis methods such as microscopy.

Human Pluripotent Stem Cell-Based Assay Predicts Developmental Toxicity Potential of ToxCast Chemicals (ACT meeting)

EPA Science Inventory

Worldwide initiatives to screen for toxicity potential among the thousands of chemicals currently in use require inexpensive and high-throughput in vitro models to meet their goals. The devTOX quickPredict platform is an in vitro human pluripotent stem cell-based assay used to as...

Robot-Based High-Throughput Engineering of Alcoholic Polymer: Fullerene Nanoparticle Inks for an Eco-Friendly Processing of Organic Solar Cells.

PubMed

Xie, Chen; Tang, Xiaofeng; Berlinghof, Marvin; Langner, Stefan; Chen, Shi; Späth, Andreas; Li, Ning; Fink, Rainer H; Unruh, Tobias; Brabec, Christoph J

2018-06-27

Development of high-quality organic nanoparticle inks is a significant scientific challenge for the industrial production of solution-processed organic photovoltaics (OPVs) with eco-friendly processing methods. In this work, we demonstrate a novel, robot-based, high-throughput procedure performing automatic poly(3-hexylthio-phene-2,5-diyl) and indene-C 60 bisadduct nanoparticle ink synthesis in nontoxic alcohols. A novel methodology to prepare particle dispersions for fully functional OPVs by manipulating the particle size and solvent system was studied in detail. The ethanol dispersion with a particle diameter of around 80-100 nm exhibits reduced degradation, yielding a power conversion efficiency of 4.52%, which is the highest performance reported so far for water/alcohol-processed OPV devices. By successfully deploying the high-throughput robot-based approach for an organic nanoparticle ink preparation, we believe that the findings demonstrated in this work will trigger more research interest and effort on eco-friendly industrial production of OPVs.

High-throughput rare cell separation from blood samples using steric hindrance and inertial microfluidics.

PubMed

Shen, Shaofei; Ma, Chao; Zhao, Lei; Wang, Yaolei; Wang, Jian-Chun; Xu, Juan; Li, Tianbao; Pang, Long; Wang, Jinyi

2014-07-21

The presence and quantity of rare cells in the bloodstream of cancer patients provide a potentially accessible source for the early detection of invasive cancer and for monitoring the treatment of advanced diseases. The separation of rare cells from peripheral blood, as a "virtual and real-time liquid biopsy", is expected to replace conventional tissue biopsies of metastatic tumors for therapy guidance. However, technical obstacles, similar to looking for a needle in a haystack, have hindered the broad clinical utility of this method. In this study, we developed a multistage microfluidic device for continuous label-free separation and enrichment of rare cells from blood samples based on cell size and deformability. We successfully separated tumor cells (MCF-7 and HeLa cells) and leukemic (K562) cells spiked in diluted whole blood using a unique complementary combination of inertial microfluidics and steric hindrance in a microfluidic system. The processing parameters of the inertial focusing and steric hindrance regions were optimized to achieve high-throughput and high-efficiency separation, significant advantages compared with existing rare cell isolation technologies. The results from experiments with rare cells spiked in 1% hematocrit blood indicated >90% cell recovery at a throughput of 2.24 × 10(7) cells min(-1). The enrichment of rare cells was >2.02 × 10(5)-fold. Thus, this microfluidic system driven by purely hydrodynamic forces has practical potential to be applied either alone or as a sample preparation platform for fundamental studies and clinical applications.

Large-scale microfluidics providing high-resolution and high-throughput screening of Caenorhabditis elegans poly-glutamine aggregation model

NASA Astrophysics Data System (ADS)

Mondal, Sudip; Hegarty, Evan; Martin, Chris; Gökçe, Sertan Kutal; Ghorashian, Navid; Ben-Yakar, Adela

2016-10-01

Next generation drug screening could benefit greatly from in vivo studies, using small animal models such as Caenorhabditis elegans for hit identification and lead optimization. Current in vivo assays can operate either at low throughput with high resolution or with low resolution at high throughput. To enable both high-throughput and high-resolution imaging of C. elegans, we developed an automated microfluidic platform. This platform can image 15 z-stacks of ~4,000 C. elegans from 96 different populations using a large-scale chip with a micron resolution in 16 min. Using this platform, we screened ~100,000 animals of the poly-glutamine aggregation model on 25 chips. We tested the efficacy of ~1,000 FDA-approved drugs in improving the aggregation phenotype of the model and identified four confirmed hits. This robust platform now enables high-content screening of various C. elegans disease models at the speed and cost of in vitro cell-based assays.

High-content profiling of cell responsiveness to graded substrates based on combinyatorially variant polymers.

PubMed

Liu, Er; Treiser, Matthew D; Patel, Hiral; Sung, Hak-Joon; Roskov, Kristen E; Kohn, Joachim; Becker, Matthew L; Moghe, Prabhas V

2009-08-01

We have developed a novel approach combining high information and high throughput analysis to characterize cell adhesive responses to biomaterial substrates possessing gradients in surface topography. These gradients were fabricated by subjecting thin film blends of tyrosine-derived polycarbonates, i.e. poly(DTE carbonate) and poly(DTO carbonate) to a gradient temperature annealing protocol. Saos-2 cells engineered with a green fluorescent protein (GFP) reporter for farnesylation (GFP-f) were cultured on the gradient substrates to assess the effects of nanoscale surface topology and roughness that arise during the phase separation process on cell attachment and adhesion strength. The high throughput imaging approach allowed us to rapidly identify the "global" and "high content" structure-property relationships between cell adhesion and biomaterial properties such as polymer chemistry and topography. This study found that cell attachment and spreading increased monotonically with DTE content and were significantly elevated at the position with intermediate regions corresponding to the highest "gradient" of surface roughness, while GFP-f farnesylation intensity descriptors were sensitively altered by surface roughness, even in cells with comparable levels of spreading.

High-throughput, label-free, single-cell, microalgal lipid screening by machine-learning-equipped optofluidic time-stretch quantitative phase microscopy.

PubMed

Guo, Baoshan; Lei, Cheng; Kobayashi, Hirofumi; Ito, Takuro; Yalikun, Yaxiaer; Jiang, Yiyue; Tanaka, Yo; Ozeki, Yasuyuki; Goda, Keisuke

2017-05-01

The development of reliable, sustainable, and economical sources of alternative fuels to petroleum is required to tackle the global energy crisis. One such alternative is microalgal biofuel, which is expected to play a key role in reducing the detrimental effects of global warming as microalgae absorb atmospheric CO 2 via photosynthesis. Unfortunately, conventional analytical methods only provide population-averaged lipid amounts and fail to characterize a diverse population of microalgal cells with single-cell resolution in a non-invasive and interference-free manner. Here high-throughput label-free single-cell screening of lipid-producing microalgal cells with optofluidic time-stretch quantitative phase microscopy was demonstrated. In particular, Euglena gracilis, an attractive microalgal species that produces wax esters (suitable for biodiesel and aviation fuel after refinement), within lipid droplets was investigated. The optofluidic time-stretch quantitative phase microscope is based on an integration of a hydrodynamic-focusing microfluidic chip, an optical time-stretch quantitative phase microscope, and a digital image processor equipped with machine learning. As a result, it provides both the opacity and phase maps of every single cell at a high throughput of 10,000 cells/s, enabling accurate cell classification without the need for fluorescent staining. Specifically, the dataset was used to characterize heterogeneous populations of E. gracilis cells under two different culture conditions (nitrogen-sufficient and nitrogen-deficient) and achieve the cell classification with an error rate of only 2.15%. The method holds promise as an effective analytical tool for microalgae-based biofuel production. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

A paper-based microbial fuel cell array for rapid and high-throughput screening of electricity-producing bacteria.

PubMed

Choi, Gihoon; Hassett, Daniel J; Choi, Seokheun

2015-06-21

There is a large global effort to improve microbial fuel cell (MFC) techniques and advance their translational potential toward practical, real-world applications. Significant boosts in MFC performance can be achieved with the development of new techniques in synthetic biology that can regulate microbial metabolic pathways or control their gene expression. For these new directions, a high-throughput and rapid screening tool for microbial biopower production is needed. In this work, a 48-well, paper-based sensing platform was developed for the high-throughput and rapid characterization of the electricity-producing capability of microbes. 48 spatially distinct wells of a sensor array were prepared by patterning 48 hydrophilic reservoirs on paper with hydrophobic wax boundaries. This paper-based platform exploited the ability of paper to quickly wick fluid and promoted bacterial attachment to the anode pads, resulting in instant current generation upon loading of the bacterial inoculum. We validated the utility of our MFC array by studying how strategic genetic modifications impacted the electrochemical activity of various Pseudomonas aeruginosa mutant strains. Within just 20 minutes, we successfully determined the electricity generation capacity of eight isogenic mutants of P. aeruginosa. These efforts demonstrate that our MFC array displays highly comparable performance characteristics and identifies genes in P. aeruginosa that can trigger a higher power density.

qFlow Cytometry-Based Receptoromic Screening: A High-Throughput Quantification Approach Informing Biomarker Selection and Nanosensor Development.

PubMed

Chen, Si; Weddell, Jared; Gupta, Pavan; Conard, Grace; Parkin, James; Imoukhuede, Princess I

2017-01-01

Nanosensor-based detection of biomarkers can improve medical diagnosis; however, a critical factor in nanosensor development is deciding which biomarker to target, as most diseases present several biomarkers. Biomarker-targeting decisions can be informed via an understanding of biomarker expression. Currently, immunohistochemistry (IHC) is the accepted standard for profiling biomarker expression. While IHC provides a relative mapping of biomarker expression, it does not provide cell-by-cell readouts of biomarker expression or absolute biomarker quantification. Flow cytometry overcomes both these IHC challenges by offering biomarker expression on a cell-by-cell basis, and when combined with calibration standards, providing quantitation of biomarker concentrations: this is known as qFlow cytometry. Here, we outline the key components for applying qFlow cytometry to detect biomarkers within the angiogenic vascular endothelial growth factor receptor family. The key aspects of the qFlow cytometry methodology include: antibody specificity testing, immunofluorescent cell labeling, saturation analysis, fluorescent microsphere calibration, and quantitative analysis of both ensemble and cell-by-cell data. Together, these methods enable high-throughput quantification of biomarker expression.

Identification of Small Molecule Inhibitors of Clostridium perfringens ε-Toxin Cytotoxicity Using a Cell-Based High-Throughput Screen.

PubMed

Lewis, Michelle; Weaver, Charles David; McClain, Mark S

2010-07-01

The Clostridium perfringens epsilon toxin, a select agent, is responsible for a severe, often fatal enterotoxemia characterized by edema in the heart, lungs, kidney, and brain. The toxin is believed to be an oligomeric pore-forming toxin. Currently, there is no effective therapy for countering the cytotoxic activity of the toxin in exposed individuals. Using a robust cell-based high-throughput screening (HTS) assay, we screened a 151,616-compound library for the ability to inhibit ε-toxin-induced cytotoxicity. Survival of MDCK cells exposed to the toxin was assessed by addition of resazurin to detect metabolic activity in surviving cells. The hit rate for this screen was 0.6%. Following a secondary screen of each hit in triplicate and assays to eliminate false positives, we focused on three structurally-distinct compounds: an N-cycloalkylbenzamide, a furo[2,3-b]quinoline, and a 6H-anthra[1,9-cd]isoxazol. None of the three compounds appeared to inhibit toxin binding to cells or the ability of the toxin to form oligomeric complexes. Additional assays demonstrated that two of the inhibitory compounds inhibited ε-toxin-induced permeabilization of MDCK cells to propidium iodide. Furthermore, the two compounds exhibited inhibitory effects on cells pre-treated with toxin. Structural analogs of one of the inhibitors identified through the high-throughput screen were analyzed and provided initial structure-activity data. These compounds should serve as the basis for further structure-activity refinement that may lead to the development of effective anti-ε-toxin therapeutics.

Identification of Small Molecule Inhibitors of Clostridium perfringens ε-Toxin Cytotoxicity Using a Cell-Based High-Throughput Screen

PubMed Central

Lewis, Michelle; Weaver, Charles David; McClain, Mark S.

2010-01-01

The Clostridium perfringens epsilon toxin, a select agent, is responsible for a severe, often fatal enterotoxemia characterized by edema in the heart, lungs, kidney, and brain. The toxin is believed to be an oligomeric pore-forming toxin. Currently, there is no effective therapy for countering the cytotoxic activity of the toxin in exposed individuals. Using a robust cell-based high-throughput screening (HTS) assay, we screened a 151,616-compound library for the ability to inhibit ε-toxin-induced cytotoxicity. Survival of MDCK cells exposed to the toxin was assessed by addition of resazurin to detect metabolic activity in surviving cells. The hit rate for this screen was 0.6%. Following a secondary screen of each hit in triplicate and assays to eliminate false positives, we focused on three structurally-distinct compounds: an N-cycloalkylbenzamide, a furo[2,3-b]quinoline, and a 6H-anthra[1,9-cd]isoxazol. None of the three compounds appeared to inhibit toxin binding to cells or the ability of the toxin to form oligomeric complexes. Additional assays demonstrated that two of the inhibitory compounds inhibited ε-toxin-induced permeabilization of MDCK cells to propidium iodide. Furthermore, the two compounds exhibited inhibitory effects on cells pre-treated with toxin. Structural analogs of one of the inhibitors identified through the high-throughput screen were analyzed and provided initial structure-activity data. These compounds should serve as the basis for further structure-activity refinement that may lead to the development of effective anti-ε-toxin therapeutics. PMID:20721308

A high-throughput three-dimensional cell migration assay for toxicity screening with mobile device-based macroscopic image analysis

PubMed Central

Timm, David M.; Chen, Jianbo; Sing, David; Gage, Jacob A.; Haisler, William L.; Neeley, Shane K.; Raphael, Robert M.; Dehghani, Mehdi; Rosenblatt, Kevin P.; Killian, T. C.; Tseng, Hubert; Souza, Glauco R.

2013-01-01

There is a growing demand for in vitro assays for toxicity screening in three-dimensional (3D) environments. In this study, 3D cell culture using magnetic levitation was used to create an assay in which cells were patterned into 3D rings that close over time. The rate of closure was determined from time-lapse images taken with a mobile device and related to drug concentration. Rings of human embryonic kidney cells (HEK293) and tracheal smooth muscle cells (SMCs) were tested with ibuprofen and sodium dodecyl sulfate (SDS). Ring closure correlated with the viability and migration of cells in two dimensions (2D). Images taken using a mobile device were similar in analysis to images taken with a microscope. Ring closure may serve as a promising label-free and quantitative assay for high-throughput in vivo toxicity in 3D cultures. PMID:24141454

Evaluation of Impermeant, DNA-Binding Dye Fluorescence as a Real-Time Readout of Eukaryotic Cell Toxicity in a High Throughput Screening Format

PubMed Central

Chiaraviglio, Lucius

2014-01-01

Abstract Interpretation of high throughput screening (HTS) data in cell-based assays may be confounded by cytotoxic properties of screening compounds. Therefore, assessing cell toxicity in real time during the HTS process itself would be highly advantageous. Here, we investigate the potential of putatively impermeant, fluorescent, DNA-binding dyes to give cell toxicity readout during HTS. Amongst 19 DNA-binding dyes examined, three classes were identified that were (1) permeant, (2) cytotoxic, or (3) neither permeant nor cytotoxic during 3-day incubation with a macrophage cell line. In the last class, four dyes (SYTOX Green, CellTox Green, GelGreen, and EvaGreen) gave highly robust cytotoxicity data in 384-well screening plates. As proof of principle, successful combination with a luminescence-based assay in HTS format was demonstrated. Here, both intracellular growth of Legionella pneumophila (luminescence) and host cell viability (SYTOX Green exclusion) were assayed in the same screening well. Incorporation of membrane-impermeant, DNA-binding, fluorescent dyes in HTS assays should prove useful by allowing evaluation of cytotoxicity in real time, eliminating reagent addition steps and effort associated with endpoint cell viability analysis, and reducing the need for follow-up cytotoxicity screening. PMID:24831788

High-Throughput Generation of Durable Droplet Arrays for Single-Cell Encapsulation, Culture, and Monitoring.

PubMed

Wu, Han; Chen, Xinlian; Gao, Xinghua; Zhang, Mengying; Wu, Jinbo; Wen, Weijia

2018-04-03

High-throughput measurements can be achieved using droplet-based assays. In this study, we exploited the principles of wetting behavior and capillarity to guide liquids sliding along a solid surface with hybrid wettability. Oil-covered droplet arrays with uniformly sized and regularly shaped picoliter droplets were successfully generated on hydrophilic-in-hydrophobic patterned substrates. More than ten thousand 31-pL droplets were generated in 5 s without any sophisticated instruments. Covering the droplet arrays with oil during generation not only isolated the droplets from each other but also effectively prevented droplet evaporation. The oil-covered droplet arrays could be stored for more than 2 days with less than 35% volume loss. Single microspheres, microbial cells, or mammalian cells were successfully captured in the droplets. We demonstrate that Escherichia coli could be encapsulated at a certain number (1-4) and cultured for 3 days in droplets. Cell population and morphology were dynamically tracked within individual droplets. Our droplet array generation method enables high-throughput processing and is facile, efficient, and low-cost; in addition, the prepared droplet arrays have enormous potential for applications in chemical and biological assays.

Analysis of JC virus DNA replication using a quantitative and high-throughput assay

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shin, Jong; Phelan, Paul J.; Chhum, Panharith

2014-11-15

Progressive Multifocal Leukoencephalopathy (PML) is caused by lytic replication of JC virus (JCV) in specific cells of the central nervous system. Like other polyomaviruses, JCV encodes a large T-antigen helicase needed for replication of the viral DNA. Here, we report the development of a luciferase-based, quantitative and high-throughput assay of JCV DNA replication in C33A cells, which, unlike the glial cell lines Hs 683 and U87, accumulate high levels of nuclear T-ag needed for robust replication. Using this assay, we investigated the requirement for different domains of T-ag, and for specific sequences within and flanking the viral origin, in JCVmore » DNA replication. Beyond providing validation of the assay, these studies revealed an important stimulatory role of the transcription factor NF1 in JCV DNA replication. Finally, we show that the assay can be used for inhibitor testing, highlighting its value for the identification of antiviral drugs targeting JCV DNA replication. - Highlights: • Development of a high-throughput screening assay for JCV DNA replication using C33A cells. • Evidence that T-ag fails to accumulate in the nuclei of established glioma cell lines. • Evidence that NF-1 directly promotes JCV DNA replication in C33A cells. • Proof-of-concept that the HTS assay can be used to identify pharmacological inhibitor of JCV DNA replication.« less

Webcam-based flow cytometer using wide-field imaging for low cell number detection at high throughput.

PubMed

Balsam, Joshua; Bruck, Hugh Alan; Rasooly, Avraham

2014-09-07

Here we describe a novel low-cost flow cytometer based on a webcam capable of low cell number detection in a large volume which may overcome the limitations of current flow cytometry. Several key elements have been combined to yield both high throughput and high sensitivity. The first element is a commercially available webcam capable of 187 frames per second video capture at a resolution of 320 × 240 pixels. The second element in this design is a 1 W 450 nm laser module for area-excitation, which combined with the webcam allows for rapid interrogation of a flow field. The final element is a 2D flow-cell which overcomes the flow limitation of hydrodynamic focusing and allows for higher sample throughput in a wider flow field. This cell allows for the linear velocity of target cells to be lower than in a conventional "1D" hydrodynamic focusing flow-cells typically used in cytometry at similar volumetric flow rates. It also allows cells to be imaged at the full frame rate of the webcam. Using this webcam-based flow cytometer with wide-field imaging, it was confirmed that the detection of fluorescently tagged 5 μm polystyrene beads in "1D" hydrodynamic focusing flow-cells was not practical for low cell number detection due to streaking from the motion of the beads, which did not occur with the 2D flow-cell design. The sensitivity and throughput of this webcam-based flow cytometer was then investigated using THP-1 human monocytes stained with SYTO-9 florescent dye in the 2D flow-cell. The flow cytometer was found to be capable of detecting fluorescently tagged cells at concentrations as low as 1 cell per mL at flow rates of 500 μL min(-1) in buffer and in blood. The effectiveness of detection was concentration dependent: at 100 cells per mL 84% of the cells were detected compared to microscopy, 10 cells per mL 79% detected and 1 cell per mL 59% of the cells were detected. With the blood samples spiked to 100 cells per mL, the average concentration for all samples was 91.4 cells per mL, with a 95% confidence interval of 86-97 cells per mL. These low cell concentrations and the large volume capabilities of the system may overcome the limitations of current cytometry, and are applicable to rare cell (such as circulating tumor cell) detection The simplicity and low cost of this device suggests that it may have a potential use in developing point-of-care clinical flow cytometry for resource-poor settings associated with global health.

High-throughput living cell-based optical biosensor for detection of bacterial lipopolysaccharide (LPS) using a red fluorescent protein reporter system.

PubMed

Jiang, Hui; Jiang, Donglei; Shao, Jingdong; Sun, Xiulan; Wang, Jiasheng

2016-11-14

Due to the high toxicity of bacterial lipopolysaccharide (LPS), resulting in sepsis and septic shock, two major causes of death worldwide, significant effort is directed toward the development of specific trace-level LPS detection systems. Here, we report sensitive, user-friendly, high-throughput LPS detection in a 96-well microplate using a transcriptional biosensor system, based on 293/hTLR4A-MD2-CD14 cells that are transformed by a red fluorescent protein (mCherry) gene under the transcriptional control of an NF-κB response element. The recognition of LPS activates the biosensor cell, TLR4, and the co-receptor-induced NF-κB signaling pathway, which results in the expression of mCherry fluorescent protein. The novel cell-based biosensor detects LPS with specificity at low concentration. The cell-based biosensor was evaluated by testing LPS isolated from 14 bacteria. Of the tested bacteria, 13 isolated Enterobacteraceous LPSs with hexa-acylated structures were found to increase red fluorescence and one penta-acylated LPS from Pseudomonadaceae appeared less potent. The proposed biosensor has potential for use in the LPS detection in foodstuff and biological products, as well as bacteria identification, assisting the control of foodborne diseases.

A continuous high-throughput bioparticle sorter based on 3D traveling-wave dielectrophoresis.

PubMed

Cheng, I-Fang; Froude, Victoria E; Zhu, Yingxi; Chang, Hsueh-Chia; Chang, Hsien-Chang

2009-11-21

We present a high throughput (maximum flow rate approximately 10 microl/min or linear velocity approximately 3 mm/s) continuous bio-particle sorter based on 3D traveling-wave dielectrophoresis (twDEP) at an optimum AC frequency of 500 kHz. The high throughput sorting is achieved with a sustained twDEP particle force normal to the continuous through-flow, which is applied over the entire chip by a single 3D electrode array. The design allows continuous fractionation of micron-sized particles into different downstream sub-channels based on differences in their twDEP mobility on both sides of the cross-over. Conventional DEP is integrated upstream to focus the particles into a single levitated queue to allow twDEP sorting by mobility difference and to minimize sedimentation and field-induced lysis. The 3D electrode array design minimizes the offsetting effect of nDEP (negative DEP with particle force towards regions with weak fields) on twDEP such that both forces increase monotonically with voltage to further increase the throughput. Effective focusing and separation of red blood cells from debris-filled heterogeneous samples are demonstrated, as well as size-based separation of poly-dispersed liposome suspensions into two distinct bands at 2.3 to 4.6 microm and 1.5 to 2.7 microm, at the highest throughput recorded in hand-held chips of 6 microl/min.

Quartz-Seq2: a high-throughput single-cell RNA-sequencing method that effectively uses limited sequence reads.

PubMed

Sasagawa, Yohei; Danno, Hiroki; Takada, Hitomi; Ebisawa, Masashi; Tanaka, Kaori; Hayashi, Tetsutaro; Kurisaki, Akira; Nikaido, Itoshi

2018-03-09

High-throughput single-cell RNA-seq methods assign limited unique molecular identifier (UMI) counts as gene expression values to single cells from shallow sequence reads and detect limited gene counts. We thus developed a high-throughput single-cell RNA-seq method, Quartz-Seq2, to overcome these issues. Our improvements in the reaction steps make it possible to effectively convert initial reads to UMI counts, at a rate of 30-50%, and detect more genes. To demonstrate the power of Quartz-Seq2, we analyzed approximately 10,000 transcriptomes from in vitro embryonic stem cells and an in vivo stromal vascular fraction with a limited number of reads.

High-throughput cell-based screening reveals a role for ZNF131 as a repressor of ERalpha signaling

PubMed Central

Han, Xiao; Guo, Jinhai; Deng, Weiwei; Zhang, Chenying; Du, Peige; Shi, Taiping; Ma, Dalong

2008-01-01

Background Estrogen receptor α (ERα) is a transcription factor whose activity is affected by multiple regulatory cofactors. In an effort to identify the human genes involved in the regulation of ERα, we constructed a high-throughput, cell-based, functional screening platform by linking a response element (ERE) with a reporter gene. This allowed the cellular activity of ERα, in cells cotransfected with the candidate gene, to be quantified in the presence or absence of its cognate ligand E2. Results From a library of 570 human cDNA clones, we identified zinc finger protein 131 (ZNF131) as a repressor of ERα mediated transactivation. ZNF131 is a typical member of the BTB/POZ family of transcription factors, and shows both ubiquitous expression and a high degree of sequence conservation. The luciferase reporter gene assay revealed that ZNF131 inhibits ligand-dependent transactivation by ERα in a dose-dependent manner. Electrophoretic mobility shift assay clearly demonstrated that the interaction between ZNF131 and ERα interrupts or prevents ERα binding to the estrogen response element (ERE). In addition, ZNF131 was able to suppress the expression of pS2, an ERα target gene. Conclusion We suggest that the functional screening platform we constructed can be applied for high-throughput genomic screening candidate ERα-related genes. This in turn may provide new insights into the underlying molecular mechanisms of ERα regulation in mammalian cells. PMID:18847501

Droplet barcoding for single-cell transcriptomics applied to embryonic stem cells.

PubMed

Klein, Allon M; Mazutis, Linas; Akartuna, Ilke; Tallapragada, Naren; Veres, Adrian; Li, Victor; Peshkin, Leonid; Weitz, David A; Kirschner, Marc W

2015-05-21

It has long been the dream of biologists to map gene expression at the single-cell level. With such data one might track heterogeneous cell sub-populations, and infer regulatory relationships between genes and pathways. Recently, RNA sequencing has achieved single-cell resolution. What is limiting is an effective way to routinely isolate and process large numbers of individual cells for quantitative in-depth sequencing. We have developed a high-throughput droplet-microfluidic approach for barcoding the RNA from thousands of individual cells for subsequent analysis by next-generation sequencing. The method shows a surprisingly low noise profile and is readily adaptable to other sequencing-based assays. We analyzed mouse embryonic stem cells, revealing in detail the population structure and the heterogeneous onset of differentiation after leukemia inhibitory factor (LIF) withdrawal. The reproducibility of these high-throughput single-cell data allowed us to deconstruct cell populations and infer gene expression relationships. VIDEO ABSTRACT. Copyright © 2015 Elsevier Inc. All rights reserved.

High-throughput linear optical stretcher for mechanical characterization of blood cells.

PubMed

Roth, Kevin B; Neeves, Keith B; Squier, Jeff; Marr, David W M

2016-04-01

This study describes a linear optical stretcher as a high-throughput mechanical property cytometer. Custom, inexpensive, and scalable optics image a linear diode bar source into a microfluidic channel, where cells are hydrodynamically focused into the optical stretcher. Upon entering the stretching region, antipodal optical forces generated by the refraction of tightly focused laser light at the cell membrane deform each cell in flow. Each cell relaxes as it flows out of the trap and is compared to the stretched state to determine deformation. The deformation response of untreated red blood cells and neutrophils were compared to chemically treated cells. Statistically significant differences were observed between normal, diamide-treated, and glutaraldehyde-treated red blood cells, as well as between normal and cytochalasin D-treated neutrophils. Based on the behavior of the pure, untreated populations of red cells and neutrophils, a mixed population of these cells was tested and the discrete populations were identified by deformability. © 2015 International Society for Advancement of Cytometry. © 2015 International Society for Advancement of Cytometry.

High-throughput and targeted in-depth mass spectrometry-based approaches for biofluid profiling and biomarker discovery.

PubMed

Jimenez, Connie R; Piersma, Sander; Pham, Thang V

2007-12-01

Proteomics aims to create a link between genomic information, biological function and disease through global studies of protein expression, modification and protein-protein interactions. Recent advances in key proteomics tools, such as mass spectrometry (MS) and (bio)informatics, provide tremendous opportunities for biomarker-related clinical applications. In this review, we focus on two complementary MS-based approaches with high potential for the discovery of biomarker patterns and low-abundant candidate biomarkers in biofluids: high-throughput matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy-based methods for peptidome profiling and label-free liquid chromatography-based methods coupled to MS for in-depth profiling of biofluids with a focus on subproteomes, including the low-molecular-weight proteome, carrier-bound proteome and N-linked glycoproteome. The two approaches differ in their aims, throughput and sensitivity. We discuss recent progress and challenges in the analysis of plasma/serum and proximal fluids using these strategies and highlight the potential of liquid chromatography-MS-based proteomics of cancer cell and tumor secretomes for the discovery of candidate blood-based biomarkers. Strategies for candidate validation are also described.

High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells.

PubMed

Zhou, Yuexin; Zhu, Shiyou; Cai, Changzu; Yuan, Pengfei; Li, Chunmei; Huang, Yanyi; Wei, Wensheng

2014-05-22

Targeted genome editing technologies are powerful tools for studying biology and disease, and have a broad range of research applications. In contrast to the rapid development of toolkits to manipulate individual genes, large-scale screening methods based on the complete loss of gene expression are only now beginning to be developed. Here we report the development of a focused CRISPR/Cas-based (clustered regularly interspaced short palindromic repeats/CRISPR-associated) lentiviral library in human cells and a method of gene identification based on functional screening and high-throughput sequencing analysis. Using knockout library screens, we successfully identified the host genes essential for the intoxication of cells by anthrax and diphtheria toxins, which were confirmed by functional validation. The broad application of this powerful genetic screening strategy will not only facilitate the rapid identification of genes important for bacterial toxicity but will also enable the discovery of genes that participate in other biological processes.

Image-based computational quantification and visualization of genetic alterations and tumour heterogeneity

PubMed Central

Zhong, Qing; Rüschoff, Jan H.; Guo, Tiannan; Gabrani, Maria; Schüffler, Peter J.; Rechsteiner, Markus; Liu, Yansheng; Fuchs, Thomas J.; Rupp, Niels J.; Fankhauser, Christian; Buhmann, Joachim M.; Perner, Sven; Poyet, Cédric; Blattner, Miriam; Soldini, Davide; Moch, Holger; Rubin, Mark A.; Noske, Aurelia; Rüschoff, Josef; Haffner, Michael C.; Jochum, Wolfram; Wild, Peter J.

2016-01-01

Recent large-scale genome analyses of human tissue samples have uncovered a high degree of genetic alterations and tumour heterogeneity in most tumour entities, independent of morphological phenotypes and histopathological characteristics. Assessment of genetic copy-number variation (CNV) and tumour heterogeneity by fluorescence in situ hybridization (ISH) provides additional tissue morphology at single-cell resolution, but it is labour intensive with limited throughput and high inter-observer variability. We present an integrative method combining bright-field dual-colour chromogenic and silver ISH assays with an image-based computational workflow (ISHProfiler), for accurate detection of molecular signals, high-throughput evaluation of CNV, expressive visualization of multi-level heterogeneity (cellular, inter- and intra-tumour heterogeneity), and objective quantification of heterogeneous genetic deletions (PTEN) and amplifications (19q12, HER2) in diverse human tumours (prostate, endometrial, ovarian and gastric), using various tissue sizes and different scanners, with unprecedented throughput and reproducibility. PMID:27052161

Image-based computational quantification and visualization of genetic alterations and tumour heterogeneity.

PubMed

Zhong, Qing; Rüschoff, Jan H; Guo, Tiannan; Gabrani, Maria; Schüffler, Peter J; Rechsteiner, Markus; Liu, Yansheng; Fuchs, Thomas J; Rupp, Niels J; Fankhauser, Christian; Buhmann, Joachim M; Perner, Sven; Poyet, Cédric; Blattner, Miriam; Soldini, Davide; Moch, Holger; Rubin, Mark A; Noske, Aurelia; Rüschoff, Josef; Haffner, Michael C; Jochum, Wolfram; Wild, Peter J

2016-04-07

Recent large-scale genome analyses of human tissue samples have uncovered a high degree of genetic alterations and tumour heterogeneity in most tumour entities, independent of morphological phenotypes and histopathological characteristics. Assessment of genetic copy-number variation (CNV) and tumour heterogeneity by fluorescence in situ hybridization (ISH) provides additional tissue morphology at single-cell resolution, but it is labour intensive with limited throughput and high inter-observer variability. We present an integrative method combining bright-field dual-colour chromogenic and silver ISH assays with an image-based computational workflow (ISHProfiler), for accurate detection of molecular signals, high-throughput evaluation of CNV, expressive visualization of multi-level heterogeneity (cellular, inter- and intra-tumour heterogeneity), and objective quantification of heterogeneous genetic deletions (PTEN) and amplifications (19q12, HER2) in diverse human tumours (prostate, endometrial, ovarian and gastric), using various tissue sizes and different scanners, with unprecedented throughput and reproducibility.

Ultra-high-throughput Production of III-V/Si Wafer for Electronic and Photonic Applications

PubMed Central

Geum, Dae-Myeong; Park, Min-Su; Lim, Ju Young; Yang, Hyun-Duk; Song, Jin Dong; Kim, Chang Zoo; Yoon, Euijoon; Kim, SangHyeon; Choi, Won Jun

2016-01-01

Si-based integrated circuits have been intensively developed over the past several decades through ultimate device scaling. However, the Si technology has reached the physical limitations of the scaling. These limitations have fuelled the search for alternative active materials (for transistors) and the introduction of optical interconnects (called “Si photonics”). A series of attempts to circumvent the Si technology limits are based on the use of III-V compound semiconductor due to their superior benefits, such as high electron mobility and direct bandgap. To use their physical properties on a Si platform, the formation of high-quality III-V films on the Si (III-V/Si) is the basic technology ; however, implementing this technology using a high-throughput process is not easy. Here, we report new concepts for an ultra-high-throughput heterogeneous integration of high-quality III-V films on the Si using the wafer bonding and epitaxial lift off (ELO) technique. We describe the ultra-fast ELO and also the re-use of the III-V donor wafer after III-V/Si formation. These approaches provide an ultra-high-throughput fabrication of III-V/Si substrates with a high-quality film, which leads to a dramatic cost reduction. As proof-of-concept devices, this paper demonstrates GaAs-based high electron mobility transistors (HEMTs), solar cells, and hetero-junction phototransistors on Si substrates. PMID:26864968

High-throughput label-free screening of euglena gracilis with optofluidic time-stretch quantitative phase microscopy

NASA Astrophysics Data System (ADS)

Guo, Baoshan; Lei, Cheng; Ito, Takuro; Yaxiaer, Yalikun; Kobayashi, Hirofumi; Jiang, Yiyue; Tanaka, Yo; Ozeki, Yasuyuki; Goda, Keisuke

2017-02-01

The development of reliable, sustainable, and economical sources of alternative fuels is an important, but challenging goal for the world. As an alternative to liquid fossil fuels, microalgal biofuel is expected to play a key role in reducing the detrimental effects of global warming since microalgae absorb atmospheric CO2 via photosynthesis. Unfortunately, conventional analytical methods only provide population-averaged lipid contents and fail to characterize a diverse population of microalgal cells with single-cell resolution in a noninvasive and interference-free manner. Here we demonstrate high-throughput label-free single-cell screening of lipid-producing microalgal cells with optofluidic time-stretch quantitative phase microscopy. In particular, we use Euglena gracilis - an attractive microalgal species that produces wax esters (suitable for biodiesel and aviation fuel after refinement) within lipid droplets. Our optofluidic time-stretch quantitative phase microscope is based on an integration of a hydrodynamic-focusing microfluidic chip, an optical time-stretch phase-contrast microscope, and a digital image processor equipped with machine learning. As a result, it provides both the opacity and phase contents of every single cell at a high throughput of 10,000 cells/s. We characterize heterogeneous populations of E. gracilis cells under two different culture conditions to evaluate their lipid production efficiency. Our method holds promise as an effective analytical tool for microalgaebased biofuel production.

Scaling and automation of a high-throughput single-cell-derived tumor sphere assay chip.

PubMed

Cheng, Yu-Heng; Chen, Yu-Chih; Brien, Riley; Yoon, Euisik

2016-10-07

Recent research suggests that cancer stem-like cells (CSCs) are the key subpopulation for tumor relapse and metastasis. Due to cancer plasticity in surface antigen and enzymatic activity markers, functional tumorsphere assays are promising alternatives for CSC identification. To reliably quantify rare CSCs (1-5%), thousands of single-cell suspension cultures are required. While microfluidics is a powerful tool in handling single cells, previous works provide limited throughput and lack automatic data analysis capability required for high-throughput studies. In this study, we present the scaling and automation of high-throughput single-cell-derived tumor sphere assay chips, facilitating the tracking of up to ∼10 000 cells on a chip with ∼76.5% capture rate. The presented cell capture scheme guarantees sampling a representative population from the bulk cells. To analyze thousands of single-cells with a variety of fluorescent intensities, a highly adaptable analysis program was developed for cell/sphere counting and size measurement. Using a Pluronic® F108 (poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol)) coating on polydimethylsiloxane (PDMS), a suspension culture environment was created to test a controversial hypothesis: whether larger or smaller cells are more stem-like defined by the capability to form single-cell-derived spheres. Different cell lines showed different correlations between sphere formation rate and initial cell size, suggesting heterogeneity in pathway regulation among breast cancer cell lines. More interestingly, by monitoring hundreds of spheres, we identified heterogeneity in sphere growth dynamics, indicating the cellular heterogeneity even within CSCs. These preliminary results highlight the power of unprecedented high-throughput and automation in CSC studies.

384 hanging drop arrays give excellent Z-factors and allow versatile formation of co-culture spheroids.

PubMed

Hsiao, Amy Y; Tung, Yi-Chung; Qu, Xianggui; Patel, Lalit R; Pienta, Kenneth J; Takayama, Shuichi

2012-05-01

We previously reported the development of a simple, user-friendly, and versatile 384 hanging drop array plate for 3D spheroid culture and the importance of utilizing 3D cellular models in anti-cancer drug sensitivity testing. The 384 hanging drop array plate allows for high-throughput capabilities and offers significant improvements over existing 3D spheroid culture methods. To allow for practical 3D cell-based high-throughput screening and enable broader use of the plate, we characterize the robustness of the 384 hanging drop array plate in terms of assay performance and demonstrate the versatility of the plate. We find that the 384 hanging drop array plate performance is robust in fluorescence- and colorimetric-based assays through Z-factor calculations. Finally, we demonstrate different plate capabilities and applications, including: spheroid transfer and retrieval for Janus spheroid formation, sequential addition of cells for concentric layer patterning of different cell types, and culture of a wide variety of cell types. Copyright © 2011 Wiley Periodicals, Inc.

384 Hanging Drop Arrays Give Excellent Z-factors and Allow Versatile Formation of Co-culture Spheroids

PubMed Central

Hsiao, Amy Y.; Tung, Yi-Chung; Qu, Xianggui; Patel, Lalit R.; Pienta, Kenneth J.; Takayama, Shuichi

2012-01-01

We previously reported the development of a simple, user-friendly, and versatile 384 hanging drop array plate for 3D spheroid culture and the importance of utilizing 3D cellular models in anti-cancer drug sensitivity testing. The 384 hanging drop array plate allows for high-throughput capabilities and offers significant improvements over existing 3D spheroid culture methods. To allow for practical 3D cell-based high-throughput screening and enable broader use of the plate, we characterize the robustness of the 384 hanging drop array plate in terms of assay performance and demonstrate the versatility of the plate. We find that the 384 hanging drop array plate performance is robust in fluorescence- and colorimetric-based assays through z-factor calculations. Finally, we demonstrate different plate capabilities and applications, including: spheroid transfer and retrieval for Janus spheroid formation, sequential addition of cells for concentric layer patterning of different cell types, and culture of a wide variety of cell types. PMID:22161651

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

PubMed Central

Gaber, Rok; Majerle, Andreja; Jerala, Roman; Benčina, Mojca

2013-01-01

To effectively fight against the human immunodeficiency virus infection/acquired immunodeficiency syndrome (HIV/AIDS) epidemic, ongoing development of novel HIV protease inhibitors is required. Inexpensive high-throughput screening assays are needed to quickly scan large sets of chemicals for potential inhibitors. We have developed a Förster resonance energy transfer (FRET)-based, HIV protease-sensitive sensor using a combination of a fluorescent protein pair, namely mCerulean and mCitrine. Through extensive in vitro characterization, we show that the FRET-HIV sensor can be used in HIV protease screening assays. Furthermore, we have used the FRET-HIV sensor for intracellular quantitative detection of HIV protease activity in living cells, which more closely resembles an actual viral infection than an in vitro assay. We have developed a high-throughput method that employs a ratiometric flow cytometry for analyzing large populations of cells that express the FRET-HIV sensor. The method enables FRET measurement of single cells with high sensitivity and speed and should be used when subpopulation-specific intracellular activity of HIV protease needs to be estimated. In addition, we have used a confocal microscopy sensitized emission FRET technique to evaluate the usefulness of the FRET-HIV sensor for spatiotemporal detection of intracellular HIV protease activity. PMID:24287545

Materials Databases Infrastructure Constructed by First Principles Calculations: A Review

DOE PAGES

Lin, Lianshan

2015-10-13

The First Principles calculations, especially the calculation based on High-Throughput Density Functional Theory, have been widely accepted as the major tools in atom scale materials design. The emerging super computers, along with the powerful First Principles calculations, have accumulated hundreds of thousands of crystal and compound records. The exponential growing of computational materials information urges the development of the materials databases, which not only provide unlimited storage for the daily increasing data, but still keep the efficiency in data storage, management, query, presentation and manipulation. This review covers the most cutting edge materials databases in materials design, and their hotmore » applications such as in fuel cells. By comparing the advantages and drawbacks of these high-throughput First Principles materials databases, the optimized computational framework can be identified to fit the needs of fuel cell applications. The further development of high-throughput DFT materials database, which in essence accelerates the materials innovation, is discussed in the summary as well.« less

Probing cellular heterogeneity in cytokine-secreting immune cells using droplet-based microfluidics.

PubMed

Chokkalingam, Venkatachalam; Tel, Jurjen; Wimmers, Florian; Liu, Xin; Semenov, Sergey; Thiele, Julian; Figdor, Carl G; Huck, Wilhelm T S

2013-12-21

Here, we present a platform to detect cytokine (IL-2, IFN-γ, TNF-α) secretion of single, activated T-cells in droplets over time. We use a novel droplet-based microfluidic approach to encapsulate cells in monodisperse agarose droplets together with functionalized cytokine-capture beads for subsequent binding and detection of secreted cytokines from single cells. This method allows high-throughput detection of cellular heterogeneity and maps subsets within cell populations with specific functions.

A biosensor generated via high throughput screening quantifies cell edge Src dynamics

PubMed Central

Gulyani, Akash; Vitriol, Eric; Allen, Richard; Wu, Jianrong; Gremyachinskiy, Dmitriy; Lewis, Steven; Dewar, Brian; Graves, Lee M.; Kay, Brian K.; Kuhlman, Brian; Elston, Tim; Hahn, Klaus M.

2011-01-01

Fluorescent biosensors for living cells currently require laborious optimization and a unique design for each target. They are limited by the availability of naturally occurring ligands with appropriate target specificity. Here we describe a biosensor based on an engineered fibronectin monobody scaffold that can be tailored to bind different targets via high throughput screening. This Src family kinase (SFK) biosensor was made by derivatizing a monobody specific for activated SFK with a bright dye whose fluorescence increases upon target binding. We identified sites for dye attachment and alterations to eliminate vesiculation in living cells, providing a generalizable scaffold for biosensor production. This approach minimizes cell perturbation because it senses endogenous, unmodified target, and because sensitivity is enhanced by direct dye excitation. Automated correlation of cell velocities and SFK activity revealed that SFK are activated specifically during protrusion. Activity correlates with velocity, and peaks 1–2 microns from the leading edge. PMID:21666688

A holistic high-throughput screening framework for biofuel feedstock assessment that characterises variations in soluble sugars and cell wall composition in Sorghum bicolor

PubMed Central

2013-01-01

Background A major hindrance to the development of high yielding biofuel feedstocks is the ability to rapidly assess large populations for fermentable sugar yields. Whilst recent advances have outlined methods for the rapid assessment of biomass saccharification efficiency, none take into account the total biomass, or the soluble sugar fraction of the plant. Here we present a holistic high-throughput methodology for assessing sweet Sorghum bicolor feedstocks at 10 days post-anthesis for total fermentable sugar yields including stalk biomass, soluble sugar concentrations, and cell wall saccharification efficiency. Results A mathematical method for assessing whole S. bicolor stalks using the fourth internode from the base of the plant proved to be an effective high-throughput strategy for assessing stalk biomass, soluble sugar concentrations, and cell wall composition and allowed calculation of total stalk fermentable sugars. A high-throughput method for measuring soluble sucrose, glucose, and fructose using partial least squares (PLS) modelling of juice Fourier transform infrared (FTIR) spectra was developed. The PLS prediction was shown to be highly accurate with each sugar attaining a coefficient of determination (R 2 ) of 0.99 with a root mean squared error of prediction (RMSEP) of 11.93, 5.52, and 3.23 mM for sucrose, glucose, and fructose, respectively, which constitutes an error of <4% in each case. The sugar PLS model correlated well with gas chromatography–mass spectrometry (GC-MS) and brix measures. Similarly, a high-throughput method for predicting enzymatic cell wall digestibility using PLS modelling of FTIR spectra obtained from S. bicolor bagasse was developed. The PLS prediction was shown to be accurate with an R 2 of 0.94 and RMSEP of 0.64 μg.mgDW-1.h-1. Conclusions This methodology has been demonstrated as an efficient and effective way to screen large biofuel feedstock populations for biomass, soluble sugar concentrations, and cell wall digestibility simultaneously allowing a total fermentable yield calculation. It unifies and simplifies previous screening methodologies to produce a holistic assessment of biofuel feedstock potential. PMID:24365407

High-throughput full-length single-cell mRNA-seq of rare cells.

PubMed

Ooi, Chin Chun; Mantalas, Gary L; Koh, Winston; Neff, Norma F; Fuchigami, Teruaki; Wong, Dawson J; Wilson, Robert J; Park, Seung-Min; Gambhir, Sanjiv S; Quake, Stephen R; Wang, Shan X

2017-01-01

Single-cell characterization techniques, such as mRNA-seq, have been applied to a diverse range of applications in cancer biology, yielding great insight into mechanisms leading to therapy resistance and tumor clonality. While single-cell techniques can yield a wealth of information, a common bottleneck is the lack of throughput, with many current processing methods being limited to the analysis of small volumes of single cell suspensions with cell densities on the order of 107 per mL. In this work, we present a high-throughput full-length mRNA-seq protocol incorporating a magnetic sifter and magnetic nanoparticle-antibody conjugates for rare cell enrichment, and Smart-seq2 chemistry for sequencing. We evaluate the efficiency and quality of this protocol with a simulated circulating tumor cell system, whereby non-small-cell lung cancer cell lines (NCI-H1650 and NCI-H1975) are spiked into whole blood, before being enriched for single-cell mRNA-seq by EpCAM-functionalized magnetic nanoparticles and the magnetic sifter. We obtain high efficiency (> 90%) capture and release of these simulated rare cells via the magnetic sifter, with reproducible transcriptome data. In addition, while mRNA-seq data is typically only used for gene expression analysis of transcriptomic data, we demonstrate the use of full-length mRNA-seq chemistries like Smart-seq2 to facilitate variant analysis of expressed genes. This enables the use of mRNA-seq data for differentiating cells in a heterogeneous population by both their phenotypic and variant profile. In a simulated heterogeneous mixture of circulating tumor cells in whole blood, we utilize this high-throughput protocol to differentiate these heterogeneous cells by both their phenotype (lung cancer versus white blood cells), and mutational profile (H1650 versus H1975 cells), in a single sequencing run. This high-throughput method can help facilitate single-cell analysis of rare cell populations, such as circulating tumor or endothelial cells, with demonstrably high-quality transcriptomic data.

Photon-Counting H33D Detector for Biological Fluorescence Imaging

PubMed Central

Michalet, X.; Siegmund, O.H.W.; Vallerga, J.V.; Jelinsky, P.; Millaud, J.E.; Weiss, S.

2010-01-01

We have developed a photon-counting High-temporal and High-spatial resolution, High-throughput 3-Dimensional detector (H33D) for biological imaging of fluorescent samples. The design is based on a 25 mm diameter S20 photocathode followed by a 3-microchannel plate stack, and a cross delay line anode. We describe the bench performance of the H33D detector, as well as preliminary imaging results obtained with fluorescent beads, quantum dots and live cells and discuss applications of future generation detectors for single-molecule imaging and high-throughput study of biomolecular interactions. PMID:20151021

Hyperchromatic laser scanning cytometry

NASA Astrophysics Data System (ADS)

Tárnok, Attila; Mittag, Anja

2007-02-01

In the emerging fields of high-content and high-throughput single cell analysis for Systems Biology and Cytomics multi- and polychromatic analysis of biological specimens has become increasingly important. Combining different technologies and staining methods polychromatic analysis (i.e. using 8 or more fluorescent colors at a time) can be pushed forward to measure anything stainable in a cell, an approach termed hyperchromatic cytometry. For cytometric cell analysis microscope based Slide Based Cytometry (SBC) technologies are ideal as, unlike flow cytometry, they are non-consumptive, i.e. the analyzed sample is fixed on the slide. Based on the feature of relocation identical cells can be subsequently reanalyzed. In this manner data on the single cell level after manipulation steps can be collected. In this overview various components for hyperchromatic cytometry are demonstrated for a SBC instrument, the Laser Scanning Cytometer (Compucyte Corp., Cambridge, MA): 1) polychromatic cytometry, 2) iterative restaining (using the same fluorochrome for restaining and subsequent reanalysis), 3) differential photobleaching (differentiating fluorochromes by their different photostability), 4) photoactivation (activating fluorescent nanoparticles or photocaged dyes), and 5) photodestruction (destruction of FRET dyes). With the intelligent combination of several of these techniques hyperchromatic cytometry allows to quantify and analyze virtually all components of relevance on the identical cell. The combination of high-throughput and high-content SBC analysis with high-resolution confocal imaging allows clear verification of phenotypically distinct subpopulations of cells with structural information. The information gained per specimen is only limited by the number of available antibodies and by sterical hindrance.

High-throughput screening in niche-based assay identifies compounds to target preleukemic stem cells

PubMed Central

Gerby, Bastien; Veiga, Diogo F.T.; Krosl, Jana; Nourreddine, Sami; Ouellette, Julianne; Haman, André; Lavoie, Geneviève; Fares, Iman; Tremblay, Mathieu; Litalien, Véronique; Ottoni, Elizabeth; Geoffrion, Dominique; Maddox, Paul S.; Chagraoui, Jalila; Hébert, Josée; Sauvageau, Guy; Kwok, Benjamin H.; Roux, Philippe P.

2016-01-01

Current chemotherapies for T cell acute lymphoblastic leukemia (T-ALL) efficiently reduce tumor mass. Nonetheless, disease relapse attributed to survival of preleukemic stem cells (pre-LSCs) is associated with poor prognosis. Herein, we provide direct evidence that pre-LSCs are much less chemosensitive to existing chemotherapy drugs than leukemic blasts because of a distinctive lower proliferative state. Improving therapies for T-ALL requires the development of strategies to target pre-LSCs that are absolutely dependent on their microenvironment. Therefore, we designed a robust protocol for high-throughput screening of compounds that target primary pre-LSCs maintained in a niche-like environment, on stromal cells that were engineered for optimal NOTCH1 activation. The multiparametric readout takes into account the intrinsic complexity of primary cells in order to specifically monitor pre-LSCs, which were induced here by the SCL/TAL1 and LMO1 oncogenes. We screened a targeted library of compounds and determined that the estrogen derivative 2-methoxyestradiol (2-ME2) disrupted both cell-autonomous and non–cell-autonomous pathways. Specifically, 2-ME2 abrogated pre-LSC viability and self-renewal activity in vivo by inhibiting translation of MYC, a downstream effector of NOTCH1, and preventing SCL/TAL1 activity. In contrast, normal hematopoietic stem/progenitor cells remained functional. These results illustrate how recapitulating tissue-like properties of primary cells in high-throughput screening is a promising avenue for innovation in cancer chemotherapy. PMID:27797342

High-Throughput Accurate Single-Cell Screening of Euglena gracilis with Fluorescence-Assisted Optofluidic Time-Stretch Microscopy.

PubMed

Guo, Baoshan; Lei, Cheng; Ito, Takuro; Jiang, Yiyue; Ozeki, Yasuyuki; Goda, Keisuke

2016-01-01

The development of reliable, sustainable, and economical sources of alternative fuels is an important, but challenging goal for the world. As an alternative to liquid fossil fuels, algal biofuel is expected to play a key role in alleviating global warming since algae absorb atmospheric CO2 via photosynthesis. Among various algae for fuel production, Euglena gracilis is an attractive microalgal species as it is known to produce wax ester (good for biodiesel and aviation fuel) within lipid droplets. To date, while there exist many techniques for inducing microalgal cells to produce and accumulate lipid with high efficiency, few analytical methods are available for characterizing a population of such lipid-accumulated microalgae including E. gracilis with high throughout, high accuracy, and single-cell resolution simultaneously. Here we demonstrate high-throughput, high-accuracy, single-cell screening of E. gracilis with fluorescence-assisted optofluidic time-stretch microscopy-a method that combines the strengths of microfluidic cell focusing, optical time-stretch microscopy, and fluorescence detection used in conventional flow cytometry. Specifically, our fluorescence-assisted optofluidic time-stretch microscope consists of an optical time-stretch microscope and a fluorescence analyzer on top of a hydrodynamically focusing microfluidic device and can detect fluorescence from every E. gracilis cell in a population and simultaneously obtain its image with a high throughput of 10,000 cells/s. With the multi-dimensional information acquired by the system, we classify nitrogen-sufficient (ordinary) and nitrogen-deficient (lipid-accumulated) E. gracilis cells with a low false positive rate of 1.0%. This method holds promise for evaluating cultivation techniques and selective breeding for microalgae-based biofuel production.

Laser-Induced Fluorescence Detection in High-Throughput Screening of Heterogeneous Catalysts and Single Cells Analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Su, Hui

2001-01-01

Laser-induced fluorescence detection is one of the most sensitive detection techniques and it has found enormous applications in various areas. The purpose of this research was to develop detection approaches based on laser-induced fluorescence detection in two different areas, heterogeneous catalysts screening and single cell study. First, the author introduced laser-induced imaging (LIFI) as a high-throughput screening technique for heterogeneous catalysts to explore the use of this high-throughput screening technique in discovery and study of various heterogeneous catalyst systems. This scheme is based on the fact that the creation or the destruction of chemical bonds alters the fluorescence properties ofmore » suitably designed molecules. By irradiating the region immediately above the catalytic surface with a laser, the fluorescence intensity of a selected product or reactant can be imaged by a charge-coupled device (CCD) camera to follow the catalytic activity as a function of time and space. By screening the catalytic activity of vanadium pentoxide catalysts in oxidation of naphthalene, they demonstrated LIFI has good detection performance and the spatial and temporal resolution needed for high-throughput screening of heterogeneous catalysts. The sample packing density can reach up to 250 x 250 subunits/cm 2 for 40-μm wells. This experimental set-up also can screen solid catalysts via near infrared thermography detection. In the second part of this dissertation, the author used laser-induced native fluorescence coupled with capillary electrophoresis (LINF-CE) and microscope imaging to study the single cell degranulation. On the basis of good temporal correlation with events observed through an optical microscope, they have identified individual peaks in the fluorescence electropherograms as serotonin released from the granular core on contact with the surrounding fluid.« less

HiCTMap: Detection and analysis of chromosome territory structure and position by high-throughput imaging.

PubMed

Jowhar, Ziad; Gudla, Prabhakar R; Shachar, Sigal; Wangsa, Darawalee; Russ, Jill L; Pegoraro, Gianluca; Ried, Thomas; Raznahan, Armin; Misteli, Tom

2018-06-01

The spatial organization of chromosomes in the nuclear space is an extensively studied field that relies on measurements of structural features and 3D positions of chromosomes with high precision and robustness. However, no tools are currently available to image and analyze chromosome territories in a high-throughput format. Here, we have developed High-throughput Chromosome Territory Mapping (HiCTMap), a method for the robust and rapid analysis of 2D and 3D chromosome territory positioning in mammalian cells. HiCTMap is a high-throughput imaging-based chromosome detection method which enables routine analysis of chromosome structure and nuclear position. Using an optimized FISH staining protocol in a 384-well plate format in conjunction with a bespoke automated image analysis workflow, HiCTMap faithfully detects chromosome territories and their position in 2D and 3D in a large population of cells per experimental condition. We apply this novel technique to visualize chromosomes 18, X, and Y in male and female primary human skin fibroblasts, and show accurate detection of the correct number of chromosomes in the respective genotypes. Given the ability to visualize and quantitatively analyze large numbers of nuclei, we use HiCTMap to measure chromosome territory area and volume with high precision and determine the radial position of chromosome territories using either centroid or equidistant-shell analysis. The HiCTMap protocol is also compatible with RNA FISH as demonstrated by simultaneous labeling of X chromosomes and Xist RNA in female cells. We suggest HiCTMap will be a useful tool for routine precision mapping of chromosome territories in a wide range of cell types and tissues. Published by Elsevier Inc.

Metabolic enzyme microarray coupled with miniaturized cell-culture array technology for high-throughput toxicity screening.

PubMed

Lee, Moo-Yeal; Dordick, Jonathan S; Clark, Douglas S

2010-01-01

Due to poor drug candidate safety profiles that are often identified late in the drug development process, the clinical progression of new chemical entities to pharmaceuticals remains hindered, thus resulting in the high cost of drug discovery. To accelerate the identification of safer drug candidates and improve the clinical progression of drug candidates to pharmaceuticals, it is important to develop high-throughput tools that can provide early-stage predictive toxicology data. In particular, in vitro cell-based systems that can accurately mimic the human in vivo response and predict the impact of drug candidates on human toxicology are needed to accelerate the assessment of drug candidate toxicity and human metabolism earlier in the drug development process. The in vitro techniques that provide a high degree of human toxicity prediction will be perhaps more important in cosmetic and chemical industries in Europe, as animal toxicity testing is being phased out entirely in the immediate future.We have developed a metabolic enzyme microarray (the Metabolizing Enzyme Toxicology Assay Chip, or MetaChip) and a miniaturized three-dimensional (3D) cell-culture array (the Data Analysis Toxicology Assay Chip, or DataChip) for high-throughput toxicity screening of target compounds and their metabolic enzyme-generated products. The human or rat MetaChip contains an array of encapsulated metabolic enzymes that is designed to emulate the metabolic reactions in the human or rat liver. The human or rat DataChip contains an array of 3D human or rat cells encapsulated in alginate gels for cell-based toxicity screening. By combining the DataChip with the complementary MetaChip, in vitro toxicity results are obtained that correlate well with in vivo rat data.

Jeffamine Derivatized TentaGel Beads and PDMS Microbead Cassettes for Ultra-high Throughput in situ Releasable Solution-Phase Cell-based Screening of OBOC Combinatorial Small Molecule Libraries

PubMed Central

Townsend, Jared B.; Shaheen, Farzana; Liu, Ruiwu; Lam, Kit S.

2011-01-01

A method to efficiently immobilize and partition large quantities of microbeads in an array format in microfabricated polydimethylsiloxane (PDMS) cassette for high-throughput in situ releasable solution-phase cell-based screening of one-bead-one-compound (OBOC) combinatorial libraries is described. Commercially available Jeffamine triamine T-403 (∼440 Da) was derivatized such that two of its amino groups were protected by Fmoc and the remaining amino group capped with succinic anhydride to generate a carboxyl group. This resulting tri-functional hydrophilic polymer was then sequentially coupled two times to the outer layer of topologically segregated bilayer TentaGel (TG) beads with solid phase peptide synthesis chemistry, resulting in beads with increased loading capacity, hydrophilicity and porosity at the outer layer. We have found that such bead configuration can facilitate ultra high-throughput in situ releasable solution-phase screening of OBOC libraries. An encoded releasable OBOC small molecule library was constructed on Jeffamine derivatized TG beads with library compounds tethered to the outer layer via a disulfide linker and coding tags in the interior of the beads. Compound-beads could be efficiently loaded (5-10 minutes) into a 5 cm diameter Petri dish containing a 10,000-well PDMS microbead cassette, such that over 90% of the microwells were each filled with only one compound-bead. Jurkat T-lymphoid cancer cells suspended in Matrigel® were then layered over the microbead cassette to immobilize the compound-beads. After 24 hours of incubation at 37°C, dithiothreitol was added to trigger the release of library compounds. Forty-eight hours later, MTT reporter assay was used to identify regions of reduced cell viability surrounding each positive bead. From a total of about 20,000 beads screened, 3 positive beads were detected and physically isolated for decoding. A strong consensus motif was identified for these three positive compounds. These compounds were re-synthesized and found to be cytotoxic (IC50 50-150 μM) against two T-lymphoma cell lines and less so against the MDA-MB 231 breast cancer cell line. This novel ultra high-throughput OBOC releasable method can potentially be adapted to many existing 96- or 384-well solution-phase cell-based or biochemical assays. PMID:20593859

High-throughput, low-loss, low-cost, and label-free cell separation using electrophysiology-activated cell enrichment.

PubMed

Faraghat, Shabnam A; Hoettges, Kai F; Steinbach, Max K; van der Veen, Daan R; Brackenbury, William J; Henslee, Erin A; Labeed, Fatima H; Hughes, Michael P

2017-05-02

Currently, cell separation occurs almost exclusively by density gradient methods and by fluorescence- and magnetic-activated cell sorting (FACS/MACS). These variously suffer from lack of specificity, high cell loss, use of labels, and high capital/operating cost. We present a dielectrophoresis (DEP)-based cell-separation method, using 3D electrodes on a low-cost disposable chip; one cell type is allowed to pass through the chip whereas the other is retained and subsequently recovered. The method advances usability and throughput of DEP separation by orders of magnitude in throughput, efficiency, purity, recovery (cells arriving in the correct output fraction), cell losses (those which are unaccounted for at the end of the separation), and cost. The system was evaluated using three example separations: live and dead yeast; human cancer cells/red blood cells; and rodent fibroblasts/red blood cells. A single-pass protocol can enrich cells with cell recovery of up to 91.3% at over 300,000 cells per second with >3% cell loss. A two-pass protocol can process 300,000,000 cells in under 30 min, with cell recovery of up to 96.4% and cell losses below 5%, an effective processing rate >160,000 cells per second. A three-step protocol is shown to be effective for removal of 99.1% of RBCs spiked with 1% cancer cells while maintaining a processing rate of ∼170,000 cells per second. Furthermore, the self-contained and low-cost nature of the separator device means that it has potential application in low-contamination applications such as cell therapies, where good manufacturing practice compatibility is of paramount importance.

3D pulsed laser-triggered high-speed microfluidic fluorescence-activated cell sorter

PubMed Central

Chen, Yue; Wu, Ting-Hsiang; Kung, Yu-Chun; Teitell, Michael A.; Chiou, Pei-Yu

2014-01-01

We report a 3D microfluidic pulsed laser-triggered fluorescence-activated cell sorter capable of sorting at a throughput of 23,000 cells sec−1 with 90% purity in high-purity mode and at a throughput of 45,000 cells sec−1 with 45% purity in enrichment mode in one stage and in a single channel. This performance is realized by exciting laser-induced cavitation bubbles in a 3D PDMS microfluidic channel to generate high-speed liquid jets that deflect detected fluorescent cells and particles focused by 3D sheath flows. The ultrafast switching mechanism (20 μsec complete on-off cycle), small liquid jet perturbation volume, and three-dimensional sheath flow focusing for accurate timing control of fast (1.5 m sec−1) passing cells and particles are three critical factors enabling high-purity sorting at high-throughput in this sorter. PMID:23844418

Engineering customized TALE nucleases (TALENs) and TALE transcription factors by fast ligation-based automatable solid-phase high-throughput (FLASH) assembly.

PubMed

Reyon, Deepak; Maeder, Morgan L; Khayter, Cyd; Tsai, Shengdar Q; Foley, Jonathan E; Sander, Jeffry D; Joung, J Keith

2013-07-01

Customized DNA-binding domains made using transcription activator-like effector (TALE) repeats are rapidly growing in importance as widely applicable research tools. TALE nucleases (TALENs), composed of an engineered array of TALE repeats fused to the FokI nuclease domain, have been used successfully for directed genome editing in various organisms and cell types. TALE transcription factors (TALE-TFs), consisting of engineered TALE repeat arrays linked to a transcriptional regulatory domain, have been used to up- or downregulate expression of endogenous genes in human cells and plants. This unit describes a detailed protocol for the recently described fast ligation-based automatable solid-phase high-throughput (FLASH) assembly method. FLASH enables automated high-throughput construction of engineered TALE repeats using an automated liquid handling robot or manually using a multichannel pipet. Using the automated approach, a single researcher can construct up to 96 DNA fragments encoding TALE repeat arrays of various lengths in a single day, and then clone these to construct sequence-verified TALEN or TALE-TF expression plasmids in a week or less. Plasmids required for FLASH are available by request from the Joung lab (http://eGenome.org). This unit also describes improvements to the Zinc Finger and TALE Targeter (ZiFiT Targeter) web server (http://ZiFiT.partners.org) that facilitate the design and construction of FLASH TALE repeat arrays in high throughput. © 2013 by John Wiley & Sons, Inc.

Engineering Customized TALE Nucleases (TALENs) and TALE Transcription Factors by Fast Ligation-based Automatable Solid-phase High-throughput (FLASH) Assembly

PubMed Central

Reyon, Deepak; Maeder, Morgan L.; Khayter, Cyd; Tsai, Shengdar Q.; Foley, Jonathan E.; Sander, Jeffry D.; Joung, J. Keith

2013-01-01

Customized DNA-binding domains made using Transcription Activator-Like Effector (TALE) repeats are rapidly growing in importance as widely applicable research tools. TALE nucleases (TALENs), composed of an engineered array of TALE repeats fused to the FokI nuclease domain, have been used successfully for directed genome editing in multiple different organisms and cell types. TALE transcription factors (TALE-TFs), consisting of engineered TALE repeat arrays linked to a transcriptional regulatory domain, have been used to up- or down-regulate expression of endogenous genes in human cells and plants. Here we describe a detailed protocol for practicing the recently described Fast Ligation-based Automatable Solid-phase High-throughput (FLASH) assembly method. FLASH enables automated high-throughput construction of engineered TALE repeats using an automated liquid handling robot or manually using a multi-channel pipet. With the automated version of FLASH, a single researcher can construct up to 96 DNA fragments encoding various length TALE repeat arrays in one day and then clone these to construct sequence-verified TALEN or TALE-TF expression plasmids in one week or less. Plas-mids required to practice FLASH are available by request from the Joung Lab (http://www.jounglab.org/). We also describe here improvements to the Zinc Finger and TALE Targeter (ZiFiT Targeter) webserver (http://ZiFiTBeta.partners.org) that facilitate the design and construction of FLASH TALE repeat arrays in high-throughput. PMID:23821439

Arrays of High-Aspect Ratio Microchannels for High-Throughput Isolation of Circulating Tumor Cells (CTCs).

PubMed

Hupert, Mateusz L; Jackson, Joshua M; Wang, Hong; Witek, Małgorzata A; Kamande, Joyce; Milowsky, Matthew I; Whang, Young E; Soper, Steven A

2014-10-01

Microsystem-based technologies are providing new opportunities in the area of in vitro diagnostics due to their ability to provide process automation enabling point-of-care operation. As an example, microsystems used for the isolation and analysis of circulating tumor cells (CTCs) from complex, heterogeneous samples in an automated fashion with improved recoveries and selectivity are providing new opportunities for this important biomarker. Unfortunately, many of the existing microfluidic systems lack the throughput capabilities and/or are too expensive to manufacture to warrant their widespread use in clinical testing scenarios. Here, we describe a disposable, all-polymer, microfluidic system for the high-throughput (HT) isolation of CTCs directly from whole blood inputs. The device employs an array of high aspect ratio (HAR), parallel, sinusoidal microchannels (25 µm × 150 µm; W × D; AR = 6.0) with walls covalently decorated with anti-EpCAM antibodies to provide affinity-based isolation of CTCs. Channel width, which is similar to an average CTC diameter (12-25 µm), plays a critical role in maximizing the probability of cell/wall interactions and allows for achieving high CTC recovery. The extended channel depth allows for increased throughput at the optimized flow velocity (2 mm/s in a microchannel); maximizes cell recovery, and prevents clogging of the microfluidic channels during blood processing. Fluidic addressing of the microchannel array with a minimal device footprint is provided by large cross-sectional area feed and exit channels poised orthogonal to the network of the sinusoidal capillary channels (so-called Z-geometry). Computational modeling was used to confirm uniform addressing of the channels in the isolation bed. Devices with various numbers of parallel microchannels ranging from 50 to 320 have been successfully constructed. Cyclic olefin copolymer (COC) was chosen as the substrate material due to its superior properties during UV-activation of the HAR microchannels surfaces prior to antibody attachment. Operation of the HT-CTC device has been validated by isolation of CTCs directly from blood secured from patients with metastatic prostate cancer. High CTC sample purities (low number of contaminating white blood cells, WBCs) allowed for direct lysis and molecular profiling of isolated CTCs.

A web platform for the network analysis of high-throughput data in melanoma and its use to investigate mechanisms of resistance to anti-PD1 immunotherapy.

PubMed

Dreyer, Florian S; Cantone, Martina; Eberhardt, Martin; Jaitly, Tanushree; Walter, Lisa; Wittmann, Jürgen; Gupta, Shailendra K; Khan, Faiz M; Wolkenhauer, Olaf; Pützer, Brigitte M; Jäck, Hans-Martin; Heinzerling, Lucie; Vera, Julio

2018-06-01

Cellular phenotypes are established and controlled by complex and precisely orchestrated molecular networks. In cancer, mutations and dysregulations of multiple molecular factors perturb the regulation of these networks and lead to malignant transformation. High-throughput technologies are a valuable source of information to establish the complex molecular relationships behind the emergence of malignancy, but full exploitation of this massive amount of data requires bioinformatics tools that rely on network-based analyses. In this report we present the Virtual Melanoma Cell, an online tool developed to facilitate the mining and interpretation of high-throughput data on melanoma by biomedical researches. The platform is based on a comprehensive, manually generated and expert-validated regulatory map composed of signaling pathways important in malignant melanoma. The Virtual Melanoma Cell is a tool designed to accept, visualize and analyze user-generated datasets. It is available at: https://www.vcells.net/melanoma. To illustrate the utilization of the web platform and the regulatory map, we have analyzed a large publicly available dataset accounting for anti-PD1 immunotherapy treatment of malignant melanoma patients. Copyright © 2018 Elsevier B.V. All rights reserved.

Identification of Isoxsuprine Hydrochloride as a Neuroprotectant in Ischemic Stroke through Cell-Based High-Throughput Screening

PubMed Central

Hill, Jeff W.; Thompson, Jeffrey F.; Carter, Mark B.; Edwards, Bruce S.; Sklar, Larry A.; Rosenberg, Gary A.

2014-01-01

Stroke is a leading cause of death and disability and treatment options are limited. A promising approach to accelerate the development of new therapeutics is the use of high-throughput screening of chemical libraries. Using a cell-based high-throughput oxygen-glucose deprivation (OGD) model, we evaluated 1,200 small molecules for repurposed application in stroke therapy. Isoxsuprine hydrochloride was identified as a potent neuroprotective compound in primary neurons exposed to OGD. Isoxsuprine, a β2-adrenergic agonist and NR2B subtype-selective N-methyl-D-aspartate (NMDA) receptor antagonist, demonstrated no loss of efficacy when administered up to an hour after reoxygenation in an in vitro stroke model. In an animal model of transient focal ischemia, isoxsuprine significantly reduced infarct volume compared to vehicle (137±18 mm3 versus 279±25 mm3, p<0.001). Isoxsuprine, a peripheral vasodilator, was FDA approved for the treatment of cerebrovascular insufficiency and peripheral vascular disease. Our demonstration of the significant and novel neuroprotective action of isoxsuprine hydrochloride in an in vivo stroke model and its history of human use suggest that isoxsuprine may be an ideal candidate for further investigation as a potential stroke therapeutic. PMID:24804769

Picking Cell Lines for High-Throughput Transcriptomic Toxicity Screening (SOT)

EPA Science Inventory

High throughput, whole genome transcriptomic profiling is a promising approach to comprehensively evaluate chemicals for potential biological effects. To be useful for in vitro toxicity screening, gene expression must be quantified in a set of representative cell types that captu...

A High-Throughput Cell-Based Screen Identified a 2-[(E)-2-Phenylvinyl]-8-Quinolinol Core Structure That Activates p53

PubMed Central

Bechill, John; Zhong, Rong; Zhang, Chen; Solomaha, Elena

2016-01-01

p53 function is frequently inhibited in cancer either through mutations or by increased degradation via MDM2 and/or E6AP E3-ubiquitin ligases. Most agents that restore p53 expression act by binding MDM2 or E6AP to prevent p53 degradation. However, fewer compounds directly bind to and activate p53. Here, we identified compounds that shared a core structure that bound p53, caused nuclear localization of p53 and caused cell death. To identify these compounds, we developed a novel cell-based screen to redirect p53 degradation to the Skip-Cullin-F-box (SCF) ubiquitin ligase complex in cells expressing high levels of p53. In a multiplexed assay, we coupled p53 targeted degradation with Rb1 targeted degradation in order to identify compounds that prevented p53 degradation while not inhibiting degradation through the SCF complex or other proteolytic machinery. High-throughput screening identified several leads that shared a common 2-[(E)-2-phenylvinyl]-8-quinolinol core structure that stabilized p53. Surface plasmon resonance analysis indicated that these compounds bound p53 with a KD of 200 ± 52 nM. Furthermore, these compounds increased p53 nuclear localization and transcription of the p53 target genes PUMA, BAX, p21 and FAS in cancer cells. Although p53-null cells had a 2.5±0.5-fold greater viability compared to p53 wild type cells after treatment with core compounds, loss of p53 did not completely rescue cell viability suggesting that compounds may target both p53-dependent and p53-independent pathways to inhibit cell proliferation. Thus, we present a novel, cell-based high-throughput screen to identify a 2-[(E)-2-phenylvinyl]-8-quinolinol core structure that bound to p53 and increased p53 activity in cancer cells. These compounds may serve as anti-neoplastic agents in part by targeting p53 as well as other potential pathways. PMID:27124407

High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

PubMed Central

Chiaraviglio, Lucius; Kang, Yoon-Suk; Kirby, James E.

2016-01-01

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require several additional experimental steps prior to readout, such as cell lysis, colony forming unit determination, or reagent addition. When performing thousands of assays, for example, during high-throughput screening, the downstream effort required for these types of assays is considerable. Therefore, to facilitate high-throughput antimicrobial discovery, we developed a real-time assay to simultaneously identify inhibitors of intracellular bacterial growth and assess eukaryotic cell cytotoxicity. Specifically, real-time intracellular bacterial growth detection was enabled by marking bacterial screening strains with either a bacterial lux operon (1st generation assay) or fluorescent protein reporters (2nd generation, orthogonal assay). A non-toxic, cell membrane-impermeant, nucleic acid-binding dye was also added during initial infection of macrophages. These dyes are excluded from viable cells. However, non-viable host cells lose membrane integrity permitting entry and fluorescent labeling of nuclear DNA (deoxyribonucleic acid). Notably, DNA binding is associated with a large increase in fluorescent quantum yield that provides a solution-based readout of host cell death. We have used this combined assay to perform a high-throughput screen in microplate format, and to assess intracellular growth and cytotoxicity by microscopy. Notably, antimicrobials may demonstrate synergy in which the combined effect of two or more antimicrobials when applied together is greater than when applied separately. Testing for in vitro synergy against intracellular pathogens is normally a prodigious task as combinatorial permutations of antibiotics at different concentrations must be assessed. However, we found that our real-time assay combined with automated, digital dispensing technology permitted facile synergy testing. Using these approaches, we were able to systematically survey action of a large number of antimicrobials alone and in combination against the intracellular pathogen, Legionella pneumophila. PMID:27911388

Single cell kinase signaling assay using pinched flow coupled droplet microfluidics.

PubMed

Ramji, Ramesh; Wang, Ming; Bhagat, Ali Asgar S; Tan Shao Weng, Daniel; Thakor, Nitish V; Teck Lim, Chwee; Chen, Chia-Hung

2014-05-01

Droplet-based microfluidics has shown potential in high throughput single cell assays by encapsulating individual cells in water-in-oil emulsions. Ordering cells in a micro-channel is necessary to encapsulate individual cells into droplets further enhancing the assay efficiency. This is typically limited due to the difficulty of preparing high-density cell solutions and maintaining them without cell aggregation in long channels (>5 cm). In this study, we developed a short pinched flow channel (5 mm) to separate cell aggregates and to form a uniform cell distribution in a droplet-generating platform that encapsulated single cells with >55% encapsulation efficiency beating Poisson encapsulation statistics. Using this platform and commercially available Sox substrates (8-hydroxy-5-(N,N-dimethylsulfonamido)-2-methylquinoline), we have demonstrated a high throughput dynamic single cell signaling assay to measure the activity of receptor tyrosine kinases (RTKs) in lung cancer cells triggered by cell surface ligand binding. The phosphorylation of the substrates resulted in fluorescent emission, showing a sigmoidal increase over a 12 h period. The result exhibited a heterogeneous signaling rate in individual cells and showed various levels of drug resistance when treated with the tyrosine kinase inhibitor, gefitinib.

High throughput imaging cytometer with acoustic focussing.

PubMed

Zmijan, Robert; Jonnalagadda, Umesh S; Carugo, Dario; Kochi, Yu; Lemm, Elizabeth; Packham, Graham; Hill, Martyn; Glynne-Jones, Peter

2015-10-31

We demonstrate an imaging flow cytometer that uses acoustic levitation to assemble cells and other particles into a sheet structure. This technique enables a high resolution, low noise CMOS camera to capture images of thousands of cells with each frame. While ultrasonic focussing has previously been demonstrated for 1D cytometry systems, extending the technology to a planar, much higher throughput format and integrating imaging is non-trivial, and represents a significant jump forward in capability, leading to diagnostic possibilities not achievable with current systems. A galvo mirror is used to track the images of the moving cells permitting exposure times of 10 ms at frame rates of 50 fps with motion blur of only a few pixels. At 80 fps, we demonstrate a throughput of 208 000 beads per second. We investigate the factors affecting motion blur and throughput, and demonstrate the system with fluorescent beads, leukaemia cells and a chondrocyte cell line. Cells require more time to reach the acoustic focus than beads, resulting in lower throughputs; however a longer device would remove this constraint.

High-Throughput Cloning and Expression Library Creation for Functional Proteomics

PubMed Central

Festa, Fernanda; Steel, Jason; Bian, Xiaofang; Labaer, Joshua

2013-01-01

The study of protein function usually requires the use of a cloned version of the gene for protein expression and functional assays. This strategy is particular important when the information available regarding function is limited. The functional characterization of the thousands of newly identified proteins revealed by genomics requires faster methods than traditional single gene experiments, creating the need for fast, flexible and reliable cloning systems. These collections of open reading frame (ORF) clones can be coupled with high-throughput proteomics platforms, such as protein microarrays and cell-based assays, to answer biological questions. In this tutorial we provide the background for DNA cloning, discuss the major high-throughput cloning systems (Gateway® Technology, Flexi® Vector Systems, and Creator™ DNA Cloning System) and compare them side-by-side. We also report an example of high-throughput cloning study and its application in functional proteomics. This Tutorial is part of the International Proteomics Tutorial Programme (IPTP12). Details can be found at http://www.proteomicstutorials.org. PMID:23457047

High-throughput cloning and expression library creation for functional proteomics.

PubMed

Festa, Fernanda; Steel, Jason; Bian, Xiaofang; Labaer, Joshua

2013-05-01

The study of protein function usually requires the use of a cloned version of the gene for protein expression and functional assays. This strategy is particularly important when the information available regarding function is limited. The functional characterization of the thousands of newly identified proteins revealed by genomics requires faster methods than traditional single-gene experiments, creating the need for fast, flexible, and reliable cloning systems. These collections of ORF clones can be coupled with high-throughput proteomics platforms, such as protein microarrays and cell-based assays, to answer biological questions. In this tutorial, we provide the background for DNA cloning, discuss the major high-throughput cloning systems (Gateway® Technology, Flexi® Vector Systems, and Creator(TM) DNA Cloning System) and compare them side-by-side. We also report an example of high-throughput cloning study and its application in functional proteomics. This tutorial is part of the International Proteomics Tutorial Programme (IPTP12). © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Genome-scale deletion screening of human long non-coding RNAs using a paired-guide RNA CRISPR library

PubMed Central

Zhu, Shiyou; Li, Wei; Liu, Jingze; Chen, Chen-Hao; Liao, Qi; Xu, Ping; Xu, Han; Xiao, Tengfei; Cao, Zhongzheng; Peng, Jingyu; Yuan, Pengfei; Brown, Myles; Liu, Xiaole Shirley; Wei, Wensheng

2017-01-01

CRISPR/Cas9 screens have been widely adopted to analyse coding gene functions, but high throughput screening of non-coding elements using this method is more challenging, because indels caused by a single cut in non-coding regions are unlikely to produce a functional knockout. A high-throughput method to produce deletions of non-coding DNA is needed. Herein, we report a high throughput genomic deletion strategy to screen for functional long non-coding RNAs (lncRNAs) that is based on a lentiviral paired-guide RNA (pgRNA) library. Applying our screening method, we identified 51 lncRNAs that can positively or negatively regulate human cancer cell growth. We individually validated 9 lncRNAs using CRISPR/Cas9-mediated genomic deletion and functional rescue, CRISPR activation or inhibition, and gene expression profiling. Our high-throughput pgRNA genome deletion method should enable rapid identification of functional mammalian non-coding elements. PMID:27798563

Computational methods for evaluation of cell-based data assessment--Bioconductor.

PubMed

Le Meur, Nolwenn

2013-02-01

Recent advances in miniaturization and automation of technologies have enabled cell-based assay high-throughput screening, bringing along new challenges in data analysis. Automation, standardization, reproducibility have become requirements for qualitative research. The Bioconductor community has worked in that direction proposing several R packages to handle high-throughput data including flow cytometry (FCM) experiment. Altogether, these packages cover the main steps of a FCM analysis workflow, that is, data management, quality assessment, normalization, outlier detection, automated gating, cluster labeling, and feature extraction. Additionally, the open-source philosophy of R and Bioconductor, which offers room for new development, continuously drives research and improvement of theses analysis methods, especially in the field of clustering and data mining. This review presents the principal FCM packages currently available in R and Bioconductor, their advantages and their limits. Copyright © 2012 Elsevier Ltd. All rights reserved.

Combinatorial high-throughput optical screening of high performance Pd alloy cathode for hybrid Li-air battery.

PubMed

Jun, Young Jin; Park, Sung Hyeon; Woo, Seong Ihl

2014-12-08

Combinatorial high-throughput optical screening method was developed to find the optimum composition of highly active Pd-based catalysts at the cathode of the hybrid Li-air battery. Pd alone, which is one-third the cost of Pt, has difficulty in replacing Pt; therefore, the integration of other metals was investigated to improve its performance toward oxygen reduction reaction (ORR). Among the binary Pd-based catalysts, the composition of Pd-Ir derived catalysts had higher performance toward ORR compared to other Pd-based binary combinations. The composition at 88:12 at. % (Pd: Ir) showed the highest activity toward ORR at the cathode of the hybrid Li-air battery. The prepared Pd(88)Ir(12)/C catalyst showed a current density of -2.58 mA cm(-2) at 0.8 V (vs RHE), which was around 30% higher compared to that of Pd/C (-1.97 mA cm(-2)). When the prepared Pd(88)Ir(12)/C catalyst was applied to the hybrid Li-air battery, the polarization of the cell was reduced and the energy efficiency of the cell was about 30% higher than that of the cell with Pd/C.

Size-Based Separation of Particles and Cells Utilizing Viscoelastic Effects in Straight Microchannels.

PubMed

Liu, Chao; Xue, Chundong; Chen, Xiaodong; Shan, Lei; Tian, Yu; Hu, Guoqing

2015-06-16

Viscoelasticity-induced particle migration has recently received increasing attention due to its ability to obtain high-quality focusing over a wide range of flow rates. However, its application is limited to low throughput regime since the particles can defocus as flow rate increases. Using an engineered carrier medium with constant and low viscosity and strong elasticity, the sample flow rates are improved to be 1 order of magnitude higher than those in existing studies. Utilizing differential focusing of particles of different sizes, here, we present sheathless particle/cell separation in simple straight microchannels that possess excellent parallelizability for further throughput enhancement. The present method can be implemented over a wide range of particle/cell sizes and flow rates. We successfully separate small particles from larger particles, MCF-7 cells from red blood cells (RBCs), and Escherichia coli (E. coli) bacteria from RBCs in different straight microchannels. The proposed method could broaden the applications of viscoelastic microfluidic devices to particle/cell separation due to the enhanced sample throughput and simple channel design.

Unsupervised automated high throughput phenotyping of RNAi time-lapse movies.

PubMed

Failmezger, Henrik; Fröhlich, Holger; Tresch, Achim

2013-10-04

Gene perturbation experiments in combination with fluorescence time-lapse cell imaging are a powerful tool in reverse genetics. High content applications require tools for the automated processing of the large amounts of data. These tools include in general several image processing steps, the extraction of morphological descriptors, and the grouping of cells into phenotype classes according to their descriptors. This phenotyping can be applied in a supervised or an unsupervised manner. Unsupervised methods are suitable for the discovery of formerly unknown phenotypes, which are expected to occur in high-throughput RNAi time-lapse screens. We developed an unsupervised phenotyping approach based on Hidden Markov Models (HMMs) with multivariate Gaussian emissions for the detection of knockdown-specific phenotypes in RNAi time-lapse movies. The automated detection of abnormal cell morphologies allows us to assign a phenotypic fingerprint to each gene knockdown. By applying our method to the Mitocheck database, we show that a phenotypic fingerprint is indicative of a gene's function. Our fully unsupervised HMM-based phenotyping is able to automatically identify cell morphologies that are specific for a certain knockdown. Beyond the identification of genes whose knockdown affects cell morphology, phenotypic fingerprints can be used to find modules of functionally related genes.

Next-generation sequencing coupled with a cell-free display technology for high-throughput production of reliable interactome data

PubMed Central

Fujimori, Shigeo; Hirai, Naoya; Ohashi, Hiroyuki; Masuoka, Kazuyo; Nishikimi, Akihiko; Fukui, Yoshinori; Washio, Takanori; Oshikubo, Tomohiro; Yamashita, Tatsuhiro; Miyamoto-Sato, Etsuko

2012-01-01

Next-generation sequencing (NGS) has been applied to various kinds of omics studies, resulting in many biological and medical discoveries. However, high-throughput protein-protein interactome datasets derived from detection by sequencing are scarce, because protein-protein interaction analysis requires many cell manipulations to examine the interactions. The low reliability of the high-throughput data is also a problem. Here, we describe a cell-free display technology combined with NGS that can improve both the coverage and reliability of interactome datasets. The completely cell-free method gives a high-throughput and a large detection space, testing the interactions without using clones. The quantitative information provided by NGS reduces the number of false positives. The method is suitable for the in vitro detection of proteins that interact not only with the bait protein, but also with DNA, RNA and chemical compounds. Thus, it could become a universal approach for exploring the large space of protein sequences and interactome networks. PMID:23056904

Microreactor Cells for High-Throughput X-ray Absorption Spectroscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Beesley, Angela; Tsapatsaris, Nikolaos; Weiher, Norbert

2007-01-19

High-throughput experimentation has been applied to X-ray Absorption spectroscopy as a novel route for increasing research productivity in the catalysis community. Suitable instrumentation has been developed for the rapid determination of the local structure in the metal component of precursors for supported catalysts. An automated analytical workflow was implemented that is much faster than traditional individual spectrum analysis. It allows the generation of structural data in quasi-real time. We describe initial results obtained from the automated high throughput (HT) data reduction and analysis of a sample library implemented through the 96 well-plate industrial standard. The results show that a fullymore » automated HT-XAS technology based on existing industry standards is feasible and useful for the rapid elucidation of geometric and electronic structure of materials.« less

Correction of Microplate Data from High-Throughput Screening.

PubMed

Wang, Yuhong; Huang, Ruili

2016-01-01

High-throughput screening (HTS) makes it possible to collect cellular response data from a large number of cell lines and small molecules in a timely and cost-effective manner. The errors and noises in the microplate-formatted data from HTS have unique characteristics, and they can be generally grouped into three categories: run-wise (temporal, multiple plates), plate-wise (background pattern, single plate), and well-wise (single well). In this chapter, we describe a systematic solution for identifying and correcting such errors and noises, mainly basing on pattern recognition and digital signal processing technologies.

High-throughput screening of hybridoma supernatants using multiplexed fluorescent cell barcoding on live cells.

PubMed

Lu, Mei; Chan, Brian M; Schow, Peter W; Chang, Wesley S; King, Chadwick T

2017-12-01

With current available assay formats using either immobilized protein (ELISA, enzyme-linked immunosorbent assay) or immunostaining of fixed cells for primary monoclonal antibody (mAb) screening, researchers often fail to identify and characterize antibodies that recognize the native conformation of cell-surface antigens. Therefore, screening using live cells has become an integral and important step contributing to the successful identification of therapeutic antibody candidates. Thus the need for developing high-throughput screening (HTS) technologies using live cells has become a major priority for therapeutic mAb discovery and development. We have developed a novel technique called Multiplexed Fluorescent Cell Barcoding (MFCB), a flow cytometry-based method based upon the Fluorescent Cell Barcoding (FCB) technique and the Luminex fluorescent bead array system, but is applicable to high-through mAb screens on live cells. Using this technique in our system, we can simultaneously identify or characterize the antibody-antigen binding of up to nine unique fluorescent labeled cell populations in the time that it would normally take to process a single population. This has significantly reduced the amount of time needed for the identification of potential lead candidates. This new technology enables investigators to conduct large-scale primary hybridoma screens using flow cytometry. This in turn has allowed us to screen antibodies more efficiently than before and streamline identification and characterization of lead molecules. Copyright © 2017 Elsevier B.V. All rights reserved.

High throughput imaging cytometer with acoustic focussing† †Electronic supplementary information (ESI) available: High throughput imaging cytometer with acoustic focussing. See DOI: 10.1039/c5ra19497k Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.

PubMed Central

Zmijan, Robert; Jonnalagadda, Umesh S.; Carugo, Dario; Kochi, Yu; Lemm, Elizabeth; Packham, Graham; Hill, Martyn

2015-01-01

We demonstrate an imaging flow cytometer that uses acoustic levitation to assemble cells and other particles into a sheet structure. This technique enables a high resolution, low noise CMOS camera to capture images of thousands of cells with each frame. While ultrasonic focussing has previously been demonstrated for 1D cytometry systems, extending the technology to a planar, much higher throughput format and integrating imaging is non-trivial, and represents a significant jump forward in capability, leading to diagnostic possibilities not achievable with current systems. A galvo mirror is used to track the images of the moving cells permitting exposure times of 10 ms at frame rates of 50 fps with motion blur of only a few pixels. At 80 fps, we demonstrate a throughput of 208 000 beads per second. We investigate the factors affecting motion blur and throughput, and demonstrate the system with fluorescent beads, leukaemia cells and a chondrocyte cell line. Cells require more time to reach the acoustic focus than beads, resulting in lower throughputs; however a longer device would remove this constraint. PMID:29456838

AI-augmented time stretch microscopy

NASA Astrophysics Data System (ADS)

Mahjoubfar, Ata; Chen, Claire L.; Lin, Jiahao; Jalali, Bahram

2017-02-01

Cell reagents used in biomedical analysis often change behavior of the cells that they are attached to, inhibiting their native signaling. On the other hand, label-free cell analysis techniques have long been viewed as challenging either due to insufficient accuracy by limited features, or because of low throughput as a sacrifice of improved precision. We present a recently developed artificial-intelligence augmented microscope, which builds upon high-throughput time stretch quantitative phase imaging (TS-QPI) and deep learning to perform label-free cell classification with record high-accuracy. Our system captures quantitative optical phase and intensity images simultaneously by frequency multiplexing, extracts multiple biophysical features of the individual cells from these images fused, and feeds these features into a supervised machine learning model for classification. The enhanced performance of our system compared to other label-free assays is demonstrated by classification of white blood T-cells versus colon cancer cells and lipid accumulating algal strains for biofuel production, which is as much as five-fold reduction in inaccuracy. This system obtains the accuracy required in practical applications such as personalized drug development, while the cells remain intact and the throughput is not sacrificed. Here, we introduce a data acquisition scheme based on quadrature phase demodulation that enables interruptionless storage of TS-QPI cell images. Our proof of principle demonstration is capable of saving 40 TB of cell images in about four hours, i.e. pictures of every single cell in 10 mL of a sample.

From Lab to Fab: Developing a Nanoscale Delivery Tool for Scalable Nanomanufacturing

NASA Astrophysics Data System (ADS)

Safi, Asmahan A.

The emergence of nanomaterials with unique properties at the nanoscale over the past two decades carries a capacity to impact society and transform or create new industries ranging from nanoelectronics to nanomedicine. However, a gap in nanomanufacturing technologies has prevented the translation of nanomaterial into real-world commercialized products. Bridging this gap requires a paradigm shift in methods for fabricating structured devices with a nanoscale resolution in a repeatable fashion. This thesis explores the new paradigms for fabricating nanoscale structures devices and systems for high throughput high registration applications. We present a robust and scalable nanoscale delivery platform, the Nanofountain Probe (NFP), for parallel direct-write of functional materials. The design and microfabrication of NFP is presented. The new generation addresses the challenges of throughput, resolution and ink replenishment characterizing tip-based nanomanufacturing. To achieve these goals, optimized probe geometry is integrated to the process along with channel sealing and cantilever bending. The capabilities of the newly fabricated probes are demonstrated through two type of delivery: protein nanopatterning and single cell nanoinjection. The broad applications of the NFP for single cell delivery are investigated. An external microfluidic packaging is developed to enable delivery in liquid environment. The system is integrated to a combined atomic force microscope and inverted fluorescence microscope. Intracellular delivery is demonstrated by injecting a fluorescent dextran into Hela cells in vitro while monitoring the injection forces. Such developments enable in vitro cellular delivery for single cell studies and high throughput gene expression. The nanomanufacturing capabilities of NFPs are explored. Nanofabrication of carbon nanotube-based electronics presents all the manufacturing challenges characterizing of assembling nanomaterials precisely onto devices. The presented study combines top-down and bottom-approaches by integrating the catalyst patterning and carbon nanotube growth directly on structures. Large array of iron-rich catalyst are patterned on an substrate for subsequent carbon nanotubes synthesis. The dependence of probe geometry and substrate wetting is assessed by modeling and experimental studies. Finally preliminary results on synthesis of carbon nanotube by catalyst assisted chemical vapor deposition suggest increasing the catalyst yield is critical. Such work will enable high throughput nanomanufacturing of carbon nanotube based devices.

High-throughput screening of chemical effects on steroidogenesis using H295R human adrenocortical carcinoma cells

EPA Science Inventory

Disruption of steroidogenesis by environmental chemicals can result in altered hormone levels causing adverse reproductive and developmental effects. A high-throughput assay using H295R human adrenocortical carcinoma cells was used to evaluate the effect of 2,060 chemical samples...

CellProfiler Tracer: exploring and validating high-throughput, time-lapse microscopy image data.

PubMed

Bray, Mark-Anthony; Carpenter, Anne E

2015-11-04

Time-lapse analysis of cellular images is an important and growing need in biology. Algorithms for cell tracking are widely available; what researchers have been missing is a single open-source software package to visualize standard tracking output (from software like CellProfiler) in a way that allows convenient assessment of track quality, especially for researchers tuning tracking parameters for high-content time-lapse experiments. This makes quality assessment and algorithm adjustment a substantial challenge, particularly when dealing with hundreds of time-lapse movies collected in a high-throughput manner. We present CellProfiler Tracer, a free and open-source tool that complements the object tracking functionality of the CellProfiler biological image analysis package. Tracer allows multi-parametric morphological data to be visualized on object tracks, providing visualizations that have already been validated within the scientific community for time-lapse experiments, and combining them with simple graph-based measures for highlighting possible tracking artifacts. CellProfiler Tracer is a useful, free tool for inspection and quality control of object tracking data, available from http://www.cellprofiler.org/tracer/.

Reconstruction of metabolic networks from high-throughput metabolite profiling data: in silico analysis of red blood cell metabolism.

PubMed

Nemenman, Ilya; Escola, G Sean; Hlavacek, William S; Unkefer, Pat J; Unkefer, Clifford J; Wall, Michael E

2007-12-01

We investigate the ability of algorithms developed for reverse engineering of transcriptional regulatory networks to reconstruct metabolic networks from high-throughput metabolite profiling data. For benchmarking purposes, we generate synthetic metabolic profiles based on a well-established model for red blood cell metabolism. A variety of data sets are generated, accounting for different properties of real metabolic networks, such as experimental noise, metabolite correlations, and temporal dynamics. These data sets are made available online. We use ARACNE, a mainstream algorithm for reverse engineering of transcriptional regulatory networks from gene expression data, to predict metabolic interactions from these data sets. We find that the performance of ARACNE on metabolic data is comparable to that on gene expression data.

Identification of Novel Pro-Migratory, Cancer-Associated Genes Using Quantitative, Microscopy-Based Screening

PubMed Central

Naffar-Abu-Amara, Suha; Shay, Tal; Galun, Meirav; Cohen, Naomi; Isakoff, Steven J.; Kam, Zvi; Geiger, Benjamin

2008-01-01

Background Cell migration is a highly complex process, regulated by multiple genes, signaling pathways and external stimuli. To discover genes or pharmacological agents that can modulate the migratory activity of cells, screening strategies that enable the monitoring of diverse migratory parameters in a large number of samples are necessary. Methodology In the present study, we describe the development of a quantitative, high-throughput cell migration assay, based on a modified phagokinetic tracks (PKT) procedure, and apply it for identifying novel pro-migratory genes in a cancer-related gene library. In brief, cells are seeded on fibronectin-coated 96-well plates, covered with a monolayer of carboxylated latex beads. Motile cells clear the beads, located along their migratory paths, forming tracks that are visualized using an automated, transmitted-light screening microscope. The tracks are then segmented and characterized by multi-parametric, morphometric analysis, resolving a variety of morphological and kinetic features. Conclusions In this screen we identified 4 novel genes derived from breast carcinoma related cDNA library, whose over-expression induces major alteration in the migration of the stationary MCF7 cells. This approach can serve for high throughput screening for novel ways to modulate cellular migration in pathological states such as tumor metastasis and invasion. PMID:18213366

Fluorescence-based assay as a new screening tool for toxic chemicals

PubMed Central

Moczko, Ewa; Mirkes, Evgeny M.; Cáceres, César; Gorban, Alexander N.; Piletsky, Sergey

2016-01-01

Our study involves development of fluorescent cell-based diagnostic assay as a new approach in high-throughput screening method. This highly sensitive optical assay operates similarly to e-noses and e-tongues which combine semi-specific sensors and multivariate data analysis for monitoring biochemical processes. The optical assay consists of a mixture of environmental-sensitive fluorescent dyes and human skin cells that generate fluorescence spectra patterns distinctive for particular physico-chemical and physiological conditions. Using chemometric techniques the optical signal is processed providing qualitative information about analytical characteristics of the samples. This integrated approach has been successfully applied (with sensitivity of 93% and specificity of 97%) in assessing whether particular chemical agents are irritating or not for human skin. It has several advantages compared with traditional biochemical or biological assays and can impact the new way of high-throughput screening and understanding cell activity. It also can provide reliable and reproducible method for assessing a risk of exposing people to different harmful substances, identification active compounds in toxicity screening and safety assessment of drugs, cosmetic or their specific ingredients. PMID:27653274

Fluorescence-based assay as a new screening tool for toxic chemicals.

PubMed

Moczko, Ewa; Mirkes, Evgeny M; Cáceres, César; Gorban, Alexander N; Piletsky, Sergey

2016-09-22

Our study involves development of fluorescent cell-based diagnostic assay as a new approach in high-throughput screening method. This highly sensitive optical assay operates similarly to e-noses and e-tongues which combine semi-specific sensors and multivariate data analysis for monitoring biochemical processes. The optical assay consists of a mixture of environmental-sensitive fluorescent dyes and human skin cells that generate fluorescence spectra patterns distinctive for particular physico-chemical and physiological conditions. Using chemometric techniques the optical signal is processed providing qualitative information about analytical characteristics of the samples. This integrated approach has been successfully applied (with sensitivity of 93% and specificity of 97%) in assessing whether particular chemical agents are irritating or not for human skin. It has several advantages compared with traditional biochemical or biological assays and can impact the new way of high-throughput screening and understanding cell activity. It also can provide reliable and reproducible method for assessing a risk of exposing people to different harmful substances, identification active compounds in toxicity screening and safety assessment of drugs, cosmetic or their specific ingredients.

Fluorescence-based assay as a new screening tool for toxic chemicals

NASA Astrophysics Data System (ADS)

Moczko, Ewa; Mirkes, Evgeny M.; Cáceres, César; Gorban, Alexander N.; Piletsky, Sergey

2016-09-01

Our study involves development of fluorescent cell-based diagnostic assay as a new approach in high-throughput screening method. This highly sensitive optical assay operates similarly to e-noses and e-tongues which combine semi-specific sensors and multivariate data analysis for monitoring biochemical processes. The optical assay consists of a mixture of environmental-sensitive fluorescent dyes and human skin cells that generate fluorescence spectra patterns distinctive for particular physico-chemical and physiological conditions. Using chemometric techniques the optical signal is processed providing qualitative information about analytical characteristics of the samples. This integrated approach has been successfully applied (with sensitivity of 93% and specificity of 97%) in assessing whether particular chemical agents are irritating or not for human skin. It has several advantages compared with traditional biochemical or biological assays and can impact the new way of high-throughput screening and understanding cell activity. It also can provide reliable and reproducible method for assessing a risk of exposing people to different harmful substances, identification active compounds in toxicity screening and safety assessment of drugs, cosmetic or their specific ingredients.

DEVELOPMENT OF PATHWAY SELECTIVE CELLS AND ASSAYS FOR SENSITIVE DETECTION OF ENVIRONMENTAL TOXICANTS VIA HIGH THROUGHPUT LABEL-FREE CELL-BASED SCREENING - PHASE I

EPA Science Inventory

Spatial tuning of acoustofluidic pressure nodes by altering net sonic velocity enables high-throughput, efficient cell sorting

DOE PAGES

Jung, Seung-Yong; Notton, Timothy; Fong, Erika; ...

2015-01-07

Particle sorting using acoustofluidics has enormous potential but widespread adoption has been limited by complex device designs and low throughput. Here, we report high-throughput separation of particles and T lymphocytes (600 μL min -1) by altering the net sonic velocity to reposition acoustic pressure nodes in a simple two-channel device. Finally, the approach is generalizable to other microfluidic platforms for rapid, high-throughput analysis.

Real Time Detection of Protein Trafficking with High Throughput Flow Cytometry (HTFC) and Fluorogen Activating Protein (FAP) Base Biosensor

PubMed Central

Wu, Yang; Tapia, Phillip H.; Jarvik, Jonathan; Waggoner, Alan S.; Sklar, Larry A.

2014-01-01

We combined fluorogen activating protein (FAP) technology with high-throughput flow cytometry to detect real-time protein trafficking to and from the plasma membrane in living cells. The hybrid platform allows drug discovery for trafficking receptors, such as G-protein coupled receptors, receptor tyrosine kinases and ion channels, that were previously not suitable for high throughput screening by flow cytometry.. The system has been validated using the β2-adrenergic receptor (β2AR) system and extended to other GPCRs. When a chemical library containing ~1,200 off-patent drugs was screened against cells expressing FAP tagged β2AR, all known β2AR active ligands in the library were successfully identified, together with a few compounds that were later confirmed to regulate receptor internalization in a non-traditional manner. The unexpected discovery of new ligands by this approach indicates the potential of using this protocol for GPCR de-orphanization. In addition, screens of multiplexed targets promise improved efficiency with minor protocol modification. PMID:24510772

Separation of rare oligodendrocyte progenitor cells from brain using a high-throughput multilayer thermoplastic-based microfluidic device.

PubMed

Didar, Tohid Fatanat; Li, Kebin; Veres, Teodor; Tabrizian, Maryam

2013-07-01

Despite the advances made in the field of regenerative medicine, the progress in cutting-edge technologies for separating target therapeutic cells are still at early stage of development. These cells are often rare, such as stem cells or progenitor cells that their overall properties should be maintained during the separation process for their subsequent application in regenerative medicine. This work, presents separation of oligodendrocyte progenitor cells (OPCs) from rat brain primary cultures using an integrated thermoplastic elastomeric (TPE)- based multilayer microfluidic device fabricated using hot-embossing technology. OPCs are frequently used in recovery, repair and regeneration of central nervous system after injuries. Indeed, their ability to differentiate in vitro into myelinating oligodendrocytes, are extremely important for myelin repair. OPCs form 5-10% of the glial cells population. The traditional macroscale techniques for OPCs separation require pre-processing of cells and/or multiple time consuming steps with low efficiency leading very often to alteration of their properties. The proposed methodology implies to separate OPCs based on their smaller size compared to other cells from the brain tissue mixture. Using aforementioned microfluidic chip embedded with a 5 μm membrane pore size and micropumping system, a separation efficiency more than 99% was achieved. This microchip was able to operate at flow rates up to 100 μl/min, capable of separating OPCs from a confluent 75 cm(2) cell culture flask in less than 10 min, which provides us with a high-throughput and highly efficient separation expected from any cell sorting techniques. Copyright © 2013 Elsevier Ltd. All rights reserved.

Two High Throughput Screen Assays for Measurement of TNF-α in THP-1 Cells

PubMed Central

Leister, Kristin P; Huang, Ruili; Goodwin, Bonnie L; Chen, Andrew; Austin, Christopher P; Xia, Menghang

2011-01-01

Tumor Necrosis Factor-α (TNF-α), a secreted cytokine, plays an important role in inflammatory diseases and immune disorders, and is a potential target for drug development. The traditional assays for detecting TNF-α, enzyme linked immunosorbent assay (ELISA) and radioimmunoassay, are not suitable for the large size compound screens. Both assays suffer from a complicated protocol, multiple plate wash steps and/or excessive radioactive waste. A simple and quick measurement of TNF-α production in a cell based assay is needed for high throughput screening to identify the lead compounds from the compound library. We have developed and optimized two homogeneous TNF-α assays using the HTRF (homogeneous time resolved fluorescence) and AlphaLISA assay formats. We have validated the HTRF based TNF-α assay in a 1536-well plate format by screening a library of 1280 pharmacologically active compounds. The active compounds identified from the screen were confirmed in the AlphaLISA TNF-α assay using a bead-based technology. These compounds were also confirmed in a traditional ELISA assay. From this study, several beta adrenergic agonists have been identified as TNF-α inhibitors. We also identified several novel inhibitors of TNF-α, such as BTO-1, CCG-2046, ellipticine, and PD 169316. The results demonstrated that both homogeneous TNF-α assays are robust and suitable for high throughput screening. PMID:21643507

Acoustic separation of circulating tumor cells

PubMed Central

Li, Peng; Mao, Zhangming; Peng, Zhangli; Zhou, Lanlan; Chen, Yuchao; Huang, Po-Hsun; Truica, Cristina I.; Drabick, Joseph J.; El-Deiry, Wafik S.; Dao, Ming; Suresh, Subra; Huang, Tony Jun

2015-01-01

Circulating tumor cells (CTCs) are important targets for cancer biology studies. To further elucidate the role of CTCs in cancer metastasis and prognosis, effective methods for isolating extremely rare tumor cells from peripheral blood must be developed. Acoustic-based methods, which are known to preserve the integrity, functionality, and viability of biological cells using label-free and contact-free sorting, have thus far not been successfully developed to isolate rare CTCs using clinical samples from cancer patients owing to technical constraints, insufficient throughput, and lack of long-term device stability. In this work, we demonstrate the development of an acoustic-based microfluidic device that is capable of high-throughput separation of CTCs from peripheral blood samples obtained from cancer patients. Our method uses tilted-angle standing surface acoustic waves. Parametric numerical simulations were performed to design optimum device geometry, tilt angle, and cell throughput that is more than 20 times higher than previously possible for such devices. We first validated the capability of this device by successfully separating low concentrations (∼100 cells/mL) of a variety of cancer cells from cell culture lines from WBCs with a recovery rate better than 83%. We then demonstrated the isolation of CTCs in blood samples obtained from patients with breast cancer. Our acoustic-based separation method thus offers the potential to serve as an invaluable supplemental tool in cancer research, diagnostics, drug efficacy assessment, and therapeutics owing to its excellent biocompatibility, simple design, and label-free automated operation while offering the capability to isolate rare CTCs in a viable state. PMID:25848039

Acoustic separation of circulating tumor cells.

PubMed

Li, Peng; Mao, Zhangming; Peng, Zhangli; Zhou, Lanlan; Chen, Yuchao; Huang, Po-Hsun; Truica, Cristina I; Drabick, Joseph J; El-Deiry, Wafik S; Dao, Ming; Suresh, Subra; Huang, Tony Jun

2015-04-21

Circulating tumor cells (CTCs) are important targets for cancer biology studies. To further elucidate the role of CTCs in cancer metastasis and prognosis, effective methods for isolating extremely rare tumor cells from peripheral blood must be developed. Acoustic-based methods, which are known to preserve the integrity, functionality, and viability of biological cells using label-free and contact-free sorting, have thus far not been successfully developed to isolate rare CTCs using clinical samples from cancer patients owing to technical constraints, insufficient throughput, and lack of long-term device stability. In this work, we demonstrate the development of an acoustic-based microfluidic device that is capable of high-throughput separation of CTCs from peripheral blood samples obtained from cancer patients. Our method uses tilted-angle standing surface acoustic waves. Parametric numerical simulations were performed to design optimum device geometry, tilt angle, and cell throughput that is more than 20 times higher than previously possible for such devices. We first validated the capability of this device by successfully separating low concentrations (∼100 cells/mL) of a variety of cancer cells from cell culture lines from WBCs with a recovery rate better than 83%. We then demonstrated the isolation of CTCs in blood samples obtained from patients with breast cancer. Our acoustic-based separation method thus offers the potential to serve as an invaluable supplemental tool in cancer research, diagnostics, drug efficacy assessment, and therapeutics owing to its excellent biocompatibility, simple design, and label-free automated operation while offering the capability to isolate rare CTCs in a viable state.

PomBase: The Scientific Resource for Fission Yeast.

PubMed

Lock, Antonia; Rutherford, Kim; Harris, Midori A; Wood, Valerie

2018-01-01

The fission yeast Schizosaccharomyces pombe has become well established as a model species for studying conserved cell-level biological processes, especially the mechanics and regulation of cell division. PomBase integrates the S. pombe genome sequence with traditional genetic, molecular, and cell biological experimental data as well as the growing body of large datasets generated by emerging high-throughput methods. This chapter provides insight into the curation philosophy and data organization at PomBase, and provides a guide to using PomBase for infrequent visitors and anyone considering exploring S. pombe in their research.

Transfection microarray and the applications.

PubMed

Miyake, Masato; Yoshikawa, Tomohiro; Fujita, Satoshi; Miyake, Jun

2009-05-01

Microarray transfection has been extensively studied for high-throughput functional analysis of mammalian cells. However, control of efficiency and reproducibility are the critical issues for practical use. By using solid-phase transfection accelerators and nano-scaffold, we provide a highly efficient and reproducible microarray-transfection device, "transfection microarray". The device would be applied to the limited number of available primary cells and stem cells not only for large-scale functional analysis but also reporter-based time-lapse cellular event analysis.

Evaluation of High-throughput Genotoxicity Assays Used in Profiling the US EPA ToxCast Chemicals

EPA Science Inventory

Three high-throughput screening (HTS) genotoxicity assays-GreenScreen HC GADD45a-GFP (Gentronix Ltd.), CellCiphr p53 (Cellumen Inc.) and CellSensor p53RE-bla (Invitrogen Corp.)-were used to analyze the collection of 320 predominantly pesticide active compounds being tested in Pha...

The Nano-Patch-Clamp Array: Microfabricated Glass Chips for High-Throughput Electrophysiology

NASA Astrophysics Data System (ADS)

Fertig, Niels

2003-03-01

Electrophysiology (i.e. patch clamping) remains the gold standard for pharmacological testing of putative ion channel active drugs (ICADs), but suffers from low throughput. A new ion channel screening technology based on microfabricated glass chip devices will be presented. The glass chips contain very fine apertures, which are used for whole-cell voltage clamp recordings as well as single channel recordings from mammalian cell lines. Chips containing multiple patch clamp wells will be used in a first bench-top device, which will allow perfusion and electrical readout of each well. This scalable technology will allow for automated, rapid and parallel screening on ion channel drug targets.

Droplet-based microfluidic high-throughput screening of heterologous enzymes secreted by the yeast Yarrowia lipolytica.

PubMed

Beneyton, Thomas; Thomas, Stéphane; Griffiths, Andrew D; Nicaud, Jean-Marc; Drevelle, Antoine; Rossignol, Tristan

2017-01-31

Droplet-based microfluidics is becoming an increasingly attractive alternative to microtiter plate techniques for enzymatic high-throughput screening (HTS), especially for exploring large diversities with lower time and cost footprint. In this case, the assayed enzyme has to be accessible to the substrate within the water-in-oil droplet by being ideally extracellular or displayed at the cell surface. However, most of the enzymes screened to date are expressed within the cytoplasm of Escherichia coli cells, which means that a lysis step must take place inside the droplets for enzyme activity to be assayed. Here, we take advantage of the excellent secretion abilities of the yeast Yarrowia lipolytica to describe a highly efficient expression system particularly suitable for the droplet-based microfluidic HTS. Five hydrolytic genes from Aspergillus niger genome were chosen and the corresponding five Yarrowia lipolytica producing strains were constructed. Each enzyme (endo-β-1,4-xylanase B and C; 1,4-β-cellobiohydrolase A; endoglucanase A; aspartic protease) was successfully overexpressed and secreted in an active form in the crude supernatant. A droplet-based microfluidic HTS system was developed to (a) encapsulate single yeast cells; (b) grow yeast in droplets; (c) inject the relevant enzymatic substrate; (d) incubate droplets on chip; (e) detect enzymatic activity; and (f) sort droplets based on enzymatic activity. Combining this integrated microfluidic platform with gene expression in Y. lipolytica results in remarkably low variability in the enzymatic activity at the single cell level within a given monoclonal population (<5%). Xylanase, cellobiohydrolase and protease activities were successfully assayed using this system. We then used the system to screen for thermostable variants of endo-β-1,4-xylanase C in error-prone PCR libraries. Variants displaying higher thermostable xylanase activities compared to the wild-type were isolated (up to 4.7-fold improvement). Yarrowia lipolytica was used to express fungal genes encoding hydrolytic enzymes of interest. We developed a successful droplet-based microfluidic platform for the high-throughput screening (10 5 strains/h) of Y. lipolytica based on enzyme secretion and activity. This approach provides highly efficient tools for the HTS of recombinant enzymatic activities. This should be extremely useful for discovering new biocatalysts via directed evolution or protein engineering approaches and should lead to major advances in microbial cell factory development.

Mechanical phenotyping of tumor cells using a microfluidic cell squeezer device

NASA Astrophysics Data System (ADS)

Khan, Zeina S.; Kamyabi, Nabiollah; Vanapalli, Siva A.

2013-03-01

Studies have indicated that cancer cells have distinct mechanical properties compared to healthy cells. We are investigating the potential of cell mechanics as a biophysical marker for diagnostics and prognosis of cancer. To establish the significance of mechanical properties for cancer diagnostics, a high throughput method is desired. Although techniques such as atomic force microscopy are very precise, they are limited in throughput for cellular mechanical property measurements. To develop a device for high throughput mechanical characterization of tumor cells, we have fabricated a microfludic cell squeezer device that contains narrow micrometer-scale pores. Fluid flow is used to drive cells into these pores mimicking the flow-induced passage of circulating tumor cells through microvasculature. By integrating high speed imaging, the device allows for the simultaneous characterization of five different parameters including the blockage pressure, cell velocity, cell size, elongation and the entry time into squeezer. We have tested a variety of in vitro cell lines, including brain and prostate cancer cell lines, and have found that the entry time is the most sensitive measurement capable of differentiating between cell lines with differing invasiveness.

Dynamic analysis of apoptosis using cyanine SYTO probes: From classical to microfluidic cytometry

PubMed Central

Wlodkowic, Donald; Skommer, Joanna; Faley, Shannon; Darzynkiewicz, Zbigniew; Cooper, Jonathan M.

2013-01-01

Cell death is a stochastic process, often initiated and/or executed in a multi-pathway/multi-organelle fashion. Therefore, high-throughput single-cell analysis platforms are required to provide detailed characterization of kinetics and mechanisms of cell death in heterogeneous cell populations. However, there is still a largely unmet need for inert fluorescent probes, suitable for prolonged kinetic studies. Here, we compare the use of innovative adaptation of unsymmetrical SYTO dyes for dynamic real-time analysis of apoptosis in conventional as well as microfluidic chip-based systems. We show that cyanine SYTO probes allow non-invasive tracking of intracellular events over extended time. Easy handling and “stain–no wash” protocols open up new opportunities for high-throughput analysis and live-cell sorting. Furthermore, SYTO probes are easily adaptable for detection of cell death using automated microfluidic chip-based cytometry. Overall, the combined use of SYTO probes and state-of-the-art Lab-on-a-Chip platform emerges as a cost effective solution for automated drug screening compared to conventional Annexin V or TUNEL assays. In particular, it should allow for dynamic analysis of samples where low cell number has so far been an obstacle, e.g. primary cancer stems cells or circulating minimal residual tumors. PMID:19298813

Novel screening techniques for ion channel targeting drugs

PubMed Central

Obergrussberger, Alison; Stölzle-Feix, Sonja; Becker, Nadine; Brüggemann, Andrea; Fertig, Niels; Möller, Clemens

2015-01-01

Ion channels are integral membrane proteins that regulate the flux of ions across the cell membrane. They are involved in nearly all physiological processes, and malfunction of ion channels has been linked to many diseases. Until recently, high-throughput screening of ion channels was limited to indirect, e.g. fluorescence-based, readout technologies. In the past years, direct label-free biophysical readout technologies by means of electrophysiology have been developed. Planar patch-clamp electrophysiology provides a direct functional label-free readout of ion channel function in medium to high throughput. Further electrophysiology features, including temperature control and higher-throughput instruments, are continually being developed. Electrophysiological screening in a 384-well format has recently become possible. Advances in chip and microfluidic design, as well as in cell preparation and handling, have allowed challenging cell types to be studied by automated patch clamp. Assays measuring action potentials in stem cell-derived cardiomyocytes, relevant for cardiac safety screening, and neuronal cells, as well as a large number of different ion channels, including fast ligand-gated ion channels, have successfully been established by automated patch clamp. Impedance and multi-electrode array measurements are particularly suitable for studying cardiomyocytes and neuronal cells within their physiological network, and to address more complex physiological questions. This article discusses recent advances in electrophysiological technologies available for screening ion channel function and regulation. PMID:26556400

Novel screening techniques for ion channel targeting drugs.

PubMed

Obergrussberger, Alison; Stölzle-Feix, Sonja; Becker, Nadine; Brüggemann, Andrea; Fertig, Niels; Möller, Clemens

2015-01-01

Ion channels are integral membrane proteins that regulate the flux of ions across the cell membrane. They are involved in nearly all physiological processes, and malfunction of ion channels has been linked to many diseases. Until recently, high-throughput screening of ion channels was limited to indirect, e.g. fluorescence-based, readout technologies. In the past years, direct label-free biophysical readout technologies by means of electrophysiology have been developed. Planar patch-clamp electrophysiology provides a direct functional label-free readout of ion channel function in medium to high throughput. Further electrophysiology features, including temperature control and higher-throughput instruments, are continually being developed. Electrophysiological screening in a 384-well format has recently become possible. Advances in chip and microfluidic design, as well as in cell preparation and handling, have allowed challenging cell types to be studied by automated patch clamp. Assays measuring action potentials in stem cell-derived cardiomyocytes, relevant for cardiac safety screening, and neuronal cells, as well as a large number of different ion channels, including fast ligand-gated ion channels, have successfully been established by automated patch clamp. Impedance and multi-electrode array measurements are particularly suitable for studying cardiomyocytes and neuronal cells within their physiological network, and to address more complex physiological questions. This article discusses recent advances in electrophysiological technologies available for screening ion channel function and regulation.

Deterministic Migration-Based Separation of White Blood Cells.

PubMed

Kim, Byeongyeon; Choi, Young Joon; Seo, Hyekyung; Shin, Eui-Cheol; Choi, Sungyoung

2016-10-01

Functional and phenotypic analyses of peripheral white blood cells provide useful clinical information. However, separation of white blood cells from peripheral blood requires a time-consuming, inconvenient process and thus analyses of separated white blood cells are limited in clinical settings. To overcome this limitation, a microfluidic separation platform is developed to enable deterministic migration of white blood cells, directing the cells into designated positions according to a ridge pattern. The platform uses slant ridge structures on the channel top to induce the deterministic migration, which allows efficient and high-throughput separation of white blood cells from unprocessed whole blood. The extent of the deterministic migration under various rheological conditions is explored, enabling highly efficient migration of white blood cells in whole blood and achieving high-throughput separation of the cells (processing 1 mL of whole blood less than 7 min). In the separated cell population, the composition of lymphocyte subpopulations is well preserved, and T cells secrete cytokines without any functional impairment. On the basis of the results, this microfluidic platform is a promising tool for the rapid enrichment of white blood cells, and it is useful for functional and phenotypic analyses of peripheral white blood cells. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Integration of ARTP mutagenesis with biosensor-mediated high-throughput screening to improve L-serine yield in Corynebacterium glutamicum.

PubMed

Zhang, Xin; Zhang, Xiaomei; Xu, Guoqiang; Zhang, Xiaojuan; Shi, Jinsong; Xu, Zhenghong

2018-05-03

L-Serine is widely used in the pharmaceutical, food, and cosmetics industries. Although direct fermentative production of L-serine from sugar in Corynebacterium glutamicum has been achieved, the L-serine yield remains relatively low. In this study, atmospheric and room temperature plasma (ARTP) mutagenesis was used to improve the L-serine yield based on engineered C. glutamicum ΔSSAAI strain. Subsequently, we developed a novel high-throughput screening method using a biosensor constructed based on NCgl0581, a transcriptional factor specifically responsive to L-serine, so that L-serine concentration within single cell of C. glutamicum can be monitored via fluorescence-activated cell sorting (FACS). Novel L-serine-producing mutants were isolated from a large library of mutagenized cells. The mutant strain A36-pDser was screened from 1.2 × 10 5 cells, and the magnesium ion concentration in the medium was optimized specifically for this mutant. C. glutamicum A36-pDser accumulated 34.78 g/L L-serine with a yield of 0.35 g/g sucrose, which were 35.9 and 66.7% higher than those of the parent C. glutamicum ΔSSAAI-pDser strain, respectively. The L-serine yield achieved in this mutant was the highest of all reported L-serine-producing strains of C. glutamicum. Moreover, the whole-genome sequencing identified 11 non-synonymous mutations of genes associated with metabolic and transport pathways, which might be responsible for the higher L-serine production and better cell growth in C. glutamicum A36-pDser. This study explored an effective mutagenesis strategy and reported a novel high-throughput screening method for the development of L-serine-producing strains.

Multiplexed mass cytometry profiling of cellular states perturbed by small-molecule regulators

PubMed Central

Bodenmiller, Bernd; Zunder, Eli R.; Finck, Rachel; Chen, Tiffany J.; Savig, Erica S.; Bruggner, Robert V.; Simonds, Erin F.; Bendall, Sean C.; Sachs, Karen; Krutzik, Peter O.; Nolan, Garry P.

2013-01-01

The ability to comprehensively explore the impact of bio-active molecules on human samples at the single-cell level can provide great insight for biomedical research. Mass cytometry enables quantitative single-cell analysis with deep dimensionality, but currently lacks high-throughput capability. Here we report a method termed mass-tag cellular barcoding (MCB) that increases mass cytometry throughput by sample multiplexing. 96-well format MCB was used to characterize human peripheral blood mononuclear cell (PBMC) signaling dynamics, cell-to-cell communication, the signaling variability between 8 donors, and to define the impact of 27 inhibitors on this system. For each compound, 14 phosphorylation sites were measured in 14 PBMC types, resulting in 18,816 quantified phosphorylation levels from each multiplexed sample. This high-dimensional systems-level inquiry allowed analysis across cell-type and signaling space, reclassified inhibitors, and revealed off-target effects. MCB enables high-content, high-throughput screening, with potential applications for drug discovery, pre-clinical testing, and mechanistic investigation of human disease. PMID:22902532

All-passive pixel super-resolution of time-stretch imaging

PubMed Central

Chan, Antony C. S.; Ng, Ho-Cheung; Bogaraju, Sharat C. V.; So, Hayden K. H.; Lam, Edmund Y.; Tsia, Kevin K.

2017-01-01

Based on image encoding in a serial-temporal format, optical time-stretch imaging entails a stringent requirement of state-of-the-art fast data acquisition unit in order to preserve high image resolution at an ultrahigh frame rate — hampering the widespread utilities of such technology. Here, we propose a pixel super-resolution (pixel-SR) technique tailored for time-stretch imaging that preserves pixel resolution at a relaxed sampling rate. It harnesses the subpixel shifts between image frames inherently introduced by asynchronous digital sampling of the continuous time-stretch imaging process. Precise pixel registration is thus accomplished without any active opto-mechanical subpixel-shift control or other additional hardware. Here, we present the experimental pixel-SR image reconstruction pipeline that restores high-resolution time-stretch images of microparticles and biological cells (phytoplankton) at a relaxed sampling rate (≈2–5 GSa/s)—more than four times lower than the originally required readout rate (20 GSa/s) — is thus effective for high-throughput label-free, morphology-based cellular classification down to single-cell precision. Upon integration with the high-throughput image processing technology, this pixel-SR time-stretch imaging technique represents a cost-effective and practical solution for large scale cell-based phenotypic screening in biomedical diagnosis and machine vision for quality control in manufacturing. PMID:28303936

Multi-crystalline II-VI based multijunction solar cells and modules

DOEpatents

Hardin, Brian E.; Connor, Stephen T.; Groves, James R.; Peters, Craig H.

2015-06-30

Multi-crystalline group II-VI solar cells and methods for fabrication of same are disclosed herein. A multi-crystalline group II-VI solar cell includes a first photovoltaic sub-cell comprising silicon, a tunnel junction, and a multi-crystalline second photovoltaic sub-cell. A plurality of the multi-crystalline group II-VI solar cells can be interconnected to form low cost, high throughput flat panel, low light concentration, and/or medium light concentration photovoltaic modules or devices.

Mapping human pluripotent stem cell differentiation pathways using high throughput single-cell RNA-sequencing.

PubMed

Han, Xiaoping; Chen, Haide; Huang, Daosheng; Chen, Huidong; Fei, Lijiang; Cheng, Chen; Huang, He; Yuan, Guo-Cheng; Guo, Guoji

2018-04-05

Human pluripotent stem cells (hPSCs) provide powerful models for studying cellular differentiations and unlimited sources of cells for regenerative medicine. However, a comprehensive single-cell level differentiation roadmap for hPSCs has not been achieved. We use high throughput single-cell RNA-sequencing (scRNA-seq), based on optimized microfluidic circuits, to profile early differentiation lineages in the human embryoid body system. We present a cellular-state landscape for hPSC early differentiation that covers multiple cellular lineages, including neural, muscle, endothelial, stromal, liver, and epithelial cells. Through pseudotime analysis, we construct the developmental trajectories of these progenitor cells and reveal the gene expression dynamics in the process of cell differentiation. We further reprogram primed H9 cells into naïve-like H9 cells to study the cellular-state transition process. We find that genes related to hemogenic endothelium development are enriched in naïve-like H9. Functionally, naïve-like H9 show higher potency for differentiation into hematopoietic lineages than primed cells. Our single-cell analysis reveals the cellular-state landscape of hPSC early differentiation, offering new insights that can be harnessed for optimization of differentiation protocols.

Metabolic cancer biology: structural-based analysis of cancer as a metabolic disease, new sights and opportunities for disease treatment.

PubMed

Masoudi-Nejad, Ali; Asgari, Yazdan

2015-02-01

The cancer cell metabolism or the Warburg effect discovery goes back to 1924 when, for the first time Otto Warburg observed, in contrast to the normal cells, cancer cells have different metabolism. With the initiation of high throughput technologies and computational systems biology, cancer cell metabolism renaissances and many attempts were performed to revise the Warburg effect. The development of experimental and analytical tools which generate high-throughput biological data including lots of information could lead to application of computational models in biological discovery and clinical medicine especially for cancer. Due to the recent availability of tissue-specific reconstructed models, new opportunities in studying metabolic alteration in various kinds of cancers open up. Structural approaches at genome-scale levels seem to be suitable for developing diagnostic and prognostic molecular signatures, as well as in identifying new drug targets. In this review, we have considered these recent advances in structural-based analysis of cancer as a metabolic disease view. Two different structural approaches have been described here: topological and constraint-based methods. The ultimate goal of this type of systems analysis is not only the discovery of novel drug targets but also the development of new systems-based therapy strategies. Copyright © 2014 Elsevier Ltd. All rights reserved.

MAPPI-DAT: data management and analysis for protein-protein interaction data from the high-throughput MAPPIT cell microarray platform.

PubMed

Gupta, Surya; De Puysseleyr, Veronic; Van der Heyden, José; Maddelein, Davy; Lemmens, Irma; Lievens, Sam; Degroeve, Sven; Tavernier, Jan; Martens, Lennart

2017-05-01

Protein-protein interaction (PPI) studies have dramatically expanded our knowledge about cellular behaviour and development in different conditions. A multitude of high-throughput PPI techniques have been developed to achieve proteome-scale coverage for PPI studies, including the microarray based Mammalian Protein-Protein Interaction Trap (MAPPIT) system. Because such high-throughput techniques typically report thousands of interactions, managing and analysing the large amounts of acquired data is a challenge. We have therefore built the MAPPIT cell microArray Protein Protein Interaction-Data management & Analysis Tool (MAPPI-DAT) as an automated data management and analysis tool for MAPPIT cell microarray experiments. MAPPI-DAT stores the experimental data and metadata in a systematic and structured way, automates data analysis and interpretation, and enables the meta-analysis of MAPPIT cell microarray data across all stored experiments. MAPPI-DAT is developed in Python, using R for data analysis and MySQL as data management system. MAPPI-DAT is cross-platform and can be ran on Microsoft Windows, Linux and OS X/macOS. The source code and a Microsoft Windows executable are freely available under the permissive Apache2 open source license at https://github.com/compomics/MAPPI-DAT. jan.tavernier@vib-ugent.be or lennart.martens@vib-ugent.be. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press.

Tracking single hematopoietic stem cells in vivo using high-throughput sequencing in conjunction with viral genetic barcoding

PubMed Central

Lu, Rong; Neff, Norma F.; Quake, Stephen R.; Weissman, Irving L.

2011-01-01

Disentangling cellular heterogeneity is a challenge in many fields, particularly in the stem cell and cancer biology fields. Here, we demonstrate how to combine viral genetic barcoding with high-throughput sequencing to track single cells in a heterogeneous population. We use this technique to track the in vivo differentiation of unitary hematopoietic stem cells (HSCs). The results are consistent with single cell transplantation studies, but require two orders of magnitude fewer mice. In addition to its high throughput, the high sensitivity of the technique allows for a direct examination of the clonality of sparse cell populations such as HSCs. We show how these capabilities offer a clonal perspective of the HSC differentiation process. In particular, our data suggests that HSCs do not equally contribute to blood cells after irradiation-mediated transplantation, and that two distinct HSC differentiation patterns co-exist in the same recipient mouse post irradiation. This technique can be applied to any viral accessible cell type for both in vitro and in vivo processes. PMID:21964413

Nanowire-based single-cell endoscopy

NASA Astrophysics Data System (ADS)

Yan, Ruoxue; Park, Ji-Ho; Choi, Yeonho; Heo, Chul-Joon; Yang, Seung-Man; Lee, Luke P.; Yang, Peidong

2012-03-01

One-dimensional smart probes based on nanowires and nanotubes that can safely penetrate the plasma membrane and enter biological cells are potentially useful in high-resolution and high-throughput gene and drug delivery, biosensing and single-cell electrophysiology. However, using such probes for optical communication across the cellular membrane at the subwavelength level remains limited. Here, we show that a nanowire waveguide attached to the tapered tip of an optical fibre can guide visible light into intracellular compartments of a living mammalian cell, and can also detect optical signals from subcellular regions with high spatial resolution. Furthermore, we show that through light-activated mechanisms the endoscope can deliver payloads into cells with spatial and temporal specificity. Moreover, insertion of the endoscope into cells and illumination of the guided laser did not induce any significant toxicity in the cells.

Establishment of an AAV Reverse Infection-Based Array

PubMed Central

Wang, Gang; Dong, Zheyue; Shen, Wei; Zheng, Gang; Wu, Xiaobing; Xue, Jinglun; Wang, Yue; Chen, Jinzhong

2010-01-01

Background The development of a convenient high-throughput gene transduction approach is critical for biological screening. Adeno-associated virus (AAV) vectors are broadly used in gene therapy studies, yet their applications in in vitro high-throughput gene transduction are limited. Principal Findings We established an AAV reverse infection (RI)-based method in which cells were transduced by quantified recombinant AAVs (rAAVs) pre-coated onto 96-well plates. The number of pre-coated rAAV particles and number of cells loaded per well, as well as the temperature stability of the rAAVs on the plates, were evaluated. As the first application of this method, six serotypes or hybrid serotypes of rAAVs (AAV1, AAV2, AAV5/5, AAV8, AAV25 m, AAV28 m) were compared for their transduction efficiencies using various cell lines, including BHK21, HEK293, BEAS-2BS, HeLaS3, Huh7, Hepa1-6, and A549. AAV2 and AAV1 displayed high transduction efficiency; thus, they were deemed to be suitable candidate vectors for the RI-based array. We next evaluated the impact of sodium butyrate (NaB) treatment on rAAV vector-mediated reporter gene expression and found it was significantly enhanced, suggesting that our system reflected the biological response of target cells to specific treatments. Conclusions/Significance Our study provides a novel method for establishing a highly efficient gene transduction array that may be developed into a platform for cell biological assays. PMID:20976058

High-throughput Screening of ToxCast™ Phase I Chemicals in a Mouse Embryonic Stem Cell (mESC) Assay Reveals Disruption of Potential Toxicity Pathways

EPA Science Inventory

Little information is available regarding the potential for many commercial chemicals to induce developmental toxicity. The mESC Adherent Cell Differentiation and Cytoxicity (ACDC) assay is a high-throughput screen used to close this data gap. Thus, ToxCast™ Phase I chemicals wer...

Large-scale generation of human iPSC-derived neural stem cells/early neural progenitor cells and their neuronal differentiation.

PubMed

D'Aiuto, Leonardo; Zhi, Yun; Kumar Das, Dhanjit; Wilcox, Madeleine R; Johnson, Jon W; McClain, Lora; MacDonald, Matthew L; Di Maio, Roberto; Schurdak, Mark E; Piazza, Paolo; Viggiano, Luigi; Sweet, Robert; Kinchington, Paul R; Bhattacharjee, Ayantika G; Yolken, Robert; Nimgaonka, Vishwajit L; Nimgaonkar, Vishwajit L

2014-01-01

Induced pluripotent stem cell (iPSC)-based technologies offer an unprecedented opportunity to perform high-throughput screening of novel drugs for neurological and neurodegenerative diseases. Such screenings require a robust and scalable method for generating large numbers of mature, differentiated neuronal cells. Currently available methods based on differentiation of embryoid bodies (EBs) or directed differentiation of adherent culture systems are either expensive or are not scalable. We developed a protocol for large-scale generation of neuronal stem cells (NSCs)/early neural progenitor cells (eNPCs) and their differentiation into neurons. Our scalable protocol allows robust and cost-effective generation of NSCs/eNPCs from iPSCs. Following culture in neurobasal medium supplemented with B27 and BDNF, NSCs/eNPCs differentiate predominantly into vesicular glutamate transporter 1 (VGLUT1) positive neurons. Targeted mass spectrometry analysis demonstrates that iPSC-derived neurons express ligand-gated channels and other synaptic proteins and whole-cell patch-clamp experiments indicate that these channels are functional. The robust and cost-effective differentiation protocol described here for large-scale generation of NSCs/eNPCs and their differentiation into neurons paves the way for automated high-throughput screening of drugs for neurological and neurodegenerative diseases.

Data for automated, high-throughput microscopy analysis of intracellular bacterial colonies using spot detection.

PubMed

Ernstsen, Christina L; Login, Frédéric H; Jensen, Helene H; Nørregaard, Rikke; Møller-Jensen, Jakob; Nejsum, Lene N

2017-10-01

Quantification of intracellular bacterial colonies is useful in strategies directed against bacterial attachment, subsequent cellular invasion and intracellular proliferation. An automated, high-throughput microscopy-method was established to quantify the number and size of intracellular bacterial colonies in infected host cells (Detection and quantification of intracellular bacterial colonies by automated, high-throughput microscopy, Ernstsen et al., 2017 [1]). The infected cells were imaged with a 10× objective and number of intracellular bacterial colonies, their size distribution and the number of cell nuclei were automatically quantified using a spot detection-tool. The spot detection-output was exported to Excel, where data analysis was performed. In this article, micrographs and spot detection data are made available to facilitate implementation of the method.

Clinical validation of an ultra high-throughput spiral microfluidics for the detection and enrichment of viable circulating tumor cells.

PubMed

Khoo, Bee Luan; Warkiani, Majid Ebrahimi; Tan, Daniel Shao-Weng; Bhagat, Ali Asgar S; Irwin, Darryl; Lau, Dawn Pingxi; Lim, Alvin S T; Lim, Kiat Hon; Krisna, Sai Sakktee; Lim, Wan-Teck; Yap, Yoon Sim; Lee, Soo Chin; Soo, Ross A; Han, Jongyoon; Lim, Chwee Teck

2014-01-01

Circulating tumor cells (CTCs) are cancer cells that can be isolated via liquid biopsy from blood and can be phenotypically and genetically characterized to provide critical information for guiding cancer treatment. Current analysis of CTCs is hindered by the throughput, selectivity and specificity of devices or assays used in CTC detection and isolation. Here, we enriched and characterized putative CTCs from blood samples of patients with both advanced stage metastatic breast and lung cancers using a novel multiplexed spiral microfluidic chip. This system detected putative CTCs under high sensitivity (100%, n = 56) (Breast cancer samples: 12-1275 CTCs/ml; Lung cancer samples: 10-1535 CTCs/ml) rapidly from clinically relevant blood volumes (7.5 ml under 5 min). Blood samples were completely separated into plasma, CTCs and PBMCs components and each fraction were characterized with immunophenotyping (Pan-cytokeratin/CD45, CD44/CD24, EpCAM), fluorescence in-situ hybridization (FISH) (EML4-ALK) or targeted somatic mutation analysis. We used an ultra-sensitive mass spectrometry based system to highlight the presence of an EGFR-activating mutation in both isolated CTCs and plasma cell-free DNA (cf-DNA), and demonstrate concordance with the original tumor-biopsy samples. We have clinically validated our multiplexed microfluidic chip for the ultra high-throughput, low-cost and label-free enrichment of CTCs. Retrieved cells were unlabeled and viable, enabling potential propagation and real-time downstream analysis using next generation sequencing (NGS) or proteomic analysis.

Chapter 17 Sterile Plate-Based Vitrification of Adherent Human Pluripotent Stem Cells and Their Derivatives Using the TWIST Method.

PubMed

Neubauer, Julia C; Stracke, Frank; Zimmermann, Heiko

2017-01-01

Due to their high biological complexity, e.g., their close cell-to-cell contacts, cryopreservation of human pluripotent stem cells with standard slow-rate protocols often is inefficient and can hardly be standardized. Vitrification that means ultrafast freezing already showed very good viability and recovery rates for this sensitive cell system, but is only applicable for low cell numbers, bears a high risk of contamination, and can hardly be implemented under GxP regulations. In this chapter, a sterile plate-based vitrification method for adherent pluripotent stem cells and their derivatives is presented based on a procedure and device for human embryonic stem cells developed by Beier et al. (Cryobiology 66:8-16, 2013). This protocol overcomes the limitations of conventional vitrification procedures resulting in the highly efficient preservation of ready-to-use adherent pluripotent stem cells with the possibility of vitrifying cells in multi-well formats for direct application in high-throughput screenings.

High-Density Droplet Microarray of Individually Addressable Electrochemical Cells.

PubMed

Zhang, Huijie; Oellers, Tobias; Feng, Wenqian; Abdulazim, Tarik; Saw, En Ning; Ludwig, Alfred; Levkin, Pavel A; Plumeré, Nicolas

2017-06-06

Microarray technology has shown great potential for various types of high-throughput screening applications. The main read-out methods of most microarray platforms, however, are based on optical techniques, limiting the scope of potential applications of such powerful screening technology. Electrochemical methods possess numerous complementary advantages over optical detection methods, including its label-free nature, capability of quantitative monitoring of various reporter molecules, and the ability to not only detect but also address compositions of individual compartments. However, application of electrochemical methods for the purpose of high-throughput screening remains very limited. In this work, we develop a high-density individually addressable electrochemical droplet microarray (eDMA). The eDMA allows for the detection of redox-active reporter molecules irrespective of their electrochemical reversibility in individual nanoliter-sized droplets. Orthogonal band microelectrodes are arranged to form at their intersections an array of three-electrode systems for precise control of the applied potential, which enables direct read-out of the current related to analyte detection. The band microelectrode array is covered with a layer of permeable porous polymethacrylate functionalized with a highly hydrophobic-hydrophilic pattern, forming spatially separated nanoliter-sized droplets on top of each electrochemical cell. Electrochemical characterization of single droplets demonstrates that the underlying electrode system is accessible to redox-active molecules through the hydrophilic polymeric pattern and that the nonwettable hydrophobic boundaries can spatially separate neighboring cells effectively. The eDMA technology opens the possibility to combine the high-throughput biochemical or living cell screenings using the droplet microarray platform with the sequential electrochemical read-out of individual droplets.

Multi-MHz laser-scanning single-cell fluorescence microscopy by spatiotemporally encoded virtual source array

PubMed Central

Wu, Jianglai; Tang, Anson H. L.; Mok, Aaron T. Y.; Yan, Wenwei; Chan, Godfrey C. F.; Wong, Kenneth K. Y.; Tsia, Kevin K.

2017-01-01

Apart from the spatial resolution enhancement, scaling of temporal resolution, equivalently the imaging throughput, of fluorescence microscopy is of equal importance in advancing cell biology and clinical diagnostics. Yet, this attribute has mostly been overlooked because of the inherent speed limitation of existing imaging strategies. To address the challenge, we employ an all-optical laser-scanning mechanism, enabled by an array of reconfigurable spatiotemporally-encoded virtual sources, to demonstrate ultrafast fluorescence microscopy at line-scan rate as high as 8 MHz. We show that this technique enables high-throughput single-cell microfluidic fluorescence imaging at 75,000 cells/second and high-speed cellular 2D dynamical imaging at 3,000 frames per second, outperforming the state-of-the-art high-speed cameras and the gold-standard laser scanning strategies. Together with its wide compatibility to the existing imaging modalities, this technology could empower new forms of high-throughput and high-speed biological fluorescence microscopy that was once challenged. PMID:28966855

Analysis of JC virus DNA replication using a quantitative and high-throughput assay

PubMed Central

Shin, Jong; Phelan, Paul J.; Chhum, Panharith; Bashkenova, Nazym; Yim, Sung; Parker, Robert; Gagnon, David; Gjoerup, Ole; Archambault, Jacques; Bullock, Peter A.

2015-01-01

Progressive Multifocal Leukoencephalopathy (PML) is caused by lytic replication of JC virus (JCV) in specific cells of the central nervous system. Like other polyomaviruses, JCV encodes a large T-antigen helicase needed for replication of the viral DNA. Here, we report the development of a luciferase-based, quantitative and high-throughput assay of JCV DNA replication in C33A cells, which, unlike the glial cell lines Hs 683 and U87, accumulate high levels of nuclear T-ag needed for robust replication. Using this assay, we investigated the requirement for different domains of T-ag, and for specific sequences within and flanking the viral origin, in JCV DNA replication. Beyond providing validation of the assay, these studies revealed an important stimulatory role of the transcription factor NF1 in JCV DNA replication. Finally, we show that the assay can be used for inhibitor testing, highlighting its value for the identification of antiviral drugs targeting JCV DNA replication. PMID:25155200

High Throughput Screening Identifies Novel Lead Compounds with Activity against Larval, Juvenile and Adult Schistosoma mansoni

PubMed Central

Gardner, J. Mark F.; Bell, Andrew S.; Parkinson, Tanya; Bickle, Quentin

2016-01-01

An estimated 600 million people are affected by the helminth disease schistosomiasis caused by parasites of the genus Schistosoma. There is currently only one drug recommended for treating schistosomiasis, praziquantel (PZQ), which is effective against adult worms but not against the juvenile stage. In an attempt to identify improved drugs for treating the disease, we have carried out high throughput screening of a number of small molecule libraries with the aim of identifying lead compounds with balanced activity against all life stages of Schistosoma. A total of almost 300,000 compounds were screened using a high throughput assay based on motility of worm larvae and image analysis of assay plates. Hits were screened against juvenile and adult worms to identify broadly active compounds and against a mammalian cell line to assess cytotoxicity. A number of compounds were identified as promising leads for further chemical optimization. PMID:27128493

Next-Generation High-Throughput Functional Annotation of Microbial Genomes.

PubMed

Baric, Ralph S; Crosson, Sean; Damania, Blossom; Miller, Samuel I; Rubin, Eric J

2016-10-04

Host infection by microbial pathogens cues global changes in microbial and host cell biology that facilitate microbial replication and disease. The complete maps of thousands of bacterial and viral genomes have recently been defined; however, the rate at which physiological or biochemical functions have been assigned to genes has greatly lagged. The National Institute of Allergy and Infectious Diseases (NIAID) addressed this gap by creating functional genomics centers dedicated to developing high-throughput approaches to assign gene function. These centers require broad-based and collaborative research programs to generate and integrate diverse data to achieve a comprehensive understanding of microbial pathogenesis. High-throughput functional genomics can lead to new therapeutics and better understanding of the next generation of emerging pathogens by rapidly defining new general mechanisms by which organisms cause disease and replicate in host tissues and by facilitating the rate at which functional data reach the scientific community. Copyright © 2016 Baric et al.

Lensless on-chip imaging of cells provides a new tool for high-throughput cell-biology and medical diagnostics.

PubMed

Mudanyali, Onur; Erlinger, Anthony; Seo, Sungkyu; Su, Ting-Wei; Tseng, Derek; Ozcan, Aydogan

2009-12-14

Conventional optical microscopes image cells by use of objective lenses that work together with other lenses and optical components. While quite effective, this classical approach has certain limitations for miniaturization of the imaging platform to make it compatible with the advanced state of the art in microfluidics. In this report, we introduce experimental details of a lensless on-chip imaging concept termed LUCAS (Lensless Ultra-wide field-of-view Cell monitoring Array platform based on Shadow imaging) that does not require any microscope objectives or other bulky optical components to image a heterogeneous cell solution over an ultra-wide field of view that can span as large as approximately 18 cm(2). Moreover, unlike conventional microscopes, LUCAS can image a heterogeneous cell solution of interest over a depth-of-field of approximately 5 mm without the need for refocusing which corresponds to up to approximately 9 mL sample volume. This imaging platform records the shadows (i.e., lensless digital holograms) of each cell of interest within its field of view, and automated digital processing of these cell shadows can determine the type, the count and the relative positions of cells within the solution. Because it does not require any bulky optical components or mechanical scanning stages it offers a significantly miniaturized platform that at the same time reduces the cost, which is quite important for especially point of care diagnostic tools. Furthermore, the imaging throughput of this platform is orders of magnitude better than conventional optical microscopes, which could be exceedingly valuable for high-throughput cell-biology experiments.

Lensless On-chip Imaging of Cells Provides a New Tool for High-throughput Cell-Biology and Medical Diagnostics

PubMed Central

Mudanyali, Onur; Erlinger, Anthony; Seo, Sungkyu; Su, Ting-Wei; Tseng, Derek; Ozcan, Aydogan

2009-01-01

Conventional optical microscopes image cells by use of objective lenses that work together with other lenses and optical components. While quite effective, this classical approach has certain limitations for miniaturization of the imaging platform to make it compatible with the advanced state of the art in microfluidics. In this report, we introduce experimental details of a lensless on-chip imaging concept termed LUCAS (Lensless Ultra-wide field-of-view Cell monitoring Array platform based on Shadow imaging) that does not require any microscope objectives or other bulky optical components to image a heterogeneous cell solution over an ultra-wide field of view that can span as large as ~18 cm2. Moreover, unlike conventional microscopes, LUCAS can image a heterogeneous cell solution of interest over a depth-of-field of ~5 mm without the need for refocusing which corresponds to up to ~9 mL sample volume. This imaging platform records the shadows (i.e., lensless digital holograms) of each cell of interest within its field of view, and automated digital processing of these cell shadows can determine the type, the count and the relative positions of cells within the solution. Because it does not require any bulky optical components or mechanical scanning stages it offers a significantly miniaturized platform that at the same time reduces the cost, which is quite important for especially point of care diagnostic tools. Furthermore, the imaging throughput of this platform is orders of magnitude better than conventional optical microscopes, which could be exceedingly valuable for high-throughput cell-biology experiments. PMID:20010542

Impact of media and antifoam selection on monoclonal antibody production and quality using a high throughput micro‐bioreactor system

PubMed Central

Velugula‐Yellela, Sai Rashmika; Williams, Abasha; Trunfio, Nicholas; Hsu, Chih‐Jung; Chavez, Brittany; Yoon, Seongkyu

2017-01-01

Monoclonal antibody production in commercial scale cell culture bioprocessing requires a thorough understanding of the engineering process and components used throughout manufacturing. It is important to identify high impact components early on during the lifecycle of a biotechnology‐derived product. While cell culture media selection is of obvious importance to the health and productivity of mammalian bioreactor operations, other components such as antifoam selection can also play an important role in bioreactor cell culture. Silicone polymer‐based antifoams were known to have negative impacts on cell health, production, and downstream filtration and purification operations. High throughput screening in micro‐scale bioreactors provides an efficient strategy to identify initial operating parameters. Here, we utilized a micro‐scale parallel bioreactor system to study an IgG1 producing CHO cell line, to screen Dynamis, ProCHO5, PowerCHO2, EX‐Cell Advanced, and OptiCHO media, and 204, C, EX‐Cell, SE‐15, and Y‐30 antifoams and their impacts on IgG1 production, cell growth, aggregation, and process control. This study found ProCHO5, EX‐Cell Advanced, and PowerCHO2 media supported strong cellular growth profiles, with an IVCD of 25‐35 × 106 cells‐d/mL, while maintaining specific antibody production (Qp > 2 pg/cell‐d) for our model cell line and a monomer percentage above 94%. Antifoams C, EX‐Cell, and SE‐15 were capable of providing adequate control of foaming while antifoam 204 and Y‐30 noticeably stunted cellular growth. This work highlights the utility of high throughput micro bioreactors and the importance of identifying both positive and negative impacts of media and antifoam selection on a model IgG1 producing CHO cell line. © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 34:262–270, 2018 PMID:29086492

High-throughput identification of antigen-specific TCRs by TCR gene capture.

PubMed

Linnemann, Carsten; Heemskerk, Bianca; Kvistborg, Pia; Kluin, Roelof J C; Bolotin, Dmitriy A; Chen, Xiaojing; Bresser, Kaspar; Nieuwland, Marja; Schotte, Remko; Michels, Samira; Gomez-Eerland, Raquel; Jahn, Lorenz; Hombrink, Pleun; Legrand, Nicolas; Shu, Chengyi Jenny; Mamedov, Ilgar Z; Velds, Arno; Blank, Christian U; Haanen, John B A G; Turchaninova, Maria A; Kerkhoven, Ron M; Spits, Hergen; Hadrup, Sine Reker; Heemskerk, Mirjam H M; Blankenstein, Thomas; Chudakov, Dmitriy M; Bendle, Gavin M; Schumacher, Ton N M

2013-11-01

The transfer of T cell receptor (TCR) genes into patient T cells is a promising approach for the treatment of both viral infections and cancer. Although efficient methods exist to identify antibodies for the treatment of these diseases, comparable strategies to identify TCRs have been lacking. We have developed a high-throughput DNA-based strategy to identify TCR sequences by the capture and sequencing of genomic DNA fragments encoding the TCR genes. We establish the value of this approach by assembling a large library of cancer germline tumor antigen-reactive TCRs. Furthermore, by exploiting the quantitative nature of TCR gene capture, we show the feasibility of identifying antigen-specific TCRs in oligoclonal T cell populations from either human material or TCR-humanized mice. Finally, we demonstrate the ability to identify tumor-reactive TCRs within intratumoral T cell subsets without knowledge of antigen specificities, which may be the first step toward the development of autologous TCR gene therapy to target patient-specific neoantigens in human cancer.

Efficient production of a gene mutant cell line through integrating TALENs and high-throughput cell cloning.

PubMed

Sun, Changhong; Fan, Yu; Li, Juan; Wang, Gancheng; Zhang, Hanshuo; Xi, Jianzhong Jeff

2015-02-01

Transcription activator-like effectors (TALEs) are becoming powerful DNA-targeting tools in a variety of mammalian cells and model organisms. However, generating a stable cell line with specific gene mutations in a simple and rapid manner remains a challenging task. Here, we report a new method to efficiently produce monoclonal cells using integrated TALE nuclease technology and a series of high-throughput cell cloning approaches. Following this method, we obtained three mTOR mutant 293T cell lines within 2 months, which included one homozygous mutant line. © 2014 Society for Laboratory Automation and Screening.

CrossCheck: an open-source web tool for high-throughput screen data analysis.

PubMed

Najafov, Jamil; Najafov, Ayaz

2017-07-19

Modern high-throughput screening methods allow researchers to generate large datasets that potentially contain important biological information. However, oftentimes, picking relevant hits from such screens and generating testable hypotheses requires training in bioinformatics and the skills to efficiently perform database mining. There are currently no tools available to general public that allow users to cross-reference their screen datasets with published screen datasets. To this end, we developed CrossCheck, an online platform for high-throughput screen data analysis. CrossCheck is a centralized database that allows effortless comparison of the user-entered list of gene symbols with 16,231 published datasets. These datasets include published data from genome-wide RNAi and CRISPR screens, interactome proteomics and phosphoproteomics screens, cancer mutation databases, low-throughput studies of major cell signaling mediators, such as kinases, E3 ubiquitin ligases and phosphatases, and gene ontological information. Moreover, CrossCheck includes a novel database of predicted protein kinase substrates, which was developed using proteome-wide consensus motif searches. CrossCheck dramatically simplifies high-throughput screen data analysis and enables researchers to dig deep into the published literature and streamline data-driven hypothesis generation. CrossCheck is freely accessible as a web-based application at http://proteinguru.com/crosscheck.

Microchip-Based Single-Cell Functional Proteomics for Biomedical Applications

PubMed Central

Lu, Yao; Yang, Liu; Wei, Wei; Shi, Qihui

2017-01-01

Cellular heterogeneity has been widely recognized but only recently have single cell tools become available that allow characterizing heterogeneity at the genomic and proteomic levels. We review the technological advances in microchip-based toolkits for single-cell functional proteomics. Each of these tools has distinct advantages and limitations, and a few have advanced toward being applied to address biological or clinical problems that fail to be addressed by traditional population-based methods. High-throughput single-cell proteomic assays generate high-dimensional data sets that contain new information and thus require developing new analytical framework to extract new biology. In this review article, we highlight a few biological and clinical applications in which the microchip-based single-cell proteomic tools provide unique advantages. The examples include resolving functional heterogeneity and dynamics of immune cells, dissecting cell-cell interaction by creating well-contolled on-chip microenvironment, capturing high-resolution snapshots of immune system functions in patients for better immunotherapy and elucidating phosphoprotein signaling networks in cancer cells for guiding effective molecularly targeted therapies. PMID:28280819

Nano-LC FTICR tandem mass spectrometry for top-down proteomics: routine baseline unit mass resolution of whole cell lysate proteins up to 72 kDa.

PubMed

Tipton, Jeremiah D; Tran, John C; Catherman, Adam D; Ahlf, Dorothy R; Durbin, Kenneth R; Lee, Ji Eun; Kellie, John F; Kelleher, Neil L; Hendrickson, Christopher L; Marshall, Alan G

2012-03-06

Current high-throughput top-down proteomic platforms provide routine identification of proteins less than 25 kDa with 4-D separations. This short communication reports the application of technological developments over the past few years that improve protein identification and characterization for masses greater than 25 kDa. Advances in separation science have allowed increased numbers of proteins to be identified, especially by nanoliquid chromatography (nLC) prior to mass spectrometry (MS) analysis. Further, a goal of high-throughput top-down proteomics is to extend the mass range for routine nLC MS analysis up to 80 kDa because gene sequence analysis predicts that ~70% of the human proteome is transcribed to be less than 80 kDa. Normally, large proteins greater than 50 kDa are identified and characterized by top-down proteomics through fraction collection and direct infusion at relatively low throughput. Further, other MS-based techniques provide top-down protein characterization, however at low resolution for intact mass measurement. Here, we present analysis of standard (up to 78 kDa) and whole cell lysate proteins by Fourier transform ion cyclotron resonance mass spectrometry (nLC electrospray ionization (ESI) FTICR MS). The separation platform reduced the complexity of the protein matrix so that, at 14.5 T, proteins from whole cell lysate up to 72 kDa are baseline mass resolved on a nano-LC chromatographic time scale. Further, the results document routine identification of proteins at improved throughput based on accurate mass measurement (less than 10 ppm mass error) of precursor and fragment ions for proteins up to 50 kDa.

An Automated High-throughput Array Microscope for Cancer Cell Mechanics

NASA Astrophysics Data System (ADS)

Cribb, Jeremy A.; Osborne, Lukas D.; Beicker, Kellie; Psioda, Matthew; Chen, Jian; O'Brien, E. Timothy; Taylor, Russell M., II; Vicci, Leandra; Hsiao, Joe Ping-Lin; Shao, Chong; Falvo, Michael; Ibrahim, Joseph G.; Wood, Kris C.; Blobe, Gerard C.; Superfine, Richard

2016-06-01

Changes in cellular mechanical properties correlate with the progression of metastatic cancer along the epithelial-to-mesenchymal transition (EMT). Few high-throughput methodologies exist that measure cell compliance, which can be used to understand the impact of genetic alterations or to screen the efficacy of chemotherapeutic agents. We have developed a novel array high-throughput microscope (AHTM) system that combines the convenience of the standard 96-well plate with the ability to image cultured cells and membrane-bound microbeads in twelve independently-focusing channels simultaneously, visiting all wells in eight steps. We use the AHTM and passive bead rheology techniques to determine the relative compliance of human pancreatic ductal epithelial (HPDE) cells, h-TERT transformed HPDE cells (HPNE), and four gain-of-function constructs related to EMT. The AHTM found HPNE, H-ras, Myr-AKT, and Bcl2 transfected cells more compliant relative to controls, consistent with parallel tests using atomic force microscopy and invasion assays, proving the AHTM capable of screening for changes in mechanical phenotype.

Laser processes and system technology for the production of high-efficient crystalline solar cells

NASA Astrophysics Data System (ADS)

Mayerhofer, R.; Hendel, R.; Zhu, Wenjie; Geiger, S.

2012-10-01

The laser as an industrial tool is an essential part of today's solar cell production. Due to the on-going efforts in the solar industry, to increase the cell efficiency, more and more laser-based processes, which have been discussed and tested at lab-scale for many years, are now being implemented in mass production lines. In order to cope with throughput requirements, standard laser concepts have to be improved continuously with respect to available average power levels, repetition rates or beam profile. Some of the laser concepts, that showed high potential in the past couple of years, will be substituted by other, more economic laser types. Furthermore, requirements for processing with less-heat affected zones fuel the development of industry-ready ultra short pulsed lasers with pulse widths even below the picosecond range. In 2011, the German Ministry of Education and Research (BMBF) had launched the program "PV-Innovation Alliance", with the aim to support the rapid transfer of high-efficiency processes out of development departments and research institutes into solar cell production lines. Here, lasers play an important role as production tools, allowing the fast implementation of high-performance solar cell concepts. We will report on the results achieved within the joint project FUTUREFAB, where efficiency optimization, throughput enhancement and cost reduction are the main goals. Here, the presentation will focus on laser processes like selective emitter doping and ablation of dielectric layers. An indispensable part of the efforts towards cost reduction in solar cell production is the improvement of wafer handling and throughput capabilities of the laser processing system. Therefore, the presentation will also elaborate on new developments in the design of complete production machines.

High-Throughput Screening of Myometrial Calcium-Mobilization to Identify Modulators of Uterine Contractility

PubMed Central

Herington, Jennifer L.; Swale, Daniel R.; Brown, Naoko; Shelton, Elaine L.; Choi, Hyehun; Williams, Charles H.; Hong, Charles C.; Paria, Bibhash C.; Denton, Jerod S.; Reese, Jeff

2015-01-01

The uterine myometrium (UT-myo) is a therapeutic target for preterm labor, labor induction, and postpartum hemorrhage. Stimulation of intracellular Ca2+-release in UT-myo cells by oxytocin is a final pathway controlling myometrial contractions. The goal of this study was to develop a dual-addition assay for high-throughput screening of small molecular compounds, which could regulate Ca2+-mobilization in UT-myo cells, and hence, myometrial contractions. Primary murine UT-myo cells in 384-well plates were loaded with a Ca2+-sensitive fluorescent probe, and then screened for inducers of Ca2+-mobilization and inhibitors of oxytocin-induced Ca2+-mobilization. The assay exhibited robust screening statistics (Z´ = 0.73), DMSO-tolerance, and was validated for high-throughput screening against 2,727 small molecules from the Spectrum, NIH Clinical I and II collections of well-annotated compounds. The screen revealed a hit-rate of 1.80% for agonist and 1.39% for antagonist compounds. Concentration-dependent responses of hit-compounds demonstrated an EC50 less than 10μM for 21 hit-antagonist compounds, compared to only 7 hit-agonist compounds. Subsequent studies focused on hit-antagonist compounds. Based on the percent inhibition and functional annotation analyses, we selected 4 confirmed hit-antagonist compounds (benzbromarone, dipyridamole, fenoterol hydrobromide and nisoldipine) for further analysis. Using an ex vivo isometric contractility assay, each compound significantly inhibited uterine contractility, at different potencies (IC50). Overall, these results demonstrate for the first time that high-throughput small-molecules screening of myometrial Ca2+-mobilization is an ideal primary approach for discovering modulators of uterine contractility. PMID:26600013

Large-scale protein-protein interactions detection by integrating big biosensing data with computational model.

PubMed

You, Zhu-Hong; Li, Shuai; Gao, Xin; Luo, Xin; Ji, Zhen

2014-01-01

Protein-protein interactions are the basis of biological functions, and studying these interactions on a molecular level is of crucial importance for understanding the functionality of a living cell. During the past decade, biosensors have emerged as an important tool for the high-throughput identification of proteins and their interactions. However, the high-throughput experimental methods for identifying PPIs are both time-consuming and expensive. On the other hand, high-throughput PPI data are often associated with high false-positive and high false-negative rates. Targeting at these problems, we propose a method for PPI detection by integrating biosensor-based PPI data with a novel computational model. This method was developed based on the algorithm of extreme learning machine combined with a novel representation of protein sequence descriptor. When performed on the large-scale human protein interaction dataset, the proposed method achieved 84.8% prediction accuracy with 84.08% sensitivity at the specificity of 85.53%. We conducted more extensive experiments to compare the proposed method with the state-of-the-art techniques, support vector machine. The achieved results demonstrate that our approach is very promising for detecting new PPIs, and it can be a helpful supplement for biosensor-based PPI data detection.

High Throughput T Epitope Mapping and Vaccine Development

PubMed Central

Li Pira, Giuseppina; Ivaldi, Federico; Moretti, Paolo; Manca, Fabrizio

2010-01-01

Mapping of antigenic peptide sequences from proteins of relevant pathogens recognized by T helper (Th) and by cytolytic T lymphocytes (CTL) is crucial for vaccine development. In fact, mapping of T-cell epitopes provides useful information for the design of peptide-based vaccines and of peptide libraries to monitor specific cellular immunity in protected individuals, patients and vaccinees. Nevertheless, epitope mapping is a challenging task. In fact, large panels of overlapping peptides need to be tested with lymphocytes to identify the sequences that induce a T-cell response. Since numerous peptide panels from antigenic proteins are to be screened, lymphocytes available from human subjects are a limiting factor. To overcome this limitation, high throughput (HTP) approaches based on miniaturization and automation of T-cell assays are needed. Here we consider the most recent applications of the HTP approach to T epitope mapping. The alternative or complementary use of in silico prediction and experimental epitope definition is discussed in the context of the recent literature. The currently used methods are described with special reference to the possibility of applying the HTP concept to make epitope mapping an easier procedure in terms of time, workload, reagents, cells and overall cost. PMID:20617148

Label-free detection of aggregated platelets in blood by machine-learning-aided optofluidic time-stretch microscopy.

PubMed

Jiang, Yiyue; Lei, Cheng; Yasumoto, Atsushi; Kobayashi, Hirofumi; Aisaka, Yuri; Ito, Takuro; Guo, Baoshan; Nitta, Nao; Kutsuna, Natsumaro; Ozeki, Yasuyuki; Nakagawa, Atsuhiro; Yatomi, Yutaka; Goda, Keisuke

2017-07-11

According to WHO, about 10 million new cases of thrombotic disorders are diagnosed worldwide every year. Thrombotic disorders, including atherothrombosis (the leading cause of death in the US and Europe), are induced by occlusion of blood vessels, due to the formation of blood clots in which aggregated platelets play an important role. The presence of aggregated platelets in blood may be related to atherothrombosis (especially acute myocardial infarction) and is, hence, useful as a potential biomarker for the disease. However, conventional high-throughput blood analysers fail to accurately identify aggregated platelets in blood. Here we present an in vitro on-chip assay for label-free, single-cell image-based detection of aggregated platelets in human blood. This assay builds on a combination of optofluidic time-stretch microscopy on a microfluidic chip operating at a high throughput of 10 000 blood cells per second with machine learning, enabling morphology-based identification and enumeration of aggregated platelets in a short period of time. By performing cell classification with machine learning, we differentiate aggregated platelets from single platelets and white blood cells with a high specificity and sensitivity of 96.6% for both. Our results indicate that the assay is potentially promising as predictive diagnosis and therapeutic monitoring of thrombotic disorders in clinical settings.

[HTRF-based high-throughput PGE2 release prohibition model and application in discovering traditional Chinese medicine active ingredients].

PubMed

Bai, Zhi-Ru; Fei, Hong-Qiang; Li, Na; Cao, Liang; Zhang, Chen-Feng; Wang, Tuan-Jie; Ding, Gang; Wang, Zhen-Zhong; Xiao, Wei

2016-02-01

Prostaglandin (PG) E2 is an active substance in pathological and physiological mechanisms, such as inflammation and pain. The in vitro high-throughput assay for screening the inhibitors of reducing PEG2 production is a useful method for finding out antiphlogistic and analgesic candidates. The assay was based on LPS-induced PGE2 production model using a homogeneous time-resolved fluorescence(HTRF) PGE2 testing kit combined with liquid handling automation and detection instruments. The critical steps, including the cell density optimization and IC50 values determination of a positive compound, were taken to verify the stability and sensibility of the assay. Low intra-plate, inter-plate and day-to-day variability were observed in this 384-well, high-throughput format assay. Totally 5 121 samples were selected from the company's traditional Chinese medicine(TCM) material base library and used to screen PGE2 inhibitors. In this model, the cell plating density was 2 000 cells for each well; the average IC₅₀ value for positive compounds was (7.3±0.1) μmol; the Z' factor for test plates was more than 0.5 and averaged at 0.7. Among the 5 121 samples, 228 components exhibited a PGE2 production prohibition rate of more than 50%, and 23 components exhibited more than 80%. This model reached the expected standards in data stability and accuracy, indicating the reliability and authenticity of the screening results. The automated screening system was introduced to make the model fast and efficient, with a average daily screening amount exceeding 14 000 data points and provide a new model for discovering new anti-inflammatory and analgesic drug and quickly screening effective constituents of TCM in the early stage. Copyright© by the Chinese Pharmaceutical Association.

Moving Toward Integrating Gene Expression Profiling Into High-Throughput Testing: A Gene Expression Biomarker Accurately Predicts Estrogen Receptor α Modulation in a Microarray Compendium

PubMed Central

Ryan, Natalia; Chorley, Brian; Tice, Raymond R.; Judson, Richard; Corton, J. Christopher

2016-01-01

Microarray profiling of chemical-induced effects is being increasingly used in medium- and high-throughput formats. Computational methods are described here to identify molecular targets from whole-genome microarray data using as an example the estrogen receptor α (ERα), often modulated by potential endocrine disrupting chemicals. ERα biomarker genes were identified by their consistent expression after exposure to 7 structurally diverse ERα agonists and 3 ERα antagonists in ERα-positive MCF-7 cells. Most of the biomarker genes were shown to be directly regulated by ERα as determined by ESR1 gene knockdown using siRNA as well as through chromatin immunoprecipitation coupled with DNA sequencing analysis of ERα-DNA interactions. The biomarker was evaluated as a predictive tool using the fold-change rank-based Running Fisher algorithm by comparison to annotated gene expression datasets from experiments using MCF-7 cells, including those evaluating the transcriptional effects of hormones and chemicals. Using 141 comparisons from chemical- and hormone-treated cells, the biomarker gave a balanced accuracy for prediction of ERα activation or suppression of 94% and 93%, respectively. The biomarker was able to correctly classify 18 out of 21 (86%) ER reference chemicals including “very weak” agonists. Importantly, the biomarker predictions accurately replicated predictions based on 18 in vitro high-throughput screening assays that queried different steps in ERα signaling. For 114 chemicals, the balanced accuracies were 95% and 98% for activation or suppression, respectively. These results demonstrate that the ERα gene expression biomarker can accurately identify ERα modulators in large collections of microarray data derived from MCF-7 cells. PMID:26865669

Industrializing electrophysiology: HT automated patch clamp on SyncroPatch® 96 using instant frozen cells.

PubMed

Polonchuk, Liudmila

2014-01-01

Patch-clamping is a powerful technique for investigating the ion channel function and regulation. However, its low throughput hampered profiling of large compound series in early drug development. Fortunately, automation has revolutionized the area of experimental electrophysiology over the past decade. Whereas the first automated patch-clamp instruments using the planar patch-clamp technology demonstrated rather a moderate throughput, few second-generation automated platforms recently launched by various companies have significantly increased ability to form a high number of high-resistance seals. Among them is SyncroPatch(®) 96 (Nanion Technologies GmbH, Munich, Germany), a fully automated giga-seal patch-clamp system with the highest throughput on the market. By recording from up to 96 cells simultaneously, the SyncroPatch(®) 96 allows to substantially increase throughput without compromising data quality. This chapter describes features of the innovative automated electrophysiology system and protocols used for a successful transfer of the established hERG assay to this high-throughput automated platform.

Global Profiling of Reactive Oxygen and Nitrogen Species in Biological Systems

PubMed Central

Zielonka, Jacek; Zielonka, Monika; Sikora, Adam; Adamus, Jan; Joseph, Joy; Hardy, Micael; Ouari, Olivier; Dranka, Brian P.; Kalyanaraman, Balaraman

2012-01-01

Herein we describe a high-throughput fluorescence and HPLC-based methodology for global profiling of reactive oxygen and nitrogen species (ROS/RNS) in biological systems. The combined use of HPLC and fluorescence detection is key to successful implementation and validation of this methodology. Included here are methods to specifically detect and quantitate the products formed from interaction between the ROS/RNS species and the fluorogenic probes, as follows: superoxide using hydroethidine, peroxynitrite using boronate-based probes, nitric oxide-derived nitrosating species with 4,5-diaminofluorescein, and hydrogen peroxide and other oxidants using 10-acetyl-3,7-dihydroxyphenoxazine (Amplex® Red) with and without horseradish peroxidase, respectively. In this study, we demonstrate real-time monitoring of ROS/RNS in activated macrophages using high-throughput fluorescence and HPLC methods. This global profiling approach, simultaneous detection of multiple ROS/RNS products of fluorescent probes, developed in this study will be useful in unraveling the complex role of ROS/RNS in redox regulation, cell signaling, and cellular oxidative processes and in high-throughput screening of anti-inflammatory antioxidants. PMID:22139901

Proteomic Analysis of Metabolic Responses to Biofuels and Chemicals in Photosynthetic Cyanobacteria.

PubMed

Sun, T; Chen, L; Zhang, W

2017-01-01

Recent progresses in various "omics" technologies have enabled quantitative measurements of biological molecules in a high-throughput manner. Among them, high-throughput proteomics is a rapidly advancing field that offers a new means to quantify metabolic changes at protein level, which has significantly facilitated our understanding of cellular process, such as protein synthesis, posttranslational modifications, and degradation in responding to environmental perturbations. Cyanobacteria are autotrophic prokaryotes that can perform oxygenic photosynthesis and have recently attracted significant attentions as one promising alternative to traditionally biomass-based "microbial cell factories" to produce green fuels and chemicals. However, early studies have shown that the low tolerance to toxic biofuels and chemicals represented one major hurdle for further improving productivity of the cyanobacterial production systems. To address the issue, metabolic responses and their regulation of cyanobacterial cells to toxic end-products need to be defined. In this chapter, we discuss recent progresses in interpreting cyanobacterial responses to biofuels and chemicals using high-throughput proteomics approach, aiming to provide insights and guidelines on how to enhance tolerance and productivity of biofuels or chemicals in the renewable cyanobacteria systems in the future. © 2017 Elsevier Inc. All rights reserved.

Three-Dimensional in Vitro Cell Culture Models in Drug Discovery and Drug Repositioning

PubMed Central

Langhans, Sigrid A.

2018-01-01

Drug development is a lengthy and costly process that proceeds through several stages from target identification to lead discovery and optimization, preclinical validation and clinical trials culminating in approval for clinical use. An important step in this process is high-throughput screening (HTS) of small compound libraries for lead identification. Currently, the majority of cell-based HTS is being carried out on cultured cells propagated in two-dimensions (2D) on plastic surfaces optimized for tissue culture. At the same time, compelling evidence suggests that cells cultured in these non-physiological conditions are not representative of cells residing in the complex microenvironment of a tissue. This discrepancy is thought to be a significant contributor to the high failure rate in drug discovery, where only a low percentage of drugs investigated ever make it through the gamut of testing and approval to the market. Thus, three-dimensional (3D) cell culture technologies that more closely resemble in vivo cell environments are now being pursued with intensity as they are expected to accommodate better precision in drug discovery. Here we will review common approaches to 3D culture, discuss the significance of 3D cultures in drug resistance and drug repositioning and address some of the challenges of applying 3D cell cultures to high-throughput drug discovery. PMID:29410625

Biomimetic macroporous hydrogel scaffolds in a high-throughput screening format for cell-based assays.

PubMed

Dainiak, Maria B; Savina, Irina N; Musolino, Isabella; Kumar, Ashok; Mattiasson, Bo; Galaev, Igor Yu

2008-01-01

Macroporous hydrogels (MHs) hold great promise as scaffolds in tissue engineering and cell-based assays. In this study, the possibility of combination of three-dimensional (3D) cell culture with a miniaturized screening format was demonstrated on human colon cancer HCT116, human acute myeloid leukemia KG-1 cells, and embryonic fibroblasts cultured on MHs (12.5 mm x 7.1 mm I.D.) in a 96-minicolumn plate format. MHs were prepared by cryogelation technique and functionalized by coating with type I collagen and by copolymerization with agmatine-based mimetic of cell adhesive peptide RGD (abRGDm). Cancer cells formed multicellular aggregates while fibroblasts formed adhesions on abRGDm-containing and collagen-MHs but not on plain MHs, as was demonstrated by scanning electron microscopy. HCT116 and KG-1 cells grown as aggregates were more resistant to the treatment with cis-diaminedichloroplatinum (II) (cisplatin) and cytosine 1-beta-D-arabinofuranoside (Ara-C), respectively, during the first 18-24 h of incubation, than single cells grown on unmodified MH. HCT116 cells grown as 2D cultures in conventional 96-well tissue culture plates were 1.5- to 3.5-fold more sensitive to the treatment with 70 microM cisplatin than cells in 3D cultures in functionalized MHs. Further development of the described experimental system including matching of a specific cell type with appropriate extracellular matrix (ECM) components and 3D cocultures on ECM-modified MHs may provide a realistic in vitro experimental model for high-throughput toxicity tests.

Tracking protein aggregation and mislocalization in cells with flow cytometry.

PubMed

Ramdzan, Yasmin M; Polling, Saskia; Chia, Cheryl P Z; Ng, Ivan H W; Ormsby, Angelique R; Croft, Nathan P; Purcell, Anthony W; Bogoyevitch, Marie A; Ng, Dominic C H; Gleeson, Paul A; Hatters, Danny M

2012-03-18

We applied pulse-shape analysis (PulSA) to monitor protein localization changes in mammalian cells by flow cytometry. PulSA enabled high-throughput tracking of protein aggregation, translocation from the cytoplasm to the nucleus and trafficking from the plasma membrane to the Golgi as well as stress-granule formation. Combining PulSA with tetracysteine-based oligomer sensors in a cell model of Huntington's disease enabled further separation of cells enriched with monomers, oligomers and inclusion bodies.

Adaptation of a Simple Microfluidic Platform for High-Dimensional Quantitative Morphological Analysis of Human Mesenchymal Stromal Cells on Polystyrene-Based Substrates.

PubMed

Lam, Johnny; Marklein, Ross A; Jimenez-Torres, Jose A; Beebe, David J; Bauer, Steven R; Sung, Kyung E

2017-12-01

Multipotent stromal cells (MSCs, often called mesenchymal stem cells) have garnered significant attention within the field of regenerative medicine because of their purported ability to differentiate down musculoskeletal lineages. Given the inherent heterogeneity of MSC populations, recent studies have suggested that cell morphology may be indicative of MSC differentiation potential. Toward improving current methods and developing simple yet effective approaches for the morphological evaluation of MSCs, we combined passive pumping microfluidic technology with high-dimensional morphological characterization to produce robust tools for standardized high-throughput analysis. Using ultraviolet (UV) light as a modality for reproducible polystyrene substrate modification, we show that MSCs seeded on microfluidic straight channel devices incorporating UV-exposed substrates exhibited morphological changes that responded accordingly to the degree of substrate modification. Substrate modification also effected greater morphological changes in MSCs seeded at a lower rather than higher density within microfluidic channels. Despite largely comparable trends in morphology, MSCs seeded in microscale as opposed to traditional macroscale platforms displayed much higher sensitivity to changes in substrate properties. In summary, we adapted and qualified microfluidic cell culture platforms comprising simple straight channel arrays as a viable and robust tool for high-throughput quantitative morphological analysis to study cell-material interactions.

Microarrays for the evaluation of cell-biomaterial surface interactions

NASA Astrophysics Data System (ADS)

Thissen, H.; Johnson, G.; McFarland, G.; Verbiest, B. C. H.; Gengenbach, T.; Voelcker, N. H.

2007-01-01

The evaluation of cell-material surface interactions is important for the design of novel biomaterials which are used in a variety of biomedical applications. While traditional in vitro test methods have routinely used samples of relatively large size, microarrays representing different biomaterials offer many advantages, including high throughput and reduced sample handling. Here, we describe the simultaneous cell-based testing of matrices of polymeric biomaterials, arrayed on glass slides with a low cell-attachment background coating. Arrays were constructed using a microarray robot at 6 fold redundancy with solid pins having a diameter of 375 μm. Printed solutions contained at least one monomer, an initiator and a bifunctional crosslinker. After subsequent UV polymerisation, the arrays were washed and characterised by X-ray photoelectron spectroscopy. Cell culture experiments were carried out over 24 hours using HeLa cells. After labelling with CellTracker ® Green for the final hour of incubation and subsequent fixation, the arrays were scanned. In addition, individual spots were also viewed by fluorescence microscopy. The evaluation of cell-surface interactions in high-throughput assays as demonstrated here is a key enabling technology for the effective development of future biomaterials.

The growing impact of lyophilized cell-free protein expression systems

PubMed Central

Hunt, J. Porter; Yang, Seung Ook; Wilding, Kristen M.; Bundy, Bradley C.

2017-01-01

ABSTRACT Recently reported shelf-stable, on-demand protein synthesis platforms are enabling new possibilities in biotherapeutics, biosensing, biocatalysis, and high throughput protein expression. Lyophilized cell-free protein expression systems not only overcome cold-storage limitations, but also enable stockpiling for on-demand synthesis and completely sterilize the protein synthesis platform. Recently reported high-yield synthesis of cytotoxic protein Onconase from lyophilized E. coli extract preparations demonstrates the utility of lyophilized cell-free protein expression and its potential for creating on-demand biotherapeutics, vaccines, biosensors, biocatalysts, and high throughput protein synthesis. PMID:27791452

A High-throughput Assay for mRNA Silencing in Primary Cortical Neurons in vitro with Oligonucleotide Therapeutics.

PubMed

Alterman, Julia F; Coles, Andrew H; Hall, Lauren M; Aronin, Neil; Khvorova, Anastasia; Didiot, Marie-Cécile

2017-08-20

Primary neurons represent an ideal cellular system for the identification of therapeutic oligonucleotides for the treatment of neurodegenerative diseases. However, due to the sensitive nature of primary cells, the transfection of small interfering RNAs (siRNA) using classical methods is laborious and often shows low efficiency. Recent progress in oligonucleotide chemistry has enabled the development of stabilized and hydrophobically modified small interfering RNAs (hsiRNAs). This new class of oligonucleotide therapeutics shows extremely efficient self-delivery properties and supports potent and durable effects in vitro and in vivo . We have developed a high-throughput in vitro assay to identify and test hsiRNAs in primary neuronal cultures. To simply, rapidly, and accurately quantify the mRNA silencing of hundreds of hsiRNAs, we use the QuantiGene 2.0 quantitative gene expression assay. This high-throughput, 96-well plate-based assay can quantify mRNA levels directly from sample lysate. Here, we describe a method to prepare short-term cultures of mouse primary cortical neurons in a 96-well plate format for high-throughput testing of oligonucleotide therapeutics. This method supports the testing of hsiRNA libraries and the identification of potential therapeutics within just two weeks. We detail methodologies of our high throughput assay workflow from primary neuron preparation to data analysis. This method can help identify oligonucleotide therapeutics for treatment of various neurological diseases.

High-throughput imaging of heterogeneous cell organelles with an X-ray laser (CXIDB ID 25)

DOE Data Explorer

Hantke, Max, F.

2014-11-17

Preprocessed detector images that were used for the paper "High-throughput imaging of heterogeneous cell organelles with an X-ray laser". The CXI file contains the entire recorded data - including both hits and blanks. It also includes down-sampled images and LCLS machine parameters. Additionally, the Cheetah configuration file is attached that was used to create the pre-processed data.

Application of Signaling Pathway-Based Adverse Outcome Pathways and High Throughput Toxicokinetic-PBPK for Developmental Cardiac Malformations

EPA Science Inventory

Associating putative molecular initiating events (MIE) with downstream cell signaling pathways and modeling fetal exposure kinetics is an important challenge for integration in developmental systems toxicology. Here, we describe an integrative systems toxicology model for develop...

High-speed cell recognition algorithm for ultrafast flow cytometer imaging system.

PubMed

Zhao, Wanyue; Wang, Chao; Chen, Hongwei; Chen, Minghua; Yang, Sigang

2018-04-01

An optical time-stretch flow imaging system enables high-throughput examination of cells/particles with unprecedented high speed and resolution. A significant amount of raw image data is produced. A high-speed cell recognition algorithm is, therefore, highly demanded to analyze large amounts of data efficiently. A high-speed cell recognition algorithm consisting of two-stage cascaded detection and Gaussian mixture model (GMM) classification is proposed. The first stage of detection extracts cell regions. The second stage integrates distance transform and the watershed algorithm to separate clustered cells. Finally, the cells detected are classified by GMM. We compared the performance of our algorithm with support vector machine. Results show that our algorithm increases the running speed by over 150% without sacrificing the recognition accuracy. This algorithm provides a promising solution for high-throughput and automated cell imaging and classification in the ultrafast flow cytometer imaging platform. (2018) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).

High-speed cell recognition algorithm for ultrafast flow cytometer imaging system

NASA Astrophysics Data System (ADS)

Zhao, Wanyue; Wang, Chao; Chen, Hongwei; Chen, Minghua; Yang, Sigang

2018-04-01

An optical time-stretch flow imaging system enables high-throughput examination of cells/particles with unprecedented high speed and resolution. A significant amount of raw image data is produced. A high-speed cell recognition algorithm is, therefore, highly demanded to analyze large amounts of data efficiently. A high-speed cell recognition algorithm consisting of two-stage cascaded detection and Gaussian mixture model (GMM) classification is proposed. The first stage of detection extracts cell regions. The second stage integrates distance transform and the watershed algorithm to separate clustered cells. Finally, the cells detected are classified by GMM. We compared the performance of our algorithm with support vector machine. Results show that our algorithm increases the running speed by over 150% without sacrificing the recognition accuracy. This algorithm provides a promising solution for high-throughput and automated cell imaging and classification in the ultrafast flow cytometer imaging platform.

Pulsed laser activated cell sorter (PLACS) for high-throughput fluorescent mammalian cell sorting

NASA Astrophysics Data System (ADS)

Chen, Yue; Wu, Ting-Hsiang; Chung, Aram; Kung, Yu-Chung; Teitell, Michael A.; Di Carlo, Dino; Chiou, Pei-Yu

2014-09-01

We present a Pulsed Laser Activated Cell Sorter (PLACS) realized by exciting laser induced cavitation bubbles in a PDMS microfluidic channel to create high speed liquid jets to deflect detected fluorescent samples for high speed sorting. Pulse laser triggered cavitation bubbles can expand in few microseconds and provide a pressure higher than tens of MPa for fluid perturbation near the focused spot. This ultrafast switching mechanism has a complete on-off cycle less than 20 μsec. Two approaches have been utilized to achieve 3D sample focusing in PLACS. One is relying on multilayer PDMS channels to provide 3D hydrodynamic sheath flows. It offers accurate timing control of fast (2 m sec-1) passing particles so that synchronization with laser bubble excitation is possible, an critically important factor for high purity and high throughput sorting. PLACS with 3D hydrodynamic focusing is capable of sorting at 11,000 cells/sec with >95% purity, and 45,000 cells/sec with 45% purity using a single channel in a single step. We have also demonstrated 3D focusing using inertial flows in PLACS. This sheathless focusing approach requires 10 times lower initial cell concentration than that in sheath-based focusing and avoids severe sample dilution from high volume sheath flows. Inertia PLACS is capable of sorting at 10,000 particles sec-1 with >90% sort purity.

Droplet Microarray Based on Patterned Superhydrophobic Surfaces Prevents Stem Cell Differentiation and Enables High-Throughput Stem Cell Screening.

PubMed

Tronser, Tina; Popova, Anna A; Jaggy, Mona; Bastmeyer, Martin; Levkin, Pavel A

2017-12-01

Over the past decades, stem cells have attracted growing interest in fundamental biological and biomedical research as well as in regenerative medicine, due to their unique ability to self-renew and differentiate into various cell types. Long-term maintenance of the self-renewal ability and inhibition of spontaneous differentiation, however, still remain challenging and are not fully understood. Uncontrolled spontaneous differentiation of stem cells makes high-throughput screening of stem cells also difficult. This further hinders investigation of the underlying mechanisms of stem cell differentiation and the factors that might affect it. In this work, a dual functionality of nanoporous superhydrophobic-hydrophilic micropatterns is demonstrated in their ability to inhibit differentiation of mouse embryonic stem cells (mESCs) and at the same time enable formation of arrays of microdroplets (droplet microarray) via the effect of discontinuous dewetting. Such combination makes high-throughput screening of undifferentiated mouse embryonic stem cells possible. The droplet microarray is used to investigate the development, differentiation, and maintenance of stemness of mESC, revealing the dependence of stem cell behavior on droplet volume in nano- and microliter scale. The inhibition of spontaneous differentiation of mESCs cultured on the droplet microarray for up to 72 h is observed. In addition, up to fourfold increased cell growth rate of mESCs cultured on our platform has been observed. The difference in the behavior of mESCs is attributed to the porosity and roughness of the polymer surface. This work demonstrates that the droplet microarray possesses the potential for the screening of mESCs under conditions of prolonged inhibition of stem cells' spontaneous differentiation. Such a platform can be useful for applications in the field of stem cell research, pharmacological testing of drug efficacy and toxicity, biomedical research as well as in the field of regenerative medicine and tissue engineering. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Linear-array-based photoacoustic tomography for label-free high-throughput detection and quantification of circulating melanoma tumor cell clusters

NASA Astrophysics Data System (ADS)

Hai, Pengfei; Zhou, Yong; Zhang, Ruiying; Ma, Jun; Li, Yang; Wang, Lihong V.

2017-03-01

Circulating tumor cell (CTC) clusters arise from multicellular grouping in the primary tumor and elevate the metastatic potential by 23 to 50 fold compared to single CTCs. High throughout detection and quantification of CTC clusters is critical for understanding the tumor metastasis process and improving cancer therapy. In this work, we report a linear-array-based photoacoustic tomography (LA-PAT) system capable of label-free high-throughput CTC cluster detection and quantification in vivo. LA-PAT detects CTC clusters and quantifies the number of cells in them based on the contrast-to-noise ratios (CNRs) of photoacoustic signals. The feasibility of LA-PAT was first demonstrated by imaging CTC clusters ex vivo. LA-PAT detected CTC clusters in the blood-filled microtubes and computed the number of cells in the clusters. The size distribution of the CTC clusters measured by LA-PAT agreed well with that obtained by optical microscopy. We demonstrated the ability of LA-PAT to detect and quantify CTC clusters in vivo by imaging injected CTC clusters in rat tail veins. LA-PAT detected CTC clusters immediately after injection as well as when they were circulating in the rat bloodstreams. Similarly, the numbers of cells in the clusters were computed based on the CNRs of the photoacoustic signals. The data showed that larger CTC clusters disappear faster than the smaller ones. The results prove the potential of LA-PAT as a promising tool for both preclinical tumor metastasis studies and clinical cancer therapy evaluation.

A gas trapping method for high-throughput metabolic experiments.

PubMed

Krycer, James R; Diskin, Ciana; Nelson, Marin E; Zeng, Xiao-Yi; Fazakerley, Daniel J; James, David E

2018-01-01

Research into cellular metabolism has become more high-throughput, with typical cell-culture experiments being performed in multiwell plates (microplates). This format presents a challenge when trying to collect gaseous products, such as carbon dioxide (CO2), which requires a sealed environment and a vessel separate from the biological sample. To address this limitation, we developed a gas trapping protocol using perforated plastic lids in sealed cell-culture multiwell plates. We used this trap design to measure CO2 production from glucose and fatty acid metabolism, as well as hydrogen sulfide production from cysteine-treated cells. Our data clearly show that this gas trap can be applied to liquid and solid gas-collection media and can be used to study gaseous product generation by both adherent cells and cells in suspension. Since our gas traps can be adapted to multiwell plates of various sizes, they present a convenient, cost-effective solution that can accommodate the trend toward high-throughput measurements in metabolic research.

High-Throughput Quantification of Bacterial-Cell Interactions Using Virtual Colony Counts

PubMed Central

Hoffmann, Stefanie; Walter, Steffi; Blume, Anne-Kathrin; Fuchs, Stephan; Schmidt, Christiane; Scholz, Annemarie; Gerlach, Roman G.

2018-01-01

The quantification of bacteria in cell culture infection models is of paramount importance for the characterization of host-pathogen interactions and pathogenicity factors involved. The standard to enumerate bacteria in these assays is plating of a dilution series on solid agar and counting of the resulting colony forming units (CFU). In contrast, the virtual colony count (VCC) method is a high-throughput compatible alternative with minimized manual input. Based on the recording of quantitative growth kinetics, VCC relates the time to reach a given absorbance threshold to the initial cell count using a series of calibration curves. Here, we adapted the VCC method using the model organism Salmonella enterica sv. Typhimurium (S. Typhimurium) in combination with established cell culture-based infection models. For HeLa infections, a direct side-by-side comparison showed a good correlation of VCC with CFU counting after plating. For MDCK cells and RAW macrophages we found that VCC reproduced the expected phenotypes of different S. Typhimurium mutants. Furthermore, we demonstrated the use of VCC to test the inhibition of Salmonella invasion by the probiotic E. coli strain Nissle 1917. Taken together, VCC provides a flexible, label-free, automation-compatible methodology to quantify bacteria in in vitro infection assays. PMID:29497603

Scalable 96-well Plate Based iPSC Culture and Production Using a Robotic Liquid Handling System.

PubMed

Conway, Michael K; Gerger, Michael J; Balay, Erin E; O'Connell, Rachel; Hanson, Seth; Daily, Neil J; Wakatsuki, Tetsuro

2015-05-14

Continued advancement in pluripotent stem cell culture is closing the gap between bench and bedside for using these cells in regenerative medicine, drug discovery and safety testing. In order to produce stem cell derived biopharmaceutics and cells for tissue engineering and transplantation, a cost-effective cell-manufacturing technology is essential. Maintenance of pluripotency and stable performance of cells in downstream applications (e.g., cell differentiation) over time is paramount to large scale cell production. Yet that can be difficult to achieve especially if cells are cultured manually where the operator can introduce significant variability as well as be prohibitively expensive to scale-up. To enable high-throughput, large-scale stem cell production and remove operator influence novel stem cell culture protocols using a bench-top multi-channel liquid handling robot were developed that require minimal technician involvement or experience. With these protocols human induced pluripotent stem cells (iPSCs) were cultured in feeder-free conditions directly from a frozen stock and maintained in 96-well plates. Depending on cell line and desired scale-up rate, the operator can easily determine when to passage based on a series of images showing the optimal colony densities for splitting. Then the necessary reagents are prepared to perform a colony split to new plates without a centrifugation step. After 20 passages (~3 months), two iPSC lines maintained stable karyotypes, expressed stem cell markers, and differentiated into cardiomyocytes with high efficiency. The system can perform subsequent high-throughput screening of new differentiation protocols or genetic manipulation designed for 96-well plates. This technology will reduce the labor and technical burden to produce large numbers of identical stem cells for a myriad of applications.

Exclusion-Based Capture and Enumeration of CD4+ T Cells from Whole Blood for Low-Resource Settings.

PubMed

Howard, Alexander L; Pezzi, Hannah M; Beebe, David J; Berry, Scott M

2014-06-01

In developing countries, demand exists for a cost-effective method to evaluate human immunodeficiency virus patients' CD4(+) T-helper cell count. The TH (CD4) cell count is the current marker used to identify when an HIV patient has progressed to acquired immunodeficiency syndrome, which results when the immune system can no longer prevent certain opportunistic infections. A system to perform TH count that obviates the use of costly flow cytometry will enable physicians to more closely follow patients' disease progression and response to therapy in areas where such advanced equipment is unavailable. Our system of two serially-operated immiscible phase exclusion-based cell isolations coupled with a rapid fluorescent readout enables exclusion-based isolation and accurate counting of T-helper cells at lower cost and from a smaller volume of blood than previous methods. TH cell isolation via immiscible filtration assisted by surface tension (IFAST) compares well against the established Dynal T4 Quant Kit and is sensitive at CD4 counts representative of immunocompromised patients (less than 200 TH cells per microliter of blood). Our technique retains use of open, simple-to-operate devices that enable IFAST as a high-throughput, automatable sample preparation method, improving throughput over previous low-resource methods. © 2013 Society for Laboratory Automation and Screening.

DSSTox ToxCast and Tox21 Chemical Inventories: Laying the Foundation for the U.S. EPA’s Computational Toxicology Research Programs

EPA Science Inventory

High quality chemical structure inventories provide the foundation of the U.S. EPA’s ToxCast and Tox21 projects, which are employing high-throughput technologies to screen thousands of chemicals in hundreds of biochemical and cell-based assays, probing a wide diversity of targets...

Purification of microalgae from bacterial contamination using a disposable inertia-based microfluidic device

NASA Astrophysics Data System (ADS)

Godino, Neus; Jorde, Felix; Lawlor, Daryl; Jaeger, Magnus; Duschl, Claus

2015-08-01

Microalgae are a promising source of bioactive ingredients for the food, pharmaceutical and cosmetic industries. Every microalgae research group or production facility is facing one major problem regarding the potential contamination of the algal cell with bacteria. Prior to the storage of the microalgae in strain collections or to cultivation in bioreactors, it is necessary to carry out laborious purification procedures to separate the microalgae from the undesired bacterial cells. In this work, we present a disposable microfluidic cartridge for the high-throughput purification of microalgae samples based on inertial microfluidics. Some of the most relevant microalgae strains have a larger size than the relatively small, few micron bacterial cells, so making them distinguishable by size. The inertial microfluidic cartridge was fabricated with inexpensive materials, like pressure sensitive adhesive (PSA) and thin plastic layers, which were patterned using a simple cutting plotter. In spite of fabrication restrictions and the intrinsic difficulties of biological samples, the separation of microalgae from bacteria reached values in excess of 99%, previously only achieved using conventional high-end and high cost lithography methods. Moreover, due to the simple and high-throughput characteristic of the separation, it is possible to concatenate serial purification to exponentially decrease the absolute amount of bacteria in the final purified sample.

A high throughput spectral image microscopy system

NASA Astrophysics Data System (ADS)

Gesley, M.; Puri, R.

2018-01-01

A high throughput spectral image microscopy system is configured for rapid detection of rare cells in large populations. To overcome flow cytometry rates and use of fluorophore tags, a system architecture integrates sample mechanical handling, signal processors, and optics in a non-confocal version of light absorption and scattering spectroscopic microscopy. Spectral images with native contrast do not require the use of exogeneous stain to render cells with submicron resolution. Structure may be characterized without restriction to cell clusters of differentiation.

Recent developments in microfluidics-based chemotaxis studies.

PubMed

Wu, Jiandong; Wu, Xun; Lin, Francis

2013-07-07

Microfluidic devices can better control cellular microenvironments compared to conventional cell migration assays. Over the past few years, microfluidics-based chemotaxis studies showed a rapid growth. New strategies were developed to explore cell migration in manipulated chemical gradients. In addition to expanding the use of microfluidic devices for a broader range of cell types, microfluidic devices were used to study cell migration and chemotaxis in complex environments. Furthermore, high-throughput microfluidic chemotaxis devices and integrated microfluidic chemotaxis systems were developed for medical and commercial applications. In this article, we review recent developments in microfluidics-based chemotaxis studies and discuss the new trends in this field observed over the past few years.

Strain Library Imaging Protocol for high-throughput, automated single-cell microscopy of large bacterial collections arrayed on multiwell plates.

PubMed

Shi, Handuo; Colavin, Alexandre; Lee, Timothy K; Huang, Kerwyn Casey

2017-02-01

Single-cell microscopy is a powerful tool for studying gene functions using strain libraries, but it suffers from throughput limitations. Here we describe the Strain Library Imaging Protocol (SLIP), which is a high-throughput, automated microscopy workflow for large strain collections that requires minimal user involvement. SLIP involves transferring arrayed bacterial cultures from multiwell plates onto large agar pads using inexpensive replicator pins and automatically imaging the resulting single cells. The acquired images are subsequently reviewed and analyzed by custom MATLAB scripts that segment single-cell contours and extract quantitative metrics. SLIP yields rich data sets on cell morphology and gene expression that illustrate the function of certain genes and the connections among strains in a library. For a library arrayed on 96-well plates, image acquisition can be completed within 4 min per plate.

Building predictive in vitro pulmonary toxicity assays using high-throughput imaging and artificial intelligence.

PubMed

Lee, Jia-Ying Joey; Miller, James Alastair; Basu, Sreetama; Kee, Ting-Zhen Vanessa; Loo, Lit-Hsin

2018-06-01

Human lungs are susceptible to the toxicity induced by soluble xenobiotics. However, the direct cellular effects of many pulmonotoxic chemicals are not always clear, and thus, a general in vitro assay for testing pulmonotoxicity applicable to a wide variety of chemicals is not currently available. Here, we report a study that uses high-throughput imaging and artificial intelligence to build an in vitro pulmonotoxicity assay by automatically comparing and selecting human lung-cell lines and their associated quantitative phenotypic features most predictive of in vivo pulmonotoxicity. This approach is called "High-throughput In vitro Phenotypic Profiling for Toxicity Prediction" (HIPPTox). We found that the resulting assay based on two phenotypic features of a human bronchial epithelial cell line, BEAS-2B, can accurately classify 33 reference chemicals with human pulmonotoxicity information (88.8% balance accuracy, 84.6% sensitivity, and 93.0% specificity). In comparison, the predictivity of a standard cell-viability assay on the same set of chemicals is much lower (77.1% balanced accuracy, 84.6% sensitivity, and 69.5% specificity). We also used the assay to evaluate 17 additional test chemicals with unknown/unclear human pulmonotoxicity, and experimentally confirmed that many of the pulmonotoxic reference and predicted-positive test chemicals induce DNA strand breaks and/or activation of the DNA-damage response (DDR) pathway. Therefore, HIPPTox helps us to uncover these common modes-of-action of pulmonotoxic chemicals. HIPPTox may also be applied to other cell types or models, and accelerate the development of predictive in vitro assays for other cell-type- or organ-specific toxicities.

A High-Throughput Genetic Complementation Assay in Yeast Cells Identified Selective Inhibitors of Sphingosine Kinase 1 Not Found Using a Cell-Free Enzyme Assay.

PubMed

Kashem, Mohammed A; Kennedy, Charles A; Fogarty, Kylie E; Dimock, Janice R; Zhang, Yunlong; Sanville-Ross, Mary L; Skow, Donna J; Brunette, Steven R; Swantek, Jennifer L; Hummel, Heidi S; Swindle, John; Nelson, Richard M

2016-01-01

Sphingosine kinase 1 (SphK1) is a lipid kinase that phosphorylates sphingosine to produce the bioactive sphingolipid, sphingosine-1-phosphate (S1P), and therefore represents a potential drug target for a variety of pathological processes such as fibrosis, inflammation, and cancer. We developed two assays compatible with high-throughput screening to identify small-molecule inhibitors of SphK1: a purified component enzyme assay and a genetic complementation assay in yeast cells. The biochemical enzyme assay measures the phosphorylation of sphingosine-fluorescein to S1P-fluorescein by recombinant human full-length SphK1 using an immobilized metal affinity for phosphochemicals (IMAP) time-resolved fluorescence resonance energy transfer format. The yeast assay employs an engineered strain of Saccharomyces cerevisiae, in which the human gene encoding SphK1 replaced the yeast ortholog and quantitates cell viability by measuring intracellular adenosine 5'-triphosphate (ATP) using a luciferase-based luminescent readout. In this assay, expression of human SphK1 was toxic, and the resulting yeast cell death was prevented by SphK1 inhibitors. We optimized both assays in a 384-well format and screened ∼10(6) compounds selected from the Boehringer Ingelheim library. The biochemical IMAP high-throughput screen identified 5,561 concentration-responsive hits, most of which were ATP competitive and not selective over sphingosine kinase 2 (SphK2). The yeast screen identified 205 concentration-responsive hits, including several distinct compound series that were selective against SphK2 and were not ATP competitive.

Multi-step high-throughput conjugation platform for the development of antibody-drug conjugates.

PubMed

Andris, Sebastian; Wendeler, Michaela; Wang, Xiangyang; Hubbuch, Jürgen

2018-07-20

Antibody-drug conjugates (ADCs) form a rapidly growing class of biopharmaceuticals which attracts a lot of attention throughout the industry due to its high potential for cancer therapy. They combine the specificity of a monoclonal antibody (mAb) and the cell-killing capacity of highly cytotoxic small molecule drugs. Site-specific conjugation approaches involve a multi-step process for covalent linkage of antibody and drug via a linker. Despite the range of parameters that have to be investigated, high-throughput methods are scarcely used so far in ADC development. In this work an automated high-throughput platform for a site-specific multi-step conjugation process on a liquid-handling station is presented by use of a model conjugation system. A high-throughput solid-phase buffer exchange was successfully incorporated for reagent removal by utilization of a batch cation exchange step. To ensure accurate screening of conjugation parameters, an intermediate UV/Vis-based concentration determination was established including feedback to the process. For conjugate characterization, a high-throughput compatible reversed-phase chromatography method with a runtime of 7 min and no sample preparation was developed. Two case studies illustrate the efficient use for mapping the operating space of a conjugation process. Due to the degree of automation and parallelization, the platform is capable of significantly reducing process development efforts and material demands and shorten development timelines for antibody-drug conjugates. Copyright © 2018 Elsevier B.V. All rights reserved.

Development and application of a recombination-based library versus library high- throughput yeast two-hybrid (RLL-Y2H) screening system.

PubMed

Yang, Fang; Lei, Yingying; Zhou, Meiling; Yao, Qili; Han, Yichao; Wu, Xiang; Zhong, Wanshun; Zhu, Chenghang; Xu, Weize; Tao, Ran; Chen, Xi; Lin, Da; Rahman, Khaista; Tyagi, Rohit; Habib, Zeshan; Xiao, Shaobo; Wang, Dang; Yu, Yang; Chen, Huanchun; Fu, Zhenfang; Cao, Gang

2018-02-16

Protein-protein interaction (PPI) network maintains proper function of all organisms. Simple high-throughput technologies are desperately needed to delineate the landscape of PPI networks. While recent state-of-the-art yeast two-hybrid (Y2H) systems improved screening efficiency, either individual colony isolation, library preparation arrays, gene barcoding or massive sequencing are still required. Here, we developed a recombination-based 'library vs library' Y2H system (RLL-Y2H), by which multi-library screening can be accomplished in a single pool without any individual treatment. This system is based on the phiC31 integrase-mediated integration between bait and prey plasmids. The integrated fragments were digested by MmeI and subjected to deep sequencing to decode the interaction matrix. We applied this system to decipher the trans-kingdom interactome between Mycobacterium tuberculosis and host cells and further identified Rv2427c interfering with the phagosome-lysosome fusion. This concept can also be applied to other systems to screen protein-RNA and protein-DNA interactions and delineate signaling landscape in cells.

Development of Genome Engineering Tools from Plant-Specific PPR Proteins Using Animal Cultured Cells.

PubMed

Kobayashi, Takehito; Yagi, Yusuke; Nakamura, Takahiro

2016-01-01

The pentatricopeptide repeat (PPR) motif is a sequence-specific RNA/DNA-binding module. Elucidation of the RNA/DNA recognition mechanism has enabled engineering of PPR motifs as new RNA/DNA manipulation tools in living cells, including for genome editing. However, the biochemical characteristics of PPR proteins remain unknown, mostly due to the instability and/or unfolding propensities of PPR proteins in heterologous expression systems such as bacteria and yeast. To overcome this issue, we constructed reporter systems using animal cultured cells. The cell-based system has highly attractive features for PPR engineering: robust eukaryotic gene expression; availability of various vectors, reagents, and antibodies; highly efficient DNA delivery ratio (>80 %); and rapid, high-throughput data production. In this chapter, we introduce an example of such reporter systems: a PPR-based sequence-specific translational activation system. The cell-based reporter system can be applied to characterize plant genes of interested and to PPR engineering.

Fishing on chips: up-and-coming technological advances in analysis of zebrafish and Xenopus embryos.

PubMed

Zhu, Feng; Skommer, Joanna; Huang, Yushi; Akagi, Jin; Adams, Dany; Levin, Michael; Hall, Chris J; Crosier, Philip S; Wlodkowic, Donald

2014-11-01

Biotests performed on small vertebrate model organisms provide significant investigative advantages as compared with bioassays that employ cell lines, isolated primary cells, or tissue samples. The main advantage offered by whole-organism approaches is that the effects under study occur in the context of intact physiological milieu, with all its intercellular and multisystem interactions. The gap between the high-throughput cell-based in vitro assays and low-throughput, disproportionally expensive and ethically controversial mammal in vivo tests can be closed by small model organisms such as zebrafish or Xenopus. The optical transparency of their tissues, the ease of genetic manipulation and straightforward husbandry, explain the growing popularity of these model organisms. Nevertheless, despite the potential for miniaturization, automation and subsequent increase in throughput of experimental setups, the manipulation, dispensing and analysis of living fish and frog embryos remain labor-intensive. Recently, a new generation of miniaturized chip-based devices have been developed for zebrafish and Xenopus embryo on-chip culture and experimentation. In this work, we review the critical developments in the field of Lab-on-a-Chip devices designed to alleviate the limits of traditional platforms for studies on zebrafish and clawed frog embryo and larvae. © 2014 International Society for Advancement of Cytometry. © 2014 International Society for Advancement of Cytometry.

Real-time quantitative fluorescence measurement of microscale cell culture analog systems

NASA Astrophysics Data System (ADS)

Oh, Taek-il; Kim, Donghyun; Tatosian, Daniel; Sung, Jong Hwan; Shuler, Michael

2007-02-01

A microscale cell culture analog (μCCA) is a cell-based lab-on-a-chip assay that, as an animal surrogate, is applied to pharmacological studies for toxicology tests. A μCCA typically comprises multiple chambers and microfluidics that connect the chambers, which represent animal organs and blood flow to mimic animal metabolism more realistically. A μCCA is expected to provide a tool for high-throughput drug discovery. Previously, a portable fluorescence detection system was investigated for a single μCCA device in real-time. In this study, we present a fluorescence-based imaging system that provides quantitative real-time data of the metabolic interactions in μCCAs with an emphasis on measuring multiple μCCA samples simultaneously for high-throughput screening. The detection system is based on discrete optics components, with a high-power LED and a charge-coupled device (CCD) camera as a light source and a detector, for monitoring cellular status on the chambers of each μCCA sample. Multiple samples are characterized mechanically on a motorized linear stage, which is fully-automated. Each μCCA sample has four chambers, where cell lines MES-SA/DX- 5, and MES-SA (tumor cells of human uterus) have been cultured. All cell-lines have been transfected to express the fusion protein H2B-GFP, which is a human histone protein fused at the amino terminus to EGFP. As a model cytotoxic drug, 10 μM doxorubicin (DOX) was used. Real-time quantitative data of the intensity loss of enhanced green fluorescent protein (EGFP) during cell death of target cells have been collected over several minutes to 40 hours. Design issues and improvements are also discussed.

QUANTITATIVE IN VITRO MEASUREMENT OF CELLULAR PROCESSES CRITICAL TO THE DEVELOPMENT OF NEURAL CONNECTIVITY USING HCA.

EPA Science Inventory

New methods are needed to screen thousands of environmental chemicals for toxicity, including developmental neurotoxicity. In vitro, cell-based assays that model key cellular events have been proposed for high throughput screening of chemicals for developmental neurotoxicity. Whi...

The Development of Novel Small Molecule Inhibitors of the Phosphoinositide-3-Kinase Pathway Through High-Throughput Cell-Based Screens

DTIC Science & Technology

2005-02-01

Akt in the P13K pathway. Given the emerging data for a positive feedback loop induced by mTOR inhibition, a bispecific 5 inhibitor might be attractive...cells relatively sensitive to rapamycin are also sensitive to thioridazine. PTEN null cells are known to be preferentially sensitized to mTOR ...a potent mTOR inhibitor, a downstream protein kinase in the Akt pathway. Rapamycin showed strong growth inhibitory effect in PTEN-null cells but 786

A droplet-based heterogeneous immunoassay for screening single cells secreting antigen-specific antibodies.

PubMed

Akbari, Samin; Pirbodaghi, Tohid

2014-09-07

High throughput heterogeneous immunoassays that screen antigen-specific antibody secreting cells are essential to accelerate monoclonal antibody discovery for therapeutic applications. Here, we introduce a heterogeneous single cell immunoassay based on alginate microparticles as permeable cell culture chambers. Using a microfluidic device, we encapsulated single antibody secreting cells in 35-40 μm diameter alginate microbeads. We functionalized the alginate to capture the secreted antibodies inside the microparticles, enabling single cell analysis and preventing the cross-talk between the neighboring encapsulated cells. We demonstrated non-covalent functionalization of alginate microparticles by adding three secondary antibodies to the alginate solution to form high molecular weight complexes that become trapped in the porous nanostructure of alginate and capture the secreted antibodies. We screened anti-TNF-alpha antibody-secreting cells from a mixture of antibody-secreting cells.

High Throughput, Polymeric Aqueous Two-Phase Printing of Tumor Spheroids

PubMed Central

Atefi, Ehsan; Lemmo, Stephanie; Fyffe, Darcy; Luker, Gary D.; Tavana, Hossein

2014-01-01

This paper presents a new 3D culture microtechnology for high throughput production of tumor spheroids and validates its utility for screening anti-cancer drugs. We use two immiscible polymeric aqueous solutions and microprint a submicroliter drop of the “patterning” phase containing cells into a bath of the “immersion” phase. Selecting proper formulations of biphasic systems using a panel of biocompatible polymers results in the formation of a round drop that confines cells to facilitate spontaneous formation of a spheroid without any external stimuli. Adapting this approach to robotic tools enables straightforward generation and maintenance of spheroids of well-defined size in standard microwell plates and biochemical analysis of spheroids in situ, which is not possible with existing techniques for spheroid culture. To enable high throughput screening, we establish a phase diagram to identify minimum cell densities within specific volumes of the patterning drop to result in a single spheroid. Spheroids show normal growth over long-term incubation and dose-dependent decrease in cellular viability when treated with drug compounds, but present significant resistance compared to monolayer cultures. The unprecedented ease of implementing this microtechnology and its robust performance will benefit high throughput studies of drug screening against cancer cells with physiologically-relevant 3D tumor models. PMID:25411577

Lessons we learned from high-throughput and top-down systems biology analyses about glioma stem cells.

PubMed

Mock, Andreas; Chiblak, Sara; Herold-Mende, Christel

2014-01-01

A growing body of evidence suggests that glioma stem cells (GSCs) account for tumor initiation, therapy resistance, and the subsequent regrowth of gliomas. Thus, continuous efforts have been undertaken to further characterize this subpopulation of less differentiated tumor cells. Although we are able to enrich GSCs, we still lack a comprehensive understanding of GSC phenotypes and behavior. The advent of high-throughput technologies raised hope that incorporation of these newly developed platforms would help to tackle such questions. Since then a couple of comparative genome-, transcriptome- and proteome-wide studies on GSCs have been conducted giving new insights in GSC biology. However, lessons had to be learned in designing high-throughput experiments and some of the resulting conclusions fell short of expectations because they were performed on only a few GSC lines or at one molecular level instead of an integrative poly-omics approach. Despite these shortcomings, our knowledge of GSC biology has markedly expanded due to a number of survival-associated biomarkers as well as glioma-relevant signaling pathways and therapeutic targets being identified. In this article we review recent findings obtained by comparative high-throughput analyses of GSCs. We further summarize fundamental concepts of systems biology as well as its applications for glioma stem cell research.

High-throughput autofluorescence flow cytometry of breast cancer metabolism (Conference Presentation)

NASA Astrophysics Data System (ADS)

Shah, Amy T.; Cannon, Taylor M.; Higginbotham, Jim N.; Skala, Melissa C.

2016-02-01

Tumor heterogeneity poses challenges for devising optimal treatment regimens for cancer patients. In particular, subpopulations of cells can escape treatment and cause relapse. There is a need for methods to characterize tumor heterogeneity of treatment response. Cell metabolism is altered in cancer (Warburg effect), and cells use the autofluorescent cofactor NADH in numerous metabolic reactions. Previous studies have shown that microscopy measurements of NADH autofluorescence are sensitive to treatment response in breast cancer, and these techniques typically assess hundreds of cells per group. An alternative approach is flow cytometry, which measures fluorescence on a single-cell level and is attractive for characterizing tumor heterogeneity because it achieves high-throughput analysis and cell sorting in millions of cells per group. Current applications for flow cytometry rely on staining with fluorophores. This study characterizes flow cytometry measurements of NADH autofluorescence in breast cancer cells. Preliminary results indicate flow cytometry of NADH is sensitive to cyanide perturbation, which inhibits oxidative phosphorylation, in nonmalignant MCF10A cells. Additionally, flow cytometry is sensitive to higher NADH intensity for HER2-positive SKBr3 cells compared with triple-negative MDA-MB-231 cells. These results agree with previous microscopy studies. Finally, a mixture of SKBr3 and MDA-MB-231 cells were sorted into each cell type using NADH intensity. Sorted cells were cultured, and microscopy validation showed the expected morphology for each cell type. Ultimately, flow cytometry could be applied to characterize tumor heterogeneity based on treatment response and sort cell subpopulations based on metabolic profile. These achievements could enable individualized treatment strategies and improved patient outcomes.

Prediction-based association control scheme in dense femtocell networks.

PubMed

Sung, Nak Woon; Pham, Ngoc-Thai; Huynh, Thong; Hwang, Won-Joo; You, Ilsun; Choo, Kim-Kwang Raymond

2017-01-01

The deployment of large number of femtocell base stations allows us to extend the coverage and efficiently utilize resources in a low cost manner. However, the small cell size of femtocell networks can result in frequent handovers to the mobile user, and consequently throughput degradation. Thus, in this paper, we propose predictive association control schemes to improve the system's effective throughput. Our design focuses on reducing handover frequency without impacting on throughput. The proposed schemes determine handover decisions that contribute most to the network throughput and are proper for distributed implementations. The simulation results show significant gains compared with existing methods in terms of handover frequency and network throughput perspective.

High-Throughput Screening to Identify Compounds That Increase Fragile X Mental Retardation Protein Expression in Neural Stem Cells Differentiated From Fragile X Syndrome Patient-Derived Induced Pluripotent Stem Cells.

PubMed

Kumari, Daman; Swaroop, Manju; Southall, Noel; Huang, Wenwei; Zheng, Wei; Usdin, Karen

2015-07-01

: Fragile X syndrome (FXS), the most common form of inherited cognitive disability, is caused by a deficiency of the fragile X mental retardation protein (FMRP). In most patients, the absence of FMRP is due to an aberrant transcriptional silencing of the fragile X mental retardation 1 (FMR1) gene. FXS has no cure, and the available treatments only provide symptomatic relief. Given that FMR1 gene silencing in FXS patient cells can be partially reversed by treatment with compounds that target repressive epigenetic marks, restoring FMRP expression could be one approach for the treatment of FXS. We describe a homogeneous and highly sensitive time-resolved fluorescence resonance energy transfer assay for FMRP detection in a 1,536-well plate format. Using neural stem cells differentiated from an FXS patient-derived induced pluripotent stem cell (iPSC) line that does not express any FMRP, we screened a collection of approximately 5,000 known tool compounds and approved drugs using this FMRP assay and identified 6 compounds that modestly increase FMR1 gene expression in FXS patient cells. Although none of these compounds resulted in clinically relevant levels of FMR1 mRNA, our data provide proof of principle that this assay combined with FXS patient-derived neural stem cells can be used in a high-throughput format to identify better lead compounds for FXS drug development. In this study, a specific and sensitive fluorescence resonance energy transfer-based assay for fragile X mental retardation protein detection was developed and optimized for high-throughput screening (HTS) of compound libraries using fragile X syndrome (FXS) patient-derived neural stem cells. The data suggest that this HTS format will be useful for the identification of better lead compounds for developing new therapeutics for FXS. This assay can also be adapted for FMRP detection in clinical and research settings. ©AlphaMed Press.

High-throughput micro-scale cultivations and chromatography modeling: Powerful tools for integrated process development.

PubMed

Baumann, Pascal; Hahn, Tobias; Hubbuch, Jürgen

2015-10-01

Upstream processes are rather complex to design and the productivity of cells under suitable cultivation conditions is hard to predict. The method of choice for examining the design space is to execute high-throughput cultivation screenings in micro-scale format. Various predictive in silico models have been developed for many downstream processes, leading to a reduction of time and material costs. This paper presents a combined optimization approach based on high-throughput micro-scale cultivation experiments and chromatography modeling. The overall optimized system must not necessarily be the one with highest product titers, but the one resulting in an overall superior process performance in up- and downstream. The methodology is presented in a case study for the Cherry-tagged enzyme Glutathione-S-Transferase from Escherichia coli SE1. The Cherry-Tag™ (Delphi Genetics, Belgium) which can be fused to any target protein allows for direct product analytics by simple VIS absorption measurements. High-throughput cultivations were carried out in a 48-well format in a BioLector micro-scale cultivation system (m2p-Labs, Germany). The downstream process optimization for a set of randomly picked upstream conditions producing high yields was performed in silico using a chromatography modeling software developed in-house (ChromX). The suggested in silico-optimized operational modes for product capturing were validated subsequently. The overall best system was chosen based on a combination of excellent up- and downstream performance. © 2015 Wiley Periodicals, Inc.

Ultra-high-throughput screening method for the directed evolution of glucose oxidase.

PubMed

Ostafe, Raluca; Prodanovic, Radivoje; Nazor, Jovana; Fischer, Rainer

2014-03-20

Glucose oxidase (GOx) is used in many industrial processes that could benefit from improved versions of the enzyme. Some improvements like higher activity under physiological conditions and thermal stability could be useful for GOx applications in biosensors and biofuel cells. Directed evolution is one of the currently available methods to engineer improved GOx variants. Here, we describe an ultra-high-throughput screening system for sorting the best enzyme variants generated by directed evolution that incorporates several methodological refinements: flow cytometry, in vitro compartmentalization, yeast surface display, fluorescent labeling of the expressed enzyme, delivery of glucose substrate to the reaction mixture through the oil phase, and covalent labeling of the cells with fluorescein-tyramide. The method enables quantitative screening of gene libraries to identify clones with improved activity and it also allows cells to be selected based not only on the overall activity but also on the specific activity of the enzyme. Copyright © 2014 Elsevier Ltd. All rights reserved.

Synthesis and high-throughput processing of polymeric hydrogels for 3D cell culture.

PubMed

Lowe, Stuart B; Tan, Vincent T G; Soeriyadi, Alexander H; Davis, Thomas P; Gooding, J Justin

2014-09-17

3D cell cultures have drawn a large amount of interest in the scientific community with their ability to closely mimic physiological conditions. Hydrogels have been used extensively in the development of extracellular matrix (ECM) mimics for 3D cell culture. Compounds such as collagen and fibrin are commonly used to synthesize natural ECM mimics; however they suffer from batch-to-batch variation. In this Review we explore the synthesis route of hydrogels; how they can be altered to give different chemical and physical properties; how different biomolecules such as arginylglycylaspartic acid (RGD) or vascular endothelial growth factor (VEGF) can be incorporated to give different biological cues; and how to create concentration gradients with UV light. There will also be emphasis on the types of techniques available in high-throughput processing such as nozzle and droplet-based biofabrication, photoenabled biofabrication, and microfluidics. The combination of these approaches and techniques allow the preparation of hydrogels which are capable of mimicking the ECM.

Microelectrode array recordings of cardiac action potentials as a high throughput method to evaluate pesticide toxicity.

PubMed

Natarajan, A; Molnar, P; Sieverdes, K; Jamshidi, A; Hickman, J J

2006-04-01

The threat of environmental pollution, biological warfare agent dissemination and new diseases in recent decades has increased research into cell-based biosensors. The creation of this class of sensors could specifically aid the detection of toxic chemicals and their effects in the environment, such as pyrethroid pesticides. Pyrethroids are synthetic pesticides that have been used increasingly over the last decade to replace other pesticides like DDT. In this study we used a high-throughput method to detect pyrethroids by using multielectrode extracellular recordings from cardiac cells. The data from this cell-electrode hybrid system was compared to published results obtained with patch-clamp electrophysiology and also used as an alternative method to further understand pyrethroid effects. Our biosensor consisted of a confluent monolayer of cardiac myocytes cultured on microelectrode arrays (MEA) composed of 60 substrate-integrated electrodes. Spontaneous activity of these beating cells produced extracellular field potentials in the range of 100 microV to nearly 1200 microV with a beating frequency of 0.5-4 Hz. All of the tested pyrethroids; alpha-Cypermethrin, Tetramethrin and Tefluthrin, produced similar changes in the electrophysiological properties of the cardiac myocytes, namely reduced beating frequency and amplitude. The sensitivity of our toxin detection method was comparable to earlier patch-clamp studies, which indicates that, in specific applications, high-throughput extracellular methods can replace single-cell studies. Moreover, the similar effect of all three pyrethroids on the measured parameters suggests, that not only detection of the toxins but, their classification might also be possible with this method. Overall our results support the idea that whole cell biosensors might be viable alternatives when compared to current toxin detection methods.

Arraycount, an algorithm for automatic cell counting in microwell arrays.

PubMed

Kachouie, Nezamoddin; Kang, Lifeng; Khademhosseini, Ali

2009-09-01

Microscale technologies have emerged as a powerful tool for studying and manipulating biological systems and miniaturizing experiments. However, the lack of software complementing these techniques has made it difficult to apply them for many high-throughput experiments. This work establishes Arraycount, an approach to automatically count cells in microwell arrays. The procedure consists of fluorescent microscope imaging of cells that are seeded in microwells of a microarray system and then analyzing images via computer to recognize the array and count cells inside each microwell. To start counting, green and red fluorescent images (representing live and dead cells, respectively) are extracted from the original image and processed separately. A template-matching algorithm is proposed in which pre-defined well and cell templates are matched against the red and green images to locate microwells and cells. Subsequently, local maxima in the correlation maps are determined and local maxima maps are thresholded. At the end, the software records the cell counts for each detected microwell on the original image in high-throughput. The automated counting was shown to be accurate compared with manual counting, with a difference of approximately 1-2 cells per microwell: based on cell concentration, the absolute difference between manual and automatic counting measurements was 2.5-13%.

High-Throughput Microfluidic Labyrinth for the Label-free Isolation of Circulating Tumor Cells.

PubMed

Lin, Eric; Rivera-Báez, Lianette; Fouladdel, Shamileh; Yoon, Hyeun Joong; Guthrie, Stephanie; Wieger, Jacob; Deol, Yadwinder; Keller, Evan; Sahai, Vaibhav; Simeone, Diane M; Burness, Monika L; Azizi, Ebrahim; Wicha, Max S; Nagrath, Sunitha

2017-09-27

We present "Labyrinth," a label-free microfluidic device to isolate circulating tumor cells (CTCs) using the combination of long loops and sharp corners to focus both CTCs and white blood cells (WBCs) at a high throughput of 2.5 mL/min. The high yield (>90%) and purity (600 WBCs/mL) of Labyrinth enabled us to profile gene expression in CTCs. As proof of principle, we used previously established cancer stem cell gene signatures to profile single cells isolated from the blood of breast cancer patients. We observed heterogeneous subpopulations of CTCs expressing genes for stem cells, epithelial cells, mesenchymal cells, and cells transitioning between epithelial and mesenchymal. Labyrinth offers a cell-surface marker-independent single-cell isolation platform to study heterogeneous CTC subpopulations. Copyright © 2017 Elsevier Inc. All rights reserved.

Living-Cell Microarrays

PubMed Central

Yarmush, Martin L.; King, Kevin R.

2011-01-01

Living cells are remarkably complex. To unravel this complexity, living-cell assays have been developed that allow delivery of experimental stimuli and measurement of the resulting cellular responses. High-throughput adaptations of these assays, known as living-cell microarrays, which are based on microtiter plates, high-density spotting, microfabrication, and microfluidics technologies, are being developed for two general applications: (a) to screen large-scale chemical and genomic libraries and (b) to systematically investigate the local cellular microenvironment. These emerging experimental platforms offer exciting opportunities to rapidly identify genetic determinants of disease, to discover modulators of cellular function, and to probe the complex and dynamic relationships between cells and their local environment. PMID:19413510

Potential of dynamically harmonized Fourier transform ion cyclotron resonance cell for high-throughput metabolomics fingerprinting: control of data quality.

PubMed

Habchi, Baninia; Alves, Sandra; Jouan-Rimbaud Bouveresse, Delphine; Appenzeller, Brice; Paris, Alain; Rutledge, Douglas N; Rathahao-Paris, Estelle

2018-01-01

Due to the presence of pollutants in the environment and food, the assessment of human exposure is required. This necessitates high-throughput approaches enabling large-scale analysis and, as a consequence, the use of high-performance analytical instruments to obtain highly informative metabolomic profiles. In this study, direct introduction mass spectrometry (DIMS) was performed using a Fourier transform ion cyclotron resonance (FT-ICR) instrument equipped with a dynamically harmonized cell. Data quality was evaluated based on mass resolving power (RP), mass measurement accuracy, and ion intensity drifts from the repeated injections of quality control sample (QC) along the analytical process. The large DIMS data size entails the use of bioinformatic tools for the automatic selection of common ions found in all QC injections and for robustness assessment and correction of eventual technical drifts. RP values greater than 10 6 and mass measurement accuracy of lower than 1 ppm were obtained using broadband mode resulting in the detection of isotopic fine structure. Hence, a very accurate relative isotopic mass defect (RΔm) value was calculated. This reduces significantly the number of elemental composition (EC) candidates and greatly improves compound annotation. A very satisfactory estimate of repeatability of both peak intensity and mass measurement was demonstrated. Although, a non negligible ion intensity drift was observed for negative ion mode data, a normalization procedure was easily applied to correct this phenomenon. This study illustrates the performance and robustness of the dynamically harmonized FT-ICR cell to perform large-scale high-throughput metabolomic analyses in routine conditions. Graphical abstract Analytical performance of FT-ICR instrument equipped with a dynamically harmonized cell.

High-performance single cell genetic analysis using microfluidic emulsion generator arrays.

PubMed

Zeng, Yong; Novak, Richard; Shuga, Joe; Smith, Martyn T; Mathies, Richard A

2010-04-15

High-throughput genetic and phenotypic analysis at the single cell level is critical to advance our understanding of the molecular mechanisms underlying cellular function and dysfunction. Here we describe a high-performance single cell genetic analysis (SCGA) technique that combines high-throughput microfluidic emulsion generation with single cell multiplex polymerase chain reaction (PCR). Microfabricated emulsion generator array (MEGA) devices containing 4, 32, and 96 channels are developed to confer a flexible capability of generating up to 3.4 x 10(6) nanoliter-volume droplets per hour. Hybrid glass-polydimethylsiloxane diaphragm micropumps integrated into the MEGA chips afford uniform droplet formation, controlled generation frequency, and effective transportation and encapsulation of primer functionalized microbeads and cells. A multiplex single cell PCR method is developed to detect and quantify both wild type and mutant/pathogenic cells. In this method, microbeads functionalized with multiple forward primers targeting specific genes from different cell types are used for solid-phase PCR in droplets. Following PCR, the droplets are lysed and the beads are pooled and rapidly analyzed by multicolor flow cytometry. Using Escherichia coli bacterial cells as a model, we show that this technique enables digital detection of pathogenic E. coli O157 cells in a high background of normal K12 cells, with a detection limit on the order of 1/10(5). This result demonstrates that multiplex SCGA is a promising tool for high-throughput quantitative digital analysis of genetic variation in complex populations.

High-Performance Single Cell Genetic Analysis Using Microfluidic Emulsion Generator Arrays

PubMed Central

Zeng, Yong; Novak, Richard; Shuga, Joe; Smith, Martyn T.; Mathies, Richard A.

2010-01-01

High-throughput genetic and phenotypic analysis at the single cell level is critical to advance our understanding of the molecular mechanisms underlying cellular function and dysfunction. Here we describe a high-performance single cell genetic analysis (SCGA) technique that combines high-throughput microfluidic emulsion generation with single cell multiplex PCR. Microfabricated emulsion generator array (MEGA) devices containing 4, 32 and 96 channels are developed to confer a flexible capability of generating up to 3.4 × 106 nanoliter-volume droplets per hour. Hybrid glass-polydimethylsiloxane diaphragm micropumps integrated into the MEGA chips afford uniform droplet formation, controlled generation frequency, and effective transportation and encapsulation of primer functionalized microbeads and cells. A multiplex single cell PCR method is developed to detect and quantify both wild type and mutant/pathogenic cells. In this method, microbeads functionalized with multiple forward primers targeting specific genes from different cell types are used for solid-phase PCR in droplets. Following PCR, the droplets are lysed, the beads are pooled and rapidly analyzed by multi-color flow cytometry. Using E. coli bacterial cells as a model, we show that this technique enables digital detection of pathogenic E. coli O157 cells in a high background of normal K12 cells, with a detection limit on the order of 1:105. This result demonstrates that multiplex SCGA is a promising tool for high-throughput quantitative digital analysis of genetic variation in complex populations. PMID:20192178

Towards High Throughput Cell Growth Screening: A New CMOS 8 × 8 Biosensor Array for Life Science Applications.

PubMed

Nabovati, Ghazal; Ghafar-Zadeh, Ebrahim; Letourneau, Antoine; Sawan, Mohamad

2017-04-01

In this paper we present a CMOS capacitive sensor array as a compact and low-cost platform for high-throughput cell growth monitoring. The proposed biosensor, consists of an array of 8 × 8 CMOS fully differential charge-based capacitive measurement sensors. A DC-input Σ∆ modulator is used to convert the sensors' signals to digital values for reading out the biological/chemical data and further signal processing. To compensate the mismatch variations between the current mirror transistors, a calibration circuitry is proposed which removes the output voltage offset with less than 8.2% error. We validate the chip functionality using various organic solvents with different dielectric constants. Moreover, we show the response of the chip to different concentrations of Polystyrene beads that have the same electrical properties as the living cells. The experimental results show that the chip allows the detection of a wide range of Polystyrene beads concentrations from as low as 10 beads/ml to 100 k beads/ml. In addition, we present the experimental results from H1299 (human lung carcinoma) cell line where we show that the chip successfully allows the detection of cell attachment and growth over capacitive electrodes in a 30 h measurement time and the results are in consistency with the standard cell-based assays. The capability of proposed device for label-free and real-time detection of cell growth with very high sensitivity opens up the important opportunity for utilizing the device in rapid screening of living cells.

A high-throughput platform for population reformatting and mammalian expression of phage display libraries to enable functional screening as full-length IgG.

PubMed

Xiao, Xiaodong; Douthwaite, Julie A; Chen, Yan; Kemp, Ben; Kidd, Sara; Percival-Alwyn, Jennifer; Smith, Alison; Goode, Kate; Swerdlow, Bonnie; Lowe, David; Wu, Herren; Dall'Acqua, William F; Chowdhury, Partha S

Phage display antibody libraries are a rich resource for discovery of potential therapeutic antibodies. Single-chain variable fragment (scFv) libraries are the most common format due to the efficient display of scFv by phage particles and the ease by which soluble scFv antibodies can be expressed for high-throughput screening. Typically, a cascade of screening and triaging activities are performed, beginning with the assessment of large numbers of E. coli-expressed scFv, and progressing through additional assays with individual reformatting of the most promising scFv to full-length IgG. However, use of high-throughput screening of scFv for the discovery of full-length IgG is not ideal because of the differences between these molecules. Furthermore, the reformatting step represents a bottle neck in the process because each antibody has to be handled individually to preserve the unique VH and VL pairing. These problems could be resolved if populations of scFv could be reformatted to full-length IgG before screening without disrupting the variable region pairing. Here, we describe a novel strategy that allows the reformatting of diverse populations of scFv from phage selections to full-length IgG in a batch format. The reformatting process maintains the diversity and variable region pairing with high fidelity, and the resulted IgG pool enables high-throughput expression of IgG in mammalian cells and cell-based functional screening. The improved process led to the discovery of potent candidates that are comparable or better than those obtained by traditional methods. This strategy should also be readily applicable to Fab-based phage libraries. Our approach, Screening in Product Format (SiPF), represents a substantial improvement in the field of antibody discovery using phage display.

Detection and quantification of intracellular bacterial colonies by automated, high-throughput microscopy.

PubMed

Ernstsen, Christina L; Login, Frédéric H; Jensen, Helene H; Nørregaard, Rikke; Møller-Jensen, Jakob; Nejsum, Lene N

2017-08-01

To target bacterial pathogens that invade and proliferate inside host cells, it is necessary to design intervention strategies directed against bacterial attachment, cellular invasion and intracellular proliferation. We present an automated microscopy-based, fast, high-throughput method for analyzing size and number of intracellular bacterial colonies in infected tissue culture cells. Cells are seeded in 48-well plates and infected with a GFP-expressing bacterial pathogen. Following gentamicin treatment to remove extracellular pathogens, cells are fixed and cell nuclei stained. This is followed by automated microscopy and subsequent semi-automated spot detection to determine the number of intracellular bacterial colonies, their size distribution, and the average number per host cell. Multiple 48-well plates can be processed sequentially and the procedure can be completed in one working day. As a model we quantified intracellular bacterial colonies formed by uropathogenic Escherichia coli (UPEC) during infection of human kidney cells (HKC-8). Urinary tract infections caused by UPEC are among the most common bacterial infectious diseases in humans. UPEC can colonize tissues of the urinary tract and is responsible for acute, chronic, and recurrent infections. In the bladder, UPEC can form intracellular quiescent reservoirs, thought to be responsible for recurrent infections. In the kidney, UPEC can colonize renal epithelial cells and pass to the blood stream, either via epithelial cell disruption or transcellular passage, to cause sepsis. Intracellular colonies are known to be clonal, originating from single invading UPEC. In our experimental setup, we found UPEC CFT073 intracellular bacterial colonies to be heterogeneous in size and present in nearly one third of the HKC-8 cells. This high-throughput experimental format substantially reduces experimental time and enables fast screening of the intracellular bacterial load and cellular distribution of multiple bacterial isolates. This will be a powerful experimental tool facilitating the study of bacterial invasion, drug resistance, and the development of new therapeutics. Copyright © 2017 Elsevier B.V. All rights reserved.

A versatile coupled cell-free transcription-translation system based on tobacco BY-2 cell lysates.

PubMed

Buntru, Matthias; Vogel, Simon; Stoff, Katrin; Spiegel, Holger; Schillberg, Stefan

2015-05-01

Cell-free protein synthesis is a powerful method for the high-throughput production of recombinant proteins, especially proteins that are difficult to express in living cells. Here we describe a coupled cell-free transcription-translation system based on tobacco BY-2 cell lysates (BYLs). Using a combination of fractional factorial designs and response surface models, we developed a cap-independent system that produces more than 250 μg/mL of functional enhanced yellow fluorescent protein (eYFP) and about 270 μg/mL of firefly luciferase using plasmid templates, and up to 180 μg/mL eYFP using linear templates (PCR products) in 18 h batch reactions. The BYL contains actively-translocating microsomal vesicles derived from the endoplasmic reticulum, promoting the formation of disulfide bonds, glycosylation and the cotranslational integration of membrane proteins. This was demonstrated by expressing a functional full-size antibody (∼ 150 μg/mL), the model enzyme glucose oxidase (GOx) (∼ 7.3 U/mL), and a transmembrane growth factor (∼ 25 μg/mL). Subsequent in vitro treatment of GOx with peptide-N-glycosidase F confirmed the presence of N-glycans. Our results show that the BYL can be used as a high-throughput expression and screening platform that is particularly suitable for complex and cytotoxic proteins. © 2014 Wiley Periodicals, Inc.

Predictive models of prenatal developmental toxicity from ToxCast high-throughput screening data

EPA Science Inventory

EPA's ToxCast™ project is profiling the in vitro bioactivity of chemicals to assess pathway-level and cell-based signatures that correlate with observed in vivo toxicity. We hypothesized that developmental toxicity in guideline animal studies captured in the ToxRefDB database wou...

Novel bacterial bioassay for a high-throughput screening of 4-hydroxyphenylpyruvate dioxygenase inhibitors

USDA-ARS?s Scientific Manuscript database

Plant 4-hydroxyphenylpyruvate dioxygenase (HPPD) is the molecular target of a range of synthetic ß-triketone herbicides that are currently used commercially. Their mode of action is based on an irreversible inhibition of HPPD. Therefore, this inhibitory capacity was used to develop a whole cell colo...

Use of High-Throughput Cell-Based and Model Organism Assays for Understanding the Potential Toxicity of Engineered Nanomaterials

EPA Science Inventory

The rapidly expanding field of nanotechnology is introducing a large number and diversity of engineered nanomaterials into research and commerce with concordant uncertainty regarding the potential adverse health and ecological effects. With costs and time of traditional animal to...

Micropillar arrays as a high-throughput screening platform for therapeutics in multiple sclerosis.

PubMed

Mei, Feng; Fancy, Stephen P J; Shen, Yun-An A; Niu, Jianqin; Zhao, Chao; Presley, Bryan; Miao, Edna; Lee, Seonok; Mayoral, Sonia R; Redmond, Stephanie A; Etxeberria, Ainhoa; Xiao, Lan; Franklin, Robin J M; Green, Ari; Hauser, Stephen L; Chan, Jonah R

2014-08-01

Functional screening for compounds that promote remyelination represents a major hurdle in the development of rational therapeutics for multiple sclerosis. Screening for remyelination is problematic, as myelination requires the presence of axons. Standard methods do not resolve cell-autonomous effects and are not suited for high-throughput formats. Here we describe a binary indicant for myelination using micropillar arrays (BIMA). Engineered with conical dimensions, micropillars permit resolution of the extent and length of membrane wrapping from a single two-dimensional image. Confocal imaging acquired from the base to the tip of the pillars allows for detection of concentric wrapping observed as 'rings' of myelin. The platform is formatted in 96-well plates, amenable to semiautomated random acquisition and automated detection and quantification. Upon screening 1,000 bioactive molecules, we identified a cluster of antimuscarinic compounds that enhance oligodendrocyte differentiation and remyelination. Our findings demonstrate a new high-throughput screening platform for potential regenerative therapeutics in multiple sclerosis.

Using constitutive activity to define appropriate high-throughput screening assays for orphan g protein-coupled receptors.

PubMed

Ngo, Tony; Coleman, James L J; Smith, Nicola J

2015-01-01

Orphan G protein-coupled receptors represent an underexploited resource for drug discovery but pose a considerable challenge for assay development because their cognate G protein signaling pathways are often unknown. In this methodological chapter, we describe the use of constitutive activity, that is, the inherent ability of receptors to couple to their cognate G proteins in the absence of ligand, to inform the development of high-throughput screening assays for a particular orphan receptor. We specifically focus on a two-step process, whereby constitutive G protein coupling is first determined using yeast Gpa1/human G protein chimeras linked to growth and β-galactosidase generation. Coupling selectivity is then confirmed in mammalian cells expressing endogenous G proteins and driving accumulation of transcription factor-fused luciferase reporters specific to each of the classes of G protein. Based on these findings, high-throughput screening campaigns can be performed on the already miniaturized mammalian reporter system.

High-throughput characterization for solar fuels materials discovery

NASA Astrophysics Data System (ADS)

Mitrovic, Slobodan; Becerra, Natalie; Cornell, Earl; Guevarra, Dan; Haber, Joel; Jin, Jian; Jones, Ryan; Kan, Kevin; Marcin, Martin; Newhouse, Paul; Soedarmadji, Edwin; Suram, Santosh; Xiang, Chengxiang; Gregoire, John; High-Throughput Experimentation Team

2014-03-01

In this talk I will present the status of the High-Throughput Experimentation (HTE) project of the Joint Center for Artificial Photosynthesis (JCAP). JCAP is an Energy Innovation Hub of the U.S. Department of Energy with a mandate to deliver a solar fuel generator based on an integrated photoelectrochemical cell (PEC). However, efficient and commercially viable catalysts or light absorbers for the PEC do not exist. The mission of HTE is to provide the accelerated discovery through combinatorial synthesis and rapid screening of material properties. The HTE pipeline also features high-throughput material characterization using x-ray diffraction and x-ray photoemission spectroscopy (XPS). In this talk I present the currently operating pipeline and focus on our combinatorial XPS efforts to build the largest free database of spectra from mixed-metal oxides, nitrides, sulfides and alloys. This work was performed at Joint Center for Artificial Photosynthesis, a DOE Energy Innovation Hub, supported through the Office of Science of the U.S. Department of Energy under Award No. DE-SC0004993.

Chemical screening identifies filastatin, a small molecule inhibitor of Candida albicans adhesion, morphogenesis, and pathogenesis.

PubMed

Fazly, Ahmed; Jain, Charu; Dehner, Amie C; Issi, Luca; Lilly, Elizabeth A; Ali, Akbar; Cao, Hong; Fidel, Paul L; Rao, Reeta P; Kaufman, Paul D

2013-08-13

Infection by pathogenic fungi, such as Candida albicans, begins with adhesion to host cells or implanted medical devices followed by biofilm formation. By high-throughput phenotypic screening of small molecules, we identified compounds that inhibit adhesion of C. albicans to polystyrene. Our lead candidate compound also inhibits binding of C. albicans to cultured human epithelial cells, the yeast-to-hyphal morphological transition, induction of the hyphal-specific HWP1 promoter, biofilm formation on silicone elastomers, and pathogenesis in a nematode infection model as well as alters fungal morphology in a mouse mucosal infection assay. We term this compound filastatin based on its strong inhibition of filamentation, and we use chemical genetic experiments to show that it acts downstream of multiple signaling pathways. These studies show that high-throughput functional assays targeting fungal adhesion can provide chemical probes for study of multiple aspects of fungal pathogenesis.

Chemical screening identifies filastatin, a small molecule inhibitor of Candida albicans adhesion, morphogenesis, and pathogenesis

PubMed Central

Fazly, Ahmed; Jain, Charu; Dehner, Amie C.; Issi, Luca; Lilly, Elizabeth A.; Ali, Akbar; Cao, Hong; Fidel, Paul L.; P. Rao, Reeta; Kaufman, Paul D.

2013-01-01

Infection by pathogenic fungi, such as Candida albicans, begins with adhesion to host cells or implanted medical devices followed by biofilm formation. By high-throughput phenotypic screening of small molecules, we identified compounds that inhibit adhesion of C. albicans to polystyrene. Our lead candidate compound also inhibits binding of C. albicans to cultured human epithelial cells, the yeast-to-hyphal morphological transition, induction of the hyphal-specific HWP1 promoter, biofilm formation on silicone elastomers, and pathogenesis in a nematode infection model as well as alters fungal morphology in a mouse mucosal infection assay. We term this compound filastatin based on its strong inhibition of filamentation, and we use chemical genetic experiments to show that it acts downstream of multiple signaling pathways. These studies show that high-throughput functional assays targeting fungal adhesion can provide chemical probes for study of multiple aspects of fungal pathogenesis. PMID:23904484

High-throughput Screening of ToxCast" Phase I Chemicals in an Embryonic Stem Cell Assay Reveals Potential Disruption of a Critical Developmental Signaling Pathway

EPA Science Inventory

Little is known about the developmental toxicity of the expansive chemical landscape in existence today. Significant efforts are being made to apply novel methods to predict developmental activity of chemicals utilizing high-throughput screening (HTS) and high-content screening (...

Comparison of Human Induced Pluripotent Stem Cell-Derived Neurons and Rat Primary CorticalNeurons as In Vitro Models of Neurite Outgrowth

EPA Science Inventory

High-throughput assays that can quantify chemical-induced changes at the cellular and molecular level have been recommended for use in chemical safety assessment. High-throughput, high content imaging assays for the key cellular events of neurodevelopment have been proposed to ra...

Analysis of Protein Expression in Cell Microarrays: A Tool for Antibody-based Proteomics

PubMed Central

Andersson, Ann-Catrin; Strömberg, Sara; Bäckvall, Helena; Kampf, Caroline; Uhlen, Mathias; Wester, Kenneth; Pontén, Fredrik

2006-01-01

Tissue microarray (TMA) technology provides a possibility to explore protein expression patterns in a multitude of normal and disease tissues in a high-throughput setting. Although TMAs have been used for analysis of tissue samples, robust methods for studying in vitro cultured cell lines and cell aspirates in a TMA format have been lacking. We have adopted a technique to homogeneously distribute cells in an agarose gel matrix, creating an artificial tissue. This enables simultaneous profiling of protein expression in suspension- and adherent-grown cell samples assembled in a microarray. In addition, the present study provides an optimized strategy for the basic laboratory steps to efficiently produce TMAs. Presented modifications resulted in an improved quality of specimens and a higher section yield compared with standard TMA production protocols. Sections from the generated cell TMAs were tested for immunohistochemical staining properties using 20 well-characterized antibodies. Comparison of immunoreactivity in cultured dispersed cells and corresponding cells in tissue samples showed congruent results for all tested antibodies. We conclude that a modified TMA technique, including cell samples, provides a valuable tool for high-throughput analysis of protein expression, and that this technique can be used for global approaches to explore the human proteome. PMID:16957166

An HTRF® Assay for the Protein Kinase ATM.

PubMed

Adams, Phillip; Clark, Jonathan; Hawdon, Simon; Hill, Jennifer; Plater, Andrew

2017-01-01

Ataxia telangiectasia mutated (ATM) is a serine/threonine kinase that plays a key role in the regulation of DNA damage pathways and checkpoint arrest. In recent years, there has been growing interest in ATM as a therapeutic target due to its association with cancer cell survival following genotoxic stress such as radio- and chemotherapy. Large-scale targeted drug screening campaigns have been hampered, however, by technical issues associated with the production of sufficient quantities of purified ATM and the availability of a suitable high-throughput assay. Using a purified, functionally active recombinant ATM and one of its physiological substrates, p53, we have developed an in vitro FRET-based activity assay that is suitable for high-throughput drug screening.

Hydrogel Droplet Microfluidics for High-Throughput Single Molecule/Cell Analysis.

PubMed

Zhu, Zhi; Yang, Chaoyong James

2017-01-17

Heterogeneity among individual molecules and cells has posed significant challenges to traditional bulk assays, due to the assumption of average behavior, which would lose important biological information in heterogeneity and result in a misleading interpretation. Single molecule/cell analysis has become an important and emerging field in biological and biomedical research for insights into heterogeneity between large populations at high resolution. Compared with the ensemble bulk method, single molecule/cell analysis explores the information on time trajectories, conformational states, and interactions of individual molecules/cells, all key factors in the study of chemical and biological reaction pathways. Various powerful techniques have been developed for single molecule/cell analysis, including flow cytometry, atomic force microscopy, optical and magnetic tweezers, single-molecule fluorescence spectroscopy, and so forth. However, some of them have the low-throughput issue that has to analyze single molecules/cells one by one. Flow cytometry is a widely used high-throughput technique for single cell analysis but lacks the ability for intercellular interaction study and local environment control. Droplet microfluidics becomes attractive for single molecule/cell manipulation because single molecules/cells can be individually encased in monodisperse microdroplets, allowing high-throughput analysis and manipulation with precise control of the local environment. Moreover, hydrogels, cross-linked polymer networks that swell in the presence of water, have been introduced into droplet microfluidic systems as hydrogel droplet microfluidics. By replacing an aqueous phase with a monomer or polymer solution, hydrogel droplets can be generated on microfluidic chips for encapsulation of single molecules/cells according to the Poisson distribution. The sol-gel transition property endows the hydrogel droplets with new functionalities and diversified applications in single molecule/cell analysis. The hydrogel can act as a 3D cell culture matrix to mimic the extracellular environment for long-term single cell culture, which allows further heterogeneity study in proliferation, drug screening, and metastasis at the single-cell level. The sol-gel transition allows reactions in solution to be performed rapidly and efficiently with product storage in the gel for flexible downstream manipulation and analysis. More importantly, controllable sol-gel regulation provides a new way to maintain phenotype-genotype linkages in the hydrogel matrix for high throughput molecular evolution. In this Account, we will review the hydrogel droplet generation on microfluidics, single molecule/cell encapsulation in hydrogel droplets, as well as the progress made by our group and others in the application of hydrogel droplet microfluidics for single molecule/cell analysis, including single cell culture, single molecule/cell detection, single cell sequencing, and molecular evolution.

A probabilistic approach to joint cell tracking and segmentation in high-throughput microscopy videos.

PubMed

Arbelle, Assaf; Reyes, Jose; Chen, Jia-Yun; Lahav, Galit; Riklin Raviv, Tammy

2018-04-22

We present a novel computational framework for the analysis of high-throughput microscopy videos of living cells. The proposed framework is generally useful and can be applied to different datasets acquired in a variety of laboratory settings. This is accomplished by tying together two fundamental aspects of cell lineage construction, namely cell segmentation and tracking, via a Bayesian inference of dynamic models. In contrast to most existing approaches, which aim to be general, no assumption of cell shape is made. Spatial, temporal, and cross-sectional variation of the analysed data are accommodated by two key contributions. First, time series analysis is exploited to estimate the temporal cell shape uncertainty in addition to cell trajectory. Second, a fast marching (FM) algorithm is used to integrate the inferred cell properties with the observed image measurements in order to obtain image likelihood for cell segmentation, and association. The proposed approach has been tested on eight different time-lapse microscopy data sets, some of which are high-throughput, demonstrating promising results for the detection, segmentation and association of planar cells. Our results surpass the state of the art for the Fluo-C2DL-MSC data set of the Cell Tracking Challenge (Maška et al., 2014). Copyright © 2018 Elsevier B.V. All rights reserved.

A miniature microbial fuel cell with conducting nanofibers-based 3D porous biofilm

NASA Astrophysics Data System (ADS)

Jiang, Huawei; Halverson, Larry J.; Dong, Liang

2015-12-01

Miniature microbial fuel cell (MFC) technology has received growing interest due to its potential applications in high-throughput screening of bacteria and mutants to elucidate mechanisms of electricity generation. This paper reports a novel miniature MFC with an improved output power density and short startup time, utilizing electrospun conducting poly(3,4-ethylenedioxythiophene) (PEDOT) nanofibers as a 3D porous anode within a 12 μl anolyte chamber. This device results in 423 μW cm-3 power density based on the volume of the anolyte chamber, using Shewanella oneidensis MR-1 as a model biocatalyst without any optimization of bacterial culture. The device also excels in a startup time of only 1hr. The high conductivity of the electrospun nanofibers makes them suitable for efficient electron transfer. The mean pore size of the conducting nanofibers is several micrometers, which is favorable for bacterial penetration and colonization of surfaces of the nanofibers. We demonstrate that S. oneidensis can fully colonize the interior region of this nanofibers-based porous anode. This work represents a new attempt to explore the use of electrospun PEDOT nanofibers as a 3D anode material for MFCs. The presented miniature MFC potentially will provide a high-sensitivity, high-throughput tool to screen suitable bacterial species and mutant strains for use in large-size MFCs.

Development and Characterization of a High Throughput Screen to investigate the delayed Effects of Radiations Commonly Encountered in Space

NASA Astrophysics Data System (ADS)

Morgan, W. F.

Astronauts based on the space station or on long-term space missions will be exposed to high Z radiations in the cosmic environment In order to evaluate the potentially deleterious effects of exposure to radiations commonly encountered in space we have developed and characterized a high throughput assay to detect mutation deletion events and or hyperrecombination in the progeny of exposed cells This assay is based on a plasmid vector containing a green fluorescence protein reporter construct We have shown that after stable transfection of the vector into human or hamster cells this construct can identify mutations specifically base changes and deletions as well as recombination events e g gene conversion or homologous recombination occurring as a result of exposure to ionizing radiation Our focus has been on those events occurring in the progeny of an irradiated cell that are potentially associated with radiation induced genomic instability rather than the more conventional assays that evaluate the direct immediate effects of radiation exposure Considerable time has been spent automating analysis of surviving colonies as a function of time after irradiation in order to determine when delayed instability is induced and the consequences of this delayed instability The assay is now automated permitting the evaluation of potentially rare events associated with low dose low dose rate radiations commonly encountered in space

High-throughput screening of small molecules in miniaturized mammalian cell-based assays involving post-translational modifications.

PubMed

Stockwell, B R; Haggarty, S J; Schreiber, S L

1999-02-01

Fully adapting a forward genetic approach to mammalian systems requires efficient methods to alter systematically gene products without prior knowledge of gene sequences, while allowing for the subsequent characterization of these alterations. Ideally, these methods would also allow function to be altered in a temporally controlled manner. We report the development of a miniaturized cell-based assay format that enables a genetic-like approach to understanding cellular pathways in mammalian systems using small molecules, rather than mutations, as the source of gene-product alterations. This whole-cell immunodetection assay can sensitively detect changes in specific cellular macromolecules in high-density arrays of mammalian cells. Furthermore, it is compatible with screening large numbers of small molecules in nanoliter to microliter culture volumes. We refer to this assay format as a 'cytoblot', and demonstrate the use of cytoblotting to monitor biosynthetic processes such as DNA synthesis, and post-translational processes such as acetylation and phosphorylation. Finally, we demonstrate the applicability of these assays to natural-product screening through the identification of marine sponge extracts exhibiting genotype-specific inhibition of 5-bromodeoxyuridine incorporation and suppression of the anti-proliferative effect of rapamycin. We show that cytoblots can be used for high-throughput screening of small molecules in cell-based assays. Together with small-molecule libraries, the cytoblot assay can be used to perform chemical genetic screens analogous to those used in classical genetics and thus should be applicable to understanding a wide variety of cellular processes, especially those involving post-transitional modifications.

Massively Parallel Rogue Cell Detection Using Serial Time-Encoded Amplified Microscopy of Inertially Ordered Cells in High Throughput Flow

DTIC Science & Technology

2011-08-01

further chemical analysis of the cells. While in our proof-of-concept demonstration, we showed high- throughput screening of budding yeast and...of 8.0 mW/cm2 through the transparency mask for 90 seconds. The wafer was baked again at 95°C for 4 minutes then developed in SU-8 developer...sonicated in isopropanol for 5 minutes, sonicated in deionized H2O for 5 minutes, and baked at 65°C for at least 30 minutes. Holes were punched

Managing evaporation for more robust microscale assays. Part 2. Characterization of convection and diffusion for cell biology.

PubMed

Berthier, Erwin; Warrick, Jay; Yu, Hongmeiy; Beebe, David J

2008-06-01

Cell based microassays allow the screening of a multitude of culture conditions in parallel, which can be used for various applications from drug screening to fundamental cell biology research. Tubeless microfluidic devices based on passive pumping are a step towards accessible high throughput microassays, however they are vulnerable to evaporation. In addition to volume loss, evaporation can lead to the generation of small flows. Here, we focus on issues of convection and diffusion for cell culture in microchannels and particularly the transport of soluble factors secreted by cells. We find that even for humidity levels as high as 95%, convection in a passive pumping channel can significantly alter distributions of these factors and that appropriate system design can prevent convection.

Natural products that reduce rotavirus infectivity identified by a cell-based moderate-throughput screening assay.

PubMed

Shaneyfelt, Mark E; Burke, Anna D; Graff, Joel W; Jutila, Mark A; Hardy, Michele E

2006-09-01

There is widespread interest in the use of innate immune modulators as a defense strategy against infectious pathogens. Using rotavirus as a model system, we developed a cell-based, moderate-throughput screening (MTS) assay to identify compounds that reduce rotavirus infectivity in vitro, toward a long-term goal of discovering immunomodulatory agents that enhance innate responses to viral infection. A natural product library consisting of 280 compounds was screened in the assay and 15 compounds that significantly reduced infectivity without cytotoxicity were identified. Time course analysis of four compounds with previously characterized effects on inflammatory gene expression inhibited replication with pre-treatment times as minimal as 2 hours. Two of these four compounds, alpha-mangostin and 18-beta-glycyrrhetinic acid, activated NFkappaB and induced IL-8 secretion. The assay is adaptable to other virus systems, and amenable to full automation and adaptation to a high-throughput format. Identification of several compounds with known effects on inflammatory and antiviral gene expression that confer resistance to rotavirus infection in vitro suggests the assay is an appropriate platform for discovery of compounds with potential to amplify innate antiviral responses.

Induced Pluripotent Stem Cells for Disease Modeling and Evaluation of Therapeutics for Niemann-Pick Disease Type A.

PubMed

Long, Yan; Xu, Miao; Li, Rong; Dai, Sheng; Beers, Jeanette; Chen, Guokai; Soheilian, Ferri; Baxa, Ulrich; Wang, Mengqiao; Marugan, Juan J; Muro, Silvia; Li, Zhiyuan; Brady, Roscoe; Zheng, Wei

2016-12-01

: Niemann-Pick disease type A (NPA) is a lysosomal storage disease caused by mutations in the SMPD1 gene that encodes acid sphingomyelinase (ASM). Deficiency in ASM function results in lysosomal accumulation of sphingomyelin and neurodegeneration. Currently, there is no effective treatment for NPA. To accelerate drug discovery for treatment of NPA, we generated induced pluripotent stem cells from two patient dermal fibroblast lines and differentiated them into neural stem cells. The NPA neural stem cells exhibit a disease phenotype of lysosomal sphingomyelin accumulation and enlarged lysosomes. By using this disease model, we also evaluated three compounds that reportedly reduced lysosomal lipid accumulation in Niemann-Pick disease type C as well as enzyme replacement therapy with ASM. We found that α-tocopherol, δ-tocopherol, hydroxypropyl-β-cyclodextrin, and ASM reduced sphingomyelin accumulation and enlarged lysosomes in NPA neural stem cells. Therefore, the NPA neural stem cells possess the characteristic NPA disease phenotype that can be ameliorated by tocopherols, cyclodextrin, and ASM. Our results demonstrate the efficacies of cyclodextrin and tocopherols in the NPA cell-based model. Our data also indicate that the NPA neural stem cells can be used as a new cell-based disease model for further study of disease pathophysiology and for high-throughput screening to identify new lead compounds for drug development. Currently, there is no effective treatment for Niemann-Pick disease type A (NPA). To accelerate drug discovery for treatment of NPA, NPA-induced pluripotent stem cells were generated from patient dermal fibroblasts and differentiated into neural stem cells. By using the differentiated NPA neuronal cells as a cell-based disease model system, α-tocopherol, δ-tocopherol, and hydroxypropyl-β-cyclodextrin significantly reduced sphingomyelin accumulation in these NPA neuronal cells. Therefore, this cell-based NPA model can be used for further study of disease pathophysiology and for high-throughput screening of compound libraries to identify lead compounds for drug development. ©AlphaMed Press.

OptoDyCE: Automated system for high-throughput all-optical dynamic cardiac electrophysiology

NASA Astrophysics Data System (ADS)

Klimas, Aleksandra; Yu, Jinzhu; Ambrosi, Christina M.; Williams, John C.; Bien, Harold; Entcheva, Emilia

2016-02-01

In the last two decades, <30% of drugs withdrawals from the market were due to cardiac toxicity, where unintended interactions with ion channels disrupt the heart's normal electrical function. Consequently, all new drugs must undergo preclinical testing for cardiac liability, adding to an already expensive and lengthy process. Recognition that proarrhythmic effects often result from drug action on multiple ion channels demonstrates a need for integrative and comprehensive measurements. Additionally, patient-specific therapies relying on emerging technologies employing stem-cell derived cardiomyocytes (e.g. induced pluripotent stem-cell-derived cardiomyocytes, iPSC-CMs) require better screening methods to become practical. However, a high-throughput, cost-effective approach for cellular cardiac electrophysiology has not been feasible. Optical techniques for manipulation and recording provide a contactless means of dynamic, high-throughput testing of cells and tissues. Here, we consider the requirements for all-optical electrophysiology for drug testing, and we implement and validate OptoDyCE, a fully automated system for all-optical cardiac electrophysiology. We demonstrate the high-throughput capabilities using multicellular samples in 96-well format by combining optogenetic actuation with simultaneous fast high-resolution optical sensing of voltage or intracellular calcium. The system can also be implemented using iPSC-CMs and other cell-types by delivery of optogenetic drivers, or through the modular use of dedicated light-sensitive somatic cells in conjunction with non-modified cells. OptoDyCE provides a truly modular and dynamic screening system, capable of fully-automated acquisition of high-content information integral for improved discovery and development of new drugs and biologics, as well as providing a means of better understanding of electrical disturbances in the heart.

A method for quantitative analysis of standard and high-throughput qPCR expression data based on input sample quantity.

PubMed

Adamski, Mateusz G; Gumann, Patryk; Baird, Alison E

2014-01-01

Over the past decade rapid advances have occurred in the understanding of RNA expression and its regulation. Quantitative polymerase chain reactions (qPCR) have become the gold standard for quantifying gene expression. Microfluidic next generation, high throughput qPCR now permits the detection of transcript copy number in thousands of reactions simultaneously, dramatically increasing the sensitivity over standard qPCR. Here we present a gene expression analysis method applicable to both standard polymerase chain reactions (qPCR) and high throughput qPCR. This technique is adjusted to the input sample quantity (e.g., the number of cells) and is independent of control gene expression. It is efficiency-corrected and with the use of a universal reference sample (commercial complementary DNA (cDNA)) permits the normalization of results between different batches and between different instruments--regardless of potential differences in transcript amplification efficiency. Modifications of the input quantity method include (1) the achievement of absolute quantification and (2) a non-efficiency corrected analysis. When compared to other commonly used algorithms the input quantity method proved to be valid. This method is of particular value for clinical studies of whole blood and circulating leukocytes where cell counts are readily available.

Prediction-based association control scheme in dense femtocell networks

PubMed Central

Pham, Ngoc-Thai; Huynh, Thong; Hwang, Won-Joo; You, Ilsun; Choo, Kim-Kwang Raymond

2017-01-01

The deployment of large number of femtocell base stations allows us to extend the coverage and efficiently utilize resources in a low cost manner. However, the small cell size of femtocell networks can result in frequent handovers to the mobile user, and consequently throughput degradation. Thus, in this paper, we propose predictive association control schemes to improve the system’s effective throughput. Our design focuses on reducing handover frequency without impacting on throughput. The proposed schemes determine handover decisions that contribute most to the network throughput and are proper for distributed implementations. The simulation results show significant gains compared with existing methods in terms of handover frequency and network throughput perspective. PMID:28328992

A conifer-friendly high-throughput α-cellulose extraction method for δ13C and δ18O stable isotope ratio analysis

NASA Astrophysics Data System (ADS)

Lin, W.; Noormets, A.; domec, J.; King, J. S.; Sun, G.; McNulty, S.

2012-12-01

Wood stable isotope ratios (δ13C and δ18O) offer insight to water source and plant water use efficiency (WUE), which in turn provide a glimpse to potential plant responses to changing climate, particularly rainfall patterns. The synthetic pathways of cell wall deposition in wood rings differ in their discrimination ratios between the light and heavy isotopes, and α-cellulose is broadly seen as the best indicator of plant water status due to its local and temporal fixation and to its high abundance within the wood. To use the effects of recent severe droughts on the WUE of loblolly pine (Pinus taeda) throughout Southeastern USA as a harbinger of future changes, an effort has been undertaken to sample the entire range of the species and to sample the isotopic composition in a consistent manner. To be able to accommodate the large number of samples required by this analysis, we have developed a new high-throughput method for α-cellulose extraction, which is the rate-limiting step in such an endeavor. Although an entire family of methods has been developed and perform well, their throughput in a typical research lab setting is limited to 16-75 samples per week with intensive labor input. The resin exclusion step in conifersis is particularly time-consuming. We have combined the recent advances of α-cellulose extraction in plant ecology and wood science, including a high-throughput extraction device developed in the Potsdam Dendro Lab and a simple chemical-based resin exclusion method. By transferring the entire extraction process to a multiport-based system allows throughputs of up to several hundred samples in two weeks, while minimizing labor requirements to 2-3 days per batch of samples.

A high-throughput assay of NK cell activity in whole blood and its clinical application

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Saet-byul; Cha, Junhoe; Kim, Im-kyung

2014-03-14

Graphical abstract: - Highlights: • We demonstrated a simple assay of NK cell activity from whole blood. • The measurement of secreted IFN-γ from NK cell enables high-throughput screening. • The NKA assay was validated by clinical results of colorectal cancer patients. - Abstract: Natural killer (NK) cells are lymphocytes of the innate immune system and have the ability to kill tumor cells and virus-infected cells without prior sensitization. Malignant tumors and viruses have developed, however, strategies to suppress NK cells to escape from their responses. Thus, the evaluation of NK cell activity (NKA) could be invaluable to estimate themore » status and the outcome of cancers, viral infections, and immune-mediated diseases. Established methods that measure NKA, such as {sup 51}Cr release assay and CD107a degranulation assay, may be used to determine NK cell function, but they are complicated and time-consuming because they require isolation of peripheral blood mononuclear cells (PBMC) or NK cells. In some cases these assays require hazardous material such as radioactive isotopes. To overcome these difficulties, we developed a simple assay that uses whole blood instead of PBMC or isolated NK cells. This novel assay is suitable for high-throughput screening and the monitoring of diseases, because it employs serum of ex vivo stimulated whole blood to detect interferon (IFN)-γ secreted from NK cells as an indicator of NKA. After the stimulation of NK cells, the determination of IFNγ concentration in serum samples by enzyme-linked immunosorbent assay (ELISA) provided a swift, uncomplicated, and high-throughput assay of NKA ex vivo. The NKA results microsatellite stable (MSS) colorectal cancer patients was showed significantly lower NKA, 263.6 ± 54.5 pg/mL compared with healthy subjects, 867.5 ± 50.2 pg/mL (p value <0.0001). Therefore, the NKA could be utilized as a supportive diagnostic marker for microsatellite stable (MSS) colorectal cancer.« less

Computational Tools for Stem Cell Biology

PubMed Central

Bian, Qin; Cahan, Patrick

2016-01-01

For over half a century, the field of developmental biology has leveraged computation to explore mechanisms of developmental processes. More recently, computational approaches have been critical in the translation of high throughput data into knowledge of both developmental and stem cell biology. In the last several years, a new sub-discipline of computational stem cell biology has emerged that synthesizes the modeling of systems-level aspects of stem cells with high-throughput molecular data. In this review, we provide an overview of this new field and pay particular attention to the impact that single-cell transcriptomics is expected to have on our understanding of development and our ability to engineer cell fate. PMID:27318512

Computational Tools for Stem Cell Biology.

PubMed

Bian, Qin; Cahan, Patrick

2016-12-01

For over half a century, the field of developmental biology has leveraged computation to explore mechanisms of developmental processes. More recently, computational approaches have been critical in the translation of high throughput data into knowledge of both developmental and stem cell biology. In the past several years, a new subdiscipline of computational stem cell biology has emerged that synthesizes the modeling of systems-level aspects of stem cells with high-throughput molecular data. In this review, we provide an overview of this new field and pay particular attention to the impact that single cell transcriptomics is expected to have on our understanding of development and our ability to engineer cell fate. Copyright © 2016 Elsevier Ltd. All rights reserved.

High-Content, High-Throughput Screening for the Identification of Cytotoxic Compounds Based on Cell Morphology and Cell Proliferation Markers

PubMed Central

Martin, Heather L.; Adams, Matthew; Higgins, Julie; Bond, Jacquelyn; Morrison, Ewan E.; Bell, Sandra M.; Warriner, Stuart; Nelson, Adam; Tomlinson, Darren C.

2014-01-01

Toxicity is a major cause of failure in drug discovery and development, and whilst robust toxicological testing occurs, efficiency could be improved if compounds with cytotoxic characteristics were identified during primary compound screening. The use of high-content imaging in primary screening is becoming more widespread, and by utilising phenotypic approaches it should be possible to incorporate cytotoxicity counter-screens into primary screens. Here we present a novel phenotypic assay that can be used as a counter-screen to identify compounds with adverse cellular effects. This assay has been developed using U2OS cells, the PerkinElmer Operetta high-content/high-throughput imaging system and Columbus image analysis software. In Columbus, algorithms were devised to identify changes in nuclear morphology, cell shape and proliferation using DAPI, TOTO-3 and phosphohistone H3 staining, respectively. The algorithms were developed and tested on cells treated with doxorubicin, taxol and nocodazole. The assay was then used to screen a novel, chemical library, rich in natural product-like molecules of over 300 compounds, 13.6% of which were identified as having adverse cellular effects. This assay provides a relatively cheap and rapid approach for identifying compounds with adverse cellular effects during screening assays, potentially reducing compound rejection due to toxicity in subsequent in vitro and in vivo assays. PMID:24505478

Toxicokinetic and Dosimetry Modeling Tools for Exposure ...

EPA Pesticide Factsheets

New technologies and in vitro testing approaches have been valuable additions to risk assessments that have historically relied solely on in vivo test results. Compared to in vivo methods, in vitro high throughput screening (HTS) assays are less expensive, faster and can provide mechanistic insights on chemical action. However, extrapolating from in vitro chemical concentrations to target tissue or blood concentrations in vivo is fraught with uncertainties, and modeling is dependent upon pharmacokinetic variables not measured in in vitro assays. To address this need, new tools have been created for characterizing, simulating, and evaluating chemical toxicokinetics. Physiologically-based pharmacokinetic (PBPK) models provide estimates of chemical exposures that produce potentially hazardous tissue concentrations, while tissue microdosimetry PK models relate whole-body chemical exposures to cell-scale concentrations. These tools rely on high-throughput in vitro measurements, and successful methods exist for pharmaceutical compounds that determine PK from limited in vitro measurements and chemical structure-derived property predictions. These high throughput (HT) methods provide a more rapid and less resource–intensive alternative to traditional PK model development. We have augmented these in vitro data with chemical structure-based descriptors and mechanistic tissue partitioning models to construct HTPBPK models for over three hundred environmental and pharmace

High-throughput microfluidic mixing and multiparametric cell sorting for bioactive compound screening.

PubMed

Young, Susan M; Curry, Mark S; Ransom, John T; Ballesteros, Juan A; Prossnitz, Eric R; Sklar, Larry A; Edwards, Bruce S

2004-03-01

HyperCyt, an automated sample handling system for flow cytometry that uses air bubbles to separate samples sequentially introduced from multiwell plates by an autosampler. In a previously documented HyperCyt configuration, air bubble separated compounds in one sample line and a continuous stream of cells in another are mixed in-line for serial flow cytometric cell response analysis. To expand capabilities for high-throughput bioactive compound screening, the authors investigated using this system configuration in combination with automated cell sorting. Peptide ligands were sampled from a 96-well plate, mixed in-line with fluo-4-loaded, formyl peptide receptor-transfected U937 cells, and screened at a rate of 3 peptide reactions per minute with approximately 10,000 cells analyzed per reaction. Cell Ca(2+) responses were detected to as little as 10(-11) M peptide with no detectable carryover between samples at up to 10(-7) M peptide. After expansion in culture, cells sort-purified from the 10% highest responders exhibited enhanced sensitivity and more sustained responses to peptide. Thus, a highly responsive cell subset was isolated under high-throughput mixing and sorting conditions in which response detection capability spanned a 1000-fold range of peptide concentration. With single-cell readout systems for protein expression libraries, this technology offers the promise of screening millions of discrete compound interactions per day.

ElectroTaxis-on-a-Chip (ETC): an integrated quantitative high-throughput screening platform for electrical field-directed cell migration.

PubMed

Zhao, Siwei; Zhu, Kan; Zhang, Yan; Zhu, Zijie; Xu, Zhengping; Zhao, Min; Pan, Tingrui

2014-11-21

Both endogenous and externally applied electrical stimulation can affect a wide range of cellular functions, including growth, migration, differentiation and division. Among those effects, the electrical field (EF)-directed cell migration, also known as electrotaxis, has received broad attention because it holds great potential in facilitating clinical wound healing. Electrotaxis experiment is conventionally conducted in centimetre-sized flow chambers built in Petri dishes. Despite the recent efforts to adapt microfluidics for electrotaxis studies, the current electrotaxis experimental setup is still cumbersome due to the needs of an external power supply and EF controlling/monitoring systems. There is also a lack of parallel experimental systems for high-throughput electrotaxis studies. In this paper, we present a first independently operable microfluidic platform for high-throughput electrotaxis studies, integrating all functional components for cell migration under EF stimulation (except microscopy) on a compact footprint (the same as a credit card), referred to as ElectroTaxis-on-a-Chip (ETC). Inspired by the R-2R resistor ladder topology in digital signal processing, we develop a systematic approach to design an infinitely expandable microfluidic generator of EF gradients for high-throughput and quantitative studies of EF-directed cell migration. Furthermore, a vacuum-assisted assembly method is utilized to allow direct and reversible attachment of our device to existing cell culture media on biological surfaces, which separates the cell culture and device preparation/fabrication steps. We have demonstrated that our ETC platform is capable of screening human cornea epithelial cell migration under the stimulation of an EF gradient spanning over three orders of magnitude. The screening results lead to the identification of the EF-sensitive range of that cell type, which can provide valuable guidance to the clinical application of EF-facilitated wound healing.

Observation of the immune response of cells and tissue through multimodal label-free microscopy

NASA Astrophysics Data System (ADS)

Pavillon, Nicolas; Smith, Nicholas I.

2017-02-01

We present applications of a label-free approach to assess the immune response based on the combination of interferometric microscopy and Raman spectroscopy, which makes it possible to simultaneously acquire morphological and molecular information of live cells. We employ this approach to derive statistical models for predicting the activation state of macrophage cells based both on morphological parameters extracted from the high-throughput full-field quantitative phase imaging, and on the molecular content information acquired through Raman spectroscopy. We also employ a system for 3D imaging based on coherence gating, enabling specific targeting of the Raman channel to structures of interest within tissue.

Asymmetric-detection time-stretch optical microscopy (ATOM) for ultrafast high-contrast cellular imaging in flow

PubMed Central

Wong, Terence T. W.; Lau, Andy K. S.; Ho, Kenneth K. Y.; Tang, Matthew Y. H.; Robles, Joseph D. F.; Wei, Xiaoming; Chan, Antony C. S.; Tang, Anson H. L.; Lam, Edmund Y.; Wong, Kenneth K. Y.; Chan, Godfrey C. F.; Shum, Ho Cheung; Tsia, Kevin K.

2014-01-01

Accelerating imaging speed in optical microscopy is often realized at the expense of image contrast, image resolution, and detection sensitivity – a common predicament for advancing high-speed and high-throughput cellular imaging. We here demonstrate a new imaging approach, called asymmetric-detection time-stretch optical microscopy (ATOM), which can deliver ultrafast label-free high-contrast flow imaging with well delineated cellular morphological resolution and in-line optical image amplification to overcome the compromised imaging sensitivity at high speed. We show that ATOM can separately reveal the enhanced phase-gradient and absorption contrast in microfluidic live-cell imaging at a flow speed as high as ~10 m/s, corresponding to an imaging throughput of ~100,000 cells/sec. ATOM could thus be the enabling platform to meet the pressing need for intercalating optical microscopy in cellular assay, e.g. imaging flow cytometry – permitting high-throughput access to the morphological information of the individual cells simultaneously with a multitude of parameters obtained in the standard assay. PMID:24413677

20180311 - Differential Gene Expression and Concentration-Response Modeling Workflow for High-Throughput Transcriptomic (HTTr) Data: Results From MCF7 Cells (SOT)

EPA Science Inventory

Increasing efficiency and declining cost of generating whole transcriptome profiles has made high-throughput transcriptomics a practical option for chemical bioactivity screening. The resulting data output provides information on the expression of thousands of genes and is amenab...

Differential Gene Expression and Concentration-Response Modeling Workflow for High-Throughput Transcriptomic (HTTr) Data: Results From MCF7 Cells

EPA Science Inventory

Increasing efficiency and declining cost of generating whole transcriptome profiles has made high-throughput transcriptomics a practical option for chemical bioactivity screening. The resulting data output provides information on the expression of thousands of genes and is amenab...

Neural Progenitor Cells as Models for High-Throughput Screens of Developmental Neurotoxicity: State of the Science

EPA Science Inventory

In vitro, high-throughput approaches have been widely recommended as an approach to screen chemicals for the potential to cause developmental neurotoxicity and prioritize them for additional testing. The choice of cellular models for such an approach will have important ramificat...

Sensitivity of neuroprogenitor cells to chemical-induced apoptosis using a multiplexed assay suitable for high-throughput screening*

EPA Science Inventory

AbstractHigh-throughput methods are useful for rapidly screening large numbers of chemicals for biological activity, including the perturbation of pathways that may lead to adverse cellular effects. In vitro assays for the key events of neurodevelopment, including apoptosis, may ...

Integrated Model of Chemical Perturbations of a Biological PathwayUsing 18 In Vitro High Throughput Screening Assays for the Estrogen Receptor

EPA Science Inventory

We demonstrate a computational network model that integrates 18 in vitro, high-throughput screening assays measuring estrogen receptor (ER) binding, dimerization, chromatin binding, transcriptional activation and ER-dependent cell proliferation. The network model uses activity pa...

Emory University: High-Throughput Protein-Protein Interaction Analysis for Hippo Pathway Profiling | Office of Cancer Genomics

Cancer.gov

The CTD2 Center at Emory University used high-throughput protein-protein interaction (PPI) mapping for Hippo signaling pathway profiling to rapidly unveil promising PPIs as potential therapeutic targets and advance functional understanding of signaling circuitry in cells. Read the abstract.

High-throughput genotoxicity assay identifies antioxidants as inducers of DNA damage response and cell death

EPA Science Inventory

Human ATAD5 is an excellent biomarker for identifying genotoxic compounds because ATADS protein levels increase post-transcriptionally following exposure to a variety of DNA damaging agents. Here we report a novel quantitative high-throughput ATAD5-Iuciferase assay that can moni...

A high throughput Cre–lox activated viral membrane fusion assay identifies pharmacological inhibitors of HIV entry

DOE Office of Scientific and Technical Information (OSTI.GOV)

Esposito, Anthony M.; Cheung, Pamela; Swartz, Talia H.

Enveloped virus entry occurs when viral and cellular membranes fuse releasing particle contents into the target cell. Human immunodeficiency virus (HIV) entry occurs by cell-free virus or virus transferred between infected and uninfected cells through structures called virological synapses. We developed a high-throughput cell-based assay to identify small molecule inhibitors of cell-free or virological synapse-mediated entry. An HIV clone carrying Cre recombinase as a Gag-internal gene fusion releases active Cre into cells upon viral entry activating a recombinatorial gene switch changing dsRed to GFP-expression. A screen of a 1998 known-biological profile small molecule library identified pharmacological HIV entry inhibitors thatmore » block both cell-free and cell-to-cell infection. Many top hits were noted as HIV inhibitors in prior studies, but not previously recognized as entry antagonists. Modest therapeutic indices for simvastatin and nigericin were observed in confirmatory HIV infection assays. This robust assay is adaptable to study HIV and heterologous viral pseudotypes. - Highlights: • Cre recombinase viral fusion assay screens cell-free or cell–cell entry inhibitors. • This Gag-iCre based assay is specific for the entry step of HIV replication. • Screened a library of known pharmacologic compounds for HIV fusion antagonists. • Many top hits were previously noted as HIV inhibitors, but here are classified as entry antagonists. Many top hits were previously noted as HIV inhibitors, but not as entry antagonists. • The assay is compatible with pseudotyping with HIV and heterologous viruses.« less

Nonlinear mixed effects dose response modeling in high throughput drug screens: application to melanoma cell line analysis.

PubMed

Ding, Kuan-Fu; Petricoin, Emanuel F; Finlay, Darren; Yin, Hongwei; Hendricks, William P D; Sereduk, Chris; Kiefer, Jeffrey; Sekulic, Aleksandar; LoRusso, Patricia M; Vuori, Kristiina; Trent, Jeffrey M; Schork, Nicholas J

2018-01-12

Cancer cell lines are often used in high throughput drug screens (HTS) to explore the relationship between cell line characteristics and responsiveness to different therapies. Many current analysis methods infer relationships by focusing on one aspect of cell line drug-specific dose-response curves (DRCs), the concentration causing 50% inhibition of a phenotypic endpoint (IC 50 ). Such methods may overlook DRC features and do not simultaneously leverage information about drug response patterns across cell lines, potentially increasing false positive and negative rates in drug response associations. We consider the application of two methods, each rooted in nonlinear mixed effects (NLME) models, that test the relationship relationships between estimated cell line DRCs and factors that might mitigate response. Both methods leverage estimation and testing techniques that consider the simultaneous analysis of different cell lines to draw inferences about any one cell line. One of the methods is designed to provide an omnibus test of the differences between cell line DRCs that is not focused on any one aspect of the DRC (such as the IC 50 value). We simulated different settings and compared the different methods on the simulated data. We also compared the proposed methods against traditional IC 50 -based methods using 40 melanoma cell lines whose transcriptomes, proteomes, and, importantly, BRAF and related mutation profiles were available. Ultimately, we find that the NLME-based methods are more robust, powerful and, for the omnibus test, more flexible, than traditional methods. Their application to the melanoma cell lines reveals insights into factors that may be clinically useful.

Human genetics in rheumatoid arthritis guides a high-throughput drug screen of the CD40 signaling pathway.

PubMed

Li, Gang; Diogo, Dorothée; Wu, Di; Spoonamore, Jim; Dancik, Vlado; Franke, Lude; Kurreeman, Fina; Rossin, Elizabeth J; Duclos, Grant; Hartland, Cathy; Zhou, Xuezhong; Li, Kejie; Liu, Jun; De Jager, Philip L; Siminovitch, Katherine A; Zhernakova, Alexandra; Raychaudhuri, Soumya; Bowes, John; Eyre, Steve; Padyukov, Leonid; Gregersen, Peter K; Worthington, Jane; Gupta, Namrata; Clemons, Paul A; Stahl, Eli; Tolliday, Nicola; Plenge, Robert M

2013-05-01

Although genetic and non-genetic studies in mouse and human implicate the CD40 pathway in rheumatoid arthritis (RA), there are no approved drugs that inhibit CD40 signaling for clinical care in RA or any other disease. Here, we sought to understand the biological consequences of a CD40 risk variant in RA discovered by a previous genome-wide association study (GWAS) and to perform a high-throughput drug screen for modulators of CD40 signaling based on human genetic findings. First, we fine-map the CD40 risk locus in 7,222 seropositive RA patients and 15,870 controls, together with deep sequencing of CD40 coding exons in 500 RA cases and 650 controls, to identify a single SNP that explains the entire signal of association (rs4810485, P = 1.4×10(-9)). Second, we demonstrate that subjects homozygous for the RA risk allele have ∼33% more CD40 on the surface of primary human CD19+ B lymphocytes than subjects homozygous for the non-risk allele (P = 10(-9)), a finding corroborated by expression quantitative trait loci (eQTL) analysis in peripheral blood mononuclear cells from 1,469 healthy control individuals. Third, we use retroviral shRNA infection to perturb the amount of CD40 on the surface of a human B lymphocyte cell line (BL2) and observe a direct correlation between amount of CD40 protein and phosphorylation of RelA (p65), a subunit of the NF-κB transcription factor. Finally, we develop a high-throughput NF-κB luciferase reporter assay in BL2 cells activated with trimerized CD40 ligand (tCD40L) and conduct an HTS of 1,982 chemical compounds and FDA-approved drugs. After a series of counter-screens and testing in primary human CD19+ B cells, we identify 2 novel chemical inhibitors not previously implicated in inflammation or CD40-mediated NF-κB signaling. Our study demonstrates proof-of-concept that human genetics can be used to guide the development of phenotype-based, high-throughput small-molecule screens to identify potential novel therapies in complex traits such as RA.

Development of a High-Content Orthopoxvirus Infectivity and Neutralization Assays

PubMed Central

Gates, Irina; Olson, Victoria; Smith, Scott; Patel, Nishi; Damon, Inger; Karem, Kevin

2015-01-01

Currently, a number of assays measure Orthopoxvirus neutralization with serum from individuals, vaccinated against smallpox. In addition to the traditional plaque reduction neutralization test (PRNT), newer higher throughput assays are based on neutralization of recombinant vaccinia virus, expressing reporter genes such as β-galactosidase or green fluorescent protein. These methods could not be used to evaluate neutralization of variola virus, since genetic manipulations of this virus are prohibited by international agreements. Currently, PRNT is the assay of choice to measure neutralization of variola virus. However, PRNT assays are time consuming, labor intensive, and require considerable volume of serum sample for testing. Here, we describe the development of a high-throughput, cell-based imaging assay that can be used to measure neutralization, and characterize replication kinetics of various Orthopoxviruses, including variola, vaccinia, monkeypox, and cowpox. PMID:26426117

Miniaturized microscope for high throughput screening of tumor spheroids in microfluidic devices

NASA Astrophysics Data System (ADS)

Uranga, Javier; Rodríguez-Pena, Alejandro; Gahigiro, Desiré; Ortiz-de-Solorzano, Carlos

2018-02-01

High-throughput in vitro screening of highly physiological three-dimensional cell cultures (3D-HTS) is rapidly gaining importance in preclinical studies, to study the effect of the microenvironment in tumor development, and to evaluate the efficacy of new anticancer drugs. Furthermore, it could also be envisioned the use of 3D-HTS systems in personalized anti-cancer treatment planning, based on tumor organoids or spheroids grown from tumor biopsies or isolated tumor circulating cells. Most commercial, multi-well plate based 3D-HTS systems are large, expensive, and are based on the use of multi-well plates that hardly provide a physiological environment and require the use of large amounts of biological material and reagents. In this paper we present a novel, miniaturized inverted microscope (hereinafter miniscospe), made up of low-cost, mass producible parts, that can be used to monitor the growth of living tumor cell spheroids within customized three-dimensional microfluidic platforms. Our 3D-HTS miniscope combines phase contrast imaging based on oblique back illumination technique with traditional widefield epi-fluorescence imaging, implemented using miniaturized electro-optical parts and gradient-index refraction lenses. This small (3x6x2cm), lightweight device can effectively image overtime the growth of (>200) tumor spheroids in a controlled and reproducible environment. Our miniscope can be used to acquire time-lapse images of cellular living spheroids over the course of several hours and captures their growth before and after drug treatment, to evaluate the effectiveness of the drug.

Advanced continuous cultivation methods for systems microbiology.

PubMed

Adamberg, Kaarel; Valgepea, Kaspar; Vilu, Raivo

2015-09-01

Increasing the throughput of systems biology-based experimental characterization of in silico-designed strains has great potential for accelerating the development of cell factories. For this, analysis of metabolism in the steady state is essential as only this enables the unequivocal definition of the physiological state of cells, which is needed for the complete description and in silico reconstruction of their phenotypes. In this review, we show that for a systems microbiology approach, high-resolution characterization of metabolism in the steady state--growth space analysis (GSA)--can be achieved by using advanced continuous cultivation methods termed changestats. In changestats, an environmental parameter is continuously changed at a constant rate within one experiment whilst maintaining cells in the physiological steady state similar to chemostats. This increases the resolution and throughput of GSA compared with chemostats, and, moreover, enables following of the dynamics of metabolism and detection of metabolic switch-points and optimal growth conditions. We also describe the concept, challenge and necessary criteria of the systematic analysis of steady-state metabolism. Finally, we propose that such systematic characterization of the steady-state growth space of cells using changestats has value not only for fundamental studies of metabolism, but also for systems biology-based metabolic engineering of cell factories.

High-Throughput Density Measurement Using Magnetic Levitation.

PubMed

Ge, Shencheng; Wang, Yunzhe; Deshler, Nicolas J; Preston, Daniel J; Whitesides, George M

2018-06-20

This work describes the development of an integrated analytical system that enables high-throughput density measurements of diamagnetic particles (including cells) using magnetic levitation (MagLev), 96-well plates, and a flatbed scanner. MagLev is a simple and useful technique with which to carry out density-based analysis and separation of a broad range of diamagnetic materials with different physical forms (e.g., liquids, solids, gels, pastes, gums, etc.); one major limitation, however, is the capacity to perform high-throughput density measurements. This work addresses this limitation by (i) re-engineering the shape of the magnetic fields so that the MagLev system is compatible with 96-well plates, and (ii) integrating a flatbed scanner (and simple optical components) to carry out imaging of the samples that levitate in the system. The resulting system is compatible with both biological samples (human erythrocytes) and nonbiological samples (simple liquids and solids, such as 3-chlorotoluene, cholesterol crystals, glass beads, copper powder, and polymer beads). The high-throughput capacity of this integrated MagLev system will enable new applications in chemistry (e.g., analysis and separation of materials) and biochemistry (e.g., cellular responses under environmental stresses) in a simple and label-free format on the basis of a universal property of all matter, i.e., density.

High-Throughput and Cost-Effective Characterization of Induced Pluripotent Stem Cells.

PubMed

D'Antonio, Matteo; Woodruff, Grace; Nathanson, Jason L; D'Antonio-Chronowska, Agnieszka; Arias, Angelo; Matsui, Hiroko; Williams, Roy; Herrera, Cheryl; Reyna, Sol M; Yeo, Gene W; Goldstein, Lawrence S B; Panopoulos, Athanasia D; Frazer, Kelly A

2017-04-11

Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) offers the possibility of studying the molecular mechanisms underlying human diseases in cell types difficult to extract from living patients, such as neurons and cardiomyocytes. To date, studies have been published that use small panels of iPSC-derived cell lines to study monogenic diseases. However, to study complex diseases, where the genetic variation underlying the disorder is unknown, a sizable number of patient-specific iPSC lines and controls need to be generated. Currently the methods for deriving and characterizing iPSCs are time consuming, expensive, and, in some cases, descriptive but not quantitative. Here we set out to develop a set of simple methods that reduce cost and increase throughput in the characterization of iPSC lines. Specifically, we outline methods for high-throughput quantification of surface markers, gene expression analysis of in vitro differentiation potential, and evaluation of karyotype with markedly reduced cost. Published by Elsevier Inc.

A microfluidic device for label-free detection of Escherichia coli in drinking water using positive dielectrophoretic focusing, capturing, and impedance measurement.

PubMed

Kim, Myounggon; Jung, Taekeon; Kim, Youngjin; Lee, Changgeun; Woo, Kyungchul; Seol, Jae Hun; Yang, Sung

2015-12-15

While sensors that allow for high-throughput enumeration of microorganisms within drinking water are useful for water quality monitoring, it is particularly challenging to accurately quantify microorganisms that are present in low numbers (<100 CFU/mL) in a high-throughput manner. Negative dielectrophoresis (nDEP) is typically utilized in DEP-based cell focusing methods; however, due to its low conductivity, drinking water cannot be analyzed by this approach. Here, we report a positive DEP (pDEP)-based Escherichia coli detection system that is integrated with a focusing and sensing electrode. By incorporating a passivation layer, we avoided issues with adhesion of E. coli to the electrode, and achieved efficient cell focusing under high flow rate conditions (1500 μL/h). The resulting focused E. coli cells were then trapped on the sensor electrode, resulting in changes in impedance. The proposed system was evaluated using four different E. coli populations (150-1500 CFU/mL). We successfully enumerated populations as low as 300 CFU/mL within 1 min, and the signal variation was 1.13±0.37%. The device introduced in this study provides the basis for the development of portable, highly sensitive microorganism sensors that enable rapid detection of bacteria in drinking water. Copyright © 2015 Elsevier B.V. All rights reserved.

A BSL-4 high-throughput screen identifies sulfonamide inhibitors of Nipah virus.

PubMed

Tigabu, Bersabeh; Rasmussen, Lynn; White, E Lucile; Tower, Nichole; Saeed, Mohammad; Bukreyev, Alexander; Rockx, Barry; LeDuc, James W; Noah, James W

2014-04-01

Nipah virus is a biosafety level 4 (BSL-4) pathogen that causes severe respiratory illness and encephalitis in humans. To identify novel small molecules that target Nipah virus replication as potential therapeutics, Southern Research Institute and Galveston National Laboratory jointly developed an automated high-throughput screening platform that is capable of testing 10,000 compounds per day within BSL-4 biocontainment. Using this platform, we screened a 10,080-compound library using a cell-based, high-throughput screen for compounds that inhibited the virus-induced cytopathic effect. From this pilot effort, 23 compounds were identified with EC50 values ranging from 3.9 to 20.0 μM and selectivities >10. Three sulfonamide compounds with EC50 values <12 μM were further characterized for their point of intervention in the viral replication cycle and for broad antiviral efficacy. Development of HTS capability under BSL-4 containment changes the paradigm for drug discovery for highly pathogenic agents because this platform can be readily modified to identify prophylactic and postexposure therapeutic candidates against other BSL-4 pathogens, particularly Ebola, Marburg, and Lassa viruses.

A BSL-4 High-Throughput Screen Identifies Sulfonamide Inhibitors of Nipah Virus

PubMed Central

Tigabu, Bersabeh; Rasmussen, Lynn; White, E. Lucile; Tower, Nichole; Saeed, Mohammad; Bukreyev, Alexander; Rockx, Barry; LeDuc, James W.

2014-01-01

Abstract Nipah virus is a biosafety level 4 (BSL-4) pathogen that causes severe respiratory illness and encephalitis in humans. To identify novel small molecules that target Nipah virus replication as potential therapeutics, Southern Research Institute and Galveston National Laboratory jointly developed an automated high-throughput screening platform that is capable of testing 10,000 compounds per day within BSL-4 biocontainment. Using this platform, we screened a 10,080-compound library using a cell-based, high-throughput screen for compounds that inhibited the virus-induced cytopathic effect. From this pilot effort, 23 compounds were identified with EC50 values ranging from 3.9 to 20.0 μM and selectivities >10. Three sulfonamide compounds with EC50 values <12 μM were further characterized for their point of intervention in the viral replication cycle and for broad antiviral efficacy. Development of HTS capability under BSL-4 containment changes the paradigm for drug discovery for highly pathogenic agents because this platform can be readily modified to identify prophylactic and postexposure therapeutic candidates against other BSL-4 pathogens, particularly Ebola, Marburg, and Lassa viruses. PMID:24735442

(Toxicological Sciences) High-throughput H295R steroidogenesis assay: utility as an alternative and a statistical approach to characterize effects on steroidogenesis

EPA Science Inventory

The U.S. Environmental Protection Agency Endocrine Disruptor Screening Program and the Organization for Economic Co-operation and Development (OECD) have used the human adrenocarcinoma (H295R) cell-based assay to predict chemical perturbation of androgen and estrogen production. ...

High-throughput sequencing in acute lymphoblastic leukemia: Follow-up of minimal residual disease and emergence of new clones.

PubMed

Salson, Mikaël; Giraud, Mathieu; Caillault, Aurélie; Grardel, Nathalie; Duployez, Nicolas; Ferret, Yann; Duez, Marc; Herbert, Ryan; Rocher, Tatiana; Sebda, Shéhérazade; Quief, Sabine; Villenet, Céline; Figeac, Martin; Preudhomme, Claude

2017-02-01

Minimal residual disease (MRD) is known to be an independent prognostic factor in patients with acute lymphoblastic leukemia (ALL). High-throughput sequencing (HTS) is currently used in routine practice for the diagnosis and follow-up of patients with hematological neoplasms. In this retrospective study, we examined the role of immunoglobulin/T-cell receptor-based MRD in patients with ALL by HTS analysis of immunoglobulin H and/or T-cell receptor gamma chain loci in bone marrow samples from 11 patients with ALL, at diagnosis and during follow-up. We assessed the clinical feasibility of using combined HTS and bioinformatics analysis with interactive visualization using Vidjil software. We discuss the advantages and drawbacks of HTS for monitoring MRD. HTS gives a more complete insight of the leukemic population than conventional real-time quantitative PCR (qPCR), and allows identification of new emerging clones at each time point of the monitoring. Thus, HTS monitoring of Ig/TR based MRD is expected to improve the management of patients with ALL. Copyright © 2016 Elsevier Ltd. All rights reserved.

Discovery of agents that eradicate leukemia stem cells using an in silico screen of public gene expression data

PubMed Central

Hassane, Duane C.; Guzman, Monica L.; Corbett, Cheryl; Li, Xiaojie; Abboud, Ramzi; Young, Fay; Liesveld, Jane L.; Carroll, Martin

2008-01-01

Increasing evidence indicates that malignant stem cells are important for the pathogenesis of acute myelogenous leukemia (AML) and represent a reservoir of cells that drive the development of AML and relapse. Therefore, new treatment regimens are necessary to prevent relapse and improve therapeutic outcomes. Previous studies have shown that the sesquiterpene lactone, parthenolide (PTL), ablates bulk, progenitor, and stem AML cells while causing no appreciable toxicity to normal hematopoietic cells. Thus, PTL must evoke cellular responses capable of mediating AML selective cell death. Given recent advances in chemical genomics such as gene expression-based high-throughput screening (GE-HTS) and the Connectivity Map, we hypothesized that the gene expression signature resulting from treatment of primary AML with PTL could be used to search for similar signatures in publicly available gene expression profiles deposited into the Gene Expression Omnibus (GEO). We therefore devised a broad in silico screen of the GEO database using the PTL gene expression signature as a template and discovered 2 new agents, celastrol and 4-hydroxy-2-nonenal, that effectively eradicate AML at the bulk, progenitor, and stem cell level. These findings suggest the use of multicenter collections of high-throughput data to facilitate discovery of leukemia drugs and drug targets. PMID:18305216

A novel approach to the discovery of anti-tumor pharmaceuticals: searching for activators of liponecrosis

PubMed Central

Arlia-Ciommo, Anthony; Svistkova, Veronika; Mohtashami, Sadaf; Titorenko, Vladimir I.

2016-01-01

A recently conducted chemical genetic screen for pharmaceuticals that can extend longevity of the yeast Saccharomyces cerevisiae has identified lithocholic acid as a potent anti-aging molecule. It was found that this hydrophobic bile acid is also a selective anti-tumor chemical compound; it kills different types of cultured cancer cells if used at concentrations that do not compromise the viability of non-cancerous cells. These studies have revealed that yeast can be successfully used as a model organism for high-throughput screens aimed at the discovery of selectively acting anti-tumor small molecules. Two metabolic traits of rapidly proliferating fermenting yeast, namely aerobic glycolysis and lipogenesis, are known to be similar to those of cancer cells. The mechanisms underlying these key metabolic features of cancer cells and fermenting yeast have been established; such mechanisms are discussed in this review. We also suggest how a yeast-based chemical genetic screen can be used for the high-throughput development of selective anti-tumor pharmaceuticals that kill only cancer cells. This screen consists of searching for chemical compounds capable of increasing the abundance of membrane lipids enriched in unsaturated fatty acids that would therefore be toxic only to rapidly proliferating cells, such as cancer cells and fermenting yeast. PMID:26636650

Miniaturization Technologies for Efficient Single-Cell Library Preparation for Next-Generation Sequencing.

PubMed

Mora-Castilla, Sergio; To, Cuong; Vaezeslami, Soheila; Morey, Robert; Srinivasan, Srimeenakshi; Dumdie, Jennifer N; Cook-Andersen, Heidi; Jenkins, Joby; Laurent, Louise C

2016-08-01

As the cost of next-generation sequencing has decreased, library preparation costs have become a more significant proportion of the total cost, especially for high-throughput applications such as single-cell RNA profiling. Here, we have applied novel technologies to scale down reaction volumes for library preparation. Our system consisted of in vitro differentiated human embryonic stem cells representing two stages of pancreatic differentiation, for which we prepared multiple biological and technical replicates. We used the Fluidigm (San Francisco, CA) C1 single-cell Autoprep System for single-cell complementary DNA (cDNA) generation and an enzyme-based tagmentation system (Nextera XT; Illumina, San Diego, CA) with a nanoliter liquid handler (mosquito HTS; TTP Labtech, Royston, UK) for library preparation, reducing the reaction volume down to 2 µL and using as little as 20 pg of input cDNA. The resulting sequencing data were bioinformatically analyzed and correlated among the different library reaction volumes. Our results showed that decreasing the reaction volume did not interfere with the quality or the reproducibility of the sequencing data, and the transcriptional data from the scaled-down libraries allowed us to distinguish between single cells. Thus, we have developed a process to enable efficient and cost-effective high-throughput single-cell transcriptome sequencing. © 2016 Society for Laboratory Automation and Screening.

Dissecting the human immunologic memory for pathogens.

PubMed

Zielinski, Christina E; Corti, Davide; Mele, Federico; Pinto, Dora; Lanzavecchia, Antonio; Sallusto, Federica

2011-03-01

Studies on immunologic memory in animal models and especially in the human system are instrumental to identify mechanisms and correlates of protection necessary for vaccine development. In this article, we provide an overview of the cellular basis of immunologic memory. We also describe experimental approaches based on high throughput cell cultures, which we have developed to interrogate human memory T cells, B cells, and plasma cells. We discuss how these approaches can provide new tools and information for vaccine design, in a process that we define as 'analytic vaccinology'. © 2011 John Wiley & Sons A/S.

Defined, serum/feeder-free conditions for expansion and drug screening of primary B-acute lymphoblastic leukemia.

PubMed

Jiang, Zhiwu; Wu, Di; Ye, Wei; Weng, Jianyu; Lai, Peilong; Shi, Pengcheng; Guo, Xutao; Huang, Guohua; Deng, Qiuhua; Tang, Yanlai; Zhao, Hongyu; Cui, Shuzhong; Lin, Simiao; Wang, Suna; Li, Baiheng; Wu, Qiting; Li, Yangqiu; Liu, Pentao; Pei, Duanqing; Du, Xin; Yao, Yao; Li, Peng

2017-12-05

Functional screening for compounds represents a major hurdle in the development of rational therapeutics for B-acute lymphoblastic leukemia (B-ALL). In addition, using cell lines as valid models for evaluating responses to novel drug therapies raises serious concerns, as cell lines are prone to genotypic/phenotypic drift and loss of heterogeneity in vitro . Here, we reported that OP9 cells, not OP9-derived adipocytes (OP9TA), support the growth of primary B-ALL cells in vitro . To identify the factors from OP9 cells that support the growth of primary B-ALL cells, we performed RNA-Seq to analyze the gene expression profiles of OP9 and OP9TA cells. We thus developed a defined, serum/feeder-free condition (FI76V) that can support the expansion of a range of clinically distinct primary B-ALL cells that still maintain their leukemia-initiating ability. We demonstrated the suitability of high-throughput drug screening based on our B-ALL cultured conditions. Upon screening 378 kinase inhibitors, we identified a cluster of 17 kinase inhibitors that can efficiently kill B-ALL cells in vitro . Importantly, we demonstrated the synergistic cytotoxicity of dinaciclib/BTG226 to B-ALL cells. Taken together, we developed a defined condition for the ex vivo expansion of primary B-ALL cells that is suitable for high-throughput screening of novel compounds.

Printed droplet microfluidics for on demand dispensing of picoliter droplets and cells

PubMed Central

Cole, Russell H.; Tang, Shi-Yang; Siltanen, Christian A.; Shahi, Payam; Zhang, Jesse Q.; Poust, Sean; Gartner, Zev J.; Abate, Adam R.

2017-01-01

Although the elementary unit of biology is the cell, high-throughput methods for the microscale manipulation of cells and reagents are limited. The existing options either are slow, lack single-cell specificity, or use fluid volumes out of scale with those of cells. Here we present printed droplet microfluidics, a technology to dispense picoliter droplets and cells with deterministic control. The core technology is a fluorescence-activated droplet sorter coupled to a specialized substrate that together act as a picoliter droplet and single-cell printer, enabling high-throughput generation of intricate arrays of droplets, cells, and microparticles. Printed droplet microfluidics provides a programmable and robust technology to construct arrays of defined cell and reagent combinations and to integrate multiple measurement modalities together in a single assay. PMID:28760972

Printed droplet microfluidics for on demand dispensing of picoliter droplets and cells.

PubMed

Cole, Russell H; Tang, Shi-Yang; Siltanen, Christian A; Shahi, Payam; Zhang, Jesse Q; Poust, Sean; Gartner, Zev J; Abate, Adam R

2017-08-15

Although the elementary unit of biology is the cell, high-throughput methods for the microscale manipulation of cells and reagents are limited. The existing options either are slow, lack single-cell specificity, or use fluid volumes out of scale with those of cells. Here we present printed droplet microfluidics, a technology to dispense picoliter droplets and cells with deterministic control. The core technology is a fluorescence-activated droplet sorter coupled to a specialized substrate that together act as a picoliter droplet and single-cell printer, enabling high-throughput generation of intricate arrays of droplets, cells, and microparticles. Printed droplet microfluidics provides a programmable and robust technology to construct arrays of defined cell and reagent combinations and to integrate multiple measurement modalities together in a single assay.

Printed droplet microfluidics for on demand dispensing of picoliter droplets and cells

NASA Astrophysics Data System (ADS)

Cole, Russell H.; Tang, Shi-Yang; Siltanen, Christian A.; Shahi, Payam; Zhang, Jesse Q.; Poust, Sean; Gartner, Zev J.; Abate, Adam R.

2017-08-01

Although the elementary unit of biology is the cell, high-throughput methods for the microscale manipulation of cells and reagents are limited. The existing options either are slow, lack single-cell specificity, or use fluid volumes out of scale with those of cells. Here we present printed droplet microfluidics, a technology to dispense picoliter droplets and cells with deterministic control. The core technology is a fluorescence-activated droplet sorter coupled to a specialized substrate that together act as a picoliter droplet and single-cell printer, enabling high-throughput generation of intricate arrays of droplets, cells, and microparticles. Printed droplet microfluidics provides a programmable and robust technology to construct arrays of defined cell and reagent combinations and to integrate multiple measurement modalities together in a single assay.

Quantitative high throughput screening identifies inhibitors of anthrax-induced cell death

PubMed Central

Zhu, Ping Jun; Hobson, Peyton; Southall, Noel; Qiu, Cunping; Thomas, Craig J.; Lu, Jiamo; Inglese, James; Zheng, Wei; Leppla, Stephen H.; Bugge, Thomas H.; Austin, Christopher P.; Liu, Shihui

2009-01-01

Here, we report the results of a quantitative high-throughput screen (qHTS) measuring the endocytosis and translocation of a β-lactamase-fused-lethal factor and the identification of small molecules capable of obstructing the process of anthrax toxin internalization. Several small molecules protect RAW264.7 macrophages and CHO cells from anthrax lethal toxin and protected cells from an LF-Pseudomonas exotoxin fusion protein and diphtheria toxin. Further efforts demonstrated that these compounds impaired the PA heptamer pre-pore to pore conversion in cells expressing the CMG2 receptor, but not the related TEM8 receptor, indicating that these compounds likely interfere with toxin internalization. PMID:19540764

Enabling systematic interrogation of protein-protein interactions in live cells with a versatile ultra-high-throughput biosensor platform | Office of Cancer Genomics

Cancer.gov

The vast datasets generated by next generation gene sequencing and expression profiling have transformed biological and translational research. However, technologies to produce large-scale functional genomics datasets, such as high-throughput detection of protein-protein interactions (PPIs), are still in early development. While a number of powerful technologies have been employed to detect PPIs, a singular PPI biosensor platform featured with both high sensitivity and robustness in a mammalian cell environment remains to be established.

Fluorescence detection-based functional assay for high-throughput screening for MraY.

PubMed

Stachyra, Thérèse; Dini, Christophe; Ferrari, Paul; Bouhss, Ahmed; van Heijenoort, Jean; Mengin-Lecreulx, Dominique; Blanot, Didier; Biton, Jacques; Le Beller, Dominique

2004-03-01

We have developed a novel assay specific to MraY, which catalyzes the first membrane step in the biosynthesis of bacterial cell wall peptidoglycan. This was accomplished by using UDP-MurNAc-N(epsilon)-dansylpentapeptide, a fluorescent derivative of the MraY nucleotide substrate, and a partially purified preparation of MraY solubilized from membranes of an Escherichia coli overproducing strain. Two versions of the assay were developed, one consisting of the high-pressure liquid chromatography separation of the substrate and product (dansylated lipid I) and the other, without separation and adapted to the high-throughput format, taking advantage of the different fluorescence properties of the nucleotide and lipid I in the reaction medium. The latter assay was validated with a set of natural and synthetic MraY inhibitors.

Analysis of high-throughput screening reveals the effect of surface topographies on cellular morphology.

PubMed

Hulsman, Marc; Hulshof, Frits; Unadkat, Hemant; Papenburg, Bernke J; Stamatialis, Dimitrios F; Truckenmüller, Roman; van Blitterswijk, Clemens; de Boer, Jan; Reinders, Marcel J T

2015-03-01

Surface topographies of materials considerably impact cellular behavior as they have been shown to affect cell growth, provide cell guidance, and even induce cell differentiation. Consequently, for successful application in tissue engineering, the contact interface of biomaterials needs to be optimized to induce the required cell behavior. However, a rational design of biomaterial surfaces is severely hampered because knowledge is lacking on the underlying biological mechanisms. Therefore, we previously developed a high-throughput screening device (TopoChip) that measures cell responses to large libraries of parameterized topographical material surfaces. Here, we introduce a computational analysis of high-throughput materiome data to capture the relationship between the surface topographies of materials and cellular morphology. We apply robust statistical techniques to find surface topographies that best promote a certain specified cellular response. By augmenting surface screening with data-driven modeling, we determine which properties of the surface topographies influence the morphological properties of the cells. With this information, we build models that predict the cellular response to surface topographies that have not yet been measured. We analyze cellular morphology on 2176 surfaces, and find that the surface topography significantly affects various cellular properties, including the roundness and size of the nucleus, as well as the perimeter and orientation of the cells. Our learned models capture and accurately predict these relationships and reveal a spectrum of topographies that induce various levels of cellular morphologies. Taken together, this novel approach of high-throughput screening of materials and subsequent analysis opens up possibilities for a rational design of biomaterial surfaces. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Robotic Patterning a Superhydrophobic Surface for Collective Cell Migration Screening.

PubMed

Pang, Yonggang; Yang, Jing; Hui, Zhixin; Grottkau, Brian E

2018-04-01

Collective cell migration, in which cells migrate as a group, is fundamental in many biological and pathological processes. There is increasing interest in studying the collective cell migration in high throughput. Cell scratching, insertion blocker, and gel-dissolving techniques are some methodologies used previously. However, these methods have the drawbacks of cell damage, substrate surface alteration, limitation in medium exchange, and solvent interference. The superhydrophobic surface, on which the water contact angle is greater than 150 degrees, has been recently utilized to generate patterned arrays. Independent cell culture areas can be generated on a substrate that functions the same as a conventional multiple well plate. However, so far there has been no report on superhydrophobic patterning for the study of cell migration. In this study, we report on the successful development of a robotically patterned superhydrophobic array for studying collective cell migration in high throughput. The array was developed on a rectangular single-well cell culture plate consisting of hydrophilic flat microwells separated by the superhydrophobic surface. The manufacturing process is robotic and includes patterning discrete protective masks to the substrate using 3D printing, robotic spray coating of silica nanoparticles, robotic mask removal, robotic mini silicone blocker patterning, automatic cell seeding, and liquid handling. Compared with a standard 96-well plate, our system increases the throughput by 2.25-fold and generates a cell-free area in each well non-destructively. Our system also demonstrates higher efficiency than conventional way of liquid handling using microwell plates, and shorter processing time than manual operating in migration assays. The superhydrophobic surface had no negative impact on cell viability. Using our system, we studied the collective migration of human umbilical vein endothelial cells and cancer cells using assays of endpoint quantification, dynamic cell tracking, and migration quantification following varied drug treatments. This system provides a versatile platform to study collective cell migration in high throughput for a broad range of applications.

A Comparison of the Performance and Application Differences Between Manual and Automated Patch-Clamp Techniques

PubMed Central

Yajuan, Xiao; Xin, Liang; Zhiyuan, Li

2012-01-01

The patch clamp technique is commonly used in electrophysiological experiments and offers direct insight into ion channel properties through the characterization of ion channel activity. This technique can be used to elucidate the interaction between a drug and a specific ion channel at different conformational states to understand the ion channel modulators’ mechanisms. The patch clamp technique is regarded as a gold standard for ion channel research; however, it suffers from low throughput and high personnel costs. In the last decade, the development of several automated electrophysiology platforms has greatly increased the screen throughput of whole cell electrophysiological recordings. New advancements in the automated patch clamp systems have aimed to provide high data quality, high content, and high throughput. However, due to the limitations noted above, automated patch clamp systems are not capable of replacing manual patch clamp systems in ion channel research. While automated patch clamp systems are useful for screening large amounts of compounds in cell lines that stably express high levels of ion channels, the manual patch clamp technique is still necessary for studying ion channel properties in some research areas and for specific cell types, including primary cells that have mixed cell types and differentiated cells that derive from induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs). Therefore, further improvements in flexibility with regard to cell types and data quality will broaden the applications of the automated patch clamp systems in both academia and industry. PMID:23346269

Cell-based dose responses from open-well microchambers.

PubMed

Hamon, Morgan; Jambovane, Sachin; Bradley, Lauren; Khademhosseini, Ali; Hong, Jong Wook

2013-05-21

Cell-based assays play a critical role in discovery of new drugs and facilitating research in cancer, immunology, and stem cells. Conventionally, they are performed in Petri dishes, tubes, or well plates, using milliliters of reagents and thousands of cells to obtain one data point. Here, we are introducing a new platform to realize cell-based assay capable of increased throughput and greater sensitivity with a limited number of cells. We integrated an array of open-well microchambers into a gradient generation system. Consequently, cell-based dose responses were examined with a single device. We measured IC50 values of three cytotoxic chemicals, Triton X-100, H2O2, and cadmium chloride, as model compounds. The present system is highly suitable for the discovery of new drugs and studying the effect of chemicals on cell viability or mortality with limited samples and cells.

A simple whole cell based high throughput screening protocol using Mycobacterium bovis BCG for inhibitors against dormant and active tubercle bacilli.

PubMed

Khan, Arshad; Sarkar, Dhiman

2008-04-01

This study aimed at developing a whole cell based high throughput screening protocol to identify inhibitors against both active and dormant tubercle bacilli. A respiratory type of nitrate reductase (NarGHJI), which was induced during dormancy, could reflect the viability of dormant bacilli of Mycobacterium bovis BCG in microplate adopted model of in vitro dormancy. Correlation between reduction in viability and nitrate reductase activity was seen clearly when dormant stage inhibitor metronidazole and itaconic anhydride were applied in this in vitro microplate model. Active replicating stage could also be monitored in the same assay by measuring the A(620) of the culture. MIC values of 0.08, 0.075, 0.3 and 3.0 microg/ml, determined through monitoring A(620) in this assay for rifampin, isoniazid, streptomycin and ethambutol respectively, were well in agreement with previously reported by BACTEC and Bio-Siv assays. S/N ratio and Z' factor for the assay were 8.5 and 0.81 respectively which indicated the robustness of the protocol. Altogether the assay provides an easy, inexpensive, rapid, robust and high content screening tool to search novel antitubercular molecules against both active and dormant bacilli.

Development of a Cell-Based Functional Assay for the Detection of Clostridium botulinum Neurotoxin Types A and E

PubMed Central

Basavanna, Uma; Muruvanda, Tim; Brown, Eric W.; Sharma, Shashi K.

2013-01-01

The standard procedure for definitive detection of BoNT-producing Clostridia is a culture method combined with neurotoxin detection using a standard mouse bioassay (MBA). The mouse bioassay is highly sensitive and specific, but it is expensive and time-consuming, and there are ethical concerns due to use of laboratory animals. Cell-based assays provide an alternative to the MBA in screening for BoNT-producing Clostridia. Here, we describe a cell-based assay utilizing a fluorescence reporter construct expressed in a neuronal cell model to study toxin activity in situ. Our data indicates that the assay can detect as little as 100 pM BoNT/A activity within living cells, and the assay is currently being evaluated for the analysis of BoNT in food matrices. Among available in vitro assays, we believe that cell-based assays are widely applicable in high-throughput screenings and have the potential to at least reduce and refine animal assays if not replace it. PMID:23533420

Re-engineering adenovirus vector systems to enable high-throughput analyses of gene function.

PubMed

Stanton, Richard J; McSharry, Brian P; Armstrong, Melanie; Tomasec, Peter; Wilkinson, Gavin W G

2008-12-01

With the enhanced capacity of bioinformatics to interrogate extensive banks of sequence data, more efficient technologies are needed to test gene function predictions. Replication-deficient recombinant adenovirus (Ad) vectors are widely used in expression analysis since they provide for extremely efficient expression of transgenes in a wide range of cell types. To facilitate rapid, high-throughput generation of recombinant viruses, we have re-engineered an adenovirus vector (designated AdZ) to allow single-step, directional gene insertion using recombineering technology. Recombineering allows for direct insertion into the Ad vector of PCR products, synthesized sequences, or oligonucleotides encoding shRNAs without requirement for a transfer vector Vectors were optimized for high-throughput applications by making them "self-excising" through incorporating the I-SceI homing endonuclease into the vector removing the need to linearize vectors prior to transfection into packaging cells. AdZ vectors allow genes to be expressed in their native form or with strep, V5, or GFP tags. Insertion of tetracycline operators downstream of the human cytomegalovirus major immediate early (HCMV MIE) promoter permits silencing of transgenes in helper cells expressing the tet repressor thus making the vector compatible with the cloning of toxic gene products. The AdZ vector system is robust, straightforward, and suited to both sporadic and high-throughput applications.

Moving Toward Integrating Gene Expression Profiling Into High-Throughput Testing: A Gene Expression Biomarker Accurately Predicts Estrogen Receptor α Modulation in a Microarray Compendium.

PubMed

Ryan, Natalia; Chorley, Brian; Tice, Raymond R; Judson, Richard; Corton, J Christopher

2016-05-01

Microarray profiling of chemical-induced effects is being increasingly used in medium- and high-throughput formats. Computational methods are described here to identify molecular targets from whole-genome microarray data using as an example the estrogen receptor α (ERα), often modulated by potential endocrine disrupting chemicals. ERα biomarker genes were identified by their consistent expression after exposure to 7 structurally diverse ERα agonists and 3 ERα antagonists in ERα-positive MCF-7 cells. Most of the biomarker genes were shown to be directly regulated by ERα as determined by ESR1 gene knockdown using siRNA as well as through chromatin immunoprecipitation coupled with DNA sequencing analysis of ERα-DNA interactions. The biomarker was evaluated as a predictive tool using the fold-change rank-based Running Fisher algorithm by comparison to annotated gene expression datasets from experiments using MCF-7 cells, including those evaluating the transcriptional effects of hormones and chemicals. Using 141 comparisons from chemical- and hormone-treated cells, the biomarker gave a balanced accuracy for prediction of ERα activation or suppression of 94% and 93%, respectively. The biomarker was able to correctly classify 18 out of 21 (86%) ER reference chemicals including "very weak" agonists. Importantly, the biomarker predictions accurately replicated predictions based on 18 in vitro high-throughput screening assays that queried different steps in ERα signaling. For 114 chemicals, the balanced accuracies were 95% and 98% for activation or suppression, respectively. These results demonstrate that the ERα gene expression biomarker can accurately identify ERα modulators in large collections of microarray data derived from MCF-7 cells. Published by Oxford University Press on behalf of the Society of Toxicology 2016. This work is written by US Government employees and is in the public domain in the US.

Simple fluorescence-based high throughput cell viability assay for filamentous fungi.

PubMed

Chadha, S; Kale, S P

2015-09-01

Filamentous fungi are important model organisms to understand the eukaryotic process and have been frequently exploited in research and industry. These fungi are also causative agents of serious diseases in plants and humans. Disease management strategies include in vitro susceptibility testing of the fungal pathogens to environmental conditions and antifungal agents. Conventional methods used for antifungal susceptibilities are cumbersome, time-consuming and are not suitable for a large-scale analysis. Here, we report a rapid, high throughput microplate-based fluorescence method for investigating the toxicity of antifungal and stress (osmotic, salt and oxidative) agents on Magnaporthe oryzae and compared it with agar dilution method. This bioassay is optimized for the resazurin reduction to fluorescent resorufin by the fungal hyphae. Resazurin bioassay showed inhibitory rates and IC50 values comparable to the agar dilution method and to previously reported IC50 or MICs for M. oryzae and other fungi. The present method can screen range of test agents from different chemical classes with different modes of action for antifungal activities in a simple, sensitive, time and cost effective manner. A simple fluorescence-based high throughput method is developed to test the effects of stress and antifungal agents on viability of filamentous fungus Magnaporthe oryzae. This resazurin fluorescence assay can detect inhibitory effects comparable to those obtained using the growth inhibition assay with added advantages of simplicity, time and cost effectiveness. This high throughput viability assay has a great potential in large-scale screening of the chemical libraries of antifungal agents, for evaluating the effects of environmental conditions and hyphal kinetic studies in mutant and natural populations of filamentous fungi. © 2015 The Society for Applied Microbiology.

First quantitative high-throughput screen in zebrafish identifies novel pathways for increasing pancreatic β-cell mass

PubMed Central

Wang, Guangliang; Rajpurohit, Surendra K; Delaspre, Fabien; Walker, Steven L; White, David T; Ceasrine, Alexis; Kuruvilla, Rejji; Li, Ruo-jing; Shim, Joong S; Liu, Jun O; Parsons, Michael J; Mumm, Jeff S

2015-01-01

Whole-organism chemical screening can circumvent bottlenecks that impede drug discovery. However, in vivo screens have not attained throughput capacities possible with in vitro assays. We therefore developed a method enabling in vivo high-throughput screening (HTS) in zebrafish, termed automated reporter quantification in vivo (ARQiv). In this study, ARQiv was combined with robotics to fully actualize whole-organism HTS (ARQiv-HTS). In a primary screen, this platform quantified cell-specific fluorescent reporters in >500,000 transgenic zebrafish larvae to identify FDA-approved (Federal Drug Administration) drugs that increased the number of insulin-producing β cells in the pancreas. 24 drugs were confirmed as inducers of endocrine differentiation and/or stimulators of β-cell proliferation. Further, we discovered novel roles for NF-κB signaling in regulating endocrine differentiation and for serotonergic signaling in selectively stimulating β-cell proliferation. These studies demonstrate the power of ARQiv-HTS for drug discovery and provide unique insights into signaling pathways controlling β-cell mass, potential therapeutic targets for treating diabetes. DOI: http://dx.doi.org/10.7554/eLife.08261.001 PMID:26218223

Automation of a Nile red staining assay enables high throughput quantification of microalgal lipid production.

PubMed

Morschett, Holger; Wiechert, Wolfgang; Oldiges, Marco

2016-02-09

Within the context of microalgal lipid production for biofuels and bulk chemical applications, specialized higher throughput devices for small scale parallelized cultivation are expected to boost the time efficiency of phototrophic bioprocess development. However, the increasing number of possible experiments is directly coupled to the demand for lipid quantification protocols that enable reliably measuring large sets of samples within short time and that can deal with the reduced sample volume typically generated at screening scale. To meet these demands, a dye based assay was established using a liquid handling robot to provide reproducible high throughput quantification of lipids with minimized hands-on-time. Lipid production was monitored using the fluorescent dye Nile red with dimethyl sulfoxide as solvent facilitating dye permeation. The staining kinetics of cells at different concentrations and physiological states were investigated to successfully down-scale the assay to 96 well microtiter plates. Gravimetric calibration against a well-established extractive protocol enabled absolute quantification of intracellular lipids improving precision from ±8 to ±2 % on average. Implementation into an automated liquid handling platform allows for measuring up to 48 samples within 6.5 h, reducing hands-on-time to a third compared to manual operation. Moreover, it was shown that automation enhances accuracy and precision compared to manual preparation. It was revealed that established protocols relying on optical density or cell number for biomass adjustion prior to staining may suffer from errors due to significant changes of the cells' optical and physiological properties during cultivation. Alternatively, the biovolume was used as a measure for biomass concentration so that errors from morphological changes can be excluded. The newly established assay proved to be applicable for absolute quantification of algal lipids avoiding limitations of currently established protocols, namely biomass adjustment and limited throughput. Automation was shown to improve data reliability, as well as experimental throughput simultaneously minimizing the needed hands-on-time to a third. Thereby, the presented protocol meets the demands for the analysis of samples generated by the upcoming generation of devices for higher throughput phototrophic cultivation and thereby contributes to boosting the time efficiency for setting up algae lipid production processes.

Phenotype-Based Screening of Small Molecules to Modify Plant Cell Walls Using BY-2 Cells.

PubMed

Okubo-Kurihara, Emiko; Matsui, Minami

2018-01-01

The plant cell wall is an important and abundant biomass with great potential for use as a modern recyclable resource. For effective utilization of this cellulosic biomass, its ability to degrade efficiently is key point. With the aim of modifying the cell wall to allow easy decomposition, we used chemical biological technology to alter its structure. As a first step toward evaluating the chemicals in the cell wall we employed a phenotype-based approach using high-throughput screening. As the plant cell wall is essential in determining cell morphology, phenotype-based screening is particularly effective in identifying compounds that bring about alterations in the cell wall. For rapid and reproducible screening, tobacco BY-2 cell is an excellent system in which to observe cell morphology. In this chapter, we provide a detailed chemical biological methodology for studying cell morphology using tobacco BY-2 cells.

High throughput two-step ultrasonic spray deposited CH3NH3PbI3 thin film layer for solar cell application

NASA Astrophysics Data System (ADS)

Lan, Ding-Hung; Hong, Shao-Huan; Chou, Li-Hui; Wang, Xiao-Feng; Liu, Cheng-Liang

2018-06-01

Organometal halide perovskite materials have demonstrated tremendous advances in the photovoltaic field recently because of their advantageous features of simple fabrication and high power conversion efficiency. To meet the high demand for high throughput and cost-effective, we present a wet process method that enables the probing of the parameters for perovskite layer deposition through two-step sequential ultrasonic spray-coating. This paper describes a detailed investigation on the effects of modification of spray precursor solution (PbI2 and CH3NH3I precursor concentration and solvents used) and post-annealing condition (temperature and time), which can be performed to create optimal film quality as well as improve device efficiency. Through the systematic optimization, the inverted planar perovskite solar cells show the reproducible photovoltaic properties with best power conversion efficiency (PCE) of 10.40% and average PCE of 9.70 ± 0.40%. A continuous spray-coating technique for rapid fabrication of total 16 pieces of perovskite films was demonstrated for providing a viable alternative for the high throughput production of the perovskite solar cells.

EZH2 and CD79B mutational status over time in B-cell non-Hodgkin lymphomas detected by high-throughput sequencing using minimal samples

PubMed Central

Saieg, Mauro Ajaj; Geddie, William R; Boerner, Scott L; Bailey, Denis; Crump, Michael; da Cunha Santos, Gilda

2013-01-01

BACKGROUND: Numerous genomic abnormalities in B-cell non-Hodgkin lymphomas (NHLs) have been revealed by novel high-throughput technologies, including recurrent mutations in EZH2 (enhancer of zeste homolog 2) and CD79B (B cell antigen receptor complex-associated protein beta chain) genes. This study sought to determine the evolution of the mutational status of EZH2 and CD79B over time in different samples from the same patient in a cohort of B-cell NHLs, through use of a customized multiplex mutation assay. METHODS: DNA that was extracted from cytological material stored on FTA cards as well as from additional specimens, including archived frozen and formalin-fixed histological specimens, archived stained smears, and cytospin preparations, were submitted to a multiplex mutation assay specifically designed for the detection of point mutations involving EZH2 and CD79B, using MassARRAY spectrometry followed by Sanger sequencing. RESULTS: All 121 samples from 80 B-cell NHL cases were successfully analyzed. Mutations in EZH2 (Y646) and CD79B (Y196) were detected in 13.2% and 8% of the samples, respectively, almost exclusively in follicular lymphomas and diffuse large B-cell lymphomas. In one-third of the positive cases, a wild type was detected in a different sample from the same patient during follow-up. CONCLUSIONS: Testing multiple minimal tissue samples using a high-throughput multiplex platform exponentially increases tissue availability for molecular analysis and might facilitate future studies of tumor progression and the related molecular events. Mutational status of EZH2 and CD79B may vary in B-cell NHL samples over time and support the concept that individualized therapy should be based on molecular findings at the time of treatment, rather than on results obtained from previous specimens. Cancer (Cancer Cytopathol) 2013;121:377–386. © 2013 American Cancer Society. PMID:23361872

Thin-film-transistor array: an exploratory attempt for high throughput cell manipulation using electrowetting principle

NASA Astrophysics Data System (ADS)

Shaik, F. Azam; Cathcart, G.; Ihida, S.; Lereau-Bernier, M.; Leclerc, E.; Sakai, Y.; Toshiyoshi, H.; Tixier-Mita, A.

2017-05-01

In lab-on-a-chip (LoC) devices, microfluidic displacement of liquids is a key component. electrowetting on dielectric (EWOD) is a technique to move fluids, with the advantage of not requiring channels, pumps or valves. Fluids are discretized into droplets on microelectrodes and moved by applying an electric field via the electrodes to manipulate the contact angle. Micro-objects, such as biological cells, can be transported inside of these droplets. However, the design of conventional microelectrodes, made by standard micro-fabrication techniques, fixes the path of the droplets, and limits the reconfigurability of paths and thus limits the parallel processing of droplets. In that respect, thin film transistor (TFT) technology presents a great opportunity as it allows infinitely reconfigurable paths, with high parallelizability. We propose here to investigate the possibility of using TFT array devices for high throughput cell manipulation using EWOD. A COMSOL based 2D simulation coupled with a MATLAB algorithm was used to simulate the contact angle modulation, displacement and mixing of droplets. These simulations were confirmed by experimental results. The EWOD technique was applied to a droplet of culture medium containing HepG2 carcinoma cells and demonstrated no negative effects on the viability of the cells. This confirms the possibility of applying EWOD techniques to cellular applications, such as parallel cell analysis.

Single Cell Profiling using Ionic Liquid Matrix-Enhanced Secondary Ion Mass Spectrometry for Neuronal Cell Type Differentiation

PubMed Central

Do, Thanh D.; Comi, Troy J.; Dunham, Sage J. B.; Rubakhin, Stanislav S.; Sweedler, Jonathan V.

2017-01-01

A high-throughput single cell profiling method has been developed for matrix-enhanced secondary ion mass spectrometry (ME-SIMS) to investigate the lipid profiles of neuronal cells. Populations of cells are dispersed onto the substrate, their locations determined using optical microscopy, and the cell locations used to guide the acquisition of SIMS spectra from the cells. Up to 2,000 cells can be assayed in one experiment at a rate of 6 s per cell. Multiple saturated and unsaturated phosphatidylcholines (PCs) and their fragments are detected and verified with tandem mass spectrometry from individual cells when ionic liquids are employed as a matrix. Optically guided single cell profiling with ME-SIMS is suitable for a range of cell sizes, from Aplysia californica neurons larger than 75 μm to 7-μm rat cerebellar neurons. ME-SIMS analysis followed by t-distributed stochastic neighbor embedding of peaks in the lipid molecular mass range (m/z 700–850) distinguishes several cell types from the rat central nervous system, largely based on the relative proportions of the four dominant lipids, PC(32:0), PC(34:1), PC(36:1), and PC(38:5). Furthermore, subpopulations within each cell type are tentatively classified consistent with their endogenous lipid ratios. The results illustrate the efficacy of a new approach to classify single cell populations and subpopulations using SIMS profiling of lipid and metabolite contents. These methods are broadly applicable for high throughput single cell chemical analyses. PMID:28194949

Single-cell-based breeding: Rational strategy for the establishment of cell lines from a single cell with the most favorable properties.

PubMed

Yoshimoto, Nobuo; Kuroda, Shun'ichi

2014-04-01

For efficient biomolecule production (e.g., antibodies, recombinant proteins), mammalian cells with high expression rates should be selected from cell libraries, propagated while maintaining a homogenous expression rate, and subsequently stabilized at their high expression rate. Clusters of isogenic cells (i.e., colonies) have been used for these processes. However, cellular heterogeneity makes it difficult to obtain cell lines with the highest expression rates by using single-colony-based breeding. Furthermore, even among the single cells in an isogenic cell population, the desired cell properties fluctuate stochastically during long-term culture. Therefore, although the molecular mechanisms underlying stochastic fluctuation are poorly understood, it is necessary to establish excellent cell lines in order to breed single cells to have higher expression, higher stability, and higher homogeneity while suppressing stochastic fluctuation (i.e., single-cell-based breeding). In this review, we describe various methods for manipulating single cells and facilitating single-cell analysis in order to better understand stochastic fluctuation. We demonstrated that single-cell-based breeding is practical and promising by using a high-throughput automated system to analyze and manipulate single cells. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

High Throughput Determination of Critical Human Dosing Parameters (SOT)

EPA Science Inventory

High throughput toxicokinetics (HTTK) is a rapid approach that uses in vitro data to estimate TK for hundreds of environmental chemicals. Reverse dosimetry (i.e., reverse toxicokinetics or RTK) based on HTTK data converts high throughput in vitro toxicity screening (HTS) data int...

High Throughput Determinations of Critical Dosing Parameters (IVIVE workshop)

EPA Science Inventory

High throughput toxicokinetics (HTTK) is an approach that allows for rapid estimations of TK for hundreds of environmental chemicals. HTTK-based reverse dosimetry (i.e, reverse toxicokinetics or RTK) is used in order to convert high throughput in vitro toxicity screening (HTS) da...

Microfabricated Electrochemical Cell-Based Biosensors for Analysis of Living Cells In Vitro

PubMed Central

Wang, Jun; Wu, Chengxiong; Hu, Ning; Zhou, Jie; Du, Liping; Wang, Ping

2012-01-01

Cellular biochemical parameters can be used to reveal the physiological and functional information of various cells. Due to demonstrated high accuracy and non-invasiveness, electrochemical detection methods have been used for cell-based investigation. When combined with improved biosensor design and advanced measurement systems, the on-line biochemical analysis of living cells in vitro has been applied for biological mechanism study, drug screening and even environmental monitoring. In recent decades, new types of miniaturized electrochemical biosensor are emerging with the development of microfabrication technology. This review aims to give an overview of the microfabricated electrochemical cell-based biosensors, such as microelectrode arrays (MEA), the electric cell-substrate impedance sensing (ECIS) technique, and the light addressable potentiometric sensor (LAPS). The details in their working principles, measurement systems, and applications in cell monitoring are covered. Driven by the need for high throughput and multi-parameter detection proposed by biomedicine, the development trends of electrochemical cell-based biosensors are also introduced, including newly developed integrated biosensors, and the application of nanotechnology and microfluidic technology. PMID:25585708

Trade-Offs in Thin Film Solar Cells with Layered Chalcostibite Photovoltaic Absorbers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Welch, Adam W.; Baranowski, Lauryn L.; Peng, Haowei

Discovery of novel semiconducting materials is needed for solar energy conversion and other optoelectronic applications. However, emerging low-dimensional solar absorbers often have unconventional crystal structures and unusual combinations of optical absorption and electrical transport properties, which considerably slows down the research and development progress. Here, the effect of stronger absorption and weaker carrier collection of 2D-like absorber materials are studied using a high-throughput combinatorial experimental approach, complemented by advanced characterization and computations. It is found that the photoexcited charge carrier collection in CuSbSe 2 solar cells is enhanced by drift in an electric field, addressing a different absorption/collection balance. Themore » resulting drift solar cells efficiency is <5% due to inherent J SC/ V OC trade-off, suggesting that improved carrier diffusion and better contacts are needed to further increase the CuSbSe 2 performance. Furthermore, this study also illustrates the advantages of high-throughput experimental methods for fast optimization of the optoelectronic devices based on emerging low-dimensional semiconductor materials.« less

Trade-Offs in Thin Film Solar Cells with Layered Chalcostibite Photovoltaic Absorbers

DOE PAGES

Welch, Adam W.; Baranowski, Lauryn L.; Peng, Haowei; ...

2017-01-25

Discovery of novel semiconducting materials is needed for solar energy conversion and other optoelectronic applications. However, emerging low-dimensional solar absorbers often have unconventional crystal structures and unusual combinations of optical absorption and electrical transport properties, which considerably slows down the research and development progress. Here, the effect of stronger absorption and weaker carrier collection of 2D-like absorber materials are studied using a high-throughput combinatorial experimental approach, complemented by advanced characterization and computations. It is found that the photoexcited charge carrier collection in CuSbSe 2 solar cells is enhanced by drift in an electric field, addressing a different absorption/collection balance. Themore » resulting drift solar cells efficiency is <5% due to inherent J SC/ V OC trade-off, suggesting that improved carrier diffusion and better contacts are needed to further increase the CuSbSe 2 performance. Furthermore, this study also illustrates the advantages of high-throughput experimental methods for fast optimization of the optoelectronic devices based on emerging low-dimensional semiconductor materials.« less

The promise and challenge of high-throughput sequencing of the antibody repertoire

PubMed Central

Georgiou, George; Ippolito, Gregory C; Beausang, John; Busse, Christian E; Wardemann, Hedda; Quake, Stephen R

2014-01-01

Efforts to determine the antibody repertoire encoded by B cells in the blood or lymphoid organs using high-throughput DNA sequencing technologies have been advancing at an extremely rapid pace and are transforming our understanding of humoral immune responses. Information gained from high-throughput DNA sequencing of immunoglobulin genes (Ig-seq) can be applied to detect B-cell malignancies with high sensitivity, to discover antibodies specific for antigens of interest, to guide vaccine development and to understand autoimmunity. Rapid progress in the development of experimental protocols and informatics analysis tools is helping to reduce sequencing artifacts, to achieve more precise quantification of clonal diversity and to extract the most pertinent biological information. That said, broader application of Ig-seq, especially in clinical settings, will require the development of a standardized experimental design framework that will enable the sharing and meta-analysis of sequencing data generated by different laboratories. PMID:24441474

Single-cell genomic profiling of acute myeloid leukemia for clinical use: A pilot study

PubMed Central

Yan, Benedict; Hu, Yongli; Ban, Kenneth H.K.; Tiang, Zenia; Ng, Christopher; Lee, Joanne; Tan, Wilson; Chiu, Lily; Tan, Tin Wee; Seah, Elaine; Ng, Chin Hin; Chng, Wee-Joo; Foo, Roger

2017-01-01

Although bulk high-throughput genomic profiling studies have led to a significant increase in the understanding of cancer biology, there is increasing awareness that bulk profiling approaches do not completely elucidate tumor heterogeneity. Single-cell genomic profiling enables the distinction of tumor heterogeneity, and may improve clinical diagnosis through the identification and characterization of putative subclonal populations. In the present study, the challenges associated with a single-cell genomics profiling workflow for clinical diagnostics were investigated. Single-cell RNA-sequencing (RNA-seq) was performed on 20 cells from an acute myeloid leukemia bone marrow sample. Putative blasts were identified based on their gene expression profiles and principal component analysis was performed to identify outlier cells. Variant calling was performed on the single-cell RNA-seq data. The present pilot study demonstrates a proof of concept for clinical single-cell genomic profiling. The recognized limitations include significant stochastic RNA loss and the relatively low throughput of the current proposed platform. Although the results of the present study are promising, further technological advances and protocol optimization are necessary for single-cell genomic profiling to be clinically viable. PMID:28454300

CSReport: A New Computational Tool Designed for Automatic Analysis of Class Switch Recombination Junctions Sequenced by High-Throughput Sequencing.

PubMed

Boyer, François; Boutouil, Hend; Dalloul, Iman; Dalloul, Zeinab; Cook-Moreau, Jeanne; Aldigier, Jean-Claude; Carrion, Claire; Herve, Bastien; Scaon, Erwan; Cogné, Michel; Péron, Sophie

2017-05-15

B cells ensure humoral immune responses due to the production of Ag-specific memory B cells and Ab-secreting plasma cells. In secondary lymphoid organs, Ag-driven B cell activation induces terminal maturation and Ig isotype class switch (class switch recombination [CSR]). CSR creates a virtually unique IgH locus in every B cell clone by intrachromosomal recombination between two switch (S) regions upstream of each C region gene. Amount and structural features of CSR junctions reveal valuable information about the CSR mechanism, and analysis of CSR junctions is useful in basic and clinical research studies of B cell functions. To provide an automated tool able to analyze large data sets of CSR junction sequences produced by high-throughput sequencing (HTS), we designed CSReport, a software program dedicated to support analysis of CSR recombination junctions sequenced with a HTS-based protocol (Ion Torrent technology). CSReport was assessed using simulated data sets of CSR junctions and then used for analysis of Sμ-Sα and Sμ-Sγ1 junctions from CH12F3 cells and primary murine B cells, respectively. CSReport identifies junction segment breakpoints on reference sequences and junction structure (blunt-ended junctions or junctions with insertions or microhomology). Besides the ability to analyze unprecedentedly large libraries of junction sequences, CSReport will provide a unified framework for CSR junction studies. Our results show that CSReport is an accurate tool for analysis of sequences from our HTS-based protocol for CSR junctions, thereby facilitating and accelerating their study. Copyright © 2017 by The American Association of Immunologists, Inc.

High-throughput Screening Identifies Aclacinomycin as a Radiosensitizer of EGFR-Mutant Non-Small Cell Lung Cancer1

PubMed Central

Bennett, Daniel C; Charest, Jonathan; Sebolt, Katrina; Lehrman, Mark; Rehemtulla, Alnawaz; Contessa, Joseph N

2013-01-01

The endoplasmic reticulum (ER) provides a specialized environment for the folding and modification of trans-membrane proteins, including receptor tyrosine kinases (RTKs), which are vital for the growth and survival of malignancies. To identify compounds which disrupt the function of the ER and thus could potentially impair cancer cell survival signaling, we adapted a set of glycosylation-sensitive luciferase reporters for the development and optimization of a cell-based high-throughput screen (HTS). Secondary screens for false-positive luciferase activation and tertiary lectin-based and biochemical analyses were also devised for compound triage. Through a pilot screen of 2802 compounds from the National Cancer Institute (NCI) chemical libraries, we identified aclacinomycin (Acm) as a compound that preferentially affects ER function. We report that Acm reduces plasma membrane expression of glycoproteins including epidermal growth factor receptor (EGFR) and Met but does not inhibit N-linked glycosylation or generalized protein translation. Fluorescence microscopy co-localization experiments were also performed and demonstrated Acm accumulation in the ER in further support of the overall HTS design. The consequences of Acm treatment on cell survival were analyzed through clonogenic survival analysis. Consistent with the reduction of EGFR levels, pretreatment with Acm sensitizes the EGFR-mutant non-small cell lung cancer (NSCLC) cell lines HCC827 and HCC2935 to ionizing radiation and did not affect the sensitivity of the RTK-independent and KRAS-mutant A549 NSCLC cell line. Thus, Acm and similar compounds targeting the ER may represent a novel approach for radiosensitizing tumor cells dependent on RTK function. PMID:23730419

History, applications, and challenges of immune repertoire research.

PubMed

Liu, Xiao; Wu, Jinghua

2018-02-27

The diversity of T and B cells in terms of their receptor sequences is huge in the vertebrate's immune system and provides broad protection against the vast diversity of pathogens. Immune repertoire is defined as the sum of T cell receptors and B cell receptors (also named immunoglobulin) that makes the organism's adaptive immune system. Before the emergence of high-throughput sequencing, the studies on immune repertoire were limited by the underdeveloped methodologies, since it was impossible to capture the whole picture by the low-throughput tools. The massive paralleled sequencing technology suits perfectly the researches on immune repertoire. In this article, we review the history of immune repertoire studies, in terms of technologies and research applications. Particularly, we discuss several aspects of challenges in this field and highlight the efforts to develop potential solutions, in the era of high-throughput sequencing of the immune repertoire.

Clinical and laboratory applications of slide-based cytometry with the LSC, SFM, and the iCYTE imaging cytometer instruments

NASA Astrophysics Data System (ADS)

Bocsi, Jozsef; Luther, Ed; Mittag, Anja; Jensen, Ingo; Sack, Ulrich; Lenz, Dominik; Trezl, Lajos; Varga, Viktor S.; Molnar, Beea; Tarnok, Attila

2004-06-01

Background: Slide based cytometry (SBC) is a technology for the rapid stoichiometric analysis of cells fixed to surfaces. Its applications are highly versatile and ranges from the clinics to high throughput drug discovery. SBC is realized in different instruments such as the Laser Scanning Cytometer (LSC) and Scanning Fluorescent Microscope (SFM) and the novel inverted microscope based iCyte image cytometer (Compucyte Corp.). Methods: Fluorochrome labeled specimens were immobilized on microscopic slides. They were placed on a conventional fluorescence microscope and analyzed by photomultiplayers or digital camera. Data comparable to flow cytometry were generated. In addition, each individual event could be visualized. Applications: The major advantage of instruments is the combination of two features: a) the minimal sample volume needed, and b) the connection of fluorescence data and morphological information. Rare cells were detected, frequency of apoptosis by myricetin formaldehyde and H2O2 mixtures was determined;. Conclusion: LSC, SFM and the novel iCyte have a wide spectrum of applicability in SBC and can be introduced as a standard technology for multiple settings. In addition, the iCyte and SFM instrument is suited for high throughput screening by automation and may be in future adapted to telepathology due to their high quality images. (This study was supported by the IZKF-Leipzig, Germany and T 034245 OTKA, Hungary)

High-throughput spectral and lifetime-based FRET screening in living cells to identify small-molecule effectors of SERCA

PubMed Central

Schaaf, Tory M.; Peterson, Kurt C.; Grant, Benjamin D.; Bawaskar, Prachi; Yuen, Samantha; Li, Ji; Muretta, Joseph M.; Gillispie, Gregory D.; Thomas, David D.

2017-01-01

A robust high-throughput screening (HTS) strategy has been developed to discover small-molecule effectors targeting the sarco/endoplasmic reticulum calcium ATPase (SERCA), based on a fluorescence microplate reader that records both the nanosecond decay waveform (lifetime mode) and the complete emission spectrum (spectral mode), with high precision and speed. This spectral unmixing plate reader (SUPR) was used to screen libraries of small molecules with a fluorescence resonance energy transfer (FRET) biosensor expressed in living cells. Ligand binding was detected by FRET associated with structural rearrangements of green (GFP, donor) and red (RFP, acceptor) fluorescent proteins fused to the cardiac-specific SERCA2a isoform. The results demonstrate accurate quantitation of FRET along with high precision of hit identification. Fluorescence lifetime analysis resolved SERCA’s distinct structural states, providing a method to classify small-molecule chemotypes on the basis of their structural effect on the target. The spectral analysis was also applied to flag interference by fluorescent compounds. FRET hits were further evaluated for functional effects on SERCA’s ATPase activity via both a coupled-enzyme assay and a FRET-based calcium sensor. Concentration-response curves indicated excellent correlation between FRET and function. These complementary spectral and lifetime FRET detection methods offer an attractive combination of precision, speed, and resolution for HTS. PMID:27899691

High-throughput combinatorial screening identifies drugs that cooperate with ibrutinib to kill activated B-cell–like diffuse large B-cell lymphoma cells

PubMed Central

Mathews Griner, Lesley A.; Guha, Rajarshi; Shinn, Paul; Young, Ryan M.; Keller, Jonathan M.; Liu, Dongbo; Goldlust, Ian S.; Yasgar, Adam; McKnight, Crystal; Boxer, Matthew B.; Duveau, Damien Y.; Jiang, Jian-Kang; Michael, Sam; Mierzwa, Tim; Huang, Wenwei; Walsh, Martin J.; Mott, Bryan T.; Patel, Paresma; Leister, William; Maloney, David J.; Leclair, Christopher A.; Rai, Ganesha; Jadhav, Ajit; Peyser, Brian D.; Austin, Christopher P.; Martin, Scott E.; Simeonov, Anton; Ferrer, Marc; Staudt, Louis M.; Thomas, Craig J.

2014-01-01

The clinical development of drug combinations is typically achieved through trial-and-error or via insight gained through a detailed molecular understanding of dysregulated signaling pathways in a specific cancer type. Unbiased small-molecule combination (matrix) screening represents a high-throughput means to explore hundreds and even thousands of drug–drug pairs for potential investigation and translation. Here, we describe a high-throughput screening platform capable of testing compounds in pairwise matrix blocks for the rapid and systematic identification of synergistic, additive, and antagonistic drug combinations. We use this platform to define potential therapeutic combinations for the activated B-cell–like subtype (ABC) of diffuse large B-cell lymphoma (DLBCL). We identify drugs with synergy, additivity, and antagonism with the Bruton’s tyrosine kinase inhibitor ibrutinib, which targets the chronic active B-cell receptor signaling that characterizes ABC DLBCL. Ibrutinib interacted favorably with a wide range of compounds, including inhibitors of the PI3K-AKT-mammalian target of rapamycin signaling cascade, other B-cell receptor pathway inhibitors, Bcl-2 family inhibitors, and several components of chemotherapy that is the standard of care for DLBCL. PMID:24469833

Identifying kinase dependency in cancer cells by integrating high-throughput drug screening and kinase inhibition data.

PubMed

Ryall, Karen A; Shin, Jimin; Yoo, Minjae; Hinz, Trista K; Kim, Jihye; Kang, Jaewoo; Heasley, Lynn E; Tan, Aik Choon

2015-12-01

Targeted kinase inhibitors have dramatically improved cancer treatment, but kinase dependency for an individual patient or cancer cell can be challenging to predict. Kinase dependency does not always correspond with gene expression and mutation status. High-throughput drug screens are powerful tools for determining kinase dependency, but drug polypharmacology can make results difficult to interpret. We developed Kinase Addiction Ranker (KAR), an algorithm that integrates high-throughput drug screening data, comprehensive kinase inhibition data and gene expression profiles to identify kinase dependency in cancer cells. We applied KAR to predict kinase dependency of 21 lung cancer cell lines and 151 leukemia patient samples using published datasets. We experimentally validated KAR predictions of FGFR and MTOR dependence in lung cancer cell line H1581, showing synergistic reduction in proliferation after combining ponatinib and AZD8055. KAR can be downloaded as a Python function or a MATLAB script along with example inputs and outputs at: http://tanlab.ucdenver.edu/KAR/. aikchoon.tan@ucdenver.edu. Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

A high brightness probe of polymer nanoparticles for biological imaging

NASA Astrophysics Data System (ADS)

Zhou, Sirong; Zhu, Jiarong; Li, Yaping; Feng, Liheng

2018-03-01

Conjugated polymer nanoparticles (CPNs) with high brightness in long wavelength region were prepared by the nano-precipitation method. Based on fluorescence resonance energy transfer (FRET) mechanism, the high brightness property of the CPNs was realized by four different emission polymers. Dynamic light scattering (DLS) and scanning electron microscopy (SEM) displayed that the CPNs possessed a spherical structure and an average diameter of 75 nm. Analysis assays showed that the CPNs had excellent biocompatibility, good photostability and low cytotoxicity. The CPNs were bio-modified with a cell penetrating peptide (Tat, a targeted element) through covalent link. Based on the entire wave fluorescence emission, the functionalized CPNs1-4 can meet multichannel and high throughput assays in cell and organ imaging. The contribution of the work lies in not only providing a new way to obtain a high brightness imaging probe in long wavelength region, but also using targeted cell and organ imaging.

WE-E-BRE-03: Biological Validation of a Novel High-Throughput Irradiator for Predictive Radiation Sensitivity Bioassays

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fowler, TL; Martin, JA; Shepard, AJ

2014-06-15

Purpose: The large dose-response variation in both tumor and normal cells between individual patients has led to the recent implementation of predictive bioassays of patient-specific radiation sensitivity in order to personalize radiation therapy. This exciting new clinical paradigm has led us to develop a novel high-throughput, variable dose-rate irradiator to accompany these efforts. Here we present the biological validation of this irradiator through the use of human cells as a relative dosimeter assessed by two metrics, DNA double-strand break repair pathway modulation and intercellular reactive oxygen species production. Methods: Immortalized human tonsilar epithelial cells were cultured in 96-well micro titermore » plates and irradiated in groups of eight wells to absorbed doses of 0, 0.5, 1, 2, 4, and 8 Gy. High-throughput immunofluorescent microscopy was used to detect γH2AX, a DNA double-strand break repair mechanism recruiter. The same analysis was performed with the cells stained with CM-H2DCFDA that produces a fluorescent adduct when exposed to reactive oxygen species during the irradiation cycle. Results: Irradiations of the immortalized human tonsilar epithelial cells at absorbed doses of 0, 0.5, 1, 2, 4, and 8 Gy produced excellent linearity in γH2AX and CM-H2DCFDA with R2 values of 0.9939 and 0.9595 respectively. Single cell gel electrophoresis experimentation for the detection of physical DNA double-strand breaks in ongoing. Conclusions: This work indicates significant potential for our high-throughput variable dose rate irradiator for patient-specific predictive radiation sensitivity bioassays. This irradiator provides a powerful tool by increasing the efficiency and number of assay techniques available to help personalize radiation therapy.« less

High-Throughput Assessment of Cellular Mechanical Properties.

PubMed

Darling, Eric M; Di Carlo, Dino

2015-01-01

Traditionally, cell analysis has focused on using molecular biomarkers for basic research, cell preparation, and clinical diagnostics; however, new microtechnologies are enabling evaluation of the mechanical properties of cells at throughputs that make them amenable to widespread use. We review the current understanding of how the mechanical characteristics of cells relate to underlying molecular and architectural changes, describe how these changes evolve with cell-state and disease processes, and propose promising biomedical applications that will be facilitated by the increased throughput of mechanical testing: from diagnosing cancer and monitoring immune states to preparing cells for regenerative medicine. We provide background about techniques that laid the groundwork for the quantitative understanding of cell mechanics and discuss current efforts to develop robust techniques for rapid analysis that aim to implement mechanophenotyping as a routine tool in biomedicine. Looking forward, we describe additional milestones that will facilitate broad adoption, as well as new directions not only in mechanically assessing cells but also in perturbing them to passively engineer cell state.

3D Nanochannel Array Platform for High-throughput Cell Manipulation and Nano-electroporation

NASA Astrophysics Data System (ADS)

Chang, Lingqian

Electroporation is one of the most common non-viral methods for gene delivery. Recent progress in gene therapy has offered special opportunities to electroporation for in vitro and in vivo applications. However, conventional bulk electroporation (BEP) inevitably causes serious cell damage and stochastic transfection between cells. Microfluidic electroporation (MEP) has been claimed to provide benign single cell transfection for the last decade. Nevertheless, the intracellular transport in both MEP and BEP systems is highly diffusion-dominant, which prevents precise dose control and high uniformity. In this Ph.D. research, we developed a 3D nanochannel-electroporation (3D NEP) platform for mass cell transfection. A silicon-based nanochannel array (3D NEP) chip was designed and fabricated for cell manipulation and electroporation. The chip, designed as Z-directional microchannel - nanochannel array, was fabricated by clean room techniques including projection photolithography and deep reactive-ion etching (DRIE). The fabricated 3D NEP chip is capable of handling 40,000 cells per 1 cm2, up to 1 million per wafer (100 mm diameter). High-throughput cell manipulation technologies were investigated for precise alignment of individual cells to the nanochannel array, a key step for NEP to achieve dose control. We developed three techniques for cell trapping in this work. (1) Magnetic tweezers (MTs) were integrated on the chip to remotely control cells under a programmed magnetic field. (2) A positive dielectrophoresis (pDEP) power system was built as an alternative to trap cells onto the nanochannel array using DEP force. (3) A novel yet simple 'dipping-trap' method was used to rapidly trap cells onto a nanochannel array, aligned by a micro-cap array pattern on the 3D NEP chip, which eventually offered 70 - 90 % trapping efficiency and 90 % specificity. 3D NEP platforms were assembled for cell transfection based on the Si-based nanochannel array chip and cell manipulation techniques. Cells were patterned on the nanochannel array and collectively were electroporated in parallel, injected with cargo in Z-direction. Controlling the dose was demonstrated with the external pulse durations at high-throughput. The 'electrophoretic'- expedited delivery of large molecular weight plasmids were demonstrated with large numbers of primary cells simultaneously, which cannot be achieved in BEP and MEP. Two clinically valuable case studies were performed with our 3D NEP for living cell sensing / interrogation. (1) In the case of in vitro transfection of primary cardiomyocytes, we studied the dose-effects of miR-29 on mitochondrial changes and the suppression of the Mcl-1 gene in adult mouse cardiomyocytes by precisely controlling the miR-29 dose injected. (2) Glioma stem cells (GSCs), a type of cell hypothesized to be highly aggressive and to lead to the relapses of gliobastoma in human brain, was studied at single cell resolution on 3D NEP platform. The developed 3D NEP system moves towards clinically oriented and user-friendly tools for life science applications. The batch-treated cells with controlled dosage delivery provide a useful tool for single cell analysis. The pioneering experiments in this work have demonstrated the 3D NEP for the applications of cell reprogramming, adoptive immunotherapy, in vitro cardiomyocytes transfection and glioma stem cells study.

Gold Nanoparticle Mediated Laser Transfection for Efficient siRNA Mediated Gene Knock Down

PubMed Central

Heinemann, Dag; Schomaker, Markus; Kalies, Stefan; Schieck, Maximilian; Carlson, Regina; Escobar, Hugo Murua; Ripken, Tammo; Meyer, Heiko; Heisterkamp, Alexander

2013-01-01

Laser based transfection methods have proven to be an efficient and gentle alternative to established molecule delivery methods like lipofection or electroporation. Among the laser based methods, gold nanoparticle mediated laser transfection bears the major advantage of high throughput and easy usability. This approach uses plasmon resonances on gold nanoparticles unspecifically attached to the cell membrane to evoke transient and spatially defined cell membrane permeabilization. In this study, we explore the parameter regime for gold nanoparticle mediated laser transfection for the delivery of molecules into cell lines and prove its suitability for siRNA mediated gene knock down. The developed setup allows easy usage and safe laser operation in a normal lab environment. We applied a 532 nm Nd:YAG microchip laser emitting 850 ps pulses at a repetition rate of 20.25 kHz. Scanning velocities of the laser spot over the sample of up to 200 mm/s were tested without a decline in perforation efficiency. This velocity leads to a process speed of ∼8 s per well of a 96 well plate. The optimal particle density was determined to be ∼6 particles per cell using environmental scanning electron microscopy. Applying the optimized parameters transfection efficiencies of 88% were achieved in canine pleomorphic adenoma ZMTH3 cells using a fluorescent labeled siRNA while maintaining a high cell viability of >90%. Gene knock down of d2-EGFP was demonstrated and validated by fluorescence repression and western blot analysis. On basis of our findings and established mathematical models we suppose a mixed transfection mechanism consisting of thermal and multiphoton near field effects. Our findings emphasize that gold nanoparticle mediated laser transfection provides an excellent tool for molecular delivery for both, high throughput purposes and the transfection of sensitive cells types. PMID:23536802

Medium-Throughput Screen of Microbially Produced Serotonin via a G-Protein-Coupled Receptor-Based Sensor.

PubMed

Ehrenworth, Amy M; Claiborne, Tauris; Peralta-Yahya, Pamela

2017-10-17

Chemical biosensors, for which chemical detection triggers a fluorescent signal, have the potential to accelerate the screening of noncolorimetric chemicals produced by microbes, enabling the high-throughput engineering of enzymes and metabolic pathways. Here, we engineer a G-protein-coupled receptor (GPCR)-based sensor to detect serotonin produced by a producer microbe in the producer microbe's supernatant. Detecting a chemical in the producer microbe's supernatant is nontrivial because of the number of other metabolites and proteins present that could interfere with sensor performance. We validate the two-cell screening system for medium-throughput applications, opening the door to the rapid engineering of microbes for the increased production of serotonin. We focus on serotonin detection as serotonin levels limit the microbial production of hydroxystrictosidine, a modified alkaloid that could accelerate the semisynthesis of camptothecin-derived anticancer pharmaceuticals. This work shows the ease of generating GPCR-based chemical sensors and their ability to detect specific chemicals in complex aqueous solutions, such as microbial spent medium. In addition, this work sets the stage for the rapid engineering of serotonin-producing microbes.

Rapid cell separation with minimal manipulation for autologous cell therapies

NASA Astrophysics Data System (ADS)

Smith, Alban J.; O'Rorke, Richard D.; Kale, Akshay; Rimsa, Roberts; Tomlinson, Matthew J.; Kirkham, Jennifer; Davies, A. Giles; Wälti, Christoph; Wood, Christopher D.

2017-02-01

The ability to isolate specific, viable cell populations from mixed ensembles with minimal manipulation and within intra-operative time would provide significant advantages for autologous, cell-based therapies in regenerative medicine. Current cell-enrichment technologies are either slow, lack specificity and/or require labelling. Thus a rapid, label-free separation technology that does not affect cell functionality, viability or phenotype is highly desirable. Here, we demonstrate separation of viable from non-viable human stromal cells using remote dielectrophoresis, in which an electric field is coupled into a microfluidic channel using shear-horizontal surface acoustic waves, producing an array of virtual electrodes within the channel. This allows high-throughput dielectrophoretic cell separation in high conductivity, physiological-like fluids, overcoming the limitations of conventional dielectrophoresis. We demonstrate viable/non-viable separation efficacy of >98% in pre-purified mesenchymal stromal cells, extracted from human dental pulp, with no adverse effects on cell viability, or on their subsequent osteogenic capabilities.

[Current applications of high-throughput DNA sequencing technology in antibody drug research].

PubMed

Yu, Xin; Liu, Qi-Gang; Wang, Ming-Rong

2012-03-01

Since the publication of a high-throughput DNA sequencing technology based on PCR reaction was carried out in oil emulsions in 2005, high-throughput DNA sequencing platforms have been evolved to a robust technology in sequencing genomes and diverse DNA libraries. Antibody libraries with vast numbers of members currently serve as a foundation of discovering novel antibody drugs, and high-throughput DNA sequencing technology makes it possible to rapidly identify functional antibody variants with desired properties. Herein we present a review of current applications of high-throughput DNA sequencing technology in the analysis of antibody library diversity, sequencing of CDR3 regions, identification of potent antibodies based on sequence frequency, discovery of functional genes, and combination with various display technologies, so as to provide an alternative approach of discovery and development of antibody drugs.

High Content Screening of a Kinase-Focused Library Reveals Compounds Broadly-Active against Dengue Viruses

PubMed Central

Li, Xiaolan; Milan Bonotto, Rafaela; No, Joo Hwan; Kim, Keum Hyun; Baek, Sungmin; Kim, Hee Young; Windisch, Marc Peter; Pamplona Mosimann, Ana Luiza; de Borba, Luana; Liuzzi, Michel; Hansen, Michael Adsetts Edberg; Nunes Duarte dos Santos, Claudia; Freitas-Junior, Lucio Holanda

2013-01-01

Dengue virus is a mosquito-borne flavivirus that has a large impact in global health. It is considered as one of the medically important arboviruses, and developing a preventive or therapeutic solution remains a top priority in the medical and scientific community. Drug discovery programs for potential dengue antivirals have increased dramatically over the last decade, largely in part to the introduction of high-throughput assays. In this study, we have developed an image-based dengue high-throughput/high-content assay (HT/HCA) using an innovative computer vision approach to screen a kinase-focused library for anti-dengue compounds. Using this dengue HT/HCA, we identified a group of compounds with a 4-(1-aminoethyl)-N-methylthiazol-2-amine as a common core structure that inhibits dengue viral infection in a human liver-derived cell line (Huh-7.5 cells). Compounds CND1201, CND1203 and CND1243 exhibited strong antiviral activities against all four dengue serotypes. Plaque reduction and time-of-addition assays suggests that these compounds interfere with the late stage of viral infection cycle. These findings demonstrate that our image-based dengue HT/HCA is a reliable tool that can be used to screen various chemical libraries for potential dengue antiviral candidates. PMID:23437413

HTS-Net: An integrated regulome-interactome approach for establishing network regulation models in high-throughput screenings

PubMed Central

Rioualen, Claire; Da Costa, Quentin; Chetrit, Bernard; Charafe-Jauffret, Emmanuelle; Ginestier, Christophe

2017-01-01

High-throughput RNAi screenings (HTS) allow quantifying the impact of the deletion of each gene in any particular function, from virus-host interactions to cell differentiation. However, there has been less development for functional analysis tools dedicated to RNAi analyses. HTS-Net, a network-based analysis program, was developed to identify gene regulatory modules impacted in high-throughput screenings, by integrating transcription factors-target genes interaction data (regulome) and protein-protein interaction networks (interactome) on top of screening z-scores. HTS-Net produces exhaustive HTML reports for results navigation and exploration. HTS-Net is a new pipeline for RNA interference screening analyses that proves better performance than simple gene rankings by z-scores, by re-prioritizing genes and replacing them in their biological context, as shown by the three studies that we reanalyzed. Formatted input data for the three studied datasets, source code and web site for testing the system are available from the companion web site at http://htsnet.marseille.inserm.fr/. We also compared our program with existing algorithms (CARD and hotnet2). PMID:28949986

Electronic-To-Optical-To-Electronic Packet-Data Conversion

NASA Technical Reports Server (NTRS)

Monacos, Steve

1996-01-01

Space-time multiplexer (STM) cell-based communication system designed to take advantage of both high throughput attainable in optical transmission links and flexibility and functionality of electronic processing, storage, and switching. Long packets segmented and transmitted optically by wavelength-division multiplexing. Performs optoelectronic and protocol conversion between electronic "store-and-forward" protocols and optical "hot-potato" protocols.

Evaluation of In Vitro Biotransformation Using HepaRG Cells to Improve High-Throughput Chemical Hazard Prediction: A Toxicogenomics Analysis (SOT)

EPA Science Inventory

The US EPA’s ToxCast program has generated a wealth of data in >600 in vitro assayson a library of 1060 environmentally relevant chemicals and failed pharmaceuticals to facilitate hazard identification. An inherent criticism of many in vitro-based strategies is the inability of a...

Developing Reverse Toxicokinetic Models to Correlate In Vitro and In Vivo Activity (ICCVAM Communities of Practice Webinar 2015)

EPA Science Inventory

Many commercial and environmental chemicals lack toxicity data necessary for users and risk assessors to make fully informed decisions about potential health effects. Generating these data using high throughput in vitro cell- or biochemical-based tests would be faster and less e...

Biological profiling of the ToxCast Phase II Chemical Library in Primary Human Cell Co-Culture Systems

EPA Science Inventory

The U.S. EPA’s ToxCast research project was developed to address the need for high-throughput testing of chemicals and a pathway-based approach to hazard screening. Phase I of ToxCast tested over 300 unique compounds (mostly pesticides and antimicrobials). With the addition of Ph...

Lead discovery for mammalian elongation of long chain fatty acids family 6 using a combination of high-throughput fluorescent-based assay and RapidFire mass spectrometry assay

DOE Office of Scientific and Technical Information (OSTI.GOV)

Takamiya, Mari; Discovery Technology Laboratories, Sohyaku, Innovative Research Division, Mitsubishi Tanabe Pharma Corporation, Kawagishi, Toda-shi, Saitama; Sakurai, Masaaki

A high-throughput RapidFire mass spectrometry assay is described for elongation of very long-chain fatty acids family 6 (Elovl6). Elovl6 is a microsomal enzyme that regulates the elongation of C12-16 saturated and monounsaturated fatty acids. Elovl6 may be a new therapeutic target for fat metabolism disorders such as obesity, type 2 diabetes, and nonalcoholic steatohepatitis. To identify new Elovl6 inhibitors, we developed a high-throughput fluorescence screening assay in 1536-well format. However, a number of false positives caused by fluorescent interference have been identified. To pick up the real active compounds among the primary hits from the fluorescence assay, we developed amore » RapidFire mass spectrometry assay and a conventional radioisotope assay. These assays have the advantage of detecting the main products directly without using fluorescent-labeled substrates. As a result, 276 compounds (30%) of the primary hits (921 compounds) in a fluorescence ultra-high-throughput screening method were identified as common active compounds in these two assays. It is concluded that both methods are very effective to eliminate false positives. Compared with the radioisotope method using an expensive {sup 14}C-labeled substrate, the RapidFire mass spectrometry method using unlabeled substrates is a high-accuracy, high-throughput method. In addition, some of the hit compounds selected from the screening inhibited cellular fatty acid elongation in HEK293 cells expressing Elovl6 transiently. This result suggests that these compounds may be promising lead candidates for therapeutic drugs. Ultra-high-throughput fluorescence screening followed by a RapidFire mass spectrometry assay was a suitable strategy for lead discovery against Elovl6. - Highlights: • A novel assay for elongation of very-long-chain fatty acids 6 (Elovl6) is proposed. • RapidFire mass spectrometry (RF-MS) assay is useful to select real screening hits. • RF-MS assay is proved to be beneficial because of its high-throughput and accuracy. • A combination of fluorescent and RF-MS assays is effective for Elovl6 inhibitors.« less

Use of FRTL-5 Cell Line as a Complementary Assay for Chemicals Identified During High-Throughput Screening as Sodium/Iodide Symporter (NIS) Inhibitors

EPA Science Inventory

Confirmation of Test Chemicals Identified by a High-Throughput Screen (HTPS) as Sodium Iodide Symporter (NIS) Inhibitors in FRTL-5 Model S. Laws1, A. Buckalew1, J. Wang2, D. Hallinger1, A. Murr1, and T. Stoker1. 1Endocrin...

DOSE RESPONSE FROM HIGH THROUGHPUT GENE EXPRESSION STUDIES AND THE INFLUENCE OF TIME AND CELL LINE ON INFERRED MODE OF ACTION BY ONTOLOGIC ENRICHMENT (SOT)

EPA Science Inventory

Gene expression with ontologic enrichment and connectivity mapping tools is widely used to infer modes of action (MOA) for therapeutic drugs. Despite progress in high-throughput (HT) genomic systems, strategies suitable to identify industrial chemical MOA are needed. The L1000 is...

High-throughput combinatorial cell co-culture using microfluidics.

PubMed

Tumarkin, Ethan; Tzadu, Lsan; Csaszar, Elizabeth; Seo, Minseok; Zhang, Hong; Lee, Anna; Peerani, Raheem; Purpura, Kelly; Zandstra, Peter W; Kumacheva, Eugenia

2011-06-01

Co-culture strategies are foundational in cell biology. These systems, which serve as mimics of in vivo tissue niches, are typically poorly defined in terms of cell ratios, local cues and supportive cell-cell interactions. In the stem cell niche, the ability to screen cell-cell interactions and identify local supportive microenvironments has a broad range of applications in transplantation, tissue engineering and wound healing. We present a microfluidic platform for the high-throughput generation of hydrogel microbeads for cell co-culture. Encapsulation of different cell populations in microgels was achieved by introducing in a microfluidic device two streams of distinct cell suspensions, emulsifying the mixed suspension, and gelling the precursor droplets. The cellular composition in the microgels was controlled by varying the volumetric flow rates of the corresponding streams. We demonstrate one of the applications of the microfluidic method by co-encapsulating factor-dependent and responsive blood progenitor cell lines (MBA2 and M07e cells, respectively) at varying ratios, and show that in-bead paracrine secretion can modulate the viability of the factor dependent cells. Furthermore, we show the application of the method as a tool to screen the impact of specific growth factors on a primary human heterogeneous cell population. Co-encapsulation of IL-3 secreting MBA2 cells with umbilical cord blood cells revealed differential sub-population responsiveness to paracrine signals (CD14+ cells were particularly responsive to locally delivered IL-3). This microfluidic co-culture platform should enable high throughput screening of cell co-culture conditions, leading to new strategies to manipulate cell fate. This journal is © The Royal Society of Chemistry 2011

Multiplexing a high-throughput liability assay to leverage efficiencies.

PubMed

Herbst, John; Anthony, Monique; Stewart, Jeremy; Connors, David; Chen, Taosheng; Banks, Martyn; Petrillo, Edward W; Agler, Michele

2009-06-01

In order to identify potential cytochrome P-450 3A4 (drug-metabolizing enzyme) inducers at an early stage of the drug discovery process, a cell-based transactivation high-throughput luciferase reporter assay for the human pregnane X receptor (PXR) in HepG2 cells has been implemented and multiplexed with a viability end point for data interpretation, as part of a Lead Profiling portfolio of assays. As a routine part of Lead Profiling operations, assays are periodically evaluated for utility as well as for potential improvements in technology or process. We used a recent evaluation of our PXR-transactivation assay as a model for the application of Lean Thinking-based process analysis to lab-bench assay optimization and automation. This resulted in the development of a 384-well multiplexed homogeneous assay simultaneously detecting PXR transactivation and HepG2 cell cytotoxicity. In order to multiplex fluorescent and luminescent read-outs, modifications to each assay were necessary, which included optimization of multiple assay parameters such as cell density, plate type, and reagent concentrations. Subsequently, a set of compounds including known cytotoxic compounds and PXR inducers were used to validate the multiplexed assay. Results from the multiplexed assay correlate well with those from the singleplexed assay formats measuring PXR transactivation and viability separately. Implementation of the multiplexed assay for routine compound profiling provides improved data quality, sample conservation, cost savings, and resource efficiencies.

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

PubMed Central

Wang, Li; Carnegie, Graeme K.

2013-01-01

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells. BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction. PMID:23979513

Flow cytometric analysis of bimolecular fluorescence complementation: a high throughput quantitative method to study protein-protein interaction.

PubMed

Wang, Li; Carnegie, Graeme K

2013-08-15

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells. BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction.

High-throughput optofluidic profiling of Euglena gracilis with morphological and chemical specificity

NASA Astrophysics Data System (ADS)

Guo, Baoshan; Lei, Cheng; Ito, Takuro; Jiang, Yiyue; Ozeki, Yasuyuki; Goda, Keisuke

2016-11-01

The world is faced with environmental problems and the energy crisis due to the combustion and depletion of fossil fuels. The development of reliable, sustainable, and economical sources of alternative fuels is an important, but challenging goal for the world. As an alternative to liquid fossil fuels, algal biofuel is expected to play a key role in alleviating global warming since algae absorb atmospheric CO2 via photosynthesis. Among various algae for fuel production, Euglena gracilis is an attractive microalgal species as it is known to produce wax ester (good for biodiesel and aviation fuel) within lipid droplets. To date, while there exist many techniques for inducing microalgal cells to produce and accumulate lipid with high efficiency, few analytical methods are available for characterizing a population of such lipid-accumulated microalgae including E. gracilis with high throughout, high accuracy, and single-cell resolution simultaneously. Here we demonstrate a high-throughput optofluidic Euglena gracilis profiler which consists of an optical time-stretch microscope and a fluorescence analyzer on top of an inertial-focusing microfluidic device that can detect fluorescence from lipid droplets in their cell body and provide images of E. gracilis cells simultaneously at a high throughput of 10,000 cells/s. With the multi-dimensional information acquired by the system, we classify nitrogen-sufficient (ordinary) and nitrogen-deficient (lipid-accumulated) E. gracilis cells with a low false positive rate of 1.0%. This method provides a promise for evaluating the efficiency of lipid-inducing techniques for biofuel production, which is also applicable for identifying biomedical samples such as blood cells and cancer cells.

Staged anticonvulsant screening for chronic epilepsy.

PubMed

Berdichevsky, Yevgeny; Saponjian, Yero; Park, Kyung-Il; Roach, Bonnie; Pouliot, Wendy; Lu, Kimberly; Swiercz, Waldemar; Dudek, F Edward; Staley, Kevin J

2016-12-01

Current anticonvulsant screening programs are based on seizures evoked in normal animals. One-third of epileptic patients do not respond to the anticonvulsants discovered with these models. We evaluated a tiered program based on chronic epilepsy and spontaneous seizures, with compounds advancing from high-throughput in vitro models to low-throughput in vivo models. Epileptogenesis in organotypic hippocampal slice cultures was quantified by lactate production and lactate dehydrogenase release into culture media as rapid assays for seizure-like activity and cell death, respectively. Compounds that reduced these biochemical measures were retested with in vitro electrophysiological confirmation (i.e., second stage). The third stage involved crossover testing in the kainate model of chronic epilepsy, with blinded analysis of spontaneous seizures after continuous electrographic recordings. We screened 407 compound-concentration combinations. The cyclooxygenase inhibitor, celecoxib, had no effect on seizures evoked in normal brain tissue but demonstrated robust antiseizure activity in all tested models of chronic epilepsy. The use of organotypic hippocampal cultures, where epileptogenesis occurs on a compressed time scale, and where seizure-like activity and seizure-induced cell death can be easily quantified with biomarker assays, allowed us to circumvent the throughput limitations of in vivo chronic epilepsy models. Ability to rapidly screen compounds in a chronic model of epilepsy allowed us to find an anticonvulsant that would be missed by screening in acute models.

A Novel Dual Expression Platform for High Throughput Functional Screening of Phage Libraries in Product like Format.

PubMed

Xiao, Xiaodong; Chen, Yan; Mugabe, Sheila; Gao, Changshou; Tkaczyk, Christine; Mazor, Yariv; Pavlik, Peter; Wu, Herren; Dall'Acqua, William; Chowdhury, Partha Sarathi

2015-01-01

High throughput screenings of single chain Fv (scFv) antibody phage display libraries are currently done as soluble scFvs produced in E.coli. Due to endotoxin contaminations from bacterial cells these preparations cannot be reliably used in mammalian cell based assays. The monovalent nature and lack of Fc in soluble scFvs prevent functional assays that are dependent on target cross linking and/or Fc functions. A convenient approach is to convert scFvs into scFv.Fc fusion proteins and express them in mammalian cell lines for screening. This approach is low throughput and is only taken after primary screening of monovalent scFvs that are expressed in bacteria. There is no platform at present that combines the benefits of both bacterial and mammalian expression system for screening phage library output. We have, therefore, developed a novel dual expression vector, called pSplice, which can be used to express scFv.Fc fusion proteins both in E.coli and mammalian cell lines. The hallmark of the vector is an engineered intron which houses the bacterial promoter and signal peptide for expression and secretion of scFv.Fc in E.coli. When the vector is transfected into a mammalian cell line, the intron is efficiently spliced out resulting in a functional operon for expression and secretion of the scFv.Fc fusion protein into the culture medium. By applying basic knowledge of mammalian introns and splisosome, we designed this vector to enable screening of phage libraries in a product like format. Like IgG, the scFv.Fc fusion protein is bi-valent for the antigen and possesses Fc effector functions. Expression in E.coli maintains the speed of the bacterial expression platform and is used to triage clones based on binding and other assays that are not sensitive to endotoxin. Triaged clones are then expressed in a mammalian cell line without the need for any additional cloning steps. Conditioned media from the mammalian cell line containing the fusion proteins are then used for different types of cell based assays. Thus this system retains the speed of the current screening system for phage libraries and adds additional functionality to it.

G-Protein Coupled Receptors: Surface Display and Biosensor Technology

NASA Astrophysics Data System (ADS)

McMurchie, Edward; Leifert, Wayne

Signal transduction by G-protein coupled receptors (GPCRs) underpins a multitude of physiological processes. Ligand recognition by the receptor leads to the activation of a generic molecular switch involving heterotrimeric G-proteins and guanine nucleotides. With growing interest and commercial investment in GPCRs in areas such as drug targets, orphan receptors, high-throughput screening of drugs and biosensors, greater attention will focus on assay development to allow for miniaturization, ultrahigh-throughput and, eventually, microarray/biochip assay formats that will require nanotechnology-based approaches. Stable, robust, cell-free signaling assemblies comprising receptor and appropriate molecular switching components will form the basis of future GPCR/G-protein platforms, which should be able to be adapted to such applications as microarrays and biosensors. This chapter focuses on cell-free GPCR assay nanotechnologies and describes some molecular biological approaches for the construction of more sophisticated, surface-immobilized, homogeneous, functional GPCR sensors. The latter points should greatly extend the range of applications to which technologies based on GPCRs could be applied.

A High-Content Live-Cell Viability Assay and Its Validation on a Diverse 12K Compound Screen.

PubMed

Chiaravalli, Jeanne; Glickman, J Fraser

2017-08-01

We have developed a new high-content cytotoxicity assay using live cells, called "ImageTOX." We used a high-throughput fluorescence microscope system, image segmentation software, and the combination of Hoechst 33342 and SYTO 17 to simultaneously score the relative size and the intensity of the nuclei, the nuclear membrane permeability, and the cell number in a 384-well microplate format. We then performed a screen of 12,668 diverse compounds and compared the results to a standard cytotoxicity assay. The ImageTOX assay identified similar sets of compounds to the standard cytotoxicity assay, while identifying more compounds having adverse effects on cell structure, earlier in treatment time. The ImageTOX assay uses inexpensive commercially available reagents and facilitates the use of live cells in toxicity screens. Furthermore, we show that we can measure the kinetic profile of compound toxicity in a high-content, high-throughput format, following the same set of cells over an extended period of time.

Cancer biomarker discovery: the entropic hallmark.

PubMed

Berretta, Regina; Moscato, Pablo

2010-08-18

It is a commonly accepted belief that cancer cells modify their transcriptional state during the progression of the disease. We propose that the progression of cancer cells towards malignant phenotypes can be efficiently tracked using high-throughput technologies that follow the gradual changes observed in the gene expression profiles by employing Shannon's mathematical theory of communication. Methods based on Information Theory can then quantify the divergence of cancer cells' transcriptional profiles from those of normally appearing cells of the originating tissues. The relevance of the proposed methods can be evaluated using microarray datasets available in the public domain but the method is in principle applicable to other high-throughput methods. Using melanoma and prostate cancer datasets we illustrate how it is possible to employ Shannon Entropy and the Jensen-Shannon divergence to trace the transcriptional changes progression of the disease. We establish how the variations of these two measures correlate with established biomarkers of cancer progression. The Information Theory measures allow us to identify novel biomarkers for both progressive and relatively more sudden transcriptional changes leading to malignant phenotypes. At the same time, the methodology was able to validate a large number of genes and processes that seem to be implicated in the progression of melanoma and prostate cancer. We thus present a quantitative guiding rule, a new unifying hallmark of cancer: the cancer cell's transcriptome changes lead to measurable observed transitions of Normalized Shannon Entropy values (as measured by high-throughput technologies). At the same time, tumor cells increment their divergence from the normal tissue profile increasing their disorder via creation of states that we might not directly measure. This unifying hallmark allows, via the the Jensen-Shannon divergence, to identify the arrow of time of the processes from the gene expression profiles, and helps to map the phenotypical and molecular hallmarks of specific cancer subtypes. The deep mathematical basis of the approach allows us to suggest that this principle is, hopefully, of general applicability for other diseases.

Androgen Receptor Functional Analyses by High Throughput Imaging: Determination of Ligand, Cell Cycle, and Mutation-Specific Effects

PubMed Central

Szafran, Adam T.; Szwarc, Maria; Marcelli, Marco; Mancini, Michael A.

2008-01-01

Background Understanding how androgen receptor (AR) function is modulated by exposure to steroids, growth factors or small molecules can have important mechanistic implications for AR-related disease therapies (e.g., prostate cancer, androgen insensitivity syndrome, AIS), and in the analysis of environmental endocrine disruptors. Methodology/Principal Findings We report the development of a high throughput (HT) image-based assay that quantifies AR subcellular and subnuclear distribution, and transcriptional reporter gene activity on a cell-by-cell basis. Furthermore, simultaneous analysis of DNA content allowed determination of cell cycle position and permitted the analysis of cell cycle dependent changes in AR function in unsynchronized cell populations. Assay quality for EC50 coefficients of variation were 5–24%, with Z' values reaching 0.91. This was achieved by the selective analysis of cells expressing physiological levels of AR, important because minor over-expression resulted in elevated nuclear speckling and decreased transcriptional reporter gene activity. A small screen of AR-binding ligands, including known agonists, antagonists, and endocrine disruptors, demonstrated that nuclear translocation and nuclear “speckling” were linked with transcriptional output, and specific ligands were noted to differentially affect measurements for wild type versus mutant AR, suggesting differing mechanisms of action. HT imaging of patient-derived AIS mutations demonstrated a proof-of-principle personalized medicine approach to rapidly identify ligands capable of restoring multiple AR functions. Conclusions/Significance HT imaging-based multiplex screening will provide a rapid, systems-level analysis of compounds/RNAi that may differentially affect wild type AR or clinically relevant AR mutations. PMID:18978937

High-throughput identification of small molecules that affect human embryonic vascular development

PubMed Central

Vazão, Helena; Rosa, Susana; Barata, Tânia; Costa, Ricardo; Pitrez, Patrícia R.; Honório, Inês; de Vries, Margreet R.; Papatsenko, Dimitri; Benedito, Rui; Saris, Daniel; Khademhosseini, Ali; Quax, Paul H. A.; Pereira, Carlos F.; Mercader, Nadia; Ferreira, Lino

2017-01-01

Birth defects, which are in part caused by exposure to environmental chemicals and pharmaceutical drugs, affect 1 in every 33 babies born in the United States each year. The current standard to screen drugs that affect embryonic development is based on prenatal animal testing; however, this approach yields low-throughput and limited mechanistic information regarding the biological pathways and potential adverse consequences in humans. To develop a screening platform for molecules that affect human embryonic development based on endothelial cells (ECs) derived from human pluripotent stem cells, we differentiated human pluripotent stem cells into embryonic ECs and induced their maturation under arterial flow conditions. These cells were then used to screen compounds that specifically affect embryonic vasculature. Using this platform, we have identified two compounds that have higher inhibitory effect in embryonic than postnatal ECs. One of them was fluphenazine (an antipsychotic), which inhibits calmodulin kinase II. The other compound was pyrrolopyrimidine (an antiinflammatory agent), which inhibits vascular endothelial growth factor receptor 2 (VEGFR2), decreases EC viability, induces an inflammatory response, and disrupts preformed vascular networks. The vascular effect of the pyrrolopyrimidine was further validated in prenatal vs. adult mouse ECs and in embryonic and adult zebrafish. We developed a platform based on human pluripotent stem cell-derived ECs for drug screening, which may open new avenues of research for the study and modulation of embryonic vasculature. PMID:28348206

High-throughput identification of small molecules that affect human embryonic vascular development.

PubMed

Vazão, Helena; Rosa, Susana; Barata, Tânia; Costa, Ricardo; Pitrez, Patrícia R; Honório, Inês; de Vries, Margreet R; Papatsenko, Dimitri; Benedito, Rui; Saris, Daniel; Khademhosseini, Ali; Quax, Paul H A; Pereira, Carlos F; Mercader, Nadia; Fernandes, Hugo; Ferreira, Lino

2017-04-11

Birth defects, which are in part caused by exposure to environmental chemicals and pharmaceutical drugs, affect 1 in every 33 babies born in the United States each year. The current standard to screen drugs that affect embryonic development is based on prenatal animal testing; however, this approach yields low-throughput and limited mechanistic information regarding the biological pathways and potential adverse consequences in humans. To develop a screening platform for molecules that affect human embryonic development based on endothelial cells (ECs) derived from human pluripotent stem cells, we differentiated human pluripotent stem cells into embryonic ECs and induced their maturation under arterial flow conditions. These cells were then used to screen compounds that specifically affect embryonic vasculature. Using this platform, we have identified two compounds that have higher inhibitory effect in embryonic than postnatal ECs. One of them was fluphenazine (an antipsychotic), which inhibits calmodulin kinase II. The other compound was pyrrolopyrimidine (an antiinflammatory agent), which inhibits vascular endothelial growth factor receptor 2 (VEGFR2), decreases EC viability, induces an inflammatory response, and disrupts preformed vascular networks. The vascular effect of the pyrrolopyrimidine was further validated in prenatal vs. adult mouse ECs and in embryonic and adult zebrafish. We developed a platform based on human pluripotent stem cell-derived ECs for drug screening, which may open new avenues of research for the study and modulation of embryonic vasculature.

Design and engineering of intracellular-metabolite-sensing/regulation gene circuits in Saccharomyces cerevisiae.

PubMed

Wang, Meng; Li, Sijin; Zhao, Huimin

2016-01-01

The development of high-throughput phenotyping tools is lagging far behind the rapid advances of genotype generation methods. To bridge this gap, we report a new strategy for design, construction, and fine-tuning of intracellular-metabolite-sensing/regulation gene circuits by repurposing bacterial transcription factors and eukaryotic promoters. As proof of concept, we systematically investigated the design and engineering of bacterial repressor-based xylose-sensing/regulation gene circuits in Saccharomyces cerevisiae. We demonstrated that numerous properties, such as induction ratio and dose-response curve, can be fine-tuned at three different nodes, including repressor expression level, operator position, and operator sequence. By applying these gene circuits, we developed a cell sorting based, rapid and robust high-throughput screening method for xylose transporter engineering and obtained a sugar transporter HXT14 mutant with 6.5-fold improvement in xylose transportation capacity. This strategy should be generally applicable and highly useful for evolutionary engineering of proteins, pathways, and genomes in S. cerevisiae. © 2015 Wiley Periodicals, Inc.

High-Throughput Screening based Identification of Small Molecule Antagonists of Integrin CD11b/CD18 Ligand Binding

PubMed Central

Faridi, Mohd Hafeez; Maiguel, Dony; Brown, Brock T.; Suyama, Eigo; Barth, Constantinos J.; Hedrick, Michael; Vasile, Stefan; Sergienko, Eduard; Schürer, Stephan; Gupta, Vineet

2010-01-01

Binding of leukocyte specific integrin CD11b/CD18 to its physiologic ligands is important for the development of normal immune response in vivo. Integrin CD11b/CD18 is also a key cellular effector of various inflammatory and autoimmune diseases. However, small molecules selectively inhibiting the function of integrin CD11b/CD18 are currently lacking. We used a newly described cell-based high throughput screening assay to identify a number of highly potent antagonists of integrin CD11b/CD18 from chemical libraries containing >100,000 unique compounds. Computational analyses suggest that the identified compounds cluster into several different chemical classes. A number of the newly identified compounds blocked adhesion of wild-type mouse neutrophils to CD11b/CD18 ligand fibrinogen. Mapping the most active compounds against chemical fingerprints of known antagonists of related integrin CD11a/CD18 shows little structural similarity, suggesting that the newly identified compounds are novel and unique. PMID:20188705

Multinode acoustic focusing for parallel flow cytometry

PubMed Central

Piyasena, Menake E.; Suthanthiraraj, Pearlson P. Austin; Applegate, Robert W.; Goumas, Andrew M.; Woods, Travis A.; López, Gabriel P.; Graves, Steven W.

2012-01-01

Flow cytometry can simultaneously measure and analyze multiple properties of single cells or particles with high sensitivity and precision. Yet, conventional flow cytometers have fundamental limitations with regards to analyzing particles larger than about 70 microns, analyzing at flow rates greater than a few hundred microliters per minute, and providing analysis rates greater than 50,000 per second. To overcome these limits, we have developed multi-node acoustic focusing flow cells that can position particles (as small as a red blood cell and as large as 107 microns in diameter) into as many as 37 parallel flow streams. We demonstrate the potential of such flow cells for the development of high throughput, parallel flow cytometers by precision focusing of flow cytometry alignment microspheres, red blood cells, and the analysis of CD4+ cellular immunophenotyping assay. This approach will have significant impact towards the creation of high throughput flow cytometers for rare cell detection applications (e.g. circulating tumor cells), applications requiring large particle analysis, and high volume flow cytometry. PMID:22239072

Ultra-High-Throughput Screening of an In Vitro-Synthesized Horseradish Peroxidase Displayed on Microbeads Using Cell Sorter

PubMed Central

Zhu, Bo; Mizoguchi, Takuro; Kojima, Takaaki; Nakano, Hideo

2015-01-01

The C1a isoenzyme of horseradish peroxidase (HRP) is an industrially important heme-containing enzyme that utilizes hydrogen peroxide to oxidize a wide variety of inorganic and organic compounds for practical applications, including synthesis of fine chemicals, medical diagnostics, and bioremediation. To develop a ultra-high-throughput screening system for HRP, we successfully produced active HRP in an Escherichia coli cell-free protein synthesis system, by adding disulfide bond isomerase DsbC and optimizing the concentrations of hemin and calcium ions and the temperature. The biosynthesized HRP was fused with a single-chain Cro (scCro) DNA-binding tag at its N-terminal and C-terminal sites. The addition of the scCro-tag at both ends increased the solubility of the protein. Next, HRP and its fusion proteins were successfully synthesized in a water droplet emulsion by using hexadecane as the oil phase and SunSoft No. 818SK as the surfactant. HRP fusion proteins were displayed on microbeads attached with double-stranded DNA (containing the scCro binding sequence) via scCro-DNA interactions. The activities of the immobilized HRP fusion proteins were detected with a tyramide-based fluorogenic assay using flow cytometry. Moreover, a model microbead library containing wild type hrp (WT) and inactive mutant (MUT) genes was screened using fluorescence-activated cell-sorting, thus efficiently enriching the WT gene from the 1:100 (WT:MUT) library. The technique described here could serve as a novel platform for the ultra-high-throughput discovery of more useful HRP mutants and other heme-containing peroxidases. PMID:25993095

Ultra-high throughput detection of single cell β-galactosidase activity in droplets using micro-optical lens array

NASA Astrophysics Data System (ADS)

Lim, Jiseok; Vrignon, Jérémy; Gruner, Philipp; Karamitros, Christos S.; Konrad, Manfred; Baret, Jean-Christophe

2013-11-01

We demonstrate the use of a hybrid microfluidic-micro-optical system for the screening of enzymatic activity at the single cell level. Escherichia coli β-galactosidase activity is revealed by a fluorogenic assay in 100 pl droplets. Individual droplets containing cells are screened by measuring their fluorescence signal using a high-speed camera. The measurement is parallelized over 100 channels equipped with microlenses and analyzed by image processing. A reinjection rate of 1 ml of emulsion per minute was reached corresponding to more than 105 droplets per second, an analytical throughput larger than those obtained using flow cytometry.

Recombinant modified vaccinia virus Ankara-based malaria vaccines.

PubMed

Sebastian, Sarah; Gilbert, Sarah C

2016-01-01

A safe and effective malaria vaccine is a crucial part of the roadmap to malaria elimination/eradication by the year 2050. Viral-vectored vaccines based on adenoviruses and modified vaccinia virus Ankara (MVA) expressing malaria immunogens are currently being used in heterologous prime-boost regimes in clinical trials for induction of strong antigen-specific T-cell responses and high-titer antibodies. Recombinant MVA is a safe and well-tolerated attenuated vector that has consistently shown significant boosting potential. Advances have been made in large-scale MVA manufacture as high-yield producer cell lines and high-throughput purification processes have recently been developed. This review describes the use of MVA as malaria vaccine vector in both preclinical and clinical studies in the past 5 years.

Organic electrochemical transistors for cell-based impedance sensing

NASA Astrophysics Data System (ADS)

Rivnay, Jonathan; Ramuz, Marc; Leleux, Pierre; Hama, Adel; Huerta, Miriam; Owens, Roisin M.

2015-01-01

Electrical impedance sensing of biological systems, especially cultured epithelial cell layers, is now a common technique to monitor cell motion, morphology, and cell layer/tissue integrity for high throughput toxicology screening. Existing methods to measure electrical impedance most often rely on a two electrode configuration, where low frequency signals are challenging to obtain for small devices and for tissues with high resistance, due to low current. Organic electrochemical transistors (OECTs) are conducting polymer-based devices, which have been shown to efficiently transduce and amplify low-level ionic fluxes in biological systems into electronic output signals. In this work, we combine OECT-based drain current measurements with simultaneous measurement of more traditional impedance sensing using the gate current to produce complex impedance traces, which show low error at both low and high frequencies. We apply this technique in vitro to a model epithelial tissue layer and show that the data can be fit to an equivalent circuit model yielding trans-epithelial resistance and cell layer capacitance values in agreement with literature. Importantly, the combined measurement allows for low biases across the cell layer, while still maintaining good broadband signal.

iScreen: Image-Based High-Content RNAi Screening Analysis Tools.

PubMed

Zhong, Rui; Dong, Xiaonan; Levine, Beth; Xie, Yang; Xiao, Guanghua

2015-09-01

High-throughput RNA interference (RNAi) screening has opened up a path to investigating functional genomics in a genome-wide pattern. However, such studies are often restricted to assays that have a single readout format. Recently, advanced image technologies have been coupled with high-throughput RNAi screening to develop high-content screening, in which one or more cell image(s), instead of a single readout, were generated from each well. This image-based high-content screening technology has led to genome-wide functional annotation in a wider spectrum of biological research studies, as well as in drug and target discovery, so that complex cellular phenotypes can be measured in a multiparametric format. Despite these advances, data analysis and visualization tools are still largely lacking for these types of experiments. Therefore, we developed iScreen (image-Based High-content RNAi Screening Analysis Tool), an R package for the statistical modeling and visualization of image-based high-content RNAi screening. Two case studies were used to demonstrate the capability and efficiency of the iScreen package. iScreen is available for download on CRAN (http://cran.cnr.berkeley.edu/web/packages/iScreen/index.html). The user manual is also available as a supplementary document. © 2014 Society for Laboratory Automation and Screening.

Multiwell cell culture plate format with integrated microfluidic perfusion system

NASA Astrophysics Data System (ADS)

Domansky, Karel; Inman, Walker; Serdy, Jim; Griffith, Linda G.

2006-01-01

A new cell culture analog has been developed. It is based on the standard multiwell cell culture plate format but it provides perfused three-dimensional cell culture capability. The new capability is achieved by integrating microfluidic valves and pumps into the plate. The system provides a means to conduct high throughput assays for target validation and predictive toxicology in the drug discovery and development process. It can be also used for evaluation of long-term exposure to drugs or environmental agents or as a model to study viral hepatitis, cancer metastasis, and other diseases and pathological conditions.

Development of a High-Throughput Gene Expression Screen for Modulators of RAS-MAPK Signaling in a Mutant RAS Cellular Context.

PubMed

Severyn, Bryan; Nguyen, Thi; Altman, Michael D; Li, Lixia; Nagashima, Kumiko; Naumov, George N; Sathyanarayanan, Sriram; Cook, Erica; Morris, Erick; Ferrer, Marc; Arthur, Bill; Benita, Yair; Watters, Jim; Loboda, Andrey; Hermes, Jeff; Gilliland, D Gary; Cleary, Michelle A; Carroll, Pamela M; Strack, Peter; Tudor, Matt; Andersen, Jannik N

2016-10-01

The RAS-MAPK pathway controls many cellular programs, including cell proliferation, differentiation, and apoptosis. In colorectal cancers, recurrent mutations in this pathway often lead to increased cell signaling that may contribute to the development of neoplasms, thereby making this pathway attractive for therapeutic intervention. To this end, we developed a 26-member gene signature of RAS-MAPK pathway activity utilizing the Affymetrix QuantiGene Plex 2.0 reagent system and performed both primary and confirmatory gene expression-based high-throughput screens (GE-HTSs) using KRAS mutant colon cancer cells (SW837) and leveraging a highly annotated chemical library. The screen achieved a hit rate of 1.4% and was able to enrich for hit compounds that target RAS-MAPK pathway members such as MEK and EGFR. Sensitivity and selectivity performance measurements were 0.84 and 1.00, respectively, indicating high true-positive and true-negative rates. Active compounds from the primary screen were confirmed in a dose-response GE-HTS assay, a GE-HTS assay using 14 additional cancer cell lines, and an in vitro colony formation assay. Altogether, our data suggest that this GE-HTS assay will be useful for larger unbiased chemical screens to identify novel compounds and mechanisms that may modulate the RAS-MAPK pathway. © 2016 Society for Laboratory Automation and Screening.

Compartmental genomics in living cells revealed by single-cell nanobiopsy.

PubMed

Actis, Paolo; Maalouf, Michelle M; Kim, Hyunsung John; Lohith, Akshar; Vilozny, Boaz; Seger, R Adam; Pourmand, Nader

2014-01-28

The ability to study the molecular biology of living single cells in heterogeneous cell populations is essential for next generation analysis of cellular circuitry and function. Here, we developed a single-cell nanobiopsy platform based on scanning ion conductance microscopy (SICM) for continuous sampling of intracellular content from individual cells. The nanobiopsy platform uses electrowetting within a nanopipette to extract cellular material from living cells with minimal disruption of the cellular milieu. We demonstrate the subcellular resolution of the nanobiopsy platform by isolating small subpopulations of mitochondria from single living cells, and quantify mutant mitochondrial genomes in those single cells with high throughput sequencing technology. These findings may provide the foundation for dynamic subcellular genomic analysis.

High-Performance Flexible Perovskite Solar Cells on Ultrathin Glass: Implications of the TCO

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dou, Benjia; Miller, Elisa M.; Christians, Jeffrey A.

For halide perovskite solar cells (PSCs) to fulfill their vast potential for combining low-cost, high efficiency, and high throughput production they must be scaled using a truly transformative method, such as roll-to-roll processing. Bringing this reality closer to fruition, the present work demonstrates flexible perovskite solar cells with 18.1% power conversion efficiency on flexible Willow Glass substrates. Here, we highlight the importance of the transparent conductive oxide (TCO) layers on device performance by studying various TCOs. And while tin-doped indium oxide (ITO) and indium zinc oxide (IZO) based PSC devices demonstrate high photovoltaic performances, aluminum-doped zinc oxide (AZO) based devicesmore » underperformed in all device parameters. Analysis of X-ray photoemission spectroscopy data shows that the stoichiometry of the perovskite film surface changes dramatically when it is fabricated on AZO, demonstrating the importance of the substrate in perovskite film formation.« less

High-Performance Flexible Perovskite Solar Cells on Ultrathin Glass: Implications of the TCO

DOE PAGES

Dou, Benjia; Miller, Elisa M.; Christians, Jeffrey A.; ...

2017-09-27

For halide perovskite solar cells (PSCs) to fulfill their vast potential for combining low-cost, high efficiency, and high throughput production they must be scaled using a truly transformative method, such as roll-to-roll processing. Bringing this reality closer to fruition, the present work demonstrates flexible perovskite solar cells with 18.1% power conversion efficiency on flexible Willow Glass substrates. Here, we highlight the importance of the transparent conductive oxide (TCO) layers on device performance by studying various TCOs. And while tin-doped indium oxide (ITO) and indium zinc oxide (IZO) based PSC devices demonstrate high photovoltaic performances, aluminum-doped zinc oxide (AZO) based devicesmore » underperformed in all device parameters. Analysis of X-ray photoemission spectroscopy data shows that the stoichiometry of the perovskite film surface changes dramatically when it is fabricated on AZO, demonstrating the importance of the substrate in perovskite film formation.« less

Characterization of three human cell line models for high-throughput neuronal cytotoxicity screening.

PubMed

Tong, Zhi-Bin; Hogberg, Helena; Kuo, David; Sakamuru, Srilatha; Xia, Menghang; Smirnova, Lena; Hartung, Thomas; Gerhold, David

2017-02-01

More than 75 000 man-made chemicals contaminate the environment; many of these have not been tested for toxicities. These chemicals demand quantitative high-throughput screening assays to assess them for causative roles in neurotoxicities, including Parkinson's disease and other neurodegenerative disorders. To facilitate high throughput screening for cytotoxicity to neurons, three human neuronal cellular models were compared: SH-SY5Y neuroblastoma cells, LUHMES conditionally-immortalized dopaminergic neurons, and Neural Stem Cells (NSC) derived from human fetal brain. These three cell lines were evaluated for rapidity and degree of differentiation, and sensitivity to 32 known or candidate neurotoxicants. First, expression of neural differentiation genes was assayed during a 7-day differentiation period. Of the three cell lines, LUHMES showed the highest gene expression of neuronal markers after differentiation. Both in the undifferentiated state and after 7 days of neuronal differentiation, LUHMES cells exhibited greater cytotoxic sensitivity to most of 32 suspected or known neurotoxicants than SH-SY5Y or NSCs. LUHMES cells were also unique in being more susceptible to several compounds in the differentiating state than in the undifferentiated state; including known neurotoxicants colchicine, methyl-mercury (II), and vincristine. Gene expression results suggest that differentiating LUHMES cells may be susceptible to apoptosis because they express low levels of anti-apoptotic genes BCL2 and BIRC5/survivin, whereas SH-SY5Y cells may be resistant to apoptosis because they express high levels of BCL2, BIRC5/survivin, and BIRC3 genes. Thus, LUHMES cells exhibited favorable characteristics for neuro-cytotoxicity screening: rapid differentiation into neurons that exhibit high level expression neuronal marker genes, and marked sensitivity of LUHMES cells to known neurotoxicants. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

High-Throughput Screening of Chemical Effects on Steroidogenesis Using H295R Human Adrenocortical Carcinoma Cells.

PubMed

Karmaus, Agnes L; Toole, Colleen M; Filer, Dayne L; Lewis, Kenneth C; Martin, Matthew T

2016-04-01

Disruption of steroidogenesis by environmental chemicals can result in altered hormone levels causing adverse reproductive and developmental effects. A high-throughput assay using H295R human adrenocortical carcinoma cells was used to evaluate the effect of 2060 chemical samples on steroidogenesis via high-performance liquid chromatography followed by tandem mass spectrometry quantification of 10 steroid hormones, including progestagens, glucocorticoids, androgens, and estrogens. The study employed a 3 stage screening strategy. The first stage established the maximum tolerated concentration (MTC; ≥ 70% viability) per sample. The second stage quantified changes in hormone levels at the MTC whereas the third stage performed concentration-response (CR) on a subset of samples. At all stages, cells were prestimulated with 10 µM forskolin for 48 h to induce steroidogenesis followed by chemical treatment for 48 h. Of the 2060 chemical samples evaluated, 524 samples were selected for 6-point CR screening, based in part on significantly altering at least 4 hormones at the MTC. CR screening identified 232 chemical samples with concentration-dependent effects on 17β-estradiol and/or testosterone, with 411 chemical samples showing an effect on at least one hormone across the steroidogenesis pathway. Clustering of the concentration-dependent chemical-mediated steroid hormone effects grouped chemical samples into 5 distinct profiles generally representing putative mechanisms of action, including CYP17A1 and HSD3B inhibition. A distinct pattern was observed between imidazole and triazole fungicides suggesting potentially distinct mechanisms of action. From a chemical testing and prioritization perspective, this assay platform provides a robust model for high-throughput screening of chemicals for effects on steroidogenesis. © The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology.

High-Throughput Screening of Chemical Effects on Steroidogenesis Using H295R Human Adrenocortical Carcinoma Cells

PubMed Central

Toole, Colleen M.; Filer, Dayne L.; Lewis, Kenneth C.; Martin, Matthew T.

2016-01-01

Disruption of steroidogenesis by environmental chemicals can result in altered hormone levels causing adverse reproductive and developmental effects. A high-throughput assay using H295R human adrenocortical carcinoma cells was used to evaluate the effect of 2060 chemical samples on steroidogenesis via high-performance liquid chromatography followed by tandem mass spectrometry quantification of 10 steroid hormones, including progestagens, glucocorticoids, androgens, and estrogens. The study employed a 3 stage screening strategy. The first stage established the maximum tolerated concentration (MTC; ≥ 70% viability) per sample. The second stage quantified changes in hormone levels at the MTC whereas the third stage performed concentration-response (CR) on a subset of samples. At all stages, cells were prestimulated with 10 µM forskolin for 48 h to induce steroidogenesis followed by chemical treatment for 48 h. Of the 2060 chemical samples evaluated, 524 samples were selected for 6-point CR screening, based in part on significantly altering at least 4 hormones at the MTC. CR screening identified 232 chemical samples with concentration-dependent effects on 17β-estradiol and/or testosterone, with 411 chemical samples showing an effect on at least one hormone across the steroidogenesis pathway. Clustering of the concentration-dependent chemical-mediated steroid hormone effects grouped chemical samples into 5 distinct profiles generally representing putative mechanisms of action, including CYP17A1 and HSD3B inhibition. A distinct pattern was observed between imidazole and triazole fungicides suggesting potentially distinct mechanisms of action. From a chemical testing and prioritization perspective, this assay platform provides a robust model for high-throughput screening of chemicals for effects on steroidogenesis. PMID:26781511

Development of a cell-based high throughput luciferase enzyme fragment complementation assay to identify nuclear-factor-e2-related transcription factor 2 activators.

PubMed

Xie, Wensheng; Pao, Christina; Graham, Taylor; Dul, Ed; Lu, Quinn; Sweitzer, Thomas D; Ames, Robert S; Li, Hu

2012-12-01

Nuclear-factor-E2-related transcription factor 2 (Nrf2) regulates a large panel of Phase II genes and plays an important role in cell survival. Nrf2 activation has been shown as preventing cigarette smoke-induced alveolar enlargement in mice. Therefore, activation of the Nrf2 protein by small-molecule activators represents an attractive therapeutic strategy that is used for chronic obstructive pulmonary disease. In this article, we describe a cell-based luciferase enzyme fragment complementation assay that identifies Nrf2 activators. This assay is based on the interaction of Nrf2 with its nuclear partner MafK or runt-related transcription factor 2 (RunX2) and is dependent on the reconstitution of a "split" luciferase. Firefly luciferase is split into two fragments, which are genetically fused to Nrf2 and MafK or RunX2, respectively. BacMam technology was used to deliver the fusion constructs into cells for expression of the tagged proteins. When the BacMam-transduced cells were treated with Nrf2 activators, the Nrf2 protein was stabilized and translocated into the nucleus where it interacted with MafK or RunX2. The interaction of Nrf2 and MafK or RunX2 brought together the two luciferase fragments that form an active luciferase. The assay was developed in a 384-well format and was optimized by titrating the BacMam concentration, transduction time, cell density, and fetal bovine serum concentration. It was further validated with known Nrf2 activators. Our data show that this assay is robust, sensitive, and amenable to high throughput screening of a large compound collection for the identification of novel Nrf2 activators.

High-throughput strategies for the discovery and engineering of enzymes for biocatalysis.

PubMed

Jacques, Philippe; Béchet, Max; Bigan, Muriel; Caly, Delphine; Chataigné, Gabrielle; Coutte, François; Flahaut, Christophe; Heuson, Egon; Leclère, Valérie; Lecouturier, Didier; Phalip, Vincent; Ravallec, Rozenn; Dhulster, Pascal; Froidevaux, Rénato

2017-02-01

Innovations in novel enzyme discoveries impact upon a wide range of industries for which biocatalysis and biotransformations represent a great challenge, i.e., food industry, polymers and chemical industry. Key tools and technologies, such as bioinformatics tools to guide mutant library design, molecular biology tools to create mutants library, microfluidics/microplates, parallel miniscale bioreactors and mass spectrometry technologies to create high-throughput screening methods and experimental design tools for screening and optimization, allow to evolve the discovery, development and implementation of enzymes and whole cells in (bio)processes. These technological innovations are also accompanied by the development and implementation of clean and sustainable integrated processes to meet the growing needs of chemical, pharmaceutical, environmental and biorefinery industries. This review gives an overview of the benefits of high-throughput screening approach from the discovery and engineering of biocatalysts to cell culture for optimizing their production in integrated processes and their extraction/purification.

High-Throughput Screening of a Luciferase Reporter of Gene Silencing on the Inactive X Chromosome.

PubMed

Keegan, Alissa; Plath, Kathrin; Damoiseaux, Robert

2018-01-01

Assays of luciferase gene activity are a sensitive and quantitative reporter system suited to high-throughput screening. We adapted a luciferase assay to a screening strategy for identifying factors that reactivate epigenetically silenced genes. This epigenetic luciferase reporter is subject to endogenous gene silencing mechanisms on the inactive X chromosome (Xi) in primary mouse cells and thus captures the multilayered nature of chromatin silencing in development. Here, we describe the optimization of an Xi-linked luciferase reactivation assay in 384-well format and adaptation of the assay for high-throughput siRNA and chemical screening. Xi-luciferase reactivation screening has applications in stem cell biology and cancer therapy. We have used the approach described here to identify chromatin-modifying proteins and to identify drug combinations that enhance the gene reactivation activity of the DNA demethylating drug 5-aza-2'-deoxycytidine.

Comprehensive analysis of the T-cell receptor beta chain gene in rhesus monkey by high throughput sequencing

PubMed Central

Li, Zhoufang; Liu, Guangjie; Tong, Yin; Zhang, Meng; Xu, Ying; Qin, Li; Wang, Zhanhui; Chen, Xiaoping; He, Jiankui

2015-01-01

Profiling immune repertoires by high throughput sequencing enhances our understanding of immune system complexity and immune-related diseases in humans. Previously, cloning and Sanger sequencing identified limited numbers of T cell receptor (TCR) nucleotide sequences in rhesus monkeys, thus their full immune repertoire is unknown. We applied multiplex PCR and Illumina high throughput sequencing to study the TCRβ of rhesus monkeys. We identified 1.26 million TCRβ sequences corresponding to 643,570 unique TCRβ sequences and 270,557 unique complementarity-determining region 3 (CDR3) gene sequences. Precise measurements of CDR3 length distribution, CDR3 amino acid distribution, length distribution of N nucleotide of junctional region, and TCRV and TCRJ gene usage preferences were performed. A comprehensive profile of rhesus monkey immune repertoire might aid human infectious disease studies using rhesus monkeys. PMID:25961410

High-throughput determination of RNA structure by proximity ligation.

PubMed

Ramani, Vijay; Qiu, Ruolan; Shendure, Jay

2015-09-01

We present an unbiased method to globally resolve RNA structures through pairwise contact measurements between interacting regions. RNA proximity ligation (RPL) uses proximity ligation of native RNA followed by deep sequencing to yield chimeric reads with ligation junctions in the vicinity of structurally proximate bases. We apply RPL in both baker's yeast (Saccharomyces cerevisiae) and human cells and generate contact probability maps for ribosomal and other abundant RNAs, including yeast snoRNAs, the RNA subunit of the signal recognition particle and the yeast U2 spliceosomal RNA homolog. RPL measurements correlate with established secondary structures for these RNA molecules, including stem-loop structures and long-range pseudoknots. We anticipate that RPL will complement the current repertoire of computational and experimental approaches in enabling the high-throughput determination of secondary and tertiary RNA structures.

Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements.

PubMed

Boutagy, Nabil E; Rogers, George W; Pyne, Emily S; Ali, Mostafa M; Hulver, Matthew W; Frisard, Madlyn I

2015-10-30

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of mitochondrial function in this tissue has become more prevalent. Although many technologies and assays exist that measure mitochondrial respiratory pathways in a variety of cells, tissue and species, there is currently a void in the literature in regards to the compilation of these assays using isolated mitochondria from mouse skeletal muscle for use in microplate based technologies. Importantly, the use of microplate based respirometric assays is growing among mitochondrial biologists as it allows for high throughput measurements using minimal quantities of isolated mitochondria. Therefore, a collection of microplate based respirometric assays were developed that are able to assess mechanistic changes/adaptations in oxygen consumption in a commonly used animal model. The methods presented herein provide step-by-step instructions to perform these assays with an optimal amount of mitochondrial protein and reagents, and high precision as evidenced by the minimal variance across the dynamic range of each assay.

Quantitative high-throughput population dynamics in continuous-culture by automated microscopy.

PubMed

Merritt, Jason; Kuehn, Seppe

2016-09-12

We present a high-throughput method to measure abundance dynamics in microbial communities sustained in continuous-culture. Our method uses custom epi-fluorescence microscopes to automatically image single cells drawn from a continuously-cultured population while precisely controlling culture conditions. For clonal populations of Escherichia coli our instrument reveals history-dependent resilience and growth rate dependent aggregation.

A high-throughput media design approach for high performance mammalian fed-batch cultures

PubMed Central

Rouiller, Yolande; Périlleux, Arnaud; Collet, Natacha; Jordan, Martin; Stettler, Matthieu; Broly, Hervé

2013-01-01

An innovative high-throughput medium development method based on media blending was successfully used to improve the performance of a Chinese hamster ovary fed-batch medium in shaking 96-deepwell plates. Starting from a proprietary chemically-defined medium, 16 formulations testing 43 of 47 components at 3 different levels were designed. Media blending was performed following a custom-made mixture design of experiments considering binary blends, resulting in 376 different blends that were tested during both cell expansion and fed-batch production phases in one single experiment. Three approaches were chosen to provide the best output of the large amount of data obtained. A simple ranking of conditions was first used as a quick approach to select new formulations with promising features. Then, prediction of the best mixes was done to maximize both growth and titer using the Design Expert software. Finally, a multivariate analysis enabled identification of individual potential critical components for further optimization. Applying this high-throughput method on a fed-batch, rather than on a simple batch, process opens new perspectives for medium and feed development that enables identification of an optimized process in a short time frame. PMID:23563583

Statistical Modeling of Single Target Cell Encapsulation

PubMed Central

Moon, SangJun; Ceyhan, Elvan; Gurkan, Umut Atakan; Demirci, Utkan

2011-01-01

High throughput drop-on-demand systems for separation and encapsulation of individual target cells from heterogeneous mixtures of multiple cell types is an emerging method in biotechnology that has broad applications in tissue engineering and regenerative medicine, genomics, and cryobiology. However, cell encapsulation in droplets is a random process that is hard to control. Statistical models can provide an understanding of the underlying processes and estimation of the relevant parameters, and enable reliable and repeatable control over the encapsulation of cells in droplets during the isolation process with high confidence level. We have modeled and experimentally verified a microdroplet-based cell encapsulation process for various combinations of cell loading and target cell concentrations. Here, we explain theoretically and validate experimentally a model to isolate and pattern single target cells from heterogeneous mixtures without using complex peripheral systems. PMID:21814548

Get to Understand More from Single-Cells: Current Studies of Microfluidic-Based Techniques for Single-Cell Analysis.

PubMed

Lo, Shih-Jie; Yao, Da-Jeng

2015-07-23

This review describes the microfluidic techniques developed for the analysis of a single cell. The characteristics of microfluidic (e.g., little sample amount required, high-throughput performance) make this tool suitable to answer and to solve biological questions of interest about a single cell. This review aims to introduce microfluidic related techniques for the isolation, trapping and manipulation of a single cell. The major approaches for detection in single-cell analysis are introduced; the applications of single-cell analysis are then summarized. The review concludes with discussions of the future directions and opportunities of microfluidic systems applied in analysis of a single cell.

Get to Understand More from Single-Cells: Current Studies of Microfluidic-Based Techniques for Single-Cell Analysis

PubMed Central

Lo, Shih-Jie; Yao, Da-Jeng

2015-01-01

This review describes the microfluidic techniques developed for the analysis of a single cell. The characteristics of microfluidic (e.g., little sample amount required, high-throughput performance) make this tool suitable to answer and to solve biological questions of interest about a single cell. This review aims to introduce microfluidic related techniques for the isolation, trapping and manipulation of a single cell. The major approaches for detection in single-cell analysis are introduced; the applications of single-cell analysis are then summarized. The review concludes with discussions of the future directions and opportunities of microfluidic systems applied in analysis of a single cell. PMID:26213918

Reproducible fashion of the HSP70B' promoter-induced cytotoxic response on a live cell-based biosensor by cell cycle synchronization.

PubMed

Migita, Satoshi; Wada, Ken-Ichi; Taniguchi, Akiyoshi

2010-10-15

Live cell-based sensors potentially provide functional information about the cytotoxic effect of reagents on various signaling cascades. Cells transfected with a reporter vector derived from a cytotoxic response promoter can be used as intelligent cytotoxicity sensors (i.e., sensor cells). We have combined sensor cells and a microfluidic cell culture system that can achieve several laminar flows, resulting in a reliable high-throughput cytotoxicity detection system. These sensor cells can also be applied to single cell arrays. However, it is difficult to detect a cellular response in a single cell array, due to the heterogeneous response of sensor cells. The objective of this study was cell homogenization with cell cycle synchronization to enhance the response of cell-based biosensors. Our previously established stable sensor cells were brought into cell cycle synchronization under serum-starved conditions and we then investigated the cadmium chloride-induced cytotoxic response at the single cell level. The GFP positive rate of synchronized cells was approximately twice as high as that of the control cells, suggesting that cell homogenization is an important step when using cell-based biosensors with microdevices, such as a single cell array. Copyright 2010 Wiley Periodicals, Inc.

Small-molecule-directed, efficient generation of retinal pigment epithelium from human pluripotent stem cells.

PubMed

Maruotti, Julien; Sripathi, Srinivas R; Bharti, Kapil; Fuller, John; Wahlin, Karl J; Ranganathan, Vinod; Sluch, Valentin M; Berlinicke, Cynthia A; Davis, Janine; Kim, Catherine; Zhao, Lijun; Wan, Jun; Qian, Jiang; Corneo, Barbara; Temple, Sally; Dubey, Ramin; Olenyuk, Bogdan Z; Bhutto, Imran; Lutty, Gerard A; Zack, Donald J

2015-09-01

Age-related macular degeneration (AMD) is associated with dysfunction and death of retinal pigment epithelial (RPE) cells. Cell-based approaches using RPE-like cells derived from human pluripotent stem cells (hPSCs) are being developed for AMD treatment. However, most efficient RPE differentiation protocols rely on complex, stepwise treatments and addition of growth factors, whereas small-molecule-only approaches developed to date display reduced yields. To identify new compounds that promote RPE differentiation, we developed and performed a high-throughput quantitative PCR screen complemented by a novel orthogonal human induced pluripotent stem cell (hiPSC)-based RPE reporter assay. Chetomin, an inhibitor of hypoxia-inducible factors, was found to strongly increase RPE differentiation; combination with nicotinamide resulted in conversion of over one-half of the differentiating cells into RPE. Single passage of the whole culture yielded a highly pure hPSC-RPE cell population that displayed many of the morphological, molecular, and functional characteristics of native RPE.

Small-molecule–directed, efficient generation of retinal pigment epithelium from human pluripotent stem cells

PubMed Central

Maruotti, Julien; Sripathi, Srinivas R.; Bharti, Kapil; Fuller, John; Wahlin, Karl J.; Ranganathan, Vinod; Sluch, Valentin M.; Berlinicke, Cynthia A.; Davis, Janine; Kim, Catherine; Zhao, Lijun; Wan, Jun; Qian, Jiang; Corneo, Barbara; Temple, Sally; Dubey, Ramin; Olenyuk, Bogdan Z.; Bhutto, Imran; Lutty, Gerard A.; Zack, Donald J.

2015-01-01

Age-related macular degeneration (AMD) is associated with dysfunction and death of retinal pigment epithelial (RPE) cells. Cell-based approaches using RPE-like cells derived from human pluripotent stem cells (hPSCs) are being developed for AMD treatment. However, most efficient RPE differentiation protocols rely on complex, stepwise treatments and addition of growth factors, whereas small-molecule–only approaches developed to date display reduced yields. To identify new compounds that promote RPE differentiation, we developed and performed a high-throughput quantitative PCR screen complemented by a novel orthogonal human induced pluripotent stem cell (hiPSC)-based RPE reporter assay. Chetomin, an inhibitor of hypoxia-inducible factors, was found to strongly increase RPE differentiation; combination with nicotinamide resulted in conversion of over one-half of the differentiating cells into RPE. Single passage of the whole culture yielded a highly pure hPSC-RPE cell population that displayed many of the morphological, molecular, and functional characteristics of native RPE. PMID:26269569

High Content Imaging (HCI) on Miniaturized Three-Dimensional (3D) Cell Cultures

PubMed Central

Joshi, Pranav; Lee, Moo-Yeal

2015-01-01

High content imaging (HCI) is a multiplexed cell staining assay developed for better understanding of complex biological functions and mechanisms of drug action, and it has become an important tool for toxicity and efficacy screening of drug candidates. Conventional HCI assays have been carried out on two-dimensional (2D) cell monolayer cultures, which in turn limit predictability of drug toxicity/efficacy in vivo; thus, there has been an urgent need to perform HCI assays on three-dimensional (3D) cell cultures. Although 3D cell cultures better mimic in vivo microenvironments of human tissues and provide an in-depth understanding of the morphological and functional features of tissues, they are also limited by having relatively low throughput and thus are not amenable to high-throughput screening (HTS). One attempt of making 3D cell culture amenable for HTS is to utilize miniaturized cell culture platforms. This review aims to highlight miniaturized 3D cell culture platforms compatible with current HCI technology. PMID:26694477

Pyrimethamine as a Potent and Selective Inhibitor of Acute Myeloid Leukemia Identified by High-throughput Drug Screening.

PubMed

Sharma, Amit; Jyotsana, Nidhi; Lai, Courteney K; Chaturvedi, Anuhar; Gabdoulline, Razif; Görlich, Kerstin; Murphy, Cecilia; Blanchard, Jan E; Ganser, Arnold; Brown, Eric; Hassell, John A; Humphries, R Keith; Morgan, Michael; Heuser, Michael

2016-01-01

Hematopoietic stem and progenitor cell differentiation are blocked in acute myeloid leukemia (AML) resulting in cytopenias and a high risk of death. Most patients with AML become resistant to treatment due to lack of effective cytotoxic and differentiation promoting compounds. High MN1 expression confers poor prognosis to AML patients and induces resistance to cytarabine and alltrans-retinoic acid (ATRA) induced differentiation. Using a high-throughput drug screening, we identified the dihydrofolate reductase (DHFR) antagonist pyrimethamine to be a potent inducer of apoptosis and differentiation in several murine and human leukemia cell lines. Oral pyrimethamine treatment was effective in two xenograft mouse models and specifically targeted leukemic cells in human AML cell lines and primary patient cells, while CD34+ cells from healthy donors were unaffected. The antileukemic effects of PMT could be partially rescued by excess folic acid, suggesting an oncogenic function of folate metabolism in AML. Thus, our study identifies pyrimethamine as a candidate drug that should be further evaluated in AML treatment.

Molecular profiling of single circulating tumor cells from lung cancer patients.

PubMed

Park, Seung-Min; Wong, Dawson J; Ooi, Chin Chun; Kurtz, David M; Vermesh, Ophir; Aalipour, Amin; Suh, Susie; Pian, Kelsey L; Chabon, Jacob J; Lee, Sang Hun; Jamali, Mehran; Say, Carmen; Carter, Justin N; Lee, Luke P; Kuschner, Ware G; Schwartz, Erich J; Shrager, Joseph B; Neal, Joel W; Wakelee, Heather A; Diehn, Maximilian; Nair, Viswam S; Wang, Shan X; Gambhir, Sanjiv S

2016-12-27

Circulating tumor cells (CTCs) are established cancer biomarkers for the "liquid biopsy" of tumors. Molecular analysis of single CTCs, which recapitulate primary and metastatic tumor biology, remains challenging because current platforms have limited throughput, are expensive, and are not easily translatable to the clinic. Here, we report a massively parallel, multigene-profiling nanoplatform to compartmentalize and analyze hundreds of single CTCs. After high-efficiency magnetic collection of CTC from blood, a single-cell nanowell array performs CTC mutation profiling using modular gene panels. Using this approach, we demonstrated multigene expression profiling of individual CTCs from non-small-cell lung cancer (NSCLC) patients with remarkable sensitivity. Thus, we report a high-throughput, multiplexed strategy for single-cell mutation profiling of individual lung cancer CTCs toward minimally invasive cancer therapy prediction and disease monitoring.

Remote detection of human toxicants in real time using a human-optimized, bioluminescent bacterial luciferase gene cassette bioreporter

NASA Astrophysics Data System (ADS)

Close, Dan; Webb, James; Ripp, Steven; Patterson, Stacey; Sayler, Gary

2012-06-01

Traditionally, human toxicant bioavailability screening has been forced to proceed in either a high throughput fashion using prokaryotic or lower eukaryotic targets with minimal applicability to humans, or in a more expensive, lower throughput manner that uses fluorescent or bioluminescent human cells to directly provide human bioavailability data. While these efforts are often sufficient for basic scientific research, they prevent the rapid and remote identification of potentially toxic chemicals required for modern biosecurity applications. To merge the advantages of high throughput, low cost screening regimens with the direct bioavailability assessment of human cell line use, we re-engineered the bioluminescent bacterial luciferase gene cassette to function autonomously (without exogenous stimulation) within human cells. Optimized cassette expression provides for fully endogenous bioluminescent production, allowing continuous, real time monitoring of the bioavailability and toxicology of various compounds in an automated fashion. To access the functionality of this system, two sets of bioluminescent human cells were developed. The first was programed to suspend bioluminescent production upon toxicological challenge to mimic the non-specific detection of a toxicant. The second induced bioluminescence upon detection of a specific compound to demonstrate autonomous remote target identification. These cells were capable of responding to μM concentrations of the toxicant n-decanal, and allowed for continuous monitoring of cellular health throughout the treatment process. Induced bioluminescence was generated through treatment with doxycycline and was detectable upon dosage at a 100 ng/ml concentration. These results demonstrate that leveraging autonomous bioluminescence allows for low-cost, high throughput direct assessment of toxicant bioavailability.

High-throughput sensing and noninvasive imaging of protein nuclear transport by using reconstitution of split Renilla luciferase.

PubMed

Kim, Sung Bae; Ozawa, Takeaki; Watanabe, Shigeaki; Umezawa, Yoshio

2004-08-10

Nucleocytoplasmic trafficking of functional proteins plays a key role in regulating gene expressions in response to extracellular signals. We developed a genetically encoded bioluminescent indicator for monitoring the nuclear trafficking of target proteins in vitro and in vivo. The principle is based on reconstitution of split fragments of Renilla reniformis (Rluc) by protein splicing with a DnaE intein (a catalytic subunit of DNA polymerase III). A target cytosolic protein fused to the N-terminal half of Rluc is expressed in mammalian cells. If the protein translocates into the nucleus, the Rluc moiety meets the C-terminal half of Rluc, and full-length Rluc is reconstituted by protein splicing. We demonstrated quantitative cell-based in vitro sensing of ligand-induced translocation of androgen receptor, which allowed high-throughput screening of exo- and endogenous agonists and antagonists. Furthermore, the indicator enabled noninvasive in vivo imaging of the androgen receptor translocation in the brains of living mice with a charge-coupled device imaging system. These rapid and quantitative analyses in vitro and in vivo provide a wide variety of applications for screening pharmacological or toxicological compounds and testing them in living animals.

Compounds with species and cell type specific toxicity identified in a 2000 compound drug screen of neural stem cells and rat mixed cortical neurons.

PubMed

Malik, Nasir; Efthymiou, Anastasia G; Mather, Karly; Chester, Nathaniel; Wang, Xiantao; Nath, Avindra; Rao, Mahendra S; Steiner, Joseph P

2014-12-01

Human primary neural tissue is a vital component for the quick and simple determination of chemical compound neurotoxicity in vitro. In particular, such tissue would be ideal for high-throughput screens that can be used to identify novel neurotoxic or neurotherapeutic compounds. We have previously established a high-throughput screening platform using human induced pluripotent stem cell (iPSC)-derived neural stem cells (NSCs) and neurons. In this study, we conducted a 2000 compound screen with human NSCs and rat cortical cells to identify compounds that are selectively toxic to each group. Approximately 100 of the tested compounds showed specific toxicity to human NSCs. A secondary screen of a small subset of compounds from the primary screen on human iPSCs, NSC-derived neurons, and fetal astrocytes validated the results from >80% of these compounds with some showing cell specific toxicity. Amongst those compounds were several cardiac glycosides, all of which were selectively toxic to the human cells. As the screen was able to reliably identify neurotoxicants, many with species and cell-type specificity, this study demonstrates the feasibility of this NSC-driven platform for higher-throughput neurotoxicity screens. Published by Elsevier B.V.

Microfluidic strategies for understanding the mechanics of cells and cell-mimetic systems

PubMed Central

Dahl, Joanna B.; Lin, Jung-Ming G.; Muller, Susan J.; Kumar, Sanjay

2016-01-01

Microfluidic systems are attracting increasing interest for the high-throughput measurement of cellular biophysical properties and for the creation of engineered cellular microenvironments. Here we review recent applications of microfluidic technologies to the mechanics of living cells and synthetic cell-mimetic systems. We begin by discussing the use of microfluidic devices to dissect the mechanics of cellular mimics such as capsules and vesicles. We then explore applications to circulating cells, including erythrocytes and other normal blood cells, and rare populations with potential disease diagnostic value, such as circulating tumor cells. We conclude by discussing how microfluidic devices have been used to investigate the mechanics, chemotaxis, and invasive migration of adherent cells. In these ways, microfluidic technologies represent an increasingly important toolbox for investigating cellular mechanics and motility at high throughput and in a format that lends itself to clinical translation. PMID:26134738

Highly Expandable Human iPS Cell-Derived Neural Progenitor Cells (NPC) and Neurons for Central Nervous System Disease Modeling and High-Throughput Screening.

PubMed

Cheng, Chialin; Fass, Daniel M; Folz-Donahue, Kat; MacDonald, Marcy E; Haggarty, Stephen J

2017-01-11

Reprogramming of human somatic cells into induced pluripotent stem (iPS) cells has greatly expanded the set of research tools available to investigate the molecular and cellular mechanisms underlying central nervous system (CNS) disorders. Realizing the promise of iPS cell technology for the identification of novel therapeutic targets and for high-throughput drug screening requires implementation of methods for the large-scale production of defined CNS cell types. Here we describe a protocol for generating stable, highly expandable, iPS cell-derived CNS neural progenitor cells (NPC) using multi-dimensional fluorescence activated cell sorting (FACS) to purify NPC defined by cell surface markers. In addition, we describe a rapid, efficient, and reproducible method for generating excitatory cortical-like neurons from these NPC through inducible expression of the pro-neural transcription factor Neurogenin 2 (iNgn2-NPC). Finally, we describe methodology for the use of iNgn2-NPC for probing human neuroplasticity and mechanisms underlying CNS disorders using high-content, single-cell-level automated microscopy assays. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

A Fully Automated High-Throughput Zebrafish Behavioral Ototoxicity Assay.

PubMed

Todd, Douglas W; Philip, Rohit C; Niihori, Maki; Ringle, Ryan A; Coyle, Kelsey R; Zehri, Sobia F; Zabala, Leanne; Mudery, Jordan A; Francis, Ross H; Rodriguez, Jeffrey J; Jacob, Abraham

2017-08-01

Zebrafish animal models lend themselves to behavioral assays that can facilitate rapid screening of ototoxic, otoprotective, and otoregenerative drugs. Structurally similar to human inner ear hair cells, the mechanosensory hair cells on their lateral line allow the zebrafish to sense water flow and orient head-to-current in a behavior called rheotaxis. This rheotaxis behavior deteriorates in a dose-dependent manner with increased exposure to the ototoxin cisplatin, thereby establishing itself as an excellent biomarker for anatomic damage to lateral line hair cells. Building on work by our group and others, we have built a new, fully automated high-throughput behavioral assay system that uses automated image analysis techniques to quantify rheotaxis behavior. This novel system consists of a custom-designed swimming apparatus and imaging system consisting of network-controlled Raspberry Pi microcomputers capturing infrared video. Automated analysis techniques detect individual zebrafish, compute their orientation, and quantify the rheotaxis behavior of a zebrafish test population, producing a powerful, high-throughput behavioral assay. Using our fully automated biological assay to test a standardized ototoxic dose of cisplatin against varying doses of compounds that protect or regenerate hair cells may facilitate rapid translation of candidate drugs into preclinical mammalian models of hearing loss.

Fourier Ptychographic Microscopy for Rapid, High-Resolution Imaging of Circulating Tumor Cells Enriched by Microfiltration.

PubMed

Williams, Anthony; Chung, Jaebum; Yang, Changhuei; Cote, Richard J

2017-01-01

Examining the hematogenous compartment for evidence of metastasis has increased significantly within the oncology research community in recent years, due to the development of technologies aimed at the enrichment of circulating tumor cells (CTCs), the subpopulation of primary tumor cells that gain access to the circulatory system and are responsible for colonization at distant sites. In contrast to other technologies, filtration-based CTC enrichment, which exploits differences in size between larger tumor cells and surrounding smaller, non-tumor blood cells, has the potential to improve CTC characterization through isolation of tumor cell populations with greater molecular heterogeneity. However, microscopic analysis of uneven filtration surfaces containing CTCs is laborious, time-consuming, and inconsistent, preventing widespread use of filtration-based enrichment technologies. Here, integrated with a microfiltration-based CTC and rare cell enrichment device we have previously described, we present a protocol for Fourier Ptychographic Microscopy (FPM), a method that, unlike many automated imaging platforms, produces high-speed, high-resolution images that can be digitally refocused, allowing users to observe objects of interest present on multiple focal planes within the same image frame. The development of a cost-effective and high-throughput CTC analysis system for filtration-based enrichment technologies could have profound clinical implications for improved CTC detection and analysis.

Stable cellular models of nuclear receptor PXR for high-throughput evaluation of small molecules.

PubMed

Negi, Seema; Singh, Shashi Kala; Kumar, Sanjay; Kumar, Subodh; Tyagi, Rakesh K

2018-06-19

Pregnane & Xenobiotic Receptor (PXR) is one of the 48 members of the ligand-modulated transcription factors belonging to nuclear receptor superfamily. Though PXR is now well-established as a 'xenosensor', regulating the central detoxification and drug metabolizing machinery, it has also emerged as a key player in several metabolic disorders. This makes PXR attractive to both, researchers and pharmaceutical industry since clinical success of small drug molecules can be pre-evaluated on PXR platform. At the early stages of drug discovery, cell-based assays are used for high-throughput screening of small molecules. The future success or failure of a drug can be predicted by this approach saving expensive resources and time. In view of this, we have developed human liver cell line-based, dual-level screening and validation protocol on PXR platform having application to assess small molecules. We have generated two different stably transfected cell lines, (i) a stable promoter-reporter cell line (HepXREM) expressing PXR and a commonly used CYP3A4 promoter-reporter i.e. XREM-luciferase; and (ii) two stable cell lines integrated with proximal PXR-promoter-reporter (Hepx-1096/+43 and Hepx-497/+43). Employing HepXREM, Hepx-1096/+43 and Hepx-497/+43 stable cell lines > 25 anti-cancer herbal drug ingredients were screened for examining their modulatory effects on a) PXR transcriptional activity and, b) PXR-promoter activity. In conclusion, the present report provides a convenient and economical, dual-level screening system to facilitate the identification of superior therapeutic small molecules. Copyright © 2018. Published by Elsevier Ltd.

An automated high throughput screening-compatible assay to identify regulators of stem cell neural differentiation.

PubMed

Casalino, Laura; Magnani, Dario; De Falco, Sandro; Filosa, Stefania; Minchiotti, Gabriella; Patriarca, Eduardo J; De Cesare, Dario

2012-03-01

The use of Embryonic Stem Cells (ESCs) holds considerable promise both for drug discovery programs and the treatment of degenerative disorders in regenerative medicine approaches. Nevertheless, the successful use of ESCs is still limited by the lack of efficient control of ESC self-renewal and differentiation capabilities. In this context, the possibility to modulate ESC biological properties and to obtain homogenous populations of correctly specified cells will help developing physiologically relevant screens, designed for the identification of stem cell modulators. Here, we developed a high throughput screening-suitable ESC neural differentiation assay by exploiting the Cell(maker) robotic platform and demonstrated that neural progenies can be generated from ESCs in complete automation, with high standards of accuracy and reliability. Moreover, we performed a pilot screening providing proof of concept that this assay allows the identification of regulators of ESC neural differentiation in full automation.

Optimizing transformations for automated, high throughput analysis of flow cytometry data

PubMed Central

2010-01-01

Background In a high throughput setting, effective flow cytometry data analysis depends heavily on proper data preprocessing. While usual preprocessing steps of quality assessment, outlier removal, normalization, and gating have received considerable scrutiny from the community, the influence of data transformation on the output of high throughput analysis has been largely overlooked. Flow cytometry measurements can vary over several orders of magnitude, cell populations can have variances that depend on their mean fluorescence intensities, and may exhibit heavily-skewed distributions. Consequently, the choice of data transformation can influence the output of automated gating. An appropriate data transformation aids in data visualization and gating of cell populations across the range of data. Experience shows that the choice of transformation is data specific. Our goal here is to compare the performance of different transformations applied to flow cytometry data in the context of automated gating in a high throughput, fully automated setting. We examine the most common transformations used in flow cytometry, including the generalized hyperbolic arcsine, biexponential, linlog, and generalized Box-Cox, all within the BioConductor flowCore framework that is widely used in high throughput, automated flow cytometry data analysis. All of these transformations have adjustable parameters whose effects upon the data are non-intuitive for most users. By making some modelling assumptions about the transformed data, we develop maximum likelihood criteria to optimize parameter choice for these different transformations. Results We compare the performance of parameter-optimized and default-parameter (in flowCore) data transformations on real and simulated data by measuring the variation in the locations of cell populations across samples, discovered via automated gating in both the scatter and fluorescence channels. We find that parameter-optimized transformations improve visualization, reduce variability in the location of discovered cell populations across samples, and decrease the misclassification (mis-gating) of individual events when compared to default-parameter counterparts. Conclusions Our results indicate that the preferred transformation for fluorescence channels is a parameter- optimized biexponential or generalized Box-Cox, in accordance with current best practices. Interestingly, for populations in the scatter channels, we find that the optimized hyperbolic arcsine may be a better choice in a high-throughput setting than current standard practice of no transformation. However, generally speaking, the choice of transformation remains data-dependent. We have implemented our algorithm in the BioConductor package, flowTrans, which is publicly available. PMID:21050468

Optimizing transformations for automated, high throughput analysis of flow cytometry data.

PubMed

Finak, Greg; Perez, Juan-Manuel; Weng, Andrew; Gottardo, Raphael

2010-11-04

In a high throughput setting, effective flow cytometry data analysis depends heavily on proper data preprocessing. While usual preprocessing steps of quality assessment, outlier removal, normalization, and gating have received considerable scrutiny from the community, the influence of data transformation on the output of high throughput analysis has been largely overlooked. Flow cytometry measurements can vary over several orders of magnitude, cell populations can have variances that depend on their mean fluorescence intensities, and may exhibit heavily-skewed distributions. Consequently, the choice of data transformation can influence the output of automated gating. An appropriate data transformation aids in data visualization and gating of cell populations across the range of data. Experience shows that the choice of transformation is data specific. Our goal here is to compare the performance of different transformations applied to flow cytometry data in the context of automated gating in a high throughput, fully automated setting. We examine the most common transformations used in flow cytometry, including the generalized hyperbolic arcsine, biexponential, linlog, and generalized Box-Cox, all within the BioConductor flowCore framework that is widely used in high throughput, automated flow cytometry data analysis. All of these transformations have adjustable parameters whose effects upon the data are non-intuitive for most users. By making some modelling assumptions about the transformed data, we develop maximum likelihood criteria to optimize parameter choice for these different transformations. We compare the performance of parameter-optimized and default-parameter (in flowCore) data transformations on real and simulated data by measuring the variation in the locations of cell populations across samples, discovered via automated gating in both the scatter and fluorescence channels. We find that parameter-optimized transformations improve visualization, reduce variability in the location of discovered cell populations across samples, and decrease the misclassification (mis-gating) of individual events when compared to default-parameter counterparts. Our results indicate that the preferred transformation for fluorescence channels is a parameter- optimized biexponential or generalized Box-Cox, in accordance with current best practices. Interestingly, for populations in the scatter channels, we find that the optimized hyperbolic arcsine may be a better choice in a high-throughput setting than current standard practice of no transformation. However, generally speaking, the choice of transformation remains data-dependent. We have implemented our algorithm in the BioConductor package, flowTrans, which is publicly available.

QPatch: the missing link between HTS and ion channel drug discovery.

PubMed

Mathes, Chris; Friis, Søren; Finley, Michael; Liu, Yi

2009-01-01

The conventional patch clamp has long been considered the best approach for studying ion channel function and pharmacology. However, its low throughput has been a major hurdle to overcome for ion channel drug discovery. The recent emergence of higher throughput, automated patch clamp technology begins to break this bottleneck by providing medicinal chemists with high-quality, information-rich data in a more timely fashion. As such, these technologies have the potential to bridge a critical missing link between high-throughput primary screening and meaningful ion channel drug discovery programs. One of these technologies, the QPatch automated patch clamp system developed by Sophion Bioscience, records whole-cell ion channel currents from 16 or 48 individual cells in a parallel fashion. Here, we review the general applicability of the QPatch to studying a wide variety of ion channel types (voltage-/ligand-gated cationic/anionic channels) in various expression systems. The success rate of gigaseals, formation of the whole-cell configuration and usable cells ranged from 40-80%, depending on a number of factors including the cell line used, ion channel expressed, assay development or optimization time and expression level in these studies. We present detailed analyses of the QPatch features and results in case studies in which secondary screening assays were successfully developed for a voltage-gated calcium channel and a ligand-gated TRP channel. The increase in throughput compared to conventional patch clamp with the same cells was approximately 10-fold. We conclude that the QPatch, combining high data quality and speed with user friendliness and suitability for a wide array of ion channels, resides on the cutting edge of automated patch clamp technology and plays a pivotal role in expediting ion channel drug discovery.

Functional screening assays with neurons generated from pluripotent stem cell-derived neural stem cells.

PubMed

Efthymiou, Anastasia; Shaltouki, Atossa; Steiner, Joseph P; Jha, Balendu; Heman-Ackah, Sabrina M; Swistowski, Andrzej; Zeng, Xianmin; Rao, Mahendra S; Malik, Nasir

2014-01-01

Rapid and effective drug discovery for neurodegenerative disease is currently impeded by an inability to source primary neural cells for high-throughput and phenotypic screens. This limitation can be addressed through the use of pluripotent stem cells (PSCs), which can be derived from patient-specific samples and differentiated to neural cells for use in identifying novel compounds for the treatment of neurodegenerative diseases. We have developed an efficient protocol to culture pure populations of neurons, as confirmed by gene expression analysis, in the 96-well format necessary for screens. These differentiated neurons were subjected to viability assays to illustrate their potential in future high-throughput screens. We have also shown that organelles such as nuclei and mitochondria could be live-labeled and visualized through fluorescence, suggesting that we should be able to monitor subcellular phenotypic changes. Neurons derived from a green fluorescent protein-expressing reporter line of PSCs were live-imaged to assess markers of neuronal maturation such as neurite length and co-cultured with astrocytes to demonstrate further maturation. These studies confirm that PSC-derived neurons can be used effectively in viability and functional assays and pave the way for high-throughput screens on neurons derived from patients with neurodegenerative disorders.

A microfluidic chip integrated with a high-density PDMS-based microfiltration membrane for rapid isolation and detection of circulating tumor cells.

PubMed

Fan, Xiaoyun; Jia, Chunping; Yang, Jun; Li, Gang; Mao, Hongju; Jin, Qinghui; Zhao, Jianlong

2015-09-15

Isolation of circulating tumor cells (CTCs) by size exclusion is a widely researched technique that offers the advantage of capturing tumor cells without reliance on cell surface expression markers. In this work, we report the development of a novel polydimethylsiloxane (PDMS) membrane filter-based microdevice for rapid and highly efficient isolation of CTCs from peripheral blood. A precise and highly porous PDMS microfilter was fabricated and integrated into the microfiltration chip by combining a sacrificial transferring film with a sandwich molding method. We achieved >90% recovery when isolating lung cancer cells from spiked blood samples, with a relatively high processing throughput of 10 mL/h. In contrast to existing CTC filtration systems, which rely on low-porosity track-etch filters or expensive lithography-based filters, our microfiltration chip does not require complex e-beam lithography or the reactive ion etching process, therefore it offers a low-cost alternative tool for highly efficient CTC enrichment and in situ analysis. Thus, this new microdevice has the potential for use in routine monitoring of cancer development and cancer therapy in a clinical setting. Copyright © 2015 Elsevier B.V. All rights reserved.