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Flow-Based Combinatorial Antibody Profiling: An Integrated Approach to Cell Characterization

Flow-Based Combinatorial Antibody Profiling: An Integrated A...

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作者： Shane Bruckner Ling Wang Ruiling Yuan Perry Haaland Amitabh Gaur

BD FACS™ CAP (CAP = combinatorial antibody profile) is a screening tool for rapid characterization of human cell surface protein expression profiles using semi-automated high-throughput flow cytometry. The current configuration consists of 229 directly conjugated antibodies arrayed in a 96-well plate as three-color cocktails, which enables the characterization of each of the 229 individual surface markers. Each individual cell type of interest is analyzed on the 96-well screening plates and the data are acquired on a flow cytometer equipped with a high-throughput sampler. The expression level of each marker for each cell type is then calculated using semiautomated custom flow cytometry software. The process of characterizing these surface markers in a highly efficient manner using BD FACS™ CAP is enabled by automated liquid handling for staining, automated flow cytometry for data acquisition, and standardized algorithms for automated data *** FACS™ CAP (CAP = combinatorial antibody profile) is a screening tool for rapid characterization of human cell surface protein expression profiles using semi-automated high-throughput flow cytometry. The current configuration consists of 229 directly conjugated antibodies arrayed in a 96-well plate as three-color cocktails, which enables the characterization of each of the 229 individual surface markers. Each individual cell type of interest is analyzed on the 96-well screening plates and the data are acquired on a flow cytometer equipped with a high-throughput sampler. The expression level of each marker for each cell type is then calculated using semiautomated custom flow cytometry software. The process of characterizing these surface markers in a highly efficient manner using BD FACS™ CAP is enabled by automated liquid handling for staining, automated flow cytometry for data acquisition, and standardized algorithms for automated data analysis.

关键词： Protein Expression Profiling Flow Cytometry Recombinant Protein Expression Liquid Handling Statistical Calculation FACS Data analysis programming language Homo sapiens HPAF-II

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Utilization of SSCprofiler to Predict a New miRNA Gene

Utilization of SSCprofiler to Predict a New miRNA Gene

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作者： Anastasis Oulas Panayiota Poirazi

Experimental identification provides a valuable yet slow and expensive method for predicting novel miRNA genes. With the advent of computational procedures, it is now possible to capture characteristic features of miRNA biogenesis in an in silico model, resulting thereafter in the fast and inexpensive prediction of multiple novel miRNA gene candidates. These computational tools provide valuable clues to experimentalists, allowing them to narrow down their search space, making experimental verification less time consuming and less costly. Furthermore, the computational model itself can provide biological information as to which are the dominant features that characterize these regulatory units. Moreover, large-scale, high-throughput techniques, such as deep sequencing and tiling arrays, require computational methods to analyze this vast amount of data. Computational miRNA gene prediction tools are often used in synergy with high-throughput methods, aiding in the discovery of putative miRNA genes. This chapter focuses on a recently developed computational tool (SSCprofiler) for identifying miRNA genes and provides an overview of the methodology undertaken by this tool, and defines a stepwise guideline on how to utilize SSCprofiler to predict novel miRNAs in the human genome. less

关键词： Deep Sequencing Gene Prediction Structure Prediction Genome Alignment Cell And Tissue Culture RNA Secondary Structure Prediction Genome Assembly programming language Homo sapiens Mus musculus Canis lupus familiaris Gallus gallus Takifugu Danio rerio Rattus norvegicus Pan troglodytes Hidden Markov model Web tool Expression data

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Super-resolution 3D microscopy of live whole cells using structured illumination

Super-resolution 3D microscopy of live whole cells using str...

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作者： Lin Shao Peter Kner E Hesper Rego Mats G L Gustafsson

Three-dimensional (3D) structured-illumination microscopy (SIM) can double the lateral and axial resolution of a wide-field fluorescence microscope but has been too slow for live imaging. Here we apply 3D SIM to living samples and record whole cells at up to 5 s per volume for >50 time points with 120-nm lateral and 360-nm axial resolution. We demonstrate the technique by imaging microtubules in S2 cells and mitochondria in HeLa cells. less

关键词： Super-resolution Microscopy Time Lapse Microscopy Microscopy 3D Structured Illumination Microscopy Live Cell Imaging X-ray Crystallography Electron Microscopy (EM) Image Analysis programming language Mitochondria Microtubules

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Analysis of High-Throughput ELISA Microarray Data

Analysis of High-Throughput ELISA Microarray Data

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作者： Amanda M. White Don S. Daly Richard C. Zangar

Our research group develops analytical methods and software for the high-throughput analysis of quantitative enzyme-linked immunosorbent assay (ELISA) microarrays. ELISA microarrays differ from DNA microarrays in several fundamental aspects and most algorithms for analysis of DNA microarray data are not applicable to ELISA microarrays. In this review, we provide an overview of the steps involved in ELISA microarray data analysis and how the statistically sound algorithms we have developed provide an integrated software suite to address the needs of each data-processing step. The algorithms discussed are available in a set of open-source software tools (http://***/statistics/ProMAT ). less

关键词： ELISA Microarray Nucleic Acid Microarray Image Analysis Sandwich ELISA Monte Carlo Simulations Statistical Calculation Data analysis programming language Bioinformatics Calibration Standard curve ProMAT ELISA-BASE

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Designing, Performing, and Interpreting a Microarray-Based Gene Expression Study

Designing, Performing, and Interpreting a Microarray-Based G...

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作者： Giovanni Coppola

Microarray-based assays have significantly expanded their scope and range of applications over the last 10 years, and – at least for gene expression – can be considered mainstream applications. High-throughput, microarray-based gene expression studies have proven particularly useful in the study of neurodegenerative diseases, for which they have provided key insights in understanding disease pathogenesis, regional and cellular specificity, and identification of therapeutic targets. Even though many experimental steps are currently performed in specialized core facilities, the key steps of a microarray study – experimental design, and data analysis and interpretation – are performed by the primary investigator. Knowledge of the issues related to these key steps is essential to properly perform and interpret a microarray experiment and constitutes the main focus of the present chapter. The basic analytical steps are covered, and annotated R code for the analysis of a published dataset is provided. less

关键词： Microarray Data Mining RNA Extraction Microarray Hybridization Gel Electrophoresis RNA Quantitation Assay Statistical Calculation Differential Expression Analysis Real-time PCR Immunohistochemistry In Situ Hybridization Data analysis programming language Mus musculus Normalization Gene expression Gene ontology Differential expression Bioconductor Pathway Review

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Setup and use of a two-laser multiphoton microscope for multichannel intravital fluorescence imaging

Setup and use of a two-laser multiphoton microscope for mult...

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作者： David Entenberg Jeffrey Wyckoff Bojana Gligorijevic Evanthia T Roussos Vladislav V Verkhusha Jeffrey W Pollard John Condeelis

Characterizing biological mechanisms dependent upon the interaction of many cell types in vivo requires both multiphoton microscope systems capable of expanding the number and types of fluorophores that can be imaged simultaneously while removing the wavelength and tunability restrictions of existing systems, and enhanced software for extracting critical cellular parameters from voluminous 4D data sets. We present a procedure for constructing a two-laser multiphoton microscope that extends the wavelength range of excitation light, expands the number of simultaneously usable fluorophores and markedly increases signal to noise via 'over-clocking' of detection. We also utilize a custom-written software plug-in that simplifies the quantitative tracking and analysis of 4D intravital image data. We begin by describing the optics, hardware, electronics and software required, and finally the use of the plug-in for analysis. We demonstrate the use of the setup and plug-in by presenting data collected via intravital imaging of a mouse model of breast cancer. The procedure may be completed in ∼24 h. less

关键词： Fluorescence Microscopy Multiphoton Microscopy Intravital Microscopy ImageJ Transillumination Cell And Tissue Culture Statistical Calculation programming language Lens Bioinformatics Cell biology

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Implementing Conceptual Spatiotemporal Model into Object DBMS with Semantic Preserving

Implementing Conceptual Spatiotemporal Model into Object DBM...

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2011 IEEE International Conference on Spatial Data Mining and Geographical Knowledge Services(ICSDM 2011)

作者： Chamseddine Zaki Myriam Servières Guillaume Moreau LUNAM Universit Ecole Centrale NantesCERMA UMR CNRS 1563 Ecole d'Architecture de Nantes6 quai Francois MitterrandBP 16202-44262 Nantes Cedex 2

Our work is developed in the context of spatiotemporal data modeling and more particularly of urban data *** goal is to propose a methodology describing the overall process of urban data modeling beginning from the design phase until the implementation in an information system. For that,we propose,within a GIS,an approach based on a conceptual model specific to spatiotemporal data modeling MADS,and on an ODBMS for storing and manipulating data. MADS uses spatial,temporal and multi-representation types that are more precise than those of programming languages on which are based ODBMS *** MADS is semantically richer than the object model,and the purpose of this paper is to present the transformation rules of this conceptual model into an object model that keeps(as close as possible) its semantics.

关键词： Spatio-temporal modelling conceptual models ODBMS transformation of models programming language geographic information systems

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Proteomic and phosphoproteomic comparison of human ES and iPS cells

Proteomic and phosphoproteomic comparison of human ES and iP...

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作者： Douglas H Phanstiel Justin Brumbaugh Craig D Wenger Shulan Tian Mitchell D Probasco Derek J Bailey Danielle L Swaney Mark A Tervo Jennifer M Bolin Victor Ruotti Ron Stewart James A Thomson Joshua J Coon

Combining high-mass-accuracy mass spectrometry, isobaric tagging and software for multiplexed, large-scale protein quantification, we report deep proteomic coverage of four human embryonic stem cell and four induced pluripotent stem cell lines in biological triplicate. This 24-sample comparison resulted in a very large set of identified proteins and phosphorylation sites in pluripotent cells. The statistical analysis afforded by our approach revealed subtle but reproducible differences in protein expression and protein phosphorylation between embryonic stem cells and induced pluripotent cells. Merging these results with RNA-seq analysis data, we found functionally related differences across each tier of regulation. We also introduce the Stem Cell–Omics Repository (SCOR), a resource to collate and display quantitative information across multiple planes of measurement, including mRNA, protein and post-translational modifications. less

关键词： Proteomics Western Blot Fractionation Mass Spectrometry Database Search Statistical Calculation Recombinant Protein Expression RNA-Seq Analysis Sonication Bowtie Cell Lysis Solid-phase Extraction (SPE) Tandem Mass Spectrometry HPLC Electron Transfer Detection Microarray programming language Homo sapiens Embryonic stem cells Induced pluripotent stem cells Anti-mouse Anti-goat

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Large-scale automated synthesis of human functional neuroimaging data

Large-scale automated synthesis of human functional neuroima...

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作者： Tal Yarkoni Russell A Poldrack Thomas E Nichols David C Van Essen Tor D Wager

The rapid growth of the literature on neuroimaging in humans has led to major advances in our understanding of human brain function but has also made it increasingly difficult to aggregate and synthesize neuroimaging findings. Here we describe and validate an automated brain-mapping framework that uses text-mining, meta-analysis and machine-learning techniques to generate a large database of mappings between neural and cognitive states. We show that our approach can be used to automatically conduct large-scale, high-quality neuroimaging meta-analyses, address long-standing inferential problems in the neuroimaging literature and support accurate 'decoding' of broad cognitive states from brain activity in both entire studies and individual human subjects. Collectively, our results have validated a powerful and generative framework for synthesizing human neuroimaging data on an unprecedented scale. less

关键词： Functional Neuroimaging Statistical Inference Neuroimaging Brain-Mapping Machine Learning Monte Carlo Simulations programming language Homo sapiens Imaging Neuroscience Bioinformatics

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Optogenetic manipulation of neural activity in freely moving

Optogenetic manipulation of neural activity in freely moving

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作者： Andrew M Leifer Christopher Fang-Yen Marc Gershow Mark J Alkema Aravinthan D T Samuel

We present an optogenetic illumination system capable of real-time light delivery with high spatial resolution to specified targets in freely moving Caenorhabditis elegans. A tracking microscope records the motion of an unrestrained worm expressing channelrhodopsin-2 or halorhodopsin in specific cell types. Image processing software analyzes the worm's position in each video frame, rapidly estimates the locations of targeted cells and instructs a digital micromirror device to illuminate targeted cells with laser light of the appropriate wavelengths to stimulate or inhibit activity. Because each cell in an unrestrained worm is a rapidly moving target, our system operates at high speed (∼50 frames per second) to provide high spatial resolution (∼30 μm). To test the accuracy, flexibility and utility of our system, we performed optogenetic analyses of the worm motor circuit, egg-laying circuit and mechanosensory circuits that have not been possible with previous methods. less

关键词： Optogenetics Microscopy Image Processing ImageJ Optical Imaging programming language Caenorhabditis elegans Imaging Neuroscience

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