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Latest Articles

A Quantitative Method for Comparing the Brightness of Antibody‐dye Reagents and Estimating Antibodies Bound per Cell
Aaron B. Kantor, Wayne A. Moore, Stephen Meehan, David R. Parks
We present a quantitative method for comparing the brightness of antibody‐dye reagents and estimating antibodies bound per cell. The method is based on complementary binding of test and fill reagents to antibody capture microspheres. Several aliquots of antibody capture beads are stained with
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Measurement and Characterization of Apoptosis by Flow Cytometry
William Telford, Karen Tamul, Jolene Bradford
Apoptosis is an important mechanism in cell biology, playing a critical regulatory role in virtually every organ system. It has been particularly well characterized in the immune system, with roles ranging from immature immune cell development and selection to down‐regulation of the mature immune
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High‐Content Microscopy Analysis of Subcellular Structures: Assay Development and Application to Focal Adhesion Quantification
Torsten Kroll, David Schmidt, Georg Schwanitz, Mubashir Ahmad, Jana Hamann, Corinne Schlosser, Yu‐Chieh Lin, Konrad J. Böhm, Jan Tuckermann, Aspasia Ploubidou
High‐content analysis (HCA) converts raw light microscopy images to quantitative data through the automated extraction, multiparametric analysis, and classification of the relevant information content. Combined with automated high‐throughput image acquisition, HCA applied to the screening of
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Quality Control for the Illumina HumanExome BeadChip
Robert P. Igo, Jessica N. Cooke Bailey, Jane Romm, Jonathan L. Haines, Janey L. Wiggs
The Illumina HumanExome BeadChip and other exome‐based genotyping arrays offer inexpensive genotyping of some 240,000 mostly nonsynonymous coding variants across the human genome. The HumanExome chip, with its highly non‐uniform distribution of markers and emphasis on rare coding variants, presents
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Sequence Variant Descriptions: HGVS Nomenclature and Mutalyzer
Johan T. den Dunnen
Consistent and unambiguous description of sequence variants is essential to report and exchange information on the analysis of a genome, in particular in DNA diagnostics. The HGVS nomenclature—recommendations for the description of sequence variants as originally proposed by the Human Genome
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Engineering Large Animal Species to Model Human Diseases
Christopher S. Rogers
Animal models are an important resource for studying human diseases. Genetically engineered mice are the most commonly used species and have made significant contributions to our understanding of basic biology, disease mechanisms, and drug development. However, they often fail to recreate important
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Fabry Disease Biomarkers: Analysis of Urinary Lyso‐Gb3 and Seven Related Analogs Using Tandem Mass Spectrometry
Pamela Lavoie, Michel Boutin, Mona Abaoui, Christiane Auray‐Blais
Fabry disease is an X‐linked lysosomal storage disorder caused by the absence or reduction of the enzyme α‐galactosidase A activity. Currently, globotriaosylsphingosine (lyso‐Gb 3 ) and globotriaosylceramide (Gb 3 ) are used as biomarkers to diagnose and monitor Fabry patients. However, recent
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Tandem Mass Spectrometry Quantitation of Lyso‐Gb3 and Six Related Analogs in Plasma for Fabry Disease Patients
Michel Boutin, Pamela Lavoie, Mona Abaoui, Christiane Auray‐Blais
Fabry disease is an X‐linked lysosomal storage disorder, caused by a deficit in α ‐galactosidase A enzyme activity, leading to the storage of sphingolipids such as globotriaosylsphingosine (lyso‐Gb 3 ), globotriaosylceramide (Gb 3 ), and galabiosylceramide (Ga 2 ) in organs, tissues and biological
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Integrative Analysis of Histone ChIP‐seq and RNA‐seq Data
Hans‐Ulrich Klein, Martin Schäfer
The R package epigenomix has been designed to detect differentially transcribed gene isoforms that, in addition, exhibit altered histone modifications at their respective genomic loci. The package provides methods to map histone ChIP‐seq profiles to isoforms and estimate their transcript abundances
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Efficient Generation of Hypothalamic Neurons from Human Pluripotent Stem Cells
Liheng Wang, Dieter Egli, Rudolph L. Leibel
The hypothalamus comprises neuronal clusters that are essential for body weight regulation and other physiological functions. Insights into the complex cellular physiology of this region of the brain are critical to understanding the pathogenesis of obesity, but human hypothalamic cells are largely
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A Microbiome DNA Enrichment Method for Next‐Generation Sequencing Sample Preparation
Erbay Yigit, George R. Feehery, Bradley W. Langhorst, Fiona J. Stewart, Eileen T. Dimalanta, Sriharsa Pradhan, Barton Slatko, Andrew F. Gardner, James McFarland, Christine Sumner, Theodore B. Davis
“Microbiome” is used to describe the communities of microorganisms and their genes in a particular environment, including communities in association with a eukaryotic host or part of a host. One challenge in microbiome analysis concerns the presence of host DNA in samples. Removal of host DNA before
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Depletion of Ribosomal RNA Sequences from Single‐Cell RNA‐Sequencing Library
Nan Fang, Rumeysa Akinci‐Tolun
Recent advances in single‐cell RNA sequencing technologies have revealed high heterogeneity of gene expression profiles in individual cells. However, most current single‐cell RNA‐seq methods use oligo‐dT priming in the reverse transcription steps and detect only polyA‐positive for more accuracy,
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Detection of Cell Proliferation Markers by Immunofluorescence Staining and Microscopy Imaging in Paraffin‐Embedded Tissue Sections
Seda Eminaga, Polakit Teekakirikul, Christine E. Seidman, Jonathan G. Seidman
This unit describes a step‐by‐step protocol to detect and quantify proliferating cells in paraffin‐embedded tissue sections. Two well‐established markers of proliferation (incorporation of BrdU into newly synthesized DNA and expression of the nuclear protein Ki67) are detected after
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AAV9 Delivery of shRNA to the Mouse Heart
Hiroko Wakimoto, J.G. Seidman, Roger S.Y. Foo, Jianming Jiang
RNA interference (RNAi) is a rapid approach to dissect loss‐of‐function phenotype for a gene of interest. However, it is challenging to perform RNAi in specific organs and tissues in vivo. Engineered viruses can provide a useful tool for delivery of small RNAs in vivo. Recombinant adeno‐associated
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Introduction to Gene Editing and Manipulation Using CRISPR/Cas9 Technology
Martin Newman, Frederick M. Ausubel
Until very recently, the prospect of introducing mutations or exogenous DNA sequences at precise locations in the genomes of plants and animals was difficult, if not impossible. This rapidly changed with the demonstration that the type II CRISPR‐Cas complex, a bacterial anti‐viral surveillance
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Practical Considerations for Using Pooled Lentiviral CRISPR Libraries
Joel R. McDade, Nicole C. Waxmonsky, Lianna E. Swanson, Melina Fan
CRISPR/Cas9 technology is ideally suited for genome‐wide screening applications due to the ease of generating guide RNAs (gRNAs) and the versatility of Cas9 or Cas9 derivatives to knockout, repress, or activate expression of target genes. Several pooled lentiviral CRISPR libraries have been
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CRISPR/Cas9‐Based Multiplex Genome Editing in Monocot and Dicot Plants
Xingliang Ma, Yao‐Guang Liu
The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9‐mediated genome targeting system has been applied to a variety of organisms, including plants. Compared to other genome‐targeting technologies such as zinc‐finger nucleases (ZFNs) and transcription activator–like effector
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CRISPR‐Cas9‐Guided Genome Engineering in C. elegans
Hyun‐Min Kim, Monica P. Colaiácovo
The CRISPR (clustered regularly interspaced short palindromic repeats)–Cas (CRISPR‐associated) system is successfully being used for efficient and targeted genome editing in various organisms, including the nematode C. elegans . Recent studies have developed various CRISPR‐Cas9 approaches to enhance
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The Importance of Titrating Antibodies for Immunocytochemical Methods
Gloria E. Hoffman, Kelley J. Murphy, Luciane V. Sita
When using immunocytochemistry, investigators may not know how to optimize staining or how to troubleshoot the method when staining fails. Lacking are guides for comparing techniques and applying information derived from one staining method to another. Newer methods amplify signal detection, but
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Extraction, Identification, and Quantification of Histones from Small Quantities of Specific Brain Tissue
Hassiba Beldjoud, Fany Messanvi, Nael Nadif Kasri, Benno Roozendaal
Histone posttranslational modifications (PTMs), by their action on the chromatin state, play a central role in the regulation of gene expression. The discovery that some PTMs in the brain are dynamically regulated by experience and environmental factors makes them an important subject for the study
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Daun02 Inactivation of Behaviorally Activated Fos‐Expressing Neuronal Ensembles
Eisuke Koya, Gabriella Margetts‐Smith, Bruce T. Hope
Learned associations about salient experiences (e.g., drug exposure, stress) and their associated environmental stimuli are mediated by a minority of sparsely distributed, behaviorally activated neurons coined ‘neuronal ensembles.’ For many years, it was not known whether these neuronal ensembles
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