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Measurement of Low‐Abundance Intracellular mRNA Using Amplified FISH Staining and Image‐Based Flow Cytometry
Andrea L. Henning, Jill N. Best Sampson, Brian Keith McFarlin
Recent advances in instrument design and reagent development have enabled the rapid progression in available measurement techniques in the field of flow cytometry. In particular, image‐based flow cytometry extends the analysis capacity found in traditional flow cytometry. Until recently, it was not
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Large Particle Sorting to Isolate Live Parasitic Nematode Eggs
Alfonso Schmidt, Tiffany Bouchery, Graham Le Gros, Kylie M. Price
Traditional jet‐in‐air cell sorters have been designed and optimized to isolate small particles such as mammalian lymphocytes with an average diameter of 10 μm. We discuss the practical considerations of setting up a conventional jet‐in‐air cell sorter, using a 200‐μm nozzle, to isolate the
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Overview of Clinical Cytogenetics
Patrick R. Gonzales, Andrew J. Carroll, Bruce R. Korf
Chromosome analysis is one of the first approaches to genetic testing and remains a key component of genetic analysis of constitutional and somatic genetic disorders. Numerical or unbalanced structural chromosome abnormalities usually lead to multiple congenital anomalies. Sometimes these are
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Using ClinVar as a Resource to Support Variant Interpretation
Steven M. Harrison, Erin R. Riggs, Donna R. Maglott, Jennifer M. Lee, Danielle R. Azzariti, Annie Niehaus, Erin M. Ramos, Christa L. Martin, Melissa J. Landrum, Heidi L. Rehm
ClinVar is a freely accessible, public archive of reports of the relationships among genomic variants and phenotypes. To facilitate evaluation of the clinical significance of each variant, ClinVar aggregates submissions of the same variant, displays supporting data from each submission, and
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An Overview of Biochemical Genetics
J. Daniel Sharer
Biochemical genetics focuses on the pathophysiology, diagnosis, and treatment of inherited metabolic disorders. While individually rare, the combined incidence of these diseases makes them a significant source of morbidity and mortality, particularly among infants and young children, and new
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Quantitative Analysis of Total Plasma Homocysteine by LC‐MS/MS
Libin Yuan, J. Daniel Sharer
Homocysteine is a nonessential, sulfur‐containing amino acid involved in one‐carbon (folate) metabolism. A number of inherited and acquired conditions cause increased accumulation of this metabolite in blood (homocysteinemia) and other biofluids. Homocysteinemia is a risk factor for cardiovascular
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Reporting of Diagnostic Cytogenetic Results
Anne B.S. Giersch, Frederick R. Bieber, Adrian M. Dubuc, Jonathan A. Fletcher, Azra H. Ligon, Heather Mason‐Suares, Cynthia C. Morton, Stanislawa Weremowicz, Sheng Xiao, Paola Dal Cin
This appendix, developed by the staff at the Center for Advanced Molecular Diagnostics in the Department of Pathology at the Brigham and Women's Hospital, includes a comprehensive list of current “macros” or standardized statements used to facilitate reporting of cytogenetic results. These are
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Measuring Bioenergetics in T Cells Using a Seahorse Extracellular Flux Analyzer
Gerritje J.W. van der Windt, Chih‐Hao Chang, Erika L. Pearce
This unit contains several protocols to determine the energy utilization of T cells in real‐time using a Seahorse Extracellular Flux Analyzer (http://www.seahorsebio.com). The advantages to using this machine over traditional metabolic assays include the simultaneous measurement of glycolysis and
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Differentiation and Characterization of Tr1 Cells
Norio Chihara, Asaf Madi, Katarzyna Karwacz, Amit Awasthi, Vijay K. Kuchroo
Regulatory T cell–mediated suppression serves as a pivotal mechanism of negative regulation of immune‐mediated inflammation. Type 1 regulatory T cells (Tr1 cells) are an important subset of CD4+ T cells that prevent excessive inflammatory responses and maintain immune tolerance. The
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In Vivo Depletion of T Lymphocytes
Karen Laky, Ada M. Kruisbeek
In vivo depletion of T lymphocytes is a means of studying the role of specific T cell populations during defined phases of in vivo immune responses. In this unit, a protocol is provided for injecting monoclonal antibodies (mAbs) into wild‐type adult mice. Depletion of the appropriate subset of cells
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Contact Hypersensitivity
Anthony A. Gaspari, Stephen I. Katz, Stefan F. Martin
Contact hypersensitivity (CHS) is a simple in vivo assay of cell‐mediated immune function in which exposure of epidermal and dermal cells to exogenous haptens results in a delayed‐type hypersensitivity (DTH) reaction that can be measured and quantified. Epidermal Langerhans cells and dermal
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Mouse Models for Immune‐Mediated Platelet Destruction or Immune Thrombocytopenia (ITP)
Anton Neschadim, Donald R. Branch
Immune thrombocytopenia (ITP) is a debilitating, life‐threatening autoimmune disorder affecting more than 4 in every 100,000 adults annually, stemming from the production of antiplatelet antibody resulting in accelerated platelet destruction and thrombocytopenia. Numerous animal models of ITP have
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Construction of a Sequencing Library from Circulating Cell‐Free DNA
Nan Fang, Dirk Löffert, Rumeysa Akinci‐Tolun, Katja Heitz, Alexander Wolf
Circulating DNA is cell‐free DNA (cfDNA) in serum or plasma that can be used for non‐invasive prenatal testing, as well as cancer diagnosis, prognosis, and stratification. High‐throughput sequence analysis of the cfDNA with next‐generation sequencing technologies has proven to be a highly sensitive
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Immunoblotting and Immunodetection
Duojiao Ni, Peng Xu, Sean Gallagher
Immunoblotting (western blotting) is used to identify specific antigens recognized by polyclonal or monoclonal antibodies. This unit provides protocols for all steps, starting with solubilization of the protein samples, usually by means of SDS and reducing agents. Following solubilization, the
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Analysis of Individual Signaling Complexes by mMAPS, a Flow‐Proteometric System
Chao‐Kai Chou, Pei‐Hsiang Tsou, Jennifer L. Hsu, Heng‐Huan Lee, Ying‐Nai Wang, Jun Kameoka, Mien‐Chie Hung
Signal transduction is essential for maintaining normal cell physiological functions, and deregulation of signaling can lead to diseases such as diabetes and cancers. Some of the major players in signal delivery are molecular complexes composed of proteins and nucleic acids. This unit describes a
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Molecular Dissection of Chromatin Maturation via Click Chemistry
Ozlem Yildirim, Robert E. Kingston
DNA synthesis and chromatin assembly are the two most critical processes of eukaryotic cell division. It is well known that their coordination is tightly regulated. Although the interplay between DNA and its higher‐order chromatin state is integral for many processes, including cell survival and
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Metabolomics by Gas Chromatography–Mass Spectrometry: Combined Targeted and Untargeted Profiling
Oliver Fiehn
Gas chromatography–mass spectrometry (GC‐MS)–based metabolomics is ideal for identifying and quantitating small‐molecule metabolites (<650 Da), including small acids, alcohols, hydroxyl acids, amino acids, sugars, fatty acids, sterols, catecholamines, drugs, and toxins, often using chemical
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Hybridization Histochemistry of Neural Transcripts
W. Scott Young, June Song, Éva Mezey
Expression of genes is manifested by the production of RNA transcripts within cells. Hybridization histochemistry (or in situ hybridization) permits localization of these transcripts with cellular resolution or better. Furthermore, the relative amounts of transcripts detected in different tissues or
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Electrophysiological Measurement of Cannabinoid‐Mediated Synaptic Modulation in Acute Mouse Brain Slices
Rita Báldi, Dipanwita Ghosh, Brad A. Grueter, Sachin Patel
Endocannabinoids (eCBs) are a class of bioactive lipids that mediate retrograde synaptic modulation at central and peripheral synapses. The highly lipophilic nature of eCBs and the pharmacological tools available to interrogate this system require unique methodological consideration, especially when
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Studying Socio‐Affective Communication in Rats through Playback of Ultrasonic Vocalizations
Markus Wöhr, Dominik Seffer, Rainer K.W. Schwarting
Rats are able to produce ultrasonic vocalizations (USVs). Such USVs are an important component of the rat social behavior repertoire and serve distinct communicative functions as socio‐affective signals. Depending on the emotional valence of the situation, juvenile and adult rats utter (1) aversive
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The Revised Neurobehavioral Severity Scale (NSS‐R) for Rodents
Angela M. Yarnell, Erin S. Barry, Andrea Mountney, Deborah Shear, Frank Tortella, Neil E. Grunberg
Motor and sensory deficits are common following traumatic brain injury (TBI). Although rodent models provide valuable insight into the biological and functional outcomes of TBI, the success of translational research is critically dependent upon proper selection of sensitive, reliable, and
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The Use of Detergents to Purify Membrane Proteins
Marcella Orwick‐Rydmark, Thomas Arnold, Dirk Linke
Extraction of membrane proteins from biological membranes is usually accomplished with the help of detergents. This unit describes the use of detergents to solubilize and purify membrane proteins. The chemical and physical properties of the different classes of detergents typically used with
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Obtaining Soluble Folded Proteins from Inclusion Bodies Using Sarkosyl, Triton X‐100, and CHAPS: Application to LB and M9 Minimal Media
Michael A. Massiah, Katharine M. Wright, Haijuan Du
This unit describes a straightforward and efficient method of using sarkosyl to solubilize and recover difficult recombinant proteins, such as GST‐ and His 6 ‐tagged fusion proteins, that are overexpressed in E. coli . This protocol is especially useful for rescuing recombinant proteins
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In Situ Proteolysis for Crystallization of Membrane Bound Cytochrome P450 17A1 and 17A2 Proteins from Zebrafish
Li Lei, Martin Egli
Fish and human cytochrome P450 (P450) 17A1 catalyze both steroid 17α‐hydroxylation and 17α,20‐lyase reactions. Fish P450 17A2 catalyzes only 17α‐hydroxylation. Both enzymes are microsomal‐type P450s, integral membrane proteins that bind to the membrane through their N‐terminal hydrophobic segment,
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Use of Protein Folding Reagents
Paul T. Wingfield
The reagents and methods for purification and use of the most commonly used denaturants, guanidine hydrochloride (guanidine‐HCl) and urea, are described. Other protein denaturants and reagents used to fold proteins are briefly mentioned. Sulfhydryl reagents (reducing agents) and “oxido‐shuffling”
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Protein Precipitation Using Ammonium Sulfate
Paul T. Wingfield
The basic theory of protein precipitation by addition of ammonium sulfate is presented, and the most common applications are listed. Tables are provided for calculating the appropriate amount of ammonium sulfate to add to a particular protein solution. © 2016 by John Wiley & Sons, Inc. Keywords:
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