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Detection of Extracellular Vesicles Using Proximity Ligation Assay with Flow Cytometry Readout—ExoPLA
Liza Löf, Linda Arngården, Tonge Ebai, Ulf Landegren, Ola Söderberg, Masood Kamali‐Moghaddam
Extracellular vesicles (EVs) are continuously released by most cells, and they carry surface markers of their cells of origin. Found in all body fluids, EVs function as conveyers of cellular information, and evidence implicates them as markers of disease. These characteristics make EVs attractive
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Measurement of Drug‐Stabilized Topoisomerase II Cleavage Complexes by Flow Cytometry
Marcelo de Campos Nebel, Micaela Palmitelli, Marcela González‐Cid
The poisoning of Topoisomerase II (Top2) has been found to be useful as a therapeutic strategy for the treatment of several tumors. The mechanism of Top2 poisons involves a drug‐mediated stabilization of a Top2‐DNA complex, termed Top2 cleavage complex (Top2cc), which maintains a 5′ end of DNA
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CyTOF Mass Cytometry for Click Proliferation Assays
Vinko Tosevski, Egor Ulashchik, Andrea Trovato, Paolo Cappella
Novel cell analyzers, including polychromatic flow cytometers and isotopical cytometry by time of flight (CyTOF) mass cytometers, enable simultaneous measurement of virtually bondless characteristics at the single‐cell level. BrdU assays for quantifying cellular proliferation are common but have
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Detection and Quantification of Mitochondrial Fusion Using Imaging Flow Cytometry
Aldo Nascimento, Joanne Lannigan, David Kashatus
Mitochondria are dynamic organelles that perform several vital cellular functions. Requisite for these functions are mitochondrial fusion and fission. Despite the increasing importance of mitochondrial dynamics in a range of cellular processes, there exist limited methods for robust quantification
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Stochastic Optical Reconstruction Microscopy (STORM)
Jianquan Xu, Hongqiang Ma, Yang Liu
Super‐resolution (SR) fluorescence microscopy, a class of optical microscopy techniques at a spatial resolution below the diffraction limit, has revolutionized the way we study biology, as recognized by the Nobel Prize in Chemistry in 2014. Stochastic optical reconstruction microscopy (STORM), a
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Overview of Admixture Mapping
Daniel Shriner
Admixture mapping is a powerful method of gene mapping for diseases or traits that show differential risk by ancestry. Admixture mapping has been applied most often to Americans who trace ancestry to various combinations of Native Americans, Europeans, and West Africans. Recent developments in
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Microscopy and Image Analysis
George McNamara, Michael Difilippantonio, Thomas Ried, Frederick R. Bieber
This unit provides an overview of light microscopy, including objectives, light sources, filters, film, and color photography for fluorescence microscopy and fluorescence in situ hybridization (FISH). We believe there are excellent opportunities for cytogeneticists, pathologists, and other
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Copy‐Number Variants Detection by Low‐Pass Whole‐Genome Sequencing
Zirui Dong, Weiwei Xie, Haixiao Chen, Jinjin Xu, Huilin Wang, Yun Li, Jun Wang, Fang Chen, Kwong Wai Choy, Hui Jiang
Emerging studies have demonstrated that whole‐genome sequencing (WGS) is an efficient tool for copy‐number variants (CNV) detection, particularly in probe‐poor regions, as compared to chromosomal microarray analysis (CMA). However, the cost of testing is beyond economical for routine usage and the
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Genetic Testing for Hereditary Nonpolyposis Colorectal Cancer (HNPCC)
Babi Ramesh Reddy Nallamilli, Madhuri Hegde
Hereditary nonpolyposis colorectal cancer (HNPCC), also called Lynch syndrome, is an autosomal dominant cancer syndrome that confers an elevated risk of early‐onset colorectal cancer (CRC) and increased lifetime risk for other cancers of the endometrium, stomach, small intestine, hepatobiliary
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1D Genome Sequencing on the Oxford Nanopore MinION
Sara Goodwin, Robert Wappel, W. Richard McCombie
Today's short‐read sequencing instruments can generate read lengths between 50 bp and 700 bp depending on the specific instrument. These high‐throughput sequencing approaches have revolutionized genomic science, allowing hundreds of thousands of full genomes to be sequenced, and have become
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Mammalian Cell Tissue Culture
Katy Phelan, Kristin M. May
Cultured mammalian cells are used extensively in the field of human genetics. It requires a number of special skills in order to be able to preserve the structure, function, behavior, and biology of the cells in culture. This unit describes the basic skills required to maintain and preserve cell
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Mitochondrial Ribosome (Mitoribosome) Profiling for Monitoring Mitochondrial Translation In Vivo
Mary T. Couvillion, L. Stirling Churchman
Translation in the mitochondria is regulated by mechanisms distinct from those acting in the cytosol and in bacteria, yet precise methods for investigating it have lagged behind. This unit describes an approach, mitochondrial ribosome (mitoribosome) profiling, to quantitatively monitor mitochondrial
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NEBNext Direct: A Novel, Rapid, Hybridization‐Based Approach for the Capture and Library Conversion of Genomic Regions of Interest
Amy B. Emerman, Sarah K. Bowman, Andrew Barry, Noa Henig, Kruti M. Patel, Andrew F. Gardner, Cynthia L. Hendrickson
Next‐generation sequencing (NGS) is a powerful tool for genomic studies, translational research, and clinical diagnostics that enables the detection of single nucleotide polymorphisms, insertions and deletions, copy number variations, and other genetic variations. Target enrichment technologies
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Automated Computational Processing of 3‐D MR Images of Mouse Brain for Phenotyping of Living Animals
Christopher S. Medina, Brett Manifold‐Wheeler, Aaron Gonzales, Elaine L. Bearer
Magnetic resonance (MR) imaging provides a method to obtain anatomical information from the brain in vivo that is not typically available by optical imaging because of this organ's opacity. MR is nondestructive and obtains deep tissue contrast with 100‐µm 3 voxel resolution or better.
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CRISPR/Cas9‐Directed Gene Editing for the Generation of Loss‐of‐Function Mutants in High‐Throughput Zebrafish F0 Screens
Sunita S. Shankaran, Timothy J. Dahlem, Brent W. Bisgrove, H. Joseph Yost, Martin Tristani‐Firouzi
The ability to perform reverse genetics in the zebrafish model organism has been greatly advanced with the advent of the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR‐associated) system. The high level of efficiency in generating mutations when using the CRISPR/Cas9
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A Guide to Creating and Testing New INTRSECT Constructs
Lief E. Fenno, Joanna Mattis, Charu Ramakrishnan, Karl Deisseroth
As the power of genetically encoded interventional and observational tools for neuroscience expands, the boundaries of experimental design are increasingly defined by limits in selectively expressing these tools in relevant cell types. Single‐recombinase‐dependent expression systems have been widely
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Multisite Electrophysiology Recordings in Mice to Study Cross‐Regional Communication During Anxiety
Alexander Z. Harris, Danielle Golder, Ekaterina Likhtik
Recording neural activity in awake, freely moving mice is a powerful and flexible technique for dissecting the neural circuit mechanisms underlying pathological behavior. This unit describes protocols for designing a drive and recording single neurons and local field potentials during
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Nicotine Vapor Method to Induce Nicotine Dependence in Rodents
Marsida Kallupi, Olivier George
Nicotine, the main addictive component of tobacco, induces potentiation of brain stimulation reward, increases locomotor activity, and induces conditioned place preference. Nicotine cessation produces a withdrawal syndrome that can be relieved by nicotine replacement therapy. In the last decade, the
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Cannabinoid‐Induced Tetrad in Mice
Mathilde Metna‐Laurent, Miguel Mondésir, Agnès Grel, Monique Vallée, Pier‐Vincenzo Piazza
Cannabinoid‐induced tetrad is a preclinical model commonly used to evaluate if a pharmacological compound is an agonist of the central type‐1 cannabinoid (CB1) receptor in rodents. The tetrad is characterized by hypolocomotion, hypothermia, catalepsy, and analgesia, four phenotypes that are induced
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Animal Model of Chronic Migraine‐Associated Pain
Laura S. Moye, Amynah A. A. Pradhan
Migraine is a debilitating condition that affects hundreds of millions of people worldwide. A subset of these patients experience chronic migraine, resulting in long‐term disability and a severely lowered quality of life. The development of novel migraine therapies has been slow, partially due to
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