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Standardization, Calibration, and Control in Flow Cytometry
Lili Wang, Robert A. Hoffman
Because flow cytometers are designed to measure particle characteristics, particles are the most common materials used to calibrate, control, and standardize the instruments. Definitions and cautions are provided for common terms to alert the reader to critical distinctions in meaning. This unit
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Assessment of Telomere Length, Phenotype, and DNA Content
Theodoros Kelesidis, Ingrid Schmid
Telomere sequences at the end of chromosomes control somatic cell division; therefore, telomere length in a given cell population provides information about its replication potential. This unit describes a method for flow cytometric measurement of telomere length in subpopulations using fluorescence
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Measurement of T‐Cell Telomere Length Using Amplified‐Signal FISH Staining and Flow Cytometry
Andrea L. Henning, Danielle E. Levitt, Jakob L. Vingren, Brian K. McFarlin
Exposure to pathogen‐associated molecular patterns (PAMPS), damage‐associated molecular patterns (DAMPS), and physiologically challenging stimuli either positively or negatively affect leukocyte maturity. Cellular maturity has implications for the effectiveness of host response to bacterial or viral
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Identification of Human Memory‐Like NK Cells
Elena I. Kovalenko, Maria A. Streltsova, Leonid M. Kanevskiy, Sophia A. Erokhina, William G. Telford
Our understanding of NK biology is increased dramatically, a product of improved flow‐cytometric techniques for analyzing these cells. NK cells undergo significant changes in repertoire during differentiation. A repeating stimulus, such as a cytomegalovirus infection, may result in accumulation of
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Quantitative Analysis of Cellular Senescence in Culture and In Vivo
Jing Zhao, Heike Fuhrmann‐Stroissnigg, Aditi U. Gurkar, Rafael R. Flores, Akaitz Dorronsoro, Donna B. Stolz, Claudette M. St. Croix, Laura J. Niedernhofer, Paul D. Robbins
Cellular senescence refers to the irreversible growth arrest of normally dividing cells in response to various types of stress. Cellular senescence is induced by telomere shortening due to repeated cell division, which causes a DNA damage response, as well as genotoxic, oxidative, and inflammatory
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Diagnosis of Spinocerebellar Ataxias Caused by Trinucleotide Repeat Expansions
Joanne E. Martindale
Spinocerebellar ataxias (SCAs) are a group of disorders that are both clinically and genetically heterogeneous. They usually demonstrate onset in adulthood, but some forms may have juvenile or infantile onset. There are many different types of SCA, demonstrating different modes of inheritance and
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Molecular Analysis of Gene Rearrangements and Mutations in Acute Leukemias and Myeloid Neoplasms
Lynette M. Sholl, Janina Longtine, Frank C. Kuo
A subset of acute leukemias and other myeloid neoplasms contains specific genetic alterations, many of which are associated with unique clinical and pathologic features. These alterations include chromosomal rearrangements leading to oncogenic fusion proteins or alteration of gene expression by
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Detecting APC Gene Mutations in Familial Adenomatous Polyposis (FAP)
Babi Ramesh Reddy Nallamilli, Madhuri Hegde
Hereditary forms of colorectal cancer (CRC) account for up to 5% of total cases. Familial adenomatous polyposis (FAP) is an autosomal dominant condition affecting nearly 1 in 5000 people and accounts for only about 1% of all CRCs. It is characterized by the progressive development of hundreds to
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Generating Exome Enriched Sequencing Libraries from Formalin‐Fixed, Paraffin‐Embedded Tissue DNA for Next‐Generation Sequencing
Beth A. Marosy, Brian D. Craig, Kurt N. Hetrick, P. Dane Witmer, Hua Ling, Sean M. Griffith, Benjamin Myers, Elaine A. Ostrander, Janet L. Stanford, Lawrence C. Brody, Kimberly F. Doheny
This unit describes a technique for generating exome‐enriched sequencing libraries using DNA extracted from formalin‐fixed paraffin‐embedded (FFPE) samples. Utilizing commercially available kits, we present a low‐input FFPE workflow starting with 50 ng of DNA. This procedure includes a repair step
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Assay for Transposase‐Accessible Chromatin Using Sequencing (ATAC‐seq) Data Analysis
Kristy L.S. Miskimen, E. Ricky Chan, Jonathan L. Haines
The study of epigenetic properties of the human genome, including structural modifications of DNA and chromatin, has increased tremendously as mounting evidence has demonstrated how much epigenetics affects human gene expression. Buenrostro et al. have developed a rapid method, requiring low
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Culturing and Neuronal Differentiation of Human Dental Pulp Stem Cells
Sarita Goorha, Lawrence T. Reiter
A major issue in studying human neurogenetic disorders, especially rare syndromes affecting the nervous system, is the ability to grow neuronal cultures that accurately represent these disorders for analysis. Although there has been some success in generating induced pluripotent stem (iPS) cells
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Human Induced Pluripotent Stem (hiPS) Cells from Urine Samples: A Non‐Integrative and Feeder‐Free Reprogramming Strategy
Clara Steichen, Karim Si‐Tayeb, Fanny Wulkan, Thayane Crestani, Graça Rosas, Rafael Dariolli, Alexandre C. Pereira, Jose E. Krieger
Human induced pluripotent stem (hiPS) cell technology has already revolutionized some aspects of fundamental and applied research such as study of disease mechanisms and pharmacology screening. The first clinical trial using hiPS cell‐derived cells began in Japan, only 10 years after the publication
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Highly Expandable Human iPS Cell–Derived Neural Progenitor Cells (NPC) and Neurons for Central Nervous System Disease Modeling and High‐Throughput Screening
Chialin Cheng, Daniel M. Fass, Kat Folz‐Donahue, Marcy E. MacDonald, Stephen J. Haggarty
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
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Next‐Generation Sequencing for Identification of EMS‐Induced Mutations in Caenorhabditis elegans
Nicolas J. Lehrbach, Fei Ji, Ruslan Sadreyev
Forward genetic analysis using chemical mutagenesis in model organisms is a powerful tool for investigation of molecular mechanisms in biological systems. In the nematode, Caenorhabditis elegans , mutagenesis screens using ethyl methanesulfonate (EMS) have led to important insights into genetic
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Immunization of Mice
Tomás Maira‐Litrán
The selection of an appropriate immunization strategy depends largely on the properties of an antigen, including its nature, purity, solubility, and availability. This unit describes the critical steps in the production of monoclonal and polyclonal antibodies against soluble molecules (such as
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Preparation of Cell Cultures and Vaccinia Virus Stocks
Catherine A. Cotter, Patricia L. Earl, Linda S. Wyatt, Bernard Moss
The culturing of cell lines used with vaccinia virus, both as monolayer and in suspension, is described. The preparation of chick embryo fibroblasts (CEF) is presented for use in the production of the highly attenuated and host range‐restricted modified vaccinia virus Ankara (MVA) strain of vaccinia
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Generation of Recombinant Vaccinia Viruses
Linda S. Wyatt, Patricia L. Earl, Bernard Moss
This unit describes how to infect cells with vaccinia virus and then transfect them with a plasmid‐transfer vector or PCR fragment to generate a recombinant virus. Selection and screening methods used to isolate recombinant viruses and a method for the amplification of recombinant viruses are
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A Simple Protoplast‐Based Method for Screening Potent Artificial miRNA for Maximal Gene Silencing in Arabidopsis
Nannan Zhang, Dandan Zhang, Jian‐Feng Li
The unpredictability of in planta silencing efficiency of artificial micro RNAs (amiRNAs) has greatly limited the application of the amiRNA technology in basic and applied plant research. This unit describes a simple and robust method called the epitope‐tagged protein‐based amiRNA (ETPamir) screen
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Scarless Cas9 Assisted Recombineering (no‐SCAR) in Escherichia coli, an Easy‐to‐Use System for Genome Editing
Christopher R. Reisch, Kristala L.J. Prather
The discovery and development of genome editing systems that leverage the site‐specific DNA endonuclease system CRISPR/Cas9 has fundamentally changed the ease and speed of genome editing in many organisms. In eukaryotes, the CRISPR/Cas9 system utilizes a “guide” RNA to enable the Cas9 nuclease to
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Mammalian Cell Tissue Culture Techniques
Katy Phelan, Kristin M. May
Cultured mammalian cells are used extensively in molecular biology studies. A number of special skills are required in order to preserve the structure, function, behavior, and biology of cells in culture. This appendix describes the basic skills required to maintain and preserve cell cultures:
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Intracerebral Injections and Ultrastructural Analysis of High‐Pressure Frozen Brain Tissue
Marie‐Theres Weil, Torben Ruhwedel, Wiebke Möbius, Mikael Simons
Intracerebral injections are an invasive method to bypass the blood brain barrier and are widely used to study molecular and cellular mechanisms of the central nervous system. The administered substances are injected directly at the site of interest, executing their effect locally. By combining
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Electroporation in the Rodent Embryonic Brain Using Whole Embryo Culture System
Takako Kikkawa, Masanori Takahashi, Noriko Osumi
This unit describes basic methods for mammalian whole embryo culture (WEC) using embryonic day 10.5 mouse embryos, including the preparation of high‐quality immediately centrifuged (IC) rat serum that is commonly used for WEC and is essential for normal growth and development of cultured mouse and
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Visualizing Changes in Neuronal Dendritic Morphology in Response to Stress and Pharmacological Challenge
Cara L. Wellman
This unit outlines a protocol for Golgi staining, which has been used extensively to reliably and quantitatively assess alterations in dendritic arborization and spine density as a result of a variety of factors, including chronic administration of glucocorticoids, chronic stress, and
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The Five‐Choice Continuous Performance Task (5C‐CPT): A Cross‐Species Relevant Paradigm for Assessment of Vigilance and Response Inhibition in Rodents
Zackary A. Cope, Jared W. Young
Deficits in the domains of attention and response inhibition are central to many psychiatric disorders. As such, animal models of disorders purporting to replicate these behavioral deficits first require tests that can accurately assess the behaviors with high fidelity. The gold‐standard clinical
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Measuring Cognitive Judgement Bias in Rats Using the Ambiguous‐Cue Interpretation Test
Justyna Papciak, Rafal Rygula
An active‐choice, operant, ambiguous‐cue interpretation (ACI) paradigm is described that can be used for measuring cognitive judgement bias in rats. In this behavioral test, animals in an operant conditioning chamber are trained to press a lever to receive a food reward when a specific tone is
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