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Culture of Escherichia coli and Related Bacteria
Sean P. Riley, Michael E. Woodman, Jonathan Holt, Brian Stevenson
This appendix provides general techniques, equipment, and media used for the growth of many commonly encountered bacteria. For specific growth conditions, readers should refer to units regarding the laboratory maintenance of the organism of interest. © 2017 by John Wiley & Sons, Inc. Keywords:
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In Situ Hybridization: Fruit Fly Embryos and Tissues
Ronit Wilk, Jack Hu, Henry M. Krause
It is well known that transcript localization controls important biological processes, including cell fate determination, cell polarity, cell migration, morphogenesis, neuronal function, and embryonic axis specification. Thus, the sub‐cellular visualization of transcripts in ‘their original place’ (
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The Advantages of Using Fluorescent Proteins for In Vivo Imaging
Robert M. Hoffman
Fluorescent proteins have revolutionized biology by enabling what was formerly invisible to be seen clearly, and potentially enable imaging of all cells in living animals. There are numerous applications for in vivo imaging with fluorescent proteins, including use in cancer, immunology, infection,
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Dictyostelium discoideum: A Model System for Cell and Developmental Biology
Sabateeshan Mathavarajah, Ana Flores, Robert J. Huber
The social amoeba Dictyostelium discoideum has long served as a model system for studying fundamental processes in cell and developmental biology. This eukaryotic microbe is also recognized as a model organism for biomedical and human disease research since the genome encodes homologs of genes
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Risk Assessment in the Research Laboratory
LouAnn C. Burnett
Risk assessment is a term that is applied to a process that we all use every day to make decisions. Conducting risk assessments while working in the laboratory should be integrated into the research culture. Reagents and equipment used in research laboratories can be hazardous if used improperly or
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Preparing and Presenting a Talk
Michael More, Christine Sjolander, Stephen Chang
How information is presented can have as much impact as the information itself. In order to ensure that you're getting your message across to the audience, you need to prepare your talk with as much care as you designed your research. This unit outlines information on how to prepare a great talk and
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Analysis of Cellular DNA Content by Flow Cytometry
Zbigniew Darzynkiewicz, Xuan Huang, Hong Zhao
Cellular DNA content can be measured by flow cytometry with the aim of : (1) revealing cell distribution within the major phases of the cell cycle, (2) estimating frequency of apoptotic cells with fractional DNA content, and/or (3) disclosing DNA ploidy of the measured cell population. In this
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High‐Dimensional Fluorescence Cytometry
Thomas Myles Ashhurst, Adrian Lloyd Smith, Nicholas Jonathan Cole King
The immune system consists of a complex network of cells, all expressing a wide range of surface and/or intracellular proteins. Using flow cytometry, these cells can be analyzed by labeling with fluorophore‐conjugated antibodies. The recent expansion of fluorescence flow cytometry technology, in
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Isolation and Functional Use of Human NKT Cells
Mark A. Exley, S. Brian Wilson, Steven P. Balk
This unit details methods for the isolation, in vitro expansion, and functional characterization of human iNKT cells. The term ‘iNKT’ derives from the fact that a large fraction of murine and some human NK marker+ T cells (‘NKT’) recognize the MHC class I–like CD1d protein and use an identical
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Mouse Eosinophils: Identification, Isolation, and Functional Analysis
Hadar Reichman, Perri Rozenberg, Ariel Munitz
Eosinophils are bone marrow–derived cells that differentiate in the bone marrow and migrate into the peripheral blood primarily under the regulation of interleukin (IL)–5. Eosinophil levels in the blood are relatively low. However, under steady‐state conditions and in settings of allergic
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The Mouse Model of Infection with Citrobacter rodentium
Nicolas Bouladoux, Oliver J. Harrison, Yasmine Belkaid
Citrobacter rodentium is a murine mucosal pathogen used as a model to elucidate the molecular and cellular pathogenesis of infection with two clinically important human gastrointestinal pathogens, enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC). C. rodentium infection
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Computational Methods for Human Microbiome Analysis
Matthieu J. Miossec, Sandro L. Valenzuela, Katterinne N. Mendez, Eduardo Castro‐Nallar
As the field of microbiomics advances, the burden of computational work that scientists need to perform in order to extract biological insight has grown accordingly. Likewise, while human microbiome analyses are increasingly shifting toward a greater integration of various high‐throughput data
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Azospirillum brasilense, a Beneficial Soil Bacterium: Isolation and Cultivation
Gladys Alexandre
Bacteria of the genus Azospirillum comprise 15 species to date, with A. brasilense the best studied species in the genus. Azospirillum are soil bacteria able to promote the growth of plants from 113 species spanning 35 botanical families. These non‐pathogenic and beneficial bacteria are ubiquitous
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Azospirillum brasilense: Laboratory Maintenance and Genetic Manipulation
Jessica Gullett, Lindsey O'Neal, Tanmoy Mukherjee, Gladys Alexandre
Bacteria of the genus Azospirillum , including the most comprehensively studied Azospirillum brasilense , are non‐pathogenic soil bacteria that promote the growth of diverse plants, making them an attractive model to understand non‐symbiotic, beneficial plant‐bacteria associations. Research into the
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Measuring pH of the Coxiella burnetii Parasitophorous Vacuole
Dhritiman Samanta, Stacey D. Gilk
Coxiella burnetii is the causative agent of human Q fever, a zoonotic disease that can cause a debilitating, flu‐like illness in acute cases, or a life‐threatening endocarditis in chronic patients. An obligate intracellular bacterial pathogen, Coxiella survives and multiplies in a large
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Leptospira: Molecular Detection of Pathogenic Species in Natural Sources
Brittney Beigel, Ashutosh Verma
This protocol describes a method for the rapid detection of leptospiral DNA in environmental water. In summary, the DNA is extracted from water samples and tested in a TaqMan‐based real‐time quantitative polymerase chain reaction (qPCR) for the presence of lipl32 , a gene that is present only in
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ELISA for Molluscum Contagiosum Virus
Subuhi Sherwani, Mohammed Chowdhury, Joachim J. Bugert
Molluscum contagiosum virus (MCV) is a common skin pathogen of children and young adults. Infection with MCV causes benign skin tumors in children and young adults and is mostly self‐limiting. In contrast to orthopoxviruses, MCV infections tend to take a subacute clinical course but may persist for
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Screening Fusion Tags for Improved Recombinant Protein Expression in E. coli with the Expresso® Solubility and Expression Screening System
Eric J. Steinmetz, Michele E. Auldridge
The simplicity, speed, and low cost of bacterial culture make E. coli the system of choice for most initial trials of recombinant protein expression. However, many heterologous proteins are either poorly expressed in bacteria, or are produced as incorrectly folded, insoluble aggregates that lack the
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Expression and Purification of Protein Complexes Suitable for Structural Studies Using Mammalian HEK 293F Cells
Irene Nigi, Louise Fairall, John W.R. Schwabe
Prokaryotic expression systems have been widely used to express proteins for structural studies. Such expression systems have the advantage of being economical, straightforward and fast. However, for many eukaryotic proteins and particularly protein complexes, bacterial expression systems do not
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Simple and Efficient Purification of Recombinant Proteins Using the Heparin‐Binding Affinity Tag
Srinivas Jayanthi, Ravi Kumar Gundampati, Thallapuranam Krishnaswamy Suresh Kumar
Heparin, a member of the glycosaminoglycan family, is known to interact with more than 400 different types of proteins. For the past few decades, significant progress has been made to understand the molecular details involved in heparin‐protein interactions. Based on the structural knowledge
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Analysis of Disulfide Bond Formation
Ineke Braakman, Lydia Lamriben, Guus van Zadelhoff, Daniel N. Hebert
In this unit, protocols are provided for detection of disulfide bond formation in cultures of intact cells and in an in vitro translation system containing isolated microsomes or semi‐permeabilized cells. First, the newly synthesized protein of interest is biosynthetically labeled with radioactive
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Screening and Identifying Membrane Proteins Favorable for Crystallization
Jared Kim, Ho Leung Ng
This unit addresses several critical challenges associated with membrane protein crystallography by screening membrane proteins from Escherichia coli , Saccharomyces cerevisiae , and Sus scrofa cerebral tissue for biochemical properties favorable for crystallization. First, a tissue sample or cell
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Visualization of Protein Interactions in Living Cells Using Bimolecular Luminescence Complementation (BiLC)
Lisette G.G.C. Verhoef, Mark Wade
The number of intracellular protein‐protein interactions (PPIs) far exceeds the total number of proteins encoded by the genome. Dynamic cellular PPI networks respond to external stimuli and endogenous metabolism in order to maintain homeostasis. Many PPIs are directly involved in disease
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Single‐Cell Functional Analysis of Stem‐Cell Derived Cardiomyocytes on Micropatterned Flexible Substrates
Jan David Kijlstra, Dongjian Hu, Peter van der Meer, Ibrahim J. Domian
Human pluripotent stem–cell derived cardiomyocytes (hPSC‐CMs) hold great promise for applications in human disease modeling, drug discovery, cardiotoxicity screening, and, ultimately, regenerative medicine. The ability to study multiple parameters of hPSC‐CM function, such as contractile and
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Rapid Screening of the Endodermal Differentiation Potential of Human Pluripotent Stem Cells
Richard Siller, Gareth J. Sullivan
Human pluripotent stem cells (hPSCs) hold tremendous promise for regenerative medicine, disease modeling, toxicology screening, and developmental biology. These applications are hindered due to inherent differences in differentiation potential observed among different hPSC lines. This is
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Mesenchymal Stem Cell Preparation and Transfection‐free Ferumoxytol Labeling for MRI Cell Tracking
Li Liu, Chien Ho
Mesenchymal stem cells (MSCs) are multipotent cells and are the most widely studied cell type for stem cell therapies. In vivo cell tracking of MSCs labeled with an FDA‐approved superparamagnetic iron‐oxide (SPIO) particle by magnetic resonance imaging (MRI) provides essential information, e.g., MSC
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Visualization and Modeling of the In Vivo Distribution of Mesenchymal Stem Cells
Haolu Wang, Camilla A. Thorling, Zhi Ping Xu, Darrell H. G. Crawford, Xiaowen Liang, Xin Liu, Michael S. Roberts
This unit describes a protocol for elucidating the in vivo disposition of administered mesenchymal stem cells (MSCs). Specifically, direct visualization of donor cell spatiotemporal distribution and assessment of donor cell quantity in recipient organs are described. Protocols for data analysis are
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Human Adipose‐Derived Stromal Cell Isolation Methods and Use in Osteogenic and Adipogenic In Vivo Applications
Elizabeth Brett, Ruth Tevlin, Adrian McArdle, Eun Young Seo, Charles K.F. Chan, Derrick C. Wan, Michael T. Longaker
Adipose tissue represents an abundant and easily accessible source of multipotent cells, which may serve as excellent building blocks for tissue engineering. This article presents a newly described protocol for isolating adipose‐derived stromal cells (ASCs) from human lipoaspirate, compared to the
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CRISPR/Cas9‐Based Safe‐Harbor Gene Editing in Rhesus iPSCs
Ravi Chandra Yada, John W. Ostrominski, Ilker Tunc, So Gun Hong, Jizhong Zou, Cynthia E. Dunbar
NHP iPSCs provide a unique opportunity to test safety and efficacy of iPSC‐derived therapies in clinically relevant NHP models. To monitor these cells in vivo, there is a need for safe and efficient labeling methods. Gene insertion into genomic safe harbors (GSHs) supports reliable transgene
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Metallic Nano‐Composite Toxicity Evaluation by Zebrafish Embryo Toxicity Test with Identification of Specific Exposure Biomarkers
Roberta Pecoraro, Antonio Salvaggio, Fabio Marino, Gianfranco Di Caro, Fabiano Capparucci, Bianca Maria Lombardo, Giuseppina Messina, Elena Maria Scalisi, Maurizio Tummino, Francesco Loreto, Giusi D'Amante, Roberto Avola, Daniele Tibullo, Maria Violetta Brundo
Nanomaterials (NM) have different shapes and can be composed of different materials such as carbon, silicon, and some metals like gold, silver, and titanium. They are used as fillers, catalysts, semiconductors, cosmetics, drug carriers in medicine, energy storage systems, and antifriction coatings.
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Gas Chromatography Mass Spectrometry (GC‐MS) for Identification of Designer Stimulants Including 2C Amines, NBOMe Compounds, and Cathinones in Urine
Hemamalini Ketha, Milad Webb, Larry Clayton, Sean Li
Phenethylamine derivatives are being increasingly exploited for recreational use as “designer” stimulants designed to mimic psychostimulant properties of amphetamine or other illicit substances like 3,4‐methylenedioxymethamphetamine (MDMA [ecstasy]). Clandestine operations meticulously design
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Measuring β‐Galactosidase Activity in Gram‐Positive Bacteria Using a Whole‐Cell Assay with MUG as a Fluorescent Reporter
Norman H. L. Chiu, Amanda L. Watson
The use of β‐galactosidase enzyme as a biomarker has the potential to determine activity levels of the microbiome of a variety of organisms due to its common presence in both eukaryotes and prokaryotes. Completing the assay in a whole‐cell format facilitates the monitoring of β‐galactosidase
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Overview of Neurotoxicology
Lucio G. Costa
The nervous system has a central and primary function in the body, and its relevance and complexity make it a target for a large number of toxic substances. The most common forms of neurotoxicity are the death of neurons (neuronopathy), the degeneration of axons (axonopathy), damage to glial cells
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Co‐Culture of Neurons and Microglia
Pamela J. Roqué, Lucio G. Costa
Microglia, the resident immune cells of the brain, have been implicated in numerous neurodegenerative and neurodevelopmental diseases. Activation of microglia by a variety of stimuli induces the release of factors, including pro‐ and anti‐inflammatory cytokines and reactive oxygen species, that
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Inter‐and Intra‐Laboratory Standardization of TUNEL Assay for Assessment of Sperm DNA Fragmentation
Sajal Gupta, Rakesh Sharma, Ashok Agarwal
The functional aspects of sperm activity such as sperm chromatin integrity and ability to fertilize cannot be characterized by routine semen parameters. Men with unexplained infertility and idiopathic infertility, as well as men with normozoospermic semen profiles, show high DNA fragmentation.
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Isolation, Cryopreservation, and Immunophenotyping of Human Peripheral Blood Mononuclear Cells
Fredine T. Lauer, Jesse L. Denson, Scott W. Burchiel
This unit describes procedures for the isolation, cryopreservation, and thawing of human peripheral blood mononuclear cells (HPBMC) and analysis of cell surface markers (CSM) for immunophenotyping using polychromatic flow cytometry. This methodology can be used to ensure that cell integrity and
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