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Enzyme‐Linked Immunosorbent Assays
Peter V. Hornbeck
This unit describes six different ELISA systems for the detection of specific antibodies, soluble antigens, or cell‐surface antigens. In all six systems, soluble reactants are removed from solution after specifically binding to solid‐phase reactants. In the first four protocols, solid‐phase
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Isolation of Mouse Neutrophils
Muthulekha Swamydas, Yi Luo, Martin E. Dorf, Michail S. Lionakis
Neutrophils represent the first line of defense against bacterial and fungal pathogens. Indeed, patients with inherited and acquired qualitative and quantitative neutrophil defects are at high risk for developing bacterial and fungal infections and suffering adverse outcomes from these infections.
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Detection of Intracellular Cytokines by Flow Cytometry
Yuzhi Yin, Alyssa Mitson‐Salazar, Calman Prussin
Intracellular cytokine staining (ICCS), employing fluorescently labeled MAbs detected by flow cytometry, has emerged as the premier technique for studying cytokine expression at the single‐cell level. Advances in polychromatic flow cytometry have dramatically enhanced the sophistication of ICCS
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Models to Study NK Cell Biology and Possible Clinical Application
Anthony E. Zamora, Steven K. Grossenbacher, Ethan G. Aguilar, William J. Murphy
Natural killer (NK) cells are large granular lymphocytes of the innate immune system, responsible for direct targeting and killing of both virally infected and transformed cells. NK cells rapidly recognize and respond to abnormal cells in the absence of prior sensitization due to their wide array of
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Infant Rabbit Model for Diarrheal Diseases
Sören Abel, Matthew K. Waldor
Vibrio cholerae is the agent of cholera, a potentially lethal diarrheal disease that remains a significant threat to populations in developing nations. The infant rabbit model of cholera is the only non‐surgical small animal model system that closely mimics human cholera. Following orogastric
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Production of Furin‐Cleaved Papillomavirus Pseudovirions and Their Use for In Vitro Neutralization Assays of L1‐ or L2‐Specific Antibodies
Joshua W. Wang, Ken Matsui, Yuanji Pan, Kihyuck Kwak, Shiwen Peng, Troy Kemp, Ligia Pinto, Richard B.S. Roden
Immunization with Human Papillomavirus (HPV) L1 virus‐like particles or L2 capsid protein elicits neutralizing antibodies that mediate protection. A high‐throughput and sensitive in vitro neutralization assay is therefore valuable for prophylactic HPV vaccine studies. Over several hours during
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Mouse Polyomavirus: Propagation, Purification, Quantification, and Storage
Lenka Horníková, Vojtěch Žíla, Hana Španielová, Jitka Forstová
Mouse polyomavirus (MPyV) is a member of the Polyomaviridae family, which comprises non‐enveloped tumorigenic viruses infecting various vertebrates including humans and causing different pathogenic responses in the infected organisms. Despite the variations in host tropism and pathogenicity, the
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In Vitro Replication Assay for Merkel Cell Polyomavirus (MCPyV)
Friederike Neumann, Manja Czech‐Sioli, Adam Grundhoff, Nicole Fischer
Merkel cell polyomavirus (MCPyV) genomes are clonally integrated in tumor cells of ∼95% of all Merkel cell carcinoma (MCC) cases. The virus is highly prevalent; however, where the virus persists and which cell types are permissive for MCPyV replication is still unknown. As a consequence, very little
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Detection and Isolation of Epichloë Species, Fungal Endophytes of Grasses
Simona Florea, Christopher L. Schardl, Walter Hollin
Epichloë species (including former Neotyphodium species) are endophytic fungi that significantly affect fitness of cool‐season grass hosts, potentially by increasing nutrient uptake and resistance to drought, parasitism and herbivory. Epichloë species are obligately biotrophic, living in the
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Overview of Probing Protein‐Ligand Interactions Using NMR
Clémentine Aguirre, Olivier Cala, Isabelle Krimm
Nuclear magnetic resonance (NMR) is a powerful technique for the study and characterization of protein‐ligand interactions. In this unit we review both experiments where the NMR spectrum of the protein is observed (protein‐observed NMR experiments) and those where the NMR spectra of the ligand is
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Using Single Lectins to Enrich Glycoproteins in Conditioned Media
Manveen K. Sethi, Susan Fanayan
Lectins are sugar‐binding proteins that can recognize and bind to carbohydrates conjugated to proteins and lipids. Coupled with mass spectrometry technologies, lectin affinity chromatography is becoming a popular approach for identification and quantification of glycoproteins in complex samples such
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Detergent Analysis in Protein Samples Using Mid‐Infrared (MIR) Spectroscopy
Chandreyee Das, Timothy Nadler, Ivona Strug
Quantitating relative levels of detergent present in protein preparations or samples derived from biological material, such as tissue or body fluids, is important because the presence of detergent may affect downstream analyses as well as protein structure/function. Especially because sample
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Applications of Lipid Nanodiscs for the Study of Membrane Proteins by Surface Plasmon Resonance
Meg Trahey, Mavis Jiarong Li, Hyewon Kwon, Erica L. Woodahl, Wynton D. McClary, William M. Atkins
Methods for the initial steps of surface plasmon resonance analysis of membrane proteins incorporated in lipid nanodiscs are described. Several types of Biacore sensor chips are available and require distinct strategies to immobilize proteonanodiscs on the chip surface. The procedures for
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Cell‐Free Expression of G Protein–Coupled Receptors
Kenneth Segers, Stefan Masure
The large‐scale production of recombinant G protein–coupled receptors (GPCRs) is one of the major bottlenecks that hamper functional and structural studies of this important class of integral membrane proteins. Heterologous overexpression of GPCRs often results in low yields of active protein,
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Isolation of Undifferentiated Female Germline Cells from Adult Drosophila Ovaries
Robyn Su May Lim, Motomi Osato, Toshie Kai
This unit describes a method for isolating undifferentiated, stem cell–like germline cells from adult Drosophila ovaries. Here, we demonstrate that this population of cells can be effectively purified from hand‐dissected ovaries in considerably large quantities. Tumor ovaries with expanded
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In Situ Hybridization to Identify Gut Stem Cells
Alex Gregorieff, Hans Clevers
In recent years, considerable effort has been directed toward identifying the repertoire of genes specifically expressed in adult stem cells. In this unit, we describe an in situ hybridization protocol adapted for the analysis of gene expression in the intestinal mucosa. This methodology allows
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Regional Cell Specific RNA Expression Profiling of FACS Isolated Drosophila Intestinal Cell Populations
Devanjali Dutta, Nicolas Buchon, Jinyi Xiang, Bruce A. Edgar
The adult Drosophila midgut is built of five distinct cell types, including stem cells, enteroblasts, enterocytes, enteroendocrine cells, and visceral muscles, and is divided into five major regions (R1 to R5), which are morphologically and functionally distinct from each other. This unit describes
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Culture and Isolation of Brain Tumor Initiating Cells
Parvez Vora, Chitra Venugopal, Nicole McFarlane, Sheila K. Singh
Brain tumors are typically composed of heterogenous cells that exhibit distinct phenotypic characteristics and proliferative potentials. Only a relatively small fraction of cells in the tumor with stem cell properties, termed brain tumor initiating cells (BTICs), possess an ability to differentiate
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TALEN‐ and CRISPR/Cas9‐Mediated Gene Editing in Human Pluripotent Stem Cells Using Lipid‐Based Transfection
William T. Hendriks, Xin Jiang, Laurence Daheron, Chad A. Cowan
Using custom‐engineered nuclease‐mediated genome editing, such as Transcription Activator–Like Effector Nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) RNA‐guided Cas9 nucleases, human pluripotent stem cell (hPSC) lines with knockout or mutant alleles can
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Alkaline Comet Assay for Assessing DNA Damage in Individual Cells
Xinzhu Pu, Zemin Wang, James E. Klaunig
Single‐cell gel electrophoresis, commonly called a comet assay, is a simple and sensitive method for assessing DNA damage at the single‐cell level. It is an important technique in genetic toxicological studies. The comet assay performed under alkaline conditions (pH >13) is considered the optimal
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Determination of Metabolic Stability Using Cryopreserved Hepatocytes from Rainbow Trout (Oncorhynchus mykiss)
Kellie A. Fay, Diane L. Nabb, Robert T. Mingoia, Ina Bischof, John W. Nichols, Helmut Segner, Karla Johanning, Xing Han
Trout provide a relatively easy source of hepatocytes that can be cryopreserved and used for a range of applications including toxicity testing and determination of intrinsic clearance. Standard protocols for isolating, cryopreserving, and thawing rainbow trout hepatocytes are described, along with
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RNASeq in C. elegans Following Manganese Exposure
Nancy L. Parmalee, Shahina B. Maqbool, Bin Ye, Brent Calder, Aaron B. Bowman, Michael Aschner
Manganese is a metal that is required for optimal biological functioning of organisms. Absorption, cellular import and export, and excretion of manganese are all tightly regulated. While some genes involved in regulation, such as DMT‐1 and ferroportin, are known, it is presumed that many more are
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Pre‐Plated Cell Lines for ADMETox Applications in the Pharmaceutical Industry
Francisco M. Torres, Zsolt Sáfár, Javier Domínguez, Anita Kurunczi, Emese Kis, Rémi Magnan, Márton Jani, Josep Oriol Nicolás, Péter Krajcsi
Membrane transporters significantly modulate membrane permeability of endobiotics and xenobiotics, such as bile acids and drugs, respectively. Various in vitro methods have been established for both ATP‐binding cassette (ABC) transporters to examine cellular efflux and uptake, and for solute
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Measuring Mitochondrial Membrane Potential with a Tetraphenylphosphonium‐Selective Electrode
António J. Moreno, Dario L. Santos, Sílvia Magalhães‐Novais, Paulo J. Oliveira
Mitochondrial bioenergetics is based on the generation of the protonmotive force by the electron transport chain. The protonmotive force is used by mitochondria for different critical aspects of its normal function, ranging from calcium accumulation to the synthesis of ATP. The transmembrane
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