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Isolation of T Cells Using Rosetting Procedures
Marjorie E. Kanof
This unit describes a procedure for separating T cells from other mononuclear cells by exploiting the unique ability of cells to bind to and form rosettes with sheep red blood cells (SRBC). This isolation method also allows recovery of the nonrosetting cell population (B lymphocytes, monocytes,
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Isolation of Human Basophils
John T. Schroeder, Anja P. Bieneman
Isolating human basophils from blood has long been hampered by the fact that these granulocytes represent just 1% or less of the circulating leukocyte population. We describe herein laboratory protocols that have been refined over the past ∼25 years that now enable investigators to prepare basophils
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Measuring Apoptosis by Microscopy and Flow Cytometry
Emilie Hollville, Seamus J. Martin
Apoptosis is a mode of programmed cell death that plays an important role during development and in the maintenance of tissue homeostasis. Numerous physiological as well as pathological stimuli trigger apoptosis such as engagement of Fas, TRAIL, or TNF receptors, growth factor deprivation, hypoxia,
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Culture, Expansion, and Differentiation of Murine Megakaryocytes from Fetal Liver, Bone Marrow, and Spleen
Harald Schulze
Megakaryocytes (MKs) are the source of circulating platelets and are readily recognized by their large size and distinctive morphology. Their poor representation in hematopoietic tissues often requires considerable ex vivo expansion to generate cells for biochemical and cell biological studies.
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Obtaining High Quality DNA from Diverse Clinical Samples
Rachael Melton‐Kreft, Tracy Spirk
Nucleic acids can be obtained in numerous ways from clinical specimens; however, the quality of the nucleic acid is only as good as the sampling and isolation protocol. While nucleic acids may be extracted they may not be representative of the original source. Large areas of tissue and explanted
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Investigation of Viral and Host Chromatin by ChIP‐PCR or ChIP‐Seq Analysis
Thomas Günther, Juliane M. Theiss, Nicole Fischer, Adam Grundhoff
Complex regulation of viral transcription patterns and DNA replication levels is a feature of many DNA viruses. This is especially true for those viruses which establish latent or persistent infections (e.g., herpesviruses, papillomaviruses, polyomaviruses, or adenovirus), as long‐term persistence
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Genetic Manipulation of Nocardia Species
Dipesh Dhakal, Amit Kumar Jha, Anaya Pokhrel, Anil Shrestha, Jae Kyung Sohng
Nocardia spp. are aerobic, Gram‐positive, catalase positive, and non‐motile actinomycetes. They are associated with human infections. However, some species produce important natural products, degrade toxic chemicals, and are involved in biotransformation of valuable products. The lack of robust
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Construction of a Transcription Map for Papillomaviruses using RACE, RNase Protection, and Primer Extension Assays
Xiaohong Wang, Zhi‐Ming Zheng
Papillomaviruses are a family of small, non‐enveloped DNA tumor viruses. Knowing a complete transcription map of each papillomavirus genome can provide guidance for various papillomavirus studies. This unit provides detailed protocols to construct a transcription map of human papillomavirus type 18.
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HT‐SPOTi: A Rapid Drug Susceptibility Test (DST) to Evaluate Antibiotic Resistance Profiles and Novel Chemicals for Anti‐Infective Drug Discovery
Cynthia A. Danquah, Arundhati Maitra, Simon Gibbons, Jane Faull, Sanjib Bhakta
Antibiotic resistance is one of the major threats to global health and well‐being. The past decade has seen an alarming rise in the evolution and spread of drug‐resistant strains of pathogenic microbes. The emergence of extensively drug resistant (XDR) strains of Mycobacterium tuberculosis and
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Selective Precipitation of Proteins
Daumantas Matulis
Selective precipitation of proteins can be used as a bulk method to recover the majority of proteins from a crude lysate, as a selective method to fractionate a subset of proteins from a protein solution, or as a very specific method to recover a single protein of interest from a purification step.
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Analysis of Protein Sumoylation
Kevin D. Sarge
Sumoylation, wherein small ubiquitin‐like modifier (SUMO) proteins are covalently attached to specific lysine residues of target proteins, plays an important role in regulating many diverse cellular processes via its control of the functional properties of the modified proteins. Identification of
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Handling Metalloproteinases
Sven Fridrich, Konstantin Karmilin, Walter Stöcker
Substrate cleavage by metalloproteinases involves nucleophilic attack on the scissile peptide bond by a water molecule that is polarized by a catalytic metal, usually a zinc ion, and a general base, usually the carboxyl group of a glutamic acid side chain. The zinc ion is most often complexed by
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Reactivation of Aggregated Proteins by the ClpB/DnaK Bi‐Chaperone System
Michal Zolkiewski, Liudmila S. Chesnokova, Stephan N. Witt
Protein aggregation is a common problem in protein biochemistry and is linked to many cellular pathologies and human diseases. The molecular chaperone ClpB can resolubilize and reactivate aggregated proteins. This unit describes the procedure for following reactivation of an aggregated enzyme
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Expression, Solubilization, and Purification of Bacterial Membrane Proteins
Constance J. Jeffery
Bacterial integral membrane proteins play many important roles, including sensing changes in the environment, transporting molecules into and out of the cell, and in the case of commensal or pathogenic bacteria, interacting with the host organism. Working with membrane proteins in the lab can be
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Derivation of Human Skin Fibroblast Lines for Feeder Cells of Human Embryonic Stem Cells
Christian Unger, Ulrika Felldin, Sergey Rodin, Agneta Nordenskjöld, Sirac Dilber, Outi Hovatta
After the first derivations of human embryonic stem cell (hESC) lines on fetal mouse feeder cell layers, the idea of using human cells instead of mouse cells as feeder cells soon arose. Mouse cells bear a risk of microbial contamination, and nonhuman immunogenic proteins are absorbed from the
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Expansion of Human Hematopoietic Stem/Progenitor Cells on Decellularized Matrix Scaffolds
Abhilasha Tiwari, Melinda L. Tursky, Lakshmi P. Nekkanti, Graham Jenkin, Mark A. Kirkland, Gopal Pande
Umbilical cord blood (UCB) is one of the richest sources for hematopoietic stem/progenitor cells (HSPCs), with more than 3000 transplantations performed each year for the treatment of leukemia and other bone marrow, immunological, and hereditary diseases. However, transplantation of single cord
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Differentiation of Mouse Embryonic Stem Cells into Ventral Foregut Precursors
Michaela Rothová, Jurriaan J. Hölzenspies, Alessandra Livigni, Santiago Nahuel Villegas, Joshua M. Brickman
Anterior definitive endoderm (ADE), the ventral foregut precursor, is both an important embryonic signaling center and a unique multipotent precursor of liver, pancreas, and other organs. Here, a method is described for the differentiation of mouse embryonic stem cells (mESCs) to definitive endoderm
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Identifying Adult Stem Cells Using Cre‐Mediated Lineage Tracing
Diana L. Carlone
Lineage‐tracing has been used for decades to establish cell fate maps during development. Recently, with the advent of genetic lineage‐tracing techniques (employing Cre‐lox recombination), it has been possible to permanently mark progenitor/stem cell populations within somatic tissues. In addition,
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Delivery of Genome Editing Reagents to Hematopoietic Stem/Progenitor Cells
Megan D. Hoban, Zulema Romero, Gregory J. Cost, Matthew Mendel, Michael Holmes, Donald B. Kohn
This unit describes the protocol for the delivery of reagents for targeted genome editing to CD34 + hematopoietic stem/progenitor cells (HSPCs). Specifically, this unit focuses on the process of thawing and pre‐stimulating CD34 + HSPCs, as well as the details of their electroporation with in
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A Simple Light Stimulation of Caenorhabditis elegans
Kun He Lee, Michael Aschner
Response via noxious stimulus can be an important indicator of sensory neuron function and overall health of an organism. If the stimulation is quick and simple, and the animal can be rescued afterwards, such a method not only allows for assays pertaining to changed sensory ability after various
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The Use of Induced Pluripotent Stem Cells for the Study and Treatment of Liver Diseases
Marc C. Hansel, Julio C. Davila, Massoud Vosough, Roberto Gramignoli, Kristen J. Skvorak, Kenneth Dorko, Fabio Marongiu, William Blake, Stephen C. Strom
Liver disease is a major global health concern. Liver cirrhosis is one of the leading causes of death in the world and currently the only therapeutic option for end‐stage liver disease (e.g., acute liver failure, cirrhosis, chronic hepatitis, cholestatic diseases, metabolic diseases, and malignant
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Associating Changes in the Immune System with Clinical Diseases for Interpretation in Risk Assessment
Jamie C. DeWitt, Dori R. Germolec, Robert W. Luebke, Victor J. Johnson
This overview is an update of the unit originally published in 2004. While the basic tenets of immunotoxicity have not changed in the past 10 years, several publications have explored the application of immunotoxicological data to the risk assessment process. Therefore, the goal of this unit is
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Evaluation of Toxicity in Mouse Bone Marrow Progenitor Cells
Peace C. Ezeh, Huan Xu, Shu Chun Wang, Sebastian Medina, Scott W. Burchiel
Development of blood cells through hematopoiesis occurs in the bone marrow (BM), and can be adversely impacted by various substances and/or conditions ranging from known therapeutic, intentionally administered xenobiotics to unintentional food additives and exposure to environmental chemicals. The
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PCR‐Based Analysis of Mitochondrial DNA Copy Number, Mitochondrial DNA Damage, and Nuclear DNA Damage
Claudia P. Gonzalez‐Hunt, John P. Rooney, Ian T. Ryde, Charumathi Anbalagan, Rashmi Joglekar, Joel N. Meyer
Because of the role that DNA damage and depletion play in human disease, it is important to develop and improve tools to assess these endpoints. This unit describes PCR‐based methods to measure nuclear and mitochondrial DNA damage and copy number. Long amplicon quantitative polymerase chain reaction
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