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Isolation of Conventional Murine Lung Dendritic Cell Subsets
Cédric Bosteels, Bart N. Lambrecht, Hamida Hammad
The lungs are continuously exposed to environmental threats, requiring an adequate and stringent immune response of a heterogeneous set of effector cells. Dendritic cells (DCs) form a dense network in the respiratory mucosa and act as the central regulators of the different components of this
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Induction and Measurement of Cytotoxic T Lymphocyte Activity
John Wonderlich, Gene Shearer, Alexandra Livingstone, Andrew Brooks, Mark J. Soloski, Matthew M. Presby
Cytotoxic T cells (CTLs) are important immune effector cells in the adaptive immune response. It has been well documented that CTLs are important in host immune responses to viral and bacterial intracellular pathogens, tumors, and transplanted tissues. The properties of CTLs have been studied
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Flow Cytometry: An Overview
Katherine M. McKinnon
Flow cytometry is a technology that provides rapid multi‐parametric analysis of single cells in solution. Flow cytometers utilize lasers as light sources to produce both scattered and fluorescent light signals that are read by detectors such as photodiodes or photomultiplier tubes. These light
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Liposome Preparation for the Analysis of Lipid‐Receptor Interaction and Efferocytosis
Oliver H. Voss, Ha‐Na Lee, Linjie Tian, Konrad Krzewski, John E. Coligan
Efficient phagocytosis of apoptotic cells (efferocytosis) is essential for immune homeostasis. Phospholipids exposed on the surface of apoptotic cells, such as phosphatidylserine, supply important “eat‐me” signals. Liposomes are lipid bilayer vesicles that can be generated from one or several types
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Autoimmune Pancreatitis Mouse Model
Ken Kamata, Tomohiro Watanabe, Kosuke Minaga, Warren Strober, Masatoshi Kudo
Autoimmune pancreatitis (AIP) is a chronic fibro‐inflammatory disorder of the pancreas. However, extensive clinico‐pathological analyses have revealed that AIP is, in reality, a pancreatic manifestation of a newly described systemic disease known as IgG4‐related disease (IgG4‐RD). IgG4‐RD is
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Thy1‐YFP‐H Mice and the Parallel Rod Floor Test to Evaluate Short‐ and Long‐Term Progression of Traumatic Brain Injury
Monique Richter, María Luciana Negro‐Demontel, Daniela Blanco‐Ocampo, Eliseo Taranto, Natalia Lago, Hugo Peluffo
Traumatic brain injury (TBI) is a leading cause of death and disability and is a risk factor for the later development of neuropsychiatric disorders and neurodegenerative diseases. Many models of TBI have been developed, but their further refinement and a more detailed long‐term follow‐up is needed.
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Intravital Imaging of Neuroimmune Interactions Through a Thinned Skull
Monica Manglani, Dorian B. McGavern
Innate and adaptive immune interactions within the central nervous system (CNS) and surrounding meninges contribute significantly to neural homeostasis as well as a variety of different neurological disorders. Two‐photon laser scanning microscopy is a deep tissue imaging technique that provides a
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Analyzing Chemotaxis and Related Behaviors of Azospirillum Brasilense
Lindsey O'Neal, Tanmoy Mukherjee, Gladys Alexandre
Bacteria of the genus A. brasilense are motile and capable of chemotaxis and aerotaxis (taxis in gradient of oxygen) using a single polar flagellum that propels the cells in aqueous environments. Responses to attractants and repellents have been described and spatial gradient assays that permit the
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Growth and Laboratory Maintenance of Paenibacillus larvae
Osama S. Mahdi, Nathan A Fisher
Paenibacillus larvae is a Gram‐positive, spore‐forming bacterium and the causative agent of American foulbrood disease (AFB), a highly contagious, fatal disease affecting managed honeybee ( Apis mellifera ) colonies. As the etiological agent of American foulbrood disease, P. larvae is the most
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Sporulation and Germination of Paenibacillus larvae Cells
Osama S. Mahdi, Nathan A. Fisher
Endospores are metabolically dormant cells formed by a variety of Gram‐positive bacteria within the phylum Firmicutes in response to nutrient limiting or otherwise unfavorable growth conditions. American foulbrood disease (AFB) is a serious disease of honeybees that is caused by the introduction of
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Poliovirus Replicon RNA Generation, Transfection, Packaging, and Quantitation of Replication
Ekaterina G. Viktorova, Sunil Khattar, Siba Samal, George A. Belov
Poliovirus is a prototype member of the Enterovirus genus of the Picornaviridae family of small positive strand RNA viruses, which include important human and animal pathogens. Quantitative assessment of viral replication is very important for investigation of the virus biology and the development
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Hepatitis E Virus: Isolation, Propagation, and Quantification
Shaoli Lin, Liping Yang, Yan‐Jin Zhang
Hepatitis E virus (HEV) predominantly causes acute liver disease in humans and is transmitted via the fecal‐oral route. HEV infection in pregnant women can result in grave consequences, with up to 30% fatality. The HEV strains infecting humans mainly belong to four genotypes. Genotypes 1 and 2 are
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Porcine Reproductive and Respiratory Syndrome Virus: Propagation and Quantification
Zexu Ma, Liping Yang, Yan‐Jin Zhang
Porcine reproductive and respiratory syndrome virus (PRRSV) is a member of the family Arteriviridae , order Nidovirale . PRRSV is an enveloped, single‐stranded, positive‐sense RNA virus with a genome around 15 kb in length. For propagation of PRRSV in vitro, the MARC‐145 cell line is the most
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Reverse Genetics for Newcastle Disease Virus as a Vaccine Vector
Shin‐Hee Kim, Siba K. Samal
Newcastle disease virus (NDV) is an economically important pathogen in the poultry industry worldwide. Recovery of infectious NDV from cDNA using reverse genetics has made it possible to manipulate the genome of NDV. This has greatly contributed to our understanding of the molecular biology and
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Molecular Genetic Manipulation of Sarcocystis neurona
Daniel K. Howe, Michelle Yeargan, Landon Simpson, Sriveny Dangoudoubiyam
Sarcocystis neurona is a member of the important phylum Apicomplexa and the primary cause of equine protozoal myeloencephalitis (EPM). Moreover, S. neurona is the best‐studied species in the genus Sarcocystis , one of the most successful parasite taxa, as virtually all vertebrate animals may be
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Overview of the Baculovirus Expression System
Adam C. Chambers, Mine Aksular, Leo P. Graves, Sarah L. Irons, Robert D. Possee, Linda A. King
This unit provides information on the replication cycle of insect baculovirus to provide an understanding of how this virus has been adapted for use as an expression vector for recombinant proteins in insect cells. We provide an overview of the virus structure and its unique bi‐phasic replication
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Protein Production Using the Baculovirus Expression System
Sarah L. Irons, Adam C. Chambers, Olga Lissina, Linda A. King, Robert D. Possee
Baculovirus expression systems are well established as an easy and reliable way to produce high quality recombinant proteins. Baculoviruses can also be used to transduce mammalian cells, termed ‘BacMam’, with considerable potential in biomedical applications. This chapter explains the process of
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A Convenient Split‐Intein Tag Method for the Purification of Tagless Target Proteins
Merideth A. Cooper, Joseph E. Taris, Changhua Shi, David W. Wood
In this work, we describe a novel self‐cleaving tag technology based on a highly modified split‐intein cleaving element. In this system, the N‐terminal segment of an engineered split intein is expressed in E. coli and covalently immobilized onto a capture resin, while the smaller C‐terminal intein
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Protein Detection in Gels Using Fixation
Lynn A. Beer, David W. Speicher
The most commonly used types of gels for separating proteins are SDS gels, either in a 1‐D format or as the second dimension of various 2‐D separations, and the most common methods of visualizing proteins in these gels use protein binding dyes after fixing the proteins in the gel matrix. In recent
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Enrichment of Methylated Peptides Using an Antibody‐free Approach for Global Methylproteomics Analysis
Keyun Wang, Mingliang Ye
Protein methylation is receiving increasing attention for its important role in regulating diverse biological processes, including epigenetic regulation of gene transcription, RNA processing, DNA damage repair, and signal transduction. Proteome level analysis of protein methylation requires the
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BioID: A Screen for Protein‐Protein Interactions
Kyle J. Roux, Dae In Kim, Brian Burke, Danielle G. May
BioID is a unique method to screen for physiologically relevant protein interactions that occur in living cells. This technique harnesses a promiscuous biotin ligase to biotinylate proteins based on proximity. The ligase is fused to a protein of interest and expressed in cells, where it biotinylates
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Generation of High‐Specificity Antibodies against Membrane Proteins Using DNA‐Gold Micronanoplexes for Gene Gun Immunization
Debra T. Hansen, Felicia M. Craciunescu, Petra Fromme, Stephen A. Johnston, Kathryn F. Sykes
Membrane proteins are the molecular interface of the cell and its environs; however, studies of membrane proteins are highly technically challenging, mainly due to instability of the isolated protein. Towards the production of antibodies that recognize properly folded and stabilized forms of
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Direct Conversion of Human Pluripotent Stem Cells to Osteoblasts With a Small Molecule
Heemin Kang, Yu‐Ru V. Shih, Shyni Varghese
Human pluripotent stem cells (hPSCs), which exhibit unlimited self‐renewal ability and can differentiate into all cell types in the human body, are a promising cell source for cell‐based therapies and regenerative medicine. Small molecules hold great potential in the derivation of tissue‐specific
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Controlling the Effective Oxygen Tension Experienced by Cells Using a Dynamic Culture Technique for Hematopoietic Ex Vivo Expansion
Abhilasha Tiwari, Cynthia S. Wong, Lakshmi P. Nekkanti, James A. Deane, Courtney McDonald, Jingang Li, Yen Pham, Amy E. Sutherland, Graham Jenkin, Mark A. Kirkland
Clinical hematopoietic stem/progenitor cell (HSPC) transplantation outcomes are strongly correlated with the number of cells infused. Hence, to generate sufficient HSPCs for transplantation, the best culture parameters for expansion are critical. It is generally assumed that the defined oxygen (O 2
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Derivation of Multipotent Mesenchymal Stromal Cells from Ovine Bone Marrow
Daniel Vivas, Marta Caminal, Irene Oliver‐Vila, Joaquim Vives
In the field of orthopedics, translational research of novel therapeutic approaches involves the use of large animal models (such as sheep, goat, pig, dog, and horse) due to the similarities with humans in weight, size, joint structure, and bone/cartilage healing mechanisms. Particularly in the
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hPSC‐derived Midbrain Dopaminergic Neurons Generated in a Scalable 3‐D Biomaterial
Maroof M. Adil, David V. Schaffer
Human pluripotent stem cell (hPSC)–derived midbrain dopaminergic (mDA) neurons may facilitate the development of therapies for Parkinson's disease via disease modeling, drug screening, and cell replacement therapy. However, large numbers of cells are typically needed for these applications, and 2‐D
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CRISPR‐Cas9‐Based Genome Editing of Human Induced Pluripotent Stem Cells
Joseph C. Giacalone, Tasneem P. Sharma, Erin R. Burnight, John F. Fingert, Robert F. Mullins, Edwin M. Stone, Budd A. Tucker
Human induced pluripotent stem cells (hiPSCs) are the ideal cell source for autologous cell replacement. However, for patients with Mendelian diseases, genetic correction of the original disease‐causing mutation is likely required prior to cellular differentiation and transplantation. The emergence
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Conditional Manipulation of Gene Function in Human Cells with Optimized Inducible shRNA
Alessandro Bertero, Loukia Yiangou, Stephanie Brown, Daniel Ortmann, Matthias Pawlowski, Ludovic Vallier
The difficulties involved in conditionally perturbing complex gene expression networks represent major challenges toward defining the mechanisms controlling human development, physiology, and disease. We developed an OPTimized inducible KnockDown (OPTiKD) platform that addresses the limitations of
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A Real‐Time Image‐Based Approach to Distinguish and Discriminate Apoptosis from Necrosis
Asha Lekshmi, Shankara Narayanan Varadarajan, Santhik Subhasingh Lupitha, Mydhily Nair, Aneesh Chandrasekharan, T.R. Santhoshkumar
Recent cell biology studies reveal that a cell can die through multiple pathways via distinct signaling mechanisms. Among these, apoptosis and necrosis are two distinct cell death pathways, and their detection and discrimination is vital in the drug discovery process and in understanding diverse
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Evaluation of Marijuana Compounds on Neuroimmune Endpoints in Experimental Autoimmune Encephalomyelitis
Barbara L. F. Kaplan
Cannabinoid compounds refer to a group of more than 60 plant‐derived compounds in Cannabis sativa , more commonly known as marijuana. Exposure to marijuana and cannabinoid compounds has been increasing due to increased societal acceptance for both recreational and possible medical use. Cannabinoid
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Use of Human Embryoid Bodies for Teratology
Anthony Flamier, Supriya Singh, Theodore P. Rasmussen
Human birth defects are relatively common and can be caused by exposure to environmental teratogens or to pharmaceuticals with teratogenic activities. Human embryonic stem cells (hESCs), by virtue of their pluripotent nature, provide an excellent cellular platform for teratogen detection and risk
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Determination of Mycophenolic Acid and Mycophenolic Acid Glucuronide Using Liquid Chromatography Tandem Mass Spectrometry (LC/MS/MS)
Uttam Garg, Ada Munar, Clinton Frazee
Mycophenolic acid (MPA) is an immunosuppressant that is used in renal, liver, and heart transplantation. Due to its narrow therapeutic range, monitoring of MPA levels is essential to avoid toxicity and organ rejection. Although immunoassays are available for the determination of MPA, due to their
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Development of an In Vitro Method to Estimate the Sensitization Induction Level of Contact Allergens
Valentina Galbiati, Sue Gibbs, Erwin Roggen, Emanuela Corsini
Recently, an in vitro procedure, which combines the epidermal equivalent potency assay with assessment of IL‐18 to provide a single test for identification and classification of skin sensitizers, was developed and validated. This unit will describe a simple in vitro method for estimation of the
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Preparation of Human Primary Colon Tissue‐Derived Organoid Using Air Liquid Interface Culture
Tatsuya Usui, Masashi Sakurai, Koji Umata, Hideyuki Yamawaki, Takashi Ohama, Koichi Sato
In vitro analysis of intestinal epithelium has been hindered by a lack of suitable culture systems useful for gastrointestinal research. To overcome the problem, an air liquid interface (ALI) method using a collagen gel was established to culture three‐dimensional primary cells containing both
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Phenotypic and Functional Evaluation of Hematopoietic Stem and Progenitor Cells in Toxicology of Heavy Metals
Qian Li, Zhengli Yang, Yifan Zhao, Xiaodong Jia, Zhijun Zhou, Yubin Zhang
All blood cells are differentiated from hematopoietic stem and progenitor cells (HSPCs), a process known as hematopoiesis that can be influenced by a variety of environmental factors. In this unit, we introduce a couple of protocols including phenotypic analysis, mixed bone marrow (BM) chimera and
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