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Current Protocols in Immunology

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https://www.readbyqxmd.com/read/30222250/bone-marrow-chimeras-to-study-neuroinflammation
#1
Nathalie Laflamme, Paul Préfontaine, Antoine Lampron, Serge Rivest
Bone marrow transplantation is the standard of care for a host of diseases such as leukemia and multiple myeloma, as well as genetically inherited metabolic diseases affecting the central nervous system. In mouse models, bone marrow transplantation has proven a valuable tool for understanding the hematopoietic system and the homing of hematopoietic cells to their target organs. Many techniques have been developed to create chimeric mice, animals with a hematopoietic system derived from a genetic background that differs from the rest of the body...
September 17, 2018: Current Protocols in Immunology
https://www.readbyqxmd.com/read/30044539/cytomegalovirus-infection-mouse-model
#2
Ilija Brizić, Berislav Lisnić, Wolfram Brune, Hartmut Hengel, Stipan Jonjić
This unit describes procedures for infecting newborn and adult mice with murine cytomegalovirus (MCMV). Methods are included for propagating MCMV in cell cultures and for preparing a more virulent form of MCMV from salivary glands of infected mice. A plaque assay is provided for determining MCMV titers of infected tissues or virus stocks. Also, a method is described for preparing the murine embryonic fibroblasts used for propagating MCMV and for the plaque assay. © 2018 by John Wiley & Sons, Inc.
July 25, 2018: Current Protocols in Immunology
https://www.readbyqxmd.com/read/30028908/pyroptosis-induction-and-detection
#3
Andreas B den Hartigh, Susan L Fink
Pyroptosis is a form of programmed pro-inflammatory cell death that plays a protective role in the host response to infection, but can also promote pathogenic inflammation. Pyroptosis is mediated by the cysteine protease, caspase-1. Caspase-1 cleaves gasdermin D, releasing the N-terminal pore-forming domain, which inserts into the plasma membrane and drives osmotic lysis. Caspase-1 also proteolytically activates the inflammatory cytokines interleukin 1β (IL-1β) and IL-18. This unit describes methods for stimulating pyroptosis and assessing subsequent loss of plasma membrane integrity...
July 20, 2018: Current Protocols in Immunology
https://www.readbyqxmd.com/read/29969195/monocyte-and-neutrophil-isolation-migration-and-phagocytosis-assays
#4
Alexander Maini, Juliet R Foote, Richard Hayhoe, Amit A Patel, Alastair O'Brien, Inbal Avraham-Davidi, Simon Yona
This article describes methods for isolating mouse monocytes and neutrophils, as well as in vitro protocols for measuring cell phagocytosis, migration, and polarization. The method employed here for the isolation of naive phagocytes overcomes many of the difficulties previously encountered concerning phagocyte activation. Three in vitro protocols are provided for the analysis of cell migration, one requiring no specialized equipment, one requiring a modified Boyden chamber, and the other employing a flow chamber, which measures cell adhesion, rolling, and migration...
July 3, 2018: Current Protocols in Immunology
https://www.readbyqxmd.com/read/29957859/isolation-of-human-innate-lymphoid-cells
#5
Lisette Krabbendam, Maho Nagasawa, Hergen Spits, Suzanne M Bal
Innate lymphoid cells (ILCs) are innate immune cells of lymphoid origin that have important effector and regulatory functions in the first line of defense against pathogens, but also regulate tissue homeostasis, remodeling, and repair. Their function mirrors T helper cells and cytotoxic CD8+ T lymphocytes, but they lack expression of rearranged antigen-specific receptors. Distinct ILC subsets are classified in group 1 ILCs (ILC1s), group 2 ILCs (ILC2s), and group 3 ILCs (ILC3s and lymphoid tissue-inducer cells), based on the expression of transcription factors and the cytokines they produce...
June 29, 2018: Current Protocols in Immunology
https://www.readbyqxmd.com/read/29927067/quantifying-microglial-phagocytosis-of-apoptotic-cells-in-the-brain-in-health-and-disease
#6
Sol Beccari, Irune Diaz-Aparicio, Amanda Sierra
Apoptosis is a ubiquitous process occurring in the brain under both physiological and pathological conditions. The central nervous system (CNS) requires an active and efficient clean-up system to prevent the spillover of intracellular contents into the surrounding parenchyma and suppress the initiation of inflammatory and immune responses. Microglia, the resident professional phagocytes of the brain, are the cells in charge of the removal of these dead cells by the process named phagocytosis. Therefore, microglial phagocytosis is a vital mechanism to maintain tissue homeostasis...
May 24, 2018: Current Protocols in Immunology
https://www.readbyqxmd.com/read/30040219/human-natural-killer-receptors-co-receptors-and-their-ligands
#7
Roberto Biassoni, Mauro S Malnati
In the last 20 years, the study of human natural killer (NK) cells has moved from the first molecular characterizations of very few receptor molecules to the identification of a plethora of receptors displaying surprisingly divergent functions. We have contributed to the description of inhibitory receptors and their signaling pathways, important in fine regulation in many cell types, but unknown until their discovery in the NK cells. Inhibitory function is central to regulating NK-mediated cytolysis, with different molecular structures evolving during speciation to assure its persistence...
April 2018: Current Protocols in Immunology
https://www.readbyqxmd.com/read/30040218/isolation-of-murine-skin-resident-and-migratory-dendritic-cells-via-enzymatic-digestion
#8
Sakeen W Kashem, Daniel H Kaplan
Dendritic cells (DCs) are a highly specialized subset of professional antigen-presenting cells (APCs) that reside in peripheral and lymphoid tissues. DCs capture antigen in the periphery and migrate to the lymph node where they prime naïve T cells. In addition, DCs have been recently appreciated to have function in innate immunity within tissues. In the skin, heterogeneous populations of DCs reside within the epidermis and the dermis. Analysis of the cutaneous DC subsets is complicated by requirements of distinct enzymatic digestion protocols for isolation of APCs from distinct anatomical compartments of the skin...
April 2018: Current Protocols in Immunology
https://www.readbyqxmd.com/read/30040211/leukocyte-isolation-from-brain-spinal-cord-and-meninges-for-flow-cytometric-analysis
#9
Monica Manglani, Selamawit Gossa, Dorian B McGavern
A continual dialogue exists between the central nervous system (CNS) and immune system that contributes to neural homeostasis as well as protection from microbes, repair following damage, autoimmune disease, and neurodegeneration. Characterization of resident and peripherally derived leukocyte populations within the central nervous system can provide valuable information regarding how these cells contribute to steady-state and inflammatory conditions. Flow cytometry provides a method to conduct detailed multi-parameter analyses of immune cells isolated from various tissues...
April 2018: Current Protocols in Immunology
https://www.readbyqxmd.com/read/30008984/gnotobiotic-and-conventional-mouse-systems-to-support-microbiota-based-studies
#10
Richard Lavin, Nicholas DiBenedetto, Vladimir Yeliseyev, Mary Delaney, Lynn Bry
Animal models are essential to dissect host-microbiota interactions that impact health and the development of disease. In addition to providing pre-clinical models for the development of novel therapeutics and diagnostic biomarkers, mouse systems actively support microbiome studies by defining microbial contributions to normal development and homeostasis, and as well as their role in promoting diseases such as inflammatory auto-immune diseases, diabetes, metabolic syndromes, and susceptibilities to infectious agents...
April 2018: Current Protocols in Immunology
https://www.readbyqxmd.com/read/30008983/meningeal-whole-mount-preparation-and-characterization-of-neural-cells-by-flow-cytometry
#11
Antoine Louveau, Anthony J Filiano, Jonathan Kipnis
Neuroimmunologists aim to understand the interactions between the central nervous system and the immune system under both homeostatic and pathological conditions. The meninges, contrary to the brain parenchyma, are populated by numerous immune cells. Soluble factors produced by these cells are capable to diffuse into the brain parenchyma and influence the brain cells within the parenchyma, including neurons. In this unit, we will describe two protocols: analysis the meningeal compartment of rodents and the use flow cytometry to study the cells of the brain parenchyma (particularly neurons)...
April 2018: Current Protocols in Immunology
https://www.readbyqxmd.com/read/29512146/intravital-imaging-of-neuroimmune-interactions-through-a-thinned-skull
#12
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 means to image immune cell dynamics and interactions in the living CNS with high spatial and temporal resolution. Optical access to the brain and meninges can be achieved through the creation of thinned skull windows, which can be made without inducing damage and inflammation in the underlying tissue...
February 21, 2018: Current Protocols in Immunology
https://www.readbyqxmd.com/read/29512145/induction-and-measurement-of-cytotoxic-t-lymphocyte-activity
#13
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 extensively in murine models, and their roles validated in the human setting. Frequently, the presence of these cells correlates well with protective immunity, so the ability to readily measure the activity of these cells is an important immunological measurement...
February 21, 2018: Current Protocols in Immunology
https://www.readbyqxmd.com/read/29512144/thy1-yfp-h-mice-and-the-parallel-rod-floor-test-to-evaluate-short-and-long-term-progression-of-traumatic-brain-injury
#14
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. We have used the Thy1-YFP-H transgenic mouse line and the parallel rod floor test to produce an unbiased and robust method for the evaluation of the multiple effects of a validated model of controlled cortical injury...
February 21, 2018: Current Protocols in Immunology
https://www.readbyqxmd.com/read/29512143/isolation-of-conventional-murine-lung-dendritic-cell-subsets
#15
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 response, both sensing the nature of the threats and precisely coordinating the effector mechanisms best suited for overcoming it. The DCs are classically subdivided in two main groups, plasmacytoid DCs (pDCs) and conventional DCs (cDCs), the latter being further subdivided into cDC1s and cDC2s based on ontogeny and their distinct non-redundant functions...
February 21, 2018: Current Protocols in Immunology
https://www.readbyqxmd.com/read/29512142/liposome-preparation-for-the-analysis-of-lipid-receptor-interaction-and-efferocytosis
#16
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 of phospholipids of interest. Thus, these vesicles offer versatility, flexibility, and, importantly, a three-dimensional structure for studying the interaction between lipids and their receptors as well as the lipid-receptor interaction-mediated signaling events controlling efferocytosis by cells like professional phagocytes...
February 21, 2018: Current Protocols in Immunology
https://www.readbyqxmd.com/read/29512141/flow-cytometry-an-overview
#17
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 signals are converted into electronic signals that are analyzed by a computer and written to a standardized format (.fcs) data file. Cell populations can be analyzed and/or purified based on their fluorescent or light scattering characteristics...
February 21, 2018: Current Protocols in Immunology
https://www.readbyqxmd.com/read/29512140/autoimmune-pancreatitis-mouse-model
#18
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 characterized by enhanced local and systemic IgG4 antibody (Ab) responses as well as inflammation involving multiple organs, including the pancreas, bile ducts, and salivary glands. Although mice lack the IgG4 Ab subtype, autoimmune-prone MRL/Mp mice treated with repeated injection with polyinosinic-polycytidylic acid (poly (I:C)) provide an experimental model of AIP...
February 21, 2018: Current Protocols in Immunology
https://www.readbyqxmd.com/read/29091265/mouse-eosinophils-identification-isolation-and-functional-analysis
#19
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 inflammation, parasite infections, or even cancer, they migrate and mainly reside in mucosal tissues where they have key effector and immune-modulating functions. Functional studies using eosinophils are not simple, since these cells are terminally differentiated and rapidly die in vitro...
November 1, 2017: Current Protocols in Immunology
https://www.readbyqxmd.com/read/29091264/analysis-of-cellular-dna-content-by-flow-cytometry
#20
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 unit, simple and universally applicable methods for staining fixed cells are presented, as are methods that utilize detergents and/or proteolytic treatment to permeabilize cells and make DNA accessible to fluorochrome...
November 1, 2017: Current Protocols in Immunology
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