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Current Protocols in Immunology

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https://www.readbyqxmd.com/read/29512146/intravital-imaging-of-neuroimmune-interactions-through-a-thinned-skull
#1
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
#2
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
#3
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
#4
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
#5
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
#6
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
#7
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
#8
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
#9
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
https://www.readbyqxmd.com/read/29091263/high-dimensional-fluorescence-cytometry
#10
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 conjunction with the ever-expanding understanding of the complexity of the immune system, has led to the generation of larger high-dimensional fluorescence flow cytometry panels. However, as panel size and complexity increases, so too does the difficulty involved in constructing high-quality panels, in addition to the challenges of analyzing such high-dimensional datasets...
November 1, 2017: Current Protocols in Immunology
https://www.readbyqxmd.com/read/29091262/isolation-and-functional-use-of-human-nkt-cells
#11
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 'invariant' TCRα chain, which is generated in humans by precise Vα24 and Jα18 rearrangements with either no N-region diversity or subsequent trimming to identical or nearly identical amino acid sequence (hence, 'iNKT' cells)...
November 1, 2017: Current Protocols in Immunology
https://www.readbyqxmd.com/read/29091261/the-mouse-model-of-infection-with-citrobacter-rodentium
#12
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 provides an excellent model to study different aspects of host-pathogen interaction in the gut, including intestinal inflammatory responses during bacteria-induced colitis, mucosal healing and epithelial repair, the induction of mucosal immune responses, and the role of the intestinal microbiota in mediating resistance to colonization by enteric pathogens...
November 1, 2017: Current Protocols in Immunology
https://www.readbyqxmd.com/read/28762488/a-real-time-cytotoxicity-assay-as-an-alternative-to-the-standard-chromium-51-release-assay-for-measurement-of-human-nk-and-t-cell-cytotoxic-activity
#13
Julien Fassy, Kyriaki Tsalkitzi, Maria Goncalves-Maia, Véronique M Braud
This unit describes the monitoring and quantification of cellular cytotoxicity using a non-radioactive and real-time cytotoxic assay. The extent of target-cell lysis is monitored over time by imaging and quantifying live fluorescent target cells using a cell-imaging multimode reader. This assay is performed in a 96 well plate in optimized culture conditions at 37°C in the presence of 5% CO2 . The basic protocol describes natural killer cell-mediated cytotoxic assay that can be adapted to include antibodies blocking inhibitory NK receptors or triggering antibody-dependent cell-mediated cytotoxicity (ADCC)...
August 1, 2017: Current Protocols in Immunology
https://www.readbyqxmd.com/read/28762487/nomenclature-and-serology-of-hla-class-i-and-class-ii-alleles
#14
James McCluskey, Carmel Kanaan, Mary Diviney
This overview presents nomenclature and serology information on human leucocyte antigens, or HLA molecules, which are encoded by a cluster of genes linked on the short arm of chromosome 6. This region is known as the major histocompatibility complex and codes for class I and class II molecules, which are distinguished from each other based upon their structure, tissue distribution, and source of peptide antigen, as well as upon their interactions with T cell subsets. © 2017 by John Wiley & Sons, Inc.
August 1, 2017: Current Protocols in Immunology
https://www.readbyqxmd.com/read/28762486/universal-precautions-necessary-safety-procedures-when-handling-human-blood-body-fluids-and-specimens
#15
Barbara E Bierer
Universal precautions are observed whenever handling human blood, body fluids, or specimens as a means of preventing exposure to blood-borne pathogens. This appendix outlines safety procedures to follow whenever undertaking research activities that involve human blood, body fluids, and specimens. © 2017 by John Wiley & Sons, Inc.
August 1, 2017: Current Protocols in Immunology
https://www.readbyqxmd.com/read/28762485/characterization-of-human-blood-monocytes-and-intestinal-macrophages
#16
Evida A Dennis, Tanya O Robinson, Lesley E Smythies, Phillip D Smith
Monocytes and macrophages play fundamental roles in defense against microbes, clearance of senescent and dead cells, and immunoregulation. Although blood monocytes are the source of intestinal macrophages in the developed mucosal immune system, blood monocytes and intestinal macrophages from healthy human subjects display distinct phenotypic and functional differences. Blood monocytes can be induced to polarize into M1 and M2 macrophages, whereas intestinal macrophages appear to be terminally differentiated and are unable to undergo such inducible polarization...
August 1, 2017: Current Protocols in Immunology
https://www.readbyqxmd.com/read/28762484/venipuncture-procedures-sources-of-human-peripheral-blood-cells-serum-and-plasma
#17
Barbara E Bierer, Warren Strober
Peripheral blood is the chief source of mononuclear cells (lymphocytes and monocytes) for immunologic studies of humans. This appendix presents protocols for obtaining blood by simple venipuncture when relatively small amounts of blood (10 to 100 ml) are necessary or by lymphapheresis when large amounts (300 to 5000 ml) are necessary. Cells collected by these procedures can be further separated by techniques described in Chapter 7. © 2017 by John Wiley & Sons, Inc.
August 1, 2017: Current Protocols in Immunology
https://www.readbyqxmd.com/read/28762483/high-dimensional-single-cell-analysis-with-mass-cytometry
#18
Tess Melinda Brodie, Vinko Tosevski
Mass cytometry is an analytical technology that combines the sample preparation workflow typical of flow cytometry and the detection capacity of atomic mass spectroscopy, allowing for highly multiplexed measurements of protein or nucleic acid targets on single cells. In 2014, the mass cytometer was adapted for the acquisition of samples from microscopy slides (termed imaging mass cytometry), greatly increasing the applicability of this technology. By using antibodies (or other probes) labeled with purified metal isotopes, the mass cytometer is able to detect up to 50 different parameters (current practical limit) at the single-cell level, enabling a deep and thorough profiling of individual cells in terms of their cell surface protein phenotype, physiological state, proliferation potential, and many other cell states or features...
August 1, 2017: Current Protocols in Immunology
https://www.readbyqxmd.com/read/28369684/gut-microbiome-standardization-in-control-and-experimental-mice
#19
Kathy D McCoy, Markus B Geuking, Francesca Ronchi
Mouse models are used extensively to study human health and to investigate the mechanisms underlying human disease. In the past, most animal studies were performed without taking into consideration the impact of the microbiota. However, the microbiota that colonizes all body surfaces, including the gastrointestinal tract, respiratory tract, genitourinary tract, and skin, heavily impacts nearly every aspect of host physiology. When performing studies utilizing mouse models it is critical to understand that the microbiome is heavily impacted by environmental factors, including (but not limited to) food, bedding, caging, and temperature...
April 3, 2017: Current Protocols in Immunology
https://www.readbyqxmd.com/read/28369683/basic-multicolor-flow-cytometry
#20
Zofia Maciorowski, Pratip K Chattopadhyay, Paresh Jain
Multicolor flow cytometry is a rapidly evolving technology that uses multiple fluorescent markers to identify and characterize cellular subpopulations of interest, allowing rapid analysis on tens of thousands of cells per second, with the possibility of isolating pure, viable populations by cell sorting for further experimentation. This unit covers the tools needed by the beginning immunologist to plan and run multicolor experiments, with information on fluorochromes and their characteristics, spectral spillover, compensation and spread, instrument and reagent variables, and the basic elements of multicolor panel design...
April 3, 2017: Current Protocols in Immunology
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