Isolation of Murine Skin Resident and Migratory Dendritic Cells via Enzymatic Digestion.
Authors: Kashem SW, Kaplan DH
Abstract
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 fr...
Source: Current Protocols in Immunology - July 25, 2018 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Human Natural Killer Receptors, Co-Receptors, and Their Ligands.
Authors: Biassoni R, Malnati MS
Abstract
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 ...
Source: Current Protocols in Immunology - July 25, 2018 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Pyroptosis Induction and Detection.
Authors: den Hartigh AB, Fink SL
Abstract
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 plas...
Source: Current Protocols in Immunology - July 22, 2018 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Meningeal whole mount preparation and characterization of neural cells by flow cytometry.
Authors: Louveau A, Filiano AJ, Kipnis J
Abstract
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 ...
Source: Current Protocols in Immunology - July 18, 2018 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Gnotobiotic and Conventional Mouse Systems to Support Microbiota Based Studies.
Authors: Lavin R, DiBenedetto N, Yeliseyev V, Delaney M, Bry L
Abstract
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. Mice provide a gen...
Source: Current Protocols in Immunology - July 18, 2018 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Monocyte and Neutrophil Isolation, Migration, and Phagocytosis Assays.
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. Three in vitro protocols to examine phagocytosis have bee...
Source: Current Protocols in Immunology - July 5, 2018 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Isolation of Human Innate Lymphoid Cells.
Authors: Krabbendam L, Nagasawa M, Spits H, Bal SM
Abstract
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 transcri...
Source: Current Protocols in Immunology - July 1, 2018 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Quantifying Microglial Phagocytosis of Apoptotic Cells in the Brain in Health and Disease.
Authors: Beccari S, Diaz-Aparicio I, Sierra A
Abstract
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 m...
Source: Current Protocols in Immunology - June 28, 2018 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Autoimmune Pancreatitis Mouse Model.
Authors: Kamata K, Watanabe T, Minaga K, Strober W, Kudo M
Abstract
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 t...
Source: Current Protocols in Immunology - March 9, 2018 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Flow Cytometry: An Overview.
Authors: McKinnon KM
Abstract
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. A variety of fluorescent reagents are util...
Source: Current Protocols in Immunology - March 9, 2018 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Liposome Preparation for the Analysis of Lipid-Receptor Interaction and Efferocytosis.
Authors: Voss OH, Lee HN, Tian L, Krzewski K, Coligan JE
Abstract
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 s...
Source: Current Protocols in Immunology - March 9, 2018 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Isolation of Conventional Murine Lung Dendritic Cell Subsets.
Authors: Bosteels C, Lambrecht BN, Hammad H
Abstract
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 su...
Source: Current Protocols in Immunology - March 9, 2018 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Thy1-YFP-H Mice and the Parallel Rod Floor Test to Evaluate Short- and Long-Term Progression of Traumatic Brain Injury.
Authors: Richter M, Negro-Demontel ML, Blanco-Ocampo D, Taranto E, Lago N, Peluffo H
Abstract
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. ...
Source: Current Protocols in Immunology - March 9, 2018 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Induction and Measurement of Cytotoxic T Lymphocyte Activity.
Authors: Wonderlich J, Shearer G, Livingstone A, Brooks A, Soloski MJ, Presby MM
Abstract
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 i...
Source: Current Protocols in Immunology - March 9, 2018 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Intravital Imaging of Neuroimmune Interactions Through a Thinned Skull.
Authors: Manglani M, McGavern DB
Abstract
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 inflammat...
Source: Current Protocols in Immunology - March 9, 2018 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
