Animal Models of Mycobacteria Infection.
Authors: Chan ED, Verma D, Ordway DJ Abstract This manuscript describes the infection of mice and guinea pigs with mycobacteria via various routes, as well as necropsy methods for the determination of mycobacterial loads within target organs. Additionally, methods for cultivating mycobacteria and preparing stocks are described. The protocols outlined are primarily used for M. tuberculosis, but can also be used for the study of other non-tuberculosis mycobacterial species. A wide variety of animal models have been used to test new vaccines, drugs, and the impact of cigarette exposure. © 2020 Wiley Periodicals L...
Source: Current Protocols in Immunology - June 6, 2020 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

Isolation and Characterization of Extracellular Vesicles from Cell Culture Conditioned Medium for Immunological Studies.
This article describes protocols for isolation of EVs from cell culture supernatants by differential ultracentrifugation and density gradient centrifugation. It also provides tools and protocols that enable characterization and validation of isolated particles, as well as analysis of interactions between EVs of interest and different subpopulations of human immune cells. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Isolation of extracellular vesicles by differential ultracentrifugation Basic Protocol 2: Isolation of extracellular vesicles by density gradient centrifugation Support Protocol 1: Imaging of extracellul...
Source: Current Protocols in Immunology - May 28, 2020 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

T Cell Receptor Engineered Lymphocytes for Cancer Therapy.
Authors: Rollins MR, Spartz EJ, Stromnes IM Abstract T lymphocytes are capable of specific recognition and elimination of target cells. Physiological antigen recognition is mediated by the T cell receptor (TCR), which is an alpha beta heterodimer comprising the products of randomly rearranged V, D, and J genes. The exquisite specificity and functionality of T cells can be leveraged for cancer therapy: specifically, the adoptive transfer of T cells that express tumor-reactive TCRs can induce regression of solid tumors in patients with advanced cancer. However, the isolation and expression of a tumor antigen-specific...
Source: Current Protocols in Immunology - May 22, 2020 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

Multispecific, Multivalent Antibody-Based Molecules Engineered on the DART ® and TRIDENTTM Platforms.
This article focuses on the design of DART and TRIDENT molecules that target two or three different antigens. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Design and generation of expression plasmids encoding DART and TRIDENT molecules Basic Protocol 2: Expression of DART and TRIDENT molecules by transient transfection of CHO cells Basic Protocol 3: Purification of DART and TRIDENT molecules from CHO cell supernatants. PMID: 32294319 [PubMed - in process] (Source: Current Protocols in Immunology)
Source: Current Protocols in Immunology - April 17, 2020 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

Antibody-Based CAR T Cells Produced by Lentiviral Transduction.
Authors: Prommersberger S, Hudecek M, Nerreter T Abstract One promising approach to treat hematologic malignancies is the usage of patient-derived CAR T cells. There are continuous efforts to improve the function of these cells, to optimize their receptor, and to use them for the treatment of additional types of cancer and especially solid tumors. In this protocol, an easy and reliable approach for CAR T cell generation is described. T cells are first isolated from peripheral blood (here: leukoreduction system chambers) and afterwards activated for one day with anti-CD3/CD28 Dynabeads. The gene transfer is per...
Source: Current Protocols in Immunology - March 11, 2020 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

Purification of T Cell Populations.
This article describes a procedure for isolating T cell subpopulations using various methods including indirect panning and immunopanning by microarray. In these methods, cells are selected by their capacity to bind to antibody-coated plates (or slides) on the basis of particular cell-surface markers. Such methods can be superior to the antibody/complement lysis method (Alternate Protocol), as they can select additional cell population for analysis. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Isolation of T cell populations by indirect panning Basic Protocol 2: Immunopanning with microarray Alternate Proto...
Source: Current Protocols in Immunology - February 11, 2020 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

Isolation and Analysis of Tumor-Derived Exosomes.
Authors: Ludwig N, Hong CS, Ludwig S, Azambuja JH, Sharma P, Theodoraki MN, Whiteside TL Abstract A method for isolation of exosomes from tumor cell supernatants or cancer patients' plasma is presented. Tumor-derived exosomes (TEX) are defined as a subset of extracellular vesicles (EVs) sized at 30 to 150 nm and originating from multivesicular bodies (MVBs). The method utilizes size exclusion chromatography (SEC) for recovery of exosomes from cell-line supernatants or cancer patients' plasma. The recovered exosomes are morphologically intact, aggregate-free, and functionally competent. Their molecular content paral...
Source: Current Protocols in Immunology - November 26, 2019 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

Characterization and Purification of Mouse Mucosal-Associated Invariant T (MAIT) Cells.
Authors: Chen Z, Wang H, D'Souza C, Koay HF, Meehan B, Zhao Z, Pediongco T, Shi M, Zhu T, Wang B, Kjer-Nielsen L, Eckle SBG, Rossjohn J, Fairlie DP, Godfrey DI, Strugnell RA, McCluskey J, Corbett AJ Abstract This unit describes the utility of various mouse models of infection and immunization for studying mucosal-associated invariant T (MAIT) cell immunity: MAIT cells can be isolated from the lungs (or from other tissues/organs) and then identified and characterized by flow cytometry using MR1 tetramers in combination with a range of antibodies. The response kinetics, cytokine profiles, and functional differentiati...
Source: Current Protocols in Immunology - November 26, 2019 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

Characterization of Human Mucosal-associated Invariant T (MAIT) Cells.
This article describes protocols to characterize the phenotype of human MAIT cells in blood and tissues by flow cytometry using fluorescently labeled human MR1 tetramers alongside antibodies specific for MAIT cell markers. © 2019 by John Wiley & Sons, Inc. The main protocols include: Basic Protocol 1: Determining the frequency and steady-state surface phenotype of human MAIT cells Basic Protocol 2: Determining the activation phenotype of human MAIT cells in blood Basic Protocol 3: Characterizing MAIT cell TCRs using TCR-positive reporter cell lines Alternate protocols are provided for determining the absolute numb...
Source: Current Protocols in Immunology - November 26, 2019 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

Isolation and Analysis of Mouse and Human Skin γδ T Cells.
Isolation and Analysis of Mouse and Human Skin γδ T Cells. Curr Protoc Immunol. 2019 Dec;127(1):e92 Authors: Gargas S, Bshara-Corson S, Cruz M, Jameson J Abstract Skin-resident and infiltrating γδ T lymphocytes are components of the cutaneous immune system that provide the first line of defense against pathogens and the environment. Research that employs the isolation and culture of T cells from murine and human skin can help delineate the molecular and cellular mechanisms utilized by T lymphocytes in skin-specific immunity. However, obtaining high numbers of T cells from epithe...
Source: Current Protocols in Immunology - November 26, 2019 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

Characterization of Immune Cells from Adipose Tissue.
This article describes the protocols to generate such AIPs. © 2019 by John Wiley & Sons, Inc. PMID: 31483101 [PubMed - in process] (Source: Current Protocols in Immunology)
Source: Current Protocols in Immunology - September 7, 2019 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

Production and Thermal Exchange of Conditional Peptide-MHC I Multimers.
Authors: Luimstra JJ, Franken KLMC, Garstka MA, Drijfhout JW, Neefjes J, Ovaa H Abstract Cytotoxic CD8+ T cells mediate cellular immunity through recognition of specific antigens presented by MHC class I on all nucleated cells. Studying T cell interactions and responses provides invaluable information on infection, autoimmunity and cancer. Fluorescently labeled multimers of MHC I can be used to quantify, characterize, and isolate specific CD8+ T cells by flow cytometry. Here we describe the production and use of conditional MHC I multimers that can be loaded with peptides of choice by incubating them at a defined t...
Source: Current Protocols in Immunology - September 7, 2019 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

Protocols for the Analysis of microRNA Expression, Biogenesis, and Function in Immune Cells.
Authors: Zhang N, Hu G, Myers TG, Williamson PR Abstract MicroRNAs (miRNAs) are short (19- to 25-nucleotide) noncoding RNA molecules that target mRNAs to repress gene expression and that play important roles in regulating many fundamental biological functions including cell differentiation, development, growth, and metabolism. They are well conserved in eukaryotic cells and are considered essential ancient elements of gene regulation. miRNA genes are transcribed by RNA polymerase II to generate primary miRNAs (pri-miRNAs), which are cleaved by microprocessor complex in the nucleus to generate stem-loop structures k...
Source: Current Protocols in Immunology - September 7, 2019 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

Genome-Wide Measurement and Computational Analysis of Transcription Factor Binding and Chromatin Accessibility in Lymphocytes.
Authors: Sadiyah MF, Roychoudhuri R Abstract Cells of the adaptive immune system, including CD4+ and CD8+ T cells, as well as B cells, possess the ability to undergo dynamic changes in population size, differentiation state, and function to counteract diverse and temporally stochastic threats from the external environment. To achieve this, lymphocytes must be able to rapidly control their gene-expression programs in a cell-type-specific manner and in response to extrinsic signals. Such capacity is provided by transcription factors (TFs), which bind to the available repertoire of regulatory DNA elements in distinct ...
Source: Current Protocols in Immunology - September 7, 2019 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

Experimental Mouse Model of Bleomycin-Induced Skin Fibrosis.
Authors: Błyszczuk P, Kozlova A, Guo Z, Kania G, Distler O Abstract Systemic sclerosis (SSc) refers to an autoimmune disease, which is manifested by inflammation, vasculopathy, and fibrosis of the skin and internal organs. There are a number of different animal models recapitulating specific aspects of SSc. The experimental mouse model of bleomycin-induced skin fibrosis is commonly used to study the pathogenesis observed in SSc. In this model, repetitive intradermal injections of the cytotoxic agent bleomycin trigger progressive skin thickening, associated with excessive accumulation of collagen, infiltration of i...
Source: Current Protocols in Immunology - September 7, 2019 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

Using Evans Blue Dye to Determine Blood-Brain Barrier Integrity in Rodents.
Authors: Goldim MPS, Della Giustina A, Petronilho F Abstract The blood-brain barrier (BBB) is an active and selective barrier that shields the brain from endogenous and exogenous insults. Different stimuli may lead to the disruption of this barrier, including inflammation and trauma. Several methods are used to evaluate BBB disruption. The most widely used method is Evans blue (EB) dye extravasation. EB cannot normally pass through the BBB and thus its presence in brain tissue indicates alterations in permeability. This protocol details the steps of EB extravasation in rodents. Important aspects regarding critical ...
Source: Current Protocols in Immunology - September 7, 2019 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

Mass Spectrometry-Based Quantitative Proteomics of Murine-Derived Polymorphonuclear Neutrophils.
Authors: Geddes-McAlister J, Gadjeva M Abstract Polymorphonuclear cells (PMNs or neutrophils) are the most abundant leukocyte in humans and represent an essential component of the innate immune system. The ability of neutrophils to initiate an immediate and non-specific host response against invading microbial species is the key to determining the outcome of infection. Neutrophils produce and secrete a plethora of immunomodulatory proteins, including major granule proteins and cytokines, as well as various enzymes, which regulate adherence, phagocytosis, chemotaxis, and cell survival. Historically, characterization...
Source: Current Protocols in Immunology - September 7, 2019 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

A Fast and Reliable Method to Isolate Human Placental Macrophages.
Authors: Mezouar S, Ben Amara A, Chartier C, Gorvel L, Mege JL Abstract Macrophages are specialized cells involved in recognition, uptake, and destruction of microorganisms. Human placental macrophages are poorly investigated because of the lack of a convenient protocol for their isolation. Here, we present a straightforward and reliable method to isolate macrophages from full-term human placentas. After enzymatic digestion of placental tissue and centrifugation of the cell suspension on a Ficoll cushion, placental macrophages are selected using magnetic beads coated with anti-CD14 antibodies. Isolated cells are ch...
Source: Current Protocols in Immunology - May 29, 2019 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

Generation of T Cell Receptor Retrogenic Mice.
This article details the materials and methods required to generate TCR retrogenic mice. It is divided into three sections and provides detailed methods for generation of stable retroviral producer cell lines, isolation and optimal transduction of hematopoietic bone marrow cells, and subsequent analysis of TCR retrogenic T cells. A detailed example of such analysis is provided. The current protocol is a culmination of many years of optimization and is the most efficient approach to date. Bone marrow transduction and transfer into recipient mice can now be achieved in a short period of four days. The protocol can be followe...
Source: Current Protocols in Immunology - May 16, 2019 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

Peptide:MHCII Tetramer-Based Cell Enrichment for the Study of Epitope-Specific CD4+ T Cells.
Authors: Kotov DI, Jenkins MK Abstract Epitope-specific CD4+ T cells can be labeled in complex cell mixtures from secondary lymphoid organs with fluorophore-labeled peptide:major histocompatibility complex class II (p:MHCII) tetramers and then detected by flow cytometry. Magnetic enrichment of tetramer-bound cells before flow cytometry increases the sensitivity of detection to the point where epitope-specific cells can be studied even when very rare at early and late times after the host has been exposed to the epitope. This method is very useful for studying polyclonal epitope-specific CD4+ T cells under physiolog...
Source: Current Protocols in Immunology - May 1, 2019 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

Identification of Group 2 Innate Lymphoid Cells in Mouse Lung, Liver, Small Intestine, Bone Marrow, and Mediastinal and Mesenteric Lymph Nodes.
Authors: Romera-Hernández M, Mathä L, Steer CA, Ghaedi M, Takei F Abstract Innate lymphoid cells (ILCs) are a heterogeneous family of lymphocytes that populate barrier and non-barrier tissues. ILCs regulate immune responses to pathogens and commensals but also sustain metabolic homeostasis, tissue remodeling after injury and establish dialogue with the nervous system. ILCs rapidly become activated in the absence of adaptive antigen receptors by responding to signaling molecules provided by hematopoietic or non-hematopoietic cells. Here we provide protocols designed for processing the lung, liver, small ...
Source: Current Protocols in Immunology - April 19, 2019 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

Generation and Analysis of Human and Murine Osteoclasts.
Authors: Steffen U, Andes FT, Schett G Abstract Osteoclasts are the only bone-resorbing cells in the body. Together with bone-forming osteoblasts, they are responsible for bone homeostasis and constant bone remodeling. Aberrant activation of osteoclasts leads to bone loss, as seen in postmenopausal osteoporosis or in autoimmune diseases like rheumatoid arthritis. Although much research has been performed to understand and prevent osteoclast-mediated bone loss, the mechanisms of osteoclast hyperactivation are not completely understood. This unit describes several protocols for ex vivo generation of murine and human ...
Source: Current Protocols in Immunology - April 9, 2019 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

Imaging Flow Cytometry to Assess Antigen-Presenting-Cell Function.
Authors: Markey KA, Gartlan KH Abstract This unit describes methods for quantifying phagocytosis and imaging the immunological synapse between T cells and antigen-presenting cells (APCs), with both techniques delivering valuable information about APC function. These aspects of APC biology have traditionally been challenging to quantify, and imaging flow cytometry, which harnesses the high-throughput nature of flow cytometry combined with the capacity of microscopy to deliver spatial localization, facilitates analysis of these APC functions in a fashion that was previously not possible. Imaging flow cytometry allows...
Source: Current Protocols in Immunology - March 7, 2019 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

IL-23- and Imiquimod-Induced Models of Experimental Psoriasis in Mice.
This article provides detailed methodologies for creating and evaluating the IL-23- and Aldara/IMQ-induced mouse models of psoriasis. The article also provides a protocol for analyzing skin leukocytes by flow cytometry. © 2019 by John Wiley & Sons, Inc. PMID: 30615272 [PubMed - as supplied by publisher] (Source: Current Protocols in Immunology)
Source: Current Protocols in Immunology - January 8, 2019 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

Isolation and Culture of Microglia.
This article describes approaches for purification of microglia by fluorescence-activated cell sorting using microglia-specific surface markers and for enrichment of microglia by magnetic sorting and immunopanning. Detailed information about culturing primary microglia at various developmental stages is also provided. Throughout, we focus on special considerations for handling microglia and compare the relative strengths or disadvantages of different protocols. © 2018 by John Wiley & Sons, Inc. PMID: 30414379 [PubMed - as supplied by publisher] (Source: Current Protocols in Immunology)
Source: Current Protocols in Immunology - November 12, 2018 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

Statistics for Immunologists
Authors: PMID: 30400720 [PubMed - in process] (Source: Current Protocols in Immunology)
Source: Current Protocols in Immunology - November 9, 2018 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

Ribonucleoprotein Transfection for CRISPR/Cas9-Mediated Gene Knockout in Primary T Cells.
Authors: Oh SA, Seki A, Rutz S Abstract CRISPR/Cas9 has enabled the rapid and efficient generation of gene knockouts across various cell types of several species. T cells are central players in adaptive immune responses. Gene editing in primary T cells not only represents a valuable research tool, but is also critical for next generation immunotherapies, such as CAR T cells. Broad application of CRIPSR/Cas9 for gene editing in primary T cells has been hampered by limitations in transfection efficiency and the requirement for TCR stimulation. In this article, we provide a detailed protocol for Cas9/gRNA ribonucleopr...
Source: Current Protocols in Immunology - October 20, 2018 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

Methods to Measure MDSC Immune Suppressive Activity In Vitro and In Vivo.
Authors: Solito S, Pinton L, De Sanctis F, Ugel S, Bronte V, Mandruzzato S, Marigo I Abstract This unit presents methods to assess the immunosuppressive properties of immunoregulatory cells of myeloid origin, such as myeloid-derived suppressor cells (MDSCs), both in vitro and in vivo in mice, as well as in biological samples from cancer patients. These methods could be adapted to test the impact of different suppressive populations on T cell activation, proliferation, and cytotoxic activity; moreover, they could be useful to assess the influence exerted by genetic modifications, chemical inhibitors, and drugs on im...
Source: Current Protocols in Immunology - October 13, 2018 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

Organotypic Brain Slice Cultures.
Authors: Humpel C Abstract Ex vivo cell culture models are of particular interest for neurobiologists, as these allow the study of brain cells in an isolated environment. Organotypic brain slice cultures allow growth of three-dimensional brain tissue that conserves the cellular architecture. This unit describes the preparation and culturing of organotypic brain slices from mice. In three basic protocols, the general procedure, the specific preparation of chopper slices, and slicing of whole-brain vibratome sections are described. Support protocols explain the use of postnatal or adult mice, the preparation of coron...
Source: Current Protocols in Immunology - October 13, 2018 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

Efficient CRISPR/Cas9-Mediated Mutagenesis in Primary Murine T Lymphocytes.
This article outlines a protocol for CRISPR/Cas9-mediated mutagenesis in primary T lymphocytes from Cas9 transgenic mice using retroviral delivery of guide RNAs. © 2018 by John Wiley & Sons, Inc. PMID: 30312021 [PubMed - as supplied by publisher] (Source: Current Protocols in Immunology)
Source: Current Protocols in Immunology - October 13, 2018 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

Neuroinflammation Quantification for Spinal Cord Injury.
Authors: Amo-Aparicio J, Martínez-Muriana A, Sánchez-Fernández A, López-Vales R Abstract Spinal cord injury (SCI) leads to irreversible devastating neurological disabilities. Accumulated evidence in the literature indicates that the inflammatory response that occurs in the spinal cord following injury contributes importantly to spread tissue damage to healthy regions adjacent to the lesion site, and consequently, to increase neurological deficits. Therefore, targeting inflammation could lead to the development of new therapies to prevent tissue damage and neurological impairments after S...
Source: Current Protocols in Immunology - September 28, 2018 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

Mouse Model for Human Vitiligo.
This article discusses the first mouse model of vitiligo that develops epidermal depigmentation, similar to disease in human patients. To achieve epidermal depigmentation, mice are genetically engineered to retain melanocytes in the skin epidermis. Induction of disease occurs by adoptive transfer of melanocyte-specific CD8+ T cells into recipient mice and the subsequent activation of these T cells using a viral vector. Depigmentation of the epidermis occurs within 5 to 7 weeks in a patchy pattern similar to patients with vitiligo. This article describes the methods of vitiligo induction, quantification of lesion progressio...
Source: Current Protocols in Immunology - September 28, 2018 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

LC3-Associated Phagocytosis and Antigen Presentation.
Authors: Ligeon LA, Loi M, Münz C Abstract LC3-associated phagocytosis (LAP) is an unconventional form of autophagy that relies on parts of the canonical autophagy machinery for its function. LAP is triggered upon receptor-mediated phagocytosis and is characterized by the formation of a single-membrane vesicle decorated with the autophagy protein LC3. In professional phagocytic cells, such as macrophages, the role of LAP in immune processes has been characterized, although how LAP functions at the molecular level remains poorly defined. It is important to point out that as for all autophagic pathways, the stud...
Source: Current Protocols in Immunology - September 28, 2018 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

Proximity Ligation Assay (PLA).
Authors: Alam MS Abstract Proximity ligation assay (PLA), also referred to as Duolink® PLA technology, permits detection of protein-protein interactions in situ (at distances
Source: Current Protocols in Immunology - September 23, 2018 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

Bone Marrow Chimeras to Study Neuroinflammation.
Authors: Laflamme N, Préfontaine P, Lampron A, Rivest S Abstract 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...
Source: Current Protocols in Immunology - September 19, 2018 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

Toll Receptors.
Authors: Takeda K, Akira S Abstract This has led to the identification of several important roles for Toll receptors in mammals. This unit discusses mammalian Toll receptors (TLR1-10) that have an essential role in the innate immune recognition of microorganisms. Also discussed are TLR-mediated signaling pathways and antibodies that are available to detect specific TLRs. PMID: 30153720 [PubMed] (Source: Current Protocols in Immunology)
Source: Current Protocols in Immunology - August 31, 2018 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

Cytomegalovirus Infection: Mouse Model.
Authors: Brizić I, Lisnić B, Brune W, Hengel H, Jonjić S Abstract 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. PMID: 30044539 [PubMed - as supplied b...
Source: Current Protocols in Immunology - July 28, 2018 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

Leukocyte Isolation from Brain, Spinal Cord, and Meninges for Flow Cytometric Analysis.
Authors: Manglani M, Gossa S, McGavern DB Abstract 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...
Source: Current Protocols in Immunology - July 25, 2018 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

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 from di...
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 assure it...
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 plasm...
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 parenchym...
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 genetically ...
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 transcription fac...
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 maintain t...
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 treated wi...
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 utilized in f...
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 ...
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 subdivided ...
Source: Current Protocols in Immunology - March 9, 2018 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research