Monoclonal Antibodies to Human Cell Surface Antigens.
Authors: Stockinger H, Majdic O, Knapp W, Swart B, Nicholson I, Zola H Abstract This appendix lists the cluster of differentiation (CD) designations for the nearly 300 different leukocyte surface molecules that have been assigned and approved by the nomenclature committee of the International Union of Immunological societies (IUIS). PMID: 33299512 [PubMed] (Source: Current Protocols in Immunology)
Source: Current Protocols in Immunology - December 13, 2020 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

Immunologic Studies in Humans.
Authors: Strober W PMID: 33299513 [PubMed] (Source: Current Protocols in Immunology)
Source: Current Protocols in Immunology - December 13, 2020 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

Immunologic Studies in Humans.
Authors: Strober W PMID: 33299514 [PubMed] (Source: Current Protocols in Immunology)
Source: Current Protocols in Immunology - December 13, 2020 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

Immunologic Studies in Humans.
Authors: Strober W PMID: 33299515 [PubMed] (Source: Current Protocols in Immunology)
Source: Current Protocols in Immunology - December 13, 2020 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

Protocols for Experimental Sj ögren's Syndrome.
This article describes the activation of specific innate immune pathways in mice to study SS salivary gland manifestations. Methodologies for evaluating salivary gland inflammation and salivary function are described. This article also describes protocols for in-house assays to measure autoantibody titers in serum. © 2020 Wiley Periodicals LLC Basic Protocol 1: Acceleration of Sjögren's syndrome by activating the toll-like receptor 3 pathway Basic Protocol 2: Induction of Sjögren's syndrome by activating the stimulator of interferon genes pathway Alternate Protocol: Acceleration of Sjögren's syndrome by...
Source: Current Protocols in Immunology - December 1, 2020 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

Development of a Rapid Focus Reduction Neutralization Test Assay for Measuring SARS-CoV-2 Neutralizing Antibodies.
Authors: Vanderheiden A, Edara VV, Floyd K, Kauffman RC, Mantus G, Anderson E, Rouphael N, Edupuganti S, Shi PY, Menachery VD, Wrammert J, Suthar MS Abstract SARS-CoV-2 is a recently emerged human coronavirus that has escalated to a pandemic. There are currently no approved vaccines for SARS-CoV-2, which causes severe respiratory illness or death. Defining the antibody response to SARS-CoV-2 will be essential for understanding disease progression, long-term immunity, and vaccine efficacy. Here we describe two methods for evaluating the neutralization capacity of SARS-CoV-2 antibodies. The basic protocol is a focus ...
Source: Current Protocols in Immunology - November 21, 2020 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

Culture of Intestinal Epithelial Cell Monolayers and Their Use in Multiplex Macromolecular Permeability Assays for In Vitro Analysis of Tight Junction Size Selectivity.
This article describes methods for culturing intestinal epithelial cell monolayers and assessing the impact of cytokine treatment on leak and unrestricted pathway permeabilities. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Generation and culture of cell monolayers in Transwells Basic Protocol 2: Assessment of cytokine (IFNγ and TNF) treatment effects on barrier function Support Protocol: Immunofluorescent staining of monolayers Basic Protocol 3: Multiplex flux assay. PMID: 33175441 [PubMed - in process] (Source: Current Protocols in Immunology)
Source: Current Protocols in Immunology - November 13, 2020 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

Co-Culture System of Human Enteroids/Colonoids with Innate Immune Cells.
Authors: Staab JF, Lemme-Dumit JM, Latanich R, Pasetti MF, Zachos NC Abstract Human intestinal enteroids derived from adult stem cells offer a relevant ex vivo system to study biological processes of the human gut. They recreate cellular and functional features of the intestinal epithelium of the small intestine (enteroids) or colon (colonoids) albeit limited by the lack of associated cell types that help maintain tissue homeostasis and respond to external challenges. In the gut, innate immune cells interact with the epithelium, support barrier function, and deploy effector functions. We have established a co-cultu...
Source: Current Protocols in Immunology - November 11, 2020 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

Coated Latex Beads as Artificial Cells for Quantitative Investigations of Receptor/Ligand Interactions.
Authors: Urlaub D, Watzl C Abstract Cellular interactions are often essential to regulate immune cell activities during an immune response. To understand the details of this process, it is necessary to study individual receptor/ligand interactions in a quantitative fashion. However, this is often very difficult or even impossible when using real cells for stimulation. Here, we present a method to use cell-sized latex beads for such studies. These beads can be coated with agonistic antibodies or specific ligands in a defined and quantifiable fashion. This creates the possibility of titrating the strength of the stim...
Source: Current Protocols in Immunology - November 5, 2020 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

Inducing Experimental Polymicrobial Sepsis by Cecal Ligation and Puncture.
Authors: Sjaastad FV, Jensen IJ, Berton RR, Badovinac VP, Griffith TS Abstract Numerous models are available for the preclinical study of sepsis, and they fall into one of three general categories: (1) administration of exogenous toxins (e.g., lipopolysaccharide, zymosan), (2) virulent bacterial or viral challenge, and (3) host barrier disruption, e.g., cecal ligation and puncture (CLP) or colon ascendens stent peritonitis (CASP). Of the murine models used to study the pathophysiology of sepsis, CLP combines tissue necrosis and polymicrobial sepsis secondary to autologous fecal leakage, as well as hemodynamic and b...
Source: Current Protocols in Immunology - October 9, 2020 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

Analysis of Antigen-Specific Human Memory B Cell Populations Based on In Vitro Polyclonal Stimulation.
This article outlines a simple and reliable strategy for efficient in vitro MBC stimulation and use of the ELISpot assay as a post-stimulation readout to determine the size of antigen-specific MBC populations. Other applications of the in vitro stimulation technique for MBC analysis are discussed. The following protocols are included. © 2020 Wiley Periodicals LLC Basic Protocol 1: Polyclonal stimulation of memory B cells using unfractionated PBMCs Alternate Protocol: Stimulation of small PBMC numbers using 96-well plates with U-bottom wells Basic Protocol 2: ELISpot assay for enumeration of memory B cell-derived antib...
Source: Current Protocols in Immunology - October 6, 2020 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

Purification, Culture, and CD19-CAR Lentiviral Transduction of Adult and Umbilical Cord Blood NK Cells.
This article outlines protocols to obtain CD19-CAR-NK cells, focusing on the importance of obtaining and culturing a purified NK cell population and how to attain good transfection efficiency. © 2020 Wiley Periodicals LLC Basic Protocol 1: Purification and culture of adult peripheral blood and umbilical cord blood NK cells Basic Protocol 2: CD19-CAR lentiviral transduction of adult peripheral blood or umbilical cord blood NK cells Support Protocol: Production of lentiviral supernatant. PMID: 33017099 [PubMed - as supplied by publisher] (Source: Current Protocols in Immunology)
Source: Current Protocols in Immunology - October 6, 2020 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

Inflammasome Assays In Vitro and in Mouse Models.
This article presents assays that allow induction and measurement of activation of different inflammasomes in mouse macrophages, human peripheral blood mononuclear cell (PBMC) cultures, and mouse peritonitis and endotoxic shock models. Basic Protocol 1 describes how to prime the inflammasome in mouse macrophages with different Toll-like receptor agonists and TNF-α; how to induce NLRP1, NLRP3, NLRC4, and AIM2 inflammasome activation by their corresponding stimuli; and how to measure inflammasome activation-mediated maturation of interleukin (IL)-1β and IL-18 and pyroptosis. Since the well-established agonists for...
Source: Current Protocols in Immunology - October 6, 2020 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

Establishment and Culture of Human Intestinal Organoids Derived from Adult Stem Cells.
Authors: Pleguezuelos-Manzano C, Puschhof J, van den Brink S, Geurts V, Beumer J, Clevers H Abstract Human intestinal organoids derived from adult stem cells are miniature ex vivo versions of the human intestinal epithelium. Intestinal organoids are useful tools for the study of intestinal physiology as well as many disease conditions. These organoids present numerous advantages compared to immortalized cell lines, but working with them requires dedicated techniques. The protocols described in this article provide a basic guide to establishment and maintenance of human intestinal organoids derived from small intest...
Source: Current Protocols in Immunology - September 19, 2020 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

Interrogating Adaptive Immunity Using LCMV.
Authors: Dangi T, Chung YR, Palacio N, Penaloza-MacMaster P Abstract In this invited article, we explain technical aspects of the lymphocytic choriomeningitis virus (LCMV) system, providing an update of a prior contribution by Matthias von Herrath and J. Lindsay Whitton. We provide an explanation of the LCMV infection models, highlighting the importance of selecting an appropriate route and viral strain. We also describe how to quantify virus-specific immune responses, followed by an explanation of useful transgenic systems. Specifically, our article will focus on the following protocols. © 2020 Wiley Periodic...
Source: Current Protocols in Immunology - September 19, 2020 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

Immunopharmacology and Quantitative Analysis of Tyrosine Kinase Signaling.
Authors: Brian BF, Guerrero CR, Freedman TS Abstract In this article we describe the use of pharmacological and genetic tools coupled with immunoblotting (Western blotting) and targeted mass spectrometry to quantify immune signaling and cell activation mediated by tyrosine kinases. Transfer of the ATP γ phosphate to a protein tyrosine residue activates signaling cascades regulating the differentiation, survival, and effector functions of all cells, with unique roles in immune antigen receptor, polarization, and other signaling pathways. Defining the substrates and scaffolding interactions of tyrosine kinases ...
Source: Current Protocols in Immunology - September 18, 2020 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

Phosphoproteome Analysis in Immune Cell Signaling.
Authors: Rathore D, Nita-Lazar A Abstract Immune cell signaling is largely regulated by protein phosphorylation. Stimulation of toll-like receptors (TLRs) by pathogen-associated ligands drives the cascade of immune response, which can be influenced by differences in phosphoprotein abundance. Therefore, the analysis of phosphorylation signatures at a global level is central to understanding the complex and integrated signaling in macrophages upon pathogen attack. Here, we describe a mass spectrometry-based approach to identify and quantify phosphoproteome changes in response to the stimulation of TLR2, TLR4, and TLR...
Source: Current Protocols in Immunology - September 18, 2020 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

In Vitro Assays to Study PD-1 Biology in Human T Cells.
We describe a phospho flow cytometry method to determine how PD-1 ligation alters the level of CD3ζ phosphorylation on Tyr142 , which can be easily applied to other proximal signaling proteins. Basic Protocol 2 describes a plate-bound assay that is useful to examine the long-term consequences of PD-1 ligation such as cytokine production and T cell proliferation. Complementary to that, Basic Protocol 3 describes an in vitro superantigen-based assay to evaluate T cell responses to therapeutic agents targeting the PD-1/PD-L axis, as well as immune synapse formation in the presence of PD-1 engagement. Finally, in Basic Pr...
Source: Current Protocols in Immunology - August 13, 2020 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

Characterization of Anaphylatoxin Receptor Expression and C3a/C5a Functions in Anaphylatoxin Receptor Reporter Mice.
Authors: Laumonnier Y, Karsten CM, Köhl G, Köhl J Abstract The anaphylatoxins (AT) C3a and C5a are effector molecules of C3 and C5 exerting multiple biologic functions through binding and activation of their cognate G protein-coupled receptors. C3a interacts with the C3a receptor (C3aR), whereas C5a and its primary degradation product C5a-desArg engage C5aR1 and C5aR2. In the past, analysis of AT expression has been hampered by cross reaction of antibodies designed to recognize the different AT receptors. Furthermore, assessment of effects mediated by cell-specific activation has been difficult. Here, flo...
Source: Current Protocols in Immunology - July 28, 2020 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

Enrichment of Neutrophils and Monocytes From the Liver Following Either Oral or Intravenous Listeria monocytogenes Infection.
This article includes detailed instructions for use of either foodborne or i.v. inoculation of mice and discusses the rationale for choosing either model. Additionally, a protocol is provided for enrichment of neutrophils and monocytes from the infected liver in a manner that allows for determination of bacterial burden while still providing sufficient cells for use in flow cytometric analysis or in vitro assays. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Foodborne L. monocytogenes infection Support Protocol 1: Preparing L. monocytogenes for foodborne infection Basic Protocol 2: Intravenous L. monocytogenes infec...
Source: Current Protocols in Immunology - July 28, 2020 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

A Novel In Vitro Mouse Model to Study Mycobacterium tuberculosis Dissemination Across Brain Vessels: A Combination Granuloma and Blood-Brain Barrier Mouse Model.
We present an in vitro mouse model of the BBB to test Mycobacterium tuberculosis (Mtb) dissemination across brain endothelial cells. One-third of the global population is infected with Mtb, and in 1%-2% of cases bacteria invade the CNS through a largely unknown process. The "Trojan horse" theory supports the role of a cellular carrier that engulfs bacteria and carries them to the brain without being recognized. We present for the first time a protocol for an in vitro BBB-granuloma model that supports the Trojan horse mechanism of Mtb dissemination into the CNS. Handling of bacterial cultures, in vivo and in vitro...
Source: Current Protocols in Immunology - July 28, 2020 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research

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 - April 30, 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