A Comprehensive Experimental Guide to Studying Cross-Presentation in Dendritic Cells In Vitro.
Authors: Sadiq BA, Mantel I, Blander JM
Abstract
Cross-presentation was first observed serendipitously in the 1970s. The importance of it was quickly realized and subsequently attracted great attention from immunologists. Since then, our knowledge of the ability of certain antigen presenting cells to internalize, process, and load exogenous antigens onto MHC-I molecules to cross-prime CD8+ T cells has increased significantly. Dendritic cells (DCs) are exceptional cross-presenters, thus making them a great tool to study cross-presentation but the relative rarity of DCs in circulation and in tissues makes it...
Source: Current Protocols in Immunology - December 15, 2020 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
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 the administrat...
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...
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...
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...
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 hemodyna...
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 antibody-...
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 NLRP1 ar...
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 sma...
Source: Current Protocols in Immunology - September 19, 2020 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
