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 number, ...
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.
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 epithelial tissue without resorting to long-term culture or transformation can be difficult. Here, specific approaches are described...
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 ...
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 str...
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 ...
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, infiltra...
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 ...
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, charact...
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 cel...
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 ...
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 i...
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 a...
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 cytomet...
Source: Current Protocols in Immunology - March 7, 2019 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
