Differentiation and Characterization of Tr1 Cells.
Authors: Chihara N, Madi A, Karwacz K, Awasthi A, Kuchroo VK
Abstract
Regulatory T cell-mediated suppression serves as a pivotal mechanism of negative regulation of immune-mediated inflammation. Type 1 regulatory T cells (Tr1 cells) are an important subset of CD4+ T cells that prevent excessive inflammatory responses and maintain immune tolerance. The anti-inflammatory role of Tr1 cells is mediated in part by their production of interleukin 10 (IL-10), which dampens the function of both antigen-presenting cells and antigen-specific effector T cells. Additionally, Tr1 cells can kill effector and myeloid cel...
Source: Current Protocols in Immunology - April 3, 2016 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
In Vivo Depletion of T Lymphocytes.
Authors: Laky K, Kruisbeek AM
Abstract
In vivo depletion of T lymphocytes is a means of studying the role of specific T cell populations during defined phases of in vivo immune responses. In this unit, a protocol is provided for injecting monoclonal antibodies (mAbs) into wild-type adult mice. Depletion of the appropriate subset of cells is verified by flow cytometry analysis of lymph node and spleen cell suspensions in pilot experiments. Once conditions have been established, depleted mice can be used to study the impact of T cell subsets on a variety of in vivo immune responses. The depleted condition ma...
Source: Current Protocols in Immunology - April 3, 2016 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Contact Hypersensitivity.
Authors: Gaspari AA, Katz SI, Martin SF
Abstract
Contact hypersensitivity (CHS) is a simple in vivo assay of cell-mediated immune function in which exposure of epidermal and dermal cells to exogenous haptens results in a delayed-type hypersensitivity (DTH) reaction that can be measured and quantified. Epidermal Langerhans cells and dermal dendritic cells are the critical antigen-presenting cells in this reaction which initiate sensitization to haptens by presenting antigens to CD4- and CD8-bearing T lymphocytes which, in turn, secrete cytokines and recruit other cells to the site of the reaction. In the pr...
Source: Current Protocols in Immunology - April 3, 2016 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Measuring Apoptosis by Microscopy and Flow Cytometry.
Authors: Hollville E, Martin SJ
Abstract
Apoptosis is a mode of programmed cell death that plays an important role during development and in the maintenance of tissue homeostasis. Numerous physiological as well as pathological stimuli trigger apoptosis such as engagement of Fas, TRAIL, or TNF receptors, growth factor deprivation, hypoxia, or exposure to cytotoxic drugs. Apoptosis is coordinated from within by members of the caspase family of cysteine proteases that, upon activation, trigger a series of morphological changes including cell shrinkage, extensive plasma membrane blebbing, chromatin condensatio...
Source: Current Protocols in Immunology - February 4, 2016 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Culture, Expansion, and Differentiation of Murine Megakaryocytes from Fetal Liver, Bone Marrow, and Spleen.
Authors: Schulze H
Abstract
Megakaryocytes (MKs) are the source of circulating platelets and are readily recognized by their large size and distinctive morphology. Their poor representation in hematopoietic tissues often requires considerable ex vivo expansion to generate cells for biochemical and cell biological studies. These experimental protocols describe the assessment of megakaryocytic potential within hematopoietic precursor cells in the bone marrow by colony-forming assays and expansion and enrichment of MKs from cultured fetal liver or spleen or bone marrow cells. Although these MKs are not synchr...
Source: Current Protocols in Immunology - February 4, 2016 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Isolation of T Cells Using Rosetting Procedures.
Authors: Kanof ME
Abstract
This unit describes a procedure for separating T cells from other mononuclear cells by exploiting the unique ability of cells to bind to and form rosettes with sheep red blood cells (SRBC). This isolation method also allows recovery of the nonrosetting cell population (B lymphocytes, monocytes, and macrophages). Neuraminidase- and 2-aminoethylisothiouronium bromide (AET)-treated SRBC are used for rosetting because of enhanced binding to T cells. It should be noted that use of the rosetting technique to obtain purified T cells or purified non-T cells by negative selection has ...
Source: Current Protocols in Immunology - February 4, 2016 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Isolation of Human Basophils.
We describe herein laboratory protocols that have been refined over the past ∼25 years that now enable investigators to prepare basophils for use in a variety of assays to assess the in vitro biology of these immune cells, both in IgE -dependent and -independent responses. © 2016 by John Wiley & Sons, Inc.
PMID: 26836512 [PubMed - as supplied by publisher] (Source: Current Protocols in Immunology)
Source: Current Protocols in Immunology - February 4, 2016 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Isolation and Functional Analysis of Human Neutrophils.
Authors: Kuhns DB, Priel DA, Chu J, Zarember KA
Abstract
This unit describes the isolation of human polymorphonuclear neutrophils (PMN) from blood using dextran sedimentation and Percoll or Ficoll-Paque density gradients. Assays of neutrophil functions including respiratory burst activation, phagocytosis, and microbial killing are also described. © 2015 by John Wiley & Sons, Inc.
PMID: 26528633 [PubMed - in process] (Source: Current Protocols in Immunology)
Source: Current Protocols in Immunology - November 7, 2015 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Trypan Blue Exclusion Test of Cell Viability.
Authors: Strober W
Abstract
The protocol described in this appendix allows for light microscopic quantitation of cell viability. Cells are suspended in PBS containing trypan blue and then examined to determine the percentage of cells that have clear cytoplasm (viable cells) versus cells that have blue cytoplasm (nonviable cells). © 2015 by John Wiley & Sons, Inc.
PMID: 26529666 [PubMed - in process] (Source: Current Protocols in Immunology)
Source: Current Protocols in Immunology - November 7, 2015 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Models to Study NK Cell Biology and Possible Clinical Application.
Authors: Zamora AE, Grossenbacher SK, Aguilar EG, Murphy WJ
Abstract
Natural killer (NK) cells are large granular lymphocytes of the innate immune system, responsible for direct targeting and killing of both virally infected and transformed cells. NK cells rapidly recognize and respond to abnormal cells in the absence of prior sensitization due to their wide array of germline-encoded inhibitory and activating receptors, which differs from the receptor diversity found in B and T lymphocytes that is due to the use of recombination-activation gene (RAG) enzymes. Although NK cells have traditionally been descr...
Source: Current Protocols in Immunology - August 4, 2015 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Enzyme-Linked Immunosorbent Assays.
Authors: Hornbeck PV
Abstract
This unit describes six different ELISA systems for the detection of specific antibodies, soluble antigens, or cell-surface antigens. In all six systems, soluble reactants are removed from solution after specifically binding to solid-phase reactants. In the first four protocols, solid-phase reactants are prepared by adsorbing an antigen or antibody onto plastic microtiter plates; in the next two protocols, the solid-phase reactants are cell-associated molecules. In all protocols, the solid-phase reagents are incubated with secondary or tertiary reactants covalently coupled to ...
Source: Current Protocols in Immunology - August 4, 2015 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Isolation of Mouse Neutrophils.
Authors: Swamydas M, Luo Y, Dorf ME, Lionakis MS
Abstract
Neutrophils represent the first line of defense against bacterial and fungal pathogens. Indeed, patients with inherited and acquired qualitative and quantitative neutrophil defects are at high risk for developing bacterial and fungal infections and suffering adverse outcomes from these infections. Therefore, research aiming at defining the molecular factors that modulate neutrophil effector function under homeostatic conditions and during infection is essential for devising strategies to augment neutrophil function and improve the outcome of infecte...
Source: Current Protocols in Immunology - August 4, 2015 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Detection of Intracellular Cytokines by Flow Cytometry.
Authors: Yin Y, Mitson-Salazar A, Prussin C
Abstract
Intracellular cytokine staining (ICCS), employing fluorescently labeled MAbs detected by flow cytometry, has emerged as the premier technique for studying cytokine expression at the single-cell level. Advances in polychromatic flow cytometry have dramatically enhanced the sophistication of ICCS investigations. ICCS can simultaneously measure multiple cytokines within a single cell, allowing the detection of complex cytokine phenotypes. Additionally, cytokines can be measured with a variety of other analytes, including transcription factors, proliferation...
Source: Current Protocols in Immunology - August 4, 2015 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Toll-like receptors.
Authors: Takeda K, Akira S
Abstract
The mammalian Toll-like receptor (TLR) family consists of 13 members, and recognizes specific patterns of microbial components, called pathogen-associated molecular patterns (PAMPs). TLR-dependent recognition of PAMPs leads to activation of the innate immune system, which subsequently leads to activation of antigen-specific adaptive immunity. The TLR-mediated signaling pathways consist of the MyD88-dependent pathway and TRIF-dependent pathway, both of which induce gene expression. This unit discusses mammalian TLRs (TLR1 to 13) that have an essential role in the innate i...
Source: Current Protocols in Immunology - April 9, 2015 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Measurement of bacterial ingestion and killing by macrophages.
Authors: Drevets DA, Canono BP, Campbell PA
Abstract
This unit presents assays that allow accurate measurement of phagocytosis and killing of bacteria by macrophages. The first basic protocol describes how to measure the ability of macrophages to ingest bacteria. Importantly, because macrophage phagocytosis entails separate binding and internalization steps, assays are described here that will also determine the extent to which bacteria bound to the macrophage are in fact internalized. Two effective methods to do this are described in alternate protocols. Both of these alternate protocols rely on enumerati...
Source: Current Protocols in Immunology - April 9, 2015 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
