Animal models for Toxoplasma gondii infection.
Authors: Subauste C
Abstract
Toxoplasma gondii is a protozoan of worldwide distribution. This unit describes murine models of acute T. gondii infection, toxoplasmic encephalitis, and Toxoplasma retinochoroiditis. T. gondii infection in SCID mice allows the study of T cell-independent mechanisms of defense. The uracil auxotroph strain cps1-1 and temperature-sensitive mutant strains of T. gondii allow studies of immunization and adoptive transfer. In vivo study of parasite host-interaction is possible with the use of parasites that express fluorescent proteins and model antigens, plus the use of transgenic m...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Single-cell phospho-protein analysis by flow cytometry.
Authors: Schulz KR, Danna EA, Krutzik PO, Nolan GP
Abstract
This protocol describes methods for monitoring intracellular phosphorylation-dependent signaling events on a single-cell basis. This approach measures cell signaling by treating cells with exogenous stimuli, fixing cells with formaldehyde, permeabilizing with methanol, and then staining with phospho-specific antibodies. Thus, cell signaling states can be determined as a measure of how cells interact with their environment. This method has applications in clinical research as well as mechanistic studies of basic biology. In clinical research, diagn...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Target-induced natural killer cell loss as a measure of NK cell responses.
Authors: Warren H
Abstract
Natural killer (NK) cells are an important effector cell of innate immunity. Their interaction with susceptible target cells triggers NK cell cytotoxicity and the release of cytokines. Immunofluorescence flow cytometry-based assays are now the preferred methods for measuring NK cell responses. For these assays, assessment is made on NK cells (CD3(-)CD56(+) CD16(+)) within the viable lymphocyte gate, defined by the parameters of size (FSC) and granularity (SSC). Accordingly, NK cells that have not dissociated from target cells at the time of measurement, or that have undergone tar...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Analysis of the targeting properties of Fc chimeric proteins and antibodies in mice in vivo.
We present procedures to prepare the reagents to be tested under endotoxin-free conditions, suitable injection sites, methodology for tissue collection, and processing, and labeling procedures to detect the injected material in situ.
PMID: 22470135 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Immunology)
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Differentiation of mast cells from embryonic stem cells.
Authors: Kovarova M, Koller B
Abstract
In this unit, we describe a simple coculture-free method for obtaining mast cells from mouse and human embryonic stem (ES) cells. Much of our knowledge regarding the mechanisms by which mast cells are activated comes from studies of mouse bone marrow-derived mast cells. Studies of human mast cells have been hampered by the limited sources from which they can be cultured, the difficulty in introducing specific genetic changes into these cells, and differences between established cultures that reflect the unique genetic makeup of the tissue donor. Derivation of mast cel...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Isolation of cells from the feto-maternal interface.
Authors: Male V, Gardner L, Moffett A
Abstract
The mucosal lining of the human uterus is host to a specialized population of leukocytes, which, during pregnancy, interact with invading placental cells (trophoblast) of fetal origin. Of particular interest are uterine natural killer cells, which account for around 70% of the leukocytes at this site during the first trimester of pregnancy, and seem to be specially adapted to recognize invading trophoblast cells. In order to understand the interactions between mucosal immune cells and trophoblast, and those among the immune cells themselves, it is useful to be...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Differentiation and characterization of dendritic cells from human embryonic stem cells.
Authors: Mohib K, Wang L
Abstract
Human embryonic stem cells (hESCs) offer great hope in regenerative medicine. Their ability to give rise to almost any type of cell present in the adult body makes them an invaluable tool in finding cures for a variety of diseases. While considerable protocols have been devised to efficiently differentiate hESCs into various cells types including cells of hematopoietic origin, this protocol will focus on the derivation of dendritic cells (DC), a potent antigen-presenting cell. DCs are a highly important arm of the immune system, as they represent one of the few cells that ...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Generation and characterization of mouse basophils from bone marrow and purification of basophils from spleen.
Authors: Yoshimoto T, Nakanishi K
Abstract
Basophils are rare circulating granulocytes that originate from progenitor cells in the bone marrow and have been considered important effector cells in IgE-mediated allergic inflammation. Basophils constitute <1% of blood leukocytes and are usually absent or present only in small numbers in tissues. They may, however, be recruited to inflammatory sites when an antigen is present and contribute immediately to hypersensitivity reactions. Basophils can therefore serve as primary effector cells in allergic disorders. Despite a large pool of experimental evidence t...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Isolation of human eosinophils.
Authors: Akuthota P, Shamri R, Weller PF
Abstract
Highly purified eosinophils can be isolated from peripheral blood by negative selection using an antibody-based magnetic negative selection protocol. The basic protocol describes a sequential fractionation of peripheral blood in which CD16+ granulocytes are enriched first from whole blood, followed by isolation of eosinophils. This technique is easy to use, fast, and highly reproducible. Support protocols describe a staining methods that can be used to evaluate the purity of eosinophils and differentiation from other leukocyte populations.
PMID: 228...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Cryopreservation and thawing of cells.
Authors: Yokoyama WM, Thompson ML, Ehrhardt RO
Abstract
Successful cryopreservation of cells requires not only that the cells be handled in a proper fashion for harvesting with equipment in place to ensure consistency, reproducibility, and sterility, but also that a correct choice and amount of cryoprotective agent is added. In general, a controlled freezing rate of 1°C/min is necessary to retain optimal viability of the recovered cells. There are many variations of cell freezing methods in use, including costly electronically regulated control rate freezers, unstandardized, passive isopropyl alcohol free...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Measurement of phagocytosis, phagosome acidification, and intracellular killing of Staphylococcus aureus.
Authors: Sokolovska A, Becker CE, Stuart LM
Abstract
Phagocytes are an important part of host defense, playing a critical role in innate immune responses against pathogens and in the initiation of adaptive immunity. One of the main characteristics of these cells is their ability to recognize and internalize invading microorganisms into a phagosome. The internalized microbe is rapidly delivered into a mature phagolysosome where it is killed and degraded. However, numerous pathogens have evolved complex mechanisms to manipulate these intracellular organelles to establish a survival niche. Here, we describe s...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Isolation of mouse lymphocytes from small intestine tissues.
Authors: Sheridan BS, Lefrançois L
Abstract
The isolation of lymphocytes and other hematopoietic-derived cells from small intestinal tissues has become increasingly relevant to immunology over the last decade. It is also becoming increasingly clear that the impact of local immunity at the mucosal barrier of the intestine has a profound impact on immune responses at distant sites, bringing a new cadre of immunologists to the mucosal frontier. Furthermore, the ability to experimentally manipulate smaller and smaller populations of immune cells has become technologically feasible and in some cases routine. T...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Isolation and generation of human dendritic cells.
Authors: Nair S, Archer GE, Tedder TF
Abstract
Dendritic cells are highly specialized antigen-presenting cells (APC), which may be isolated or generated from human blood mononuclear cells. Although mature blood dendritic cells normally represent ∼0.2% of human blood mononuclear cells, their frequency can be greatly increased using the cell enrichment methods described in this unit. More highly purified dendritic cell preparations can be obtained from these populations by sorting of fluorescence-labeled cells. Alternatively, dendritic cells can be generated from monocytes by culture with the appropriate c...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Identification of B cell and T cell epitopes using synthetic peptide combinatorial libraries.
Authors: Pinilla C, Appel JR, Judkowski V, Houghten RA
Abstract
This unit presents a combinatorial library method that consists of the synthesis and screening of mixture-based synthetic combinatorial libraries of peptide molecules. The protocols employ peptide libraries to identify peptides recognized by MAbs and T cells. The first protocol uses a positional scanning peptide library made up of hexapeptides to identify antigenic determinants recognized by MAbs. The 120 mixtures in the hexapeptide library are tested for their inhibitory activity in a competitive ELISA. The second protocol uses a decapeptide ...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Evaluation of B cell function in patients with HIV.
Authors: Buckner CM, Kardava L, Moir S
Abstract
HIV disease is associated with abnormalities in all major lymphocyte populations, including B cells. B cell dysfunction in HIV infection is largely driven by alterations in the subsets of B cells that circulate in the blood or reside in tissues. Meaningful functional assays are thus dependent on the identification and isolation of B cell subsets present in the starting material. This unit describes several assays designed to phenotype, fractionate, and assess functional properties of B cells that circulate in the blood of HIV-infected individuals. The four pr...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
