Measurement of phagocytic engulfment of apoptotic cells by macrophages using pHrodo succinimidyl ester.
Authors: Aziz M, Yang WL, Wang P
Abstract
Considerable interest has emerged towards phagocytosis of apoptotic cells, due to its intricate molecular mechanisms and important regulatory functions in development, homoeostasis, and immune tolerance. Impaired clearance of apoptotic cells leads to immune-mediated disorders. Current quantification methods of the engulfment of apoptotic cells by macrophages are potentially flawed by several limitations. Adherent macrophage populations are overlaid with apoptotic targets in suspension and then co-cultured for a definite period, which may give rise to two different ...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Comparing individual NK cell activity in vitro.
Authors: Sierich H, Eiermann T
Abstract
Natural killer (NK) cells play an important role in the innate immune system by eliminating infected and mutated cells. Their cytotoxic capacities vary markedly among individuals. The cytotoxic activity can be measured in peripheral blood mononuclear cells (PBMCs) using the NK cell-specific target cell line K562. In this chapter, we present a protocol for the standardization and normalization of cell preparation and NK cell cytotoxicity measurement in a (51)Cr-release assay. By following these protocols, it is possible to compare the NK cell activity of numerous-if n...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Experimental autoimmune myasthenia gravis in the mouse.
Authors: Wu B, Goluszko E, Huda R, Tüzün E, Christadoss P
Abstract
Myasthenia gravis (MG) is a T cell-dependent antibody-mediated autoimmune neuromuscular disease. Antibodies to the nicotinic acetylcholine receptor (AChR) destroy the AChR, thus leading to defective neuromuscular transmission of electrical impulse and to muscle weakness. This unit is a practical guide to the induction and evaluation of experimental autoimmune myasthenia gravis (EAMG) in the mouse, the animal model for MG. Protocols are provided for the extraction and purification of AChR from the electric organs of Torpedo californica, or...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Measurement of MHC/peptide interactions by gel filtration or monoclonal antibody capture.
Authors: Sidney J, Southwood S, Moore C, Oseroff C, Pinilla C, Grey HM, Sette A
Abstract
This unit describes a technique for the direct and quantitative measurement of the capacity of peptide ligands to bind Class I and Class II MHC molecules. The binding of a peptide of interest to MHC is assessed based on its ability to inhibit the binding of a radiolabeled probe peptide to purified MHC molecules. This unit includes protocols for the purification of Class I and Class II MHC molecules by affinity chromatography, and for the radiolabeling of peptides using the chloramine T method. An alternate protocol des...
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
Measuring intravascular migration of mouse Ly6C(low) monocytes in vivo using intravital microscopy.
Authors: Carlin LM, Auffray C, Geissmann F
Abstract
This unit describes methods for intravital imaging of monocytes in the vasculature of the dermis and the mesentery in vivo using fluorescent reporter mice, fluorescent dyes, and antibodies. Cx3cr1(gfp/gfp (or +)), Rag2(-/-), Il2rg(-/-) mice expressing eGFP at the locus of the Cx3cr1 gene, on the Rag2(-/-) Il2rg(-/-) C57Bl/6 background, are used. Although aimed at specifically tracking Ly6C(low) monocytes, these protocols could readily be adapted to investigate the interaction of other blood leukocytes with the vascular endothelium by use of other fluoresc...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Autoimmune myocarditis, valvulitis, and cardiomyopathy.
Authors: Myers JM, Fairweather D, Huber SA, Cunningham MW
Abstract
Myocarditis and valvulitis are inflammatory diseases affecting myocardium and valve. Myocarditis, a viral-induced disease of myocardium, may lead to dilated cardiomyopathy and loss of heart function. Valvulitis leads to deformed heart valves and altered blood flow in rheumatic heart disease. Animal models recapitulating these diseases are important in understanding the human condition. Cardiac myosin is a major autoantigen in heart, and antibodies and T cells to cardiac myosin are evident in inflammatory heart diseases. This unit is a pract...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Mouse model for pyelonephritis.
Authors: Tittel AP, Heuser C, Kurts C
Abstract
This unit describes a simple way to induce pyelonephritis in female mice using uropathogenic Escherichia coli (UPEC). Methods for culturing and preparing working stocks of UPEC are provided. Protocols to measure the bacterial load in the murine kidney following the establishment of pyelonephritis by determining the bacterial colony forming units (CFU) are also included. This assay can be performed with kidney homogenates if the bacterial load is the sole read-out or if cellular intactness is not required for subsequent assays, or from kidney single cell suspen...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Production of monoclonal antibodies.
Authors: Yokoyama WM, Christensen M, Dos Santos G, Miller D, Ho J, Wu T, Dziegelewski M, Neethling FA
Abstract
This unit describes the production of monoclonal antibodies beginning with immunization, cell fusion, and selection. Support protocols are provided for screening primary hybridoma supernatants for antibodies of desired specificity, establishment of stable hybridoma lines, cloning of these B cell lines by limiting dilution to obtain monoclonal lines, and preparation of cloning/expansion medium. An alternate protocol describes cell fusion and one-step selection and cloning of hybridomas utilizing a ...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Nonhuman primate models for HIV/AIDS vaccine development.
Authors: Sui Y, Gordon S, Franchini G, Berzofsky JA
Abstract
The development of HIV vaccines has been hampered by the lack of an animal model that can accurately predict vaccine efficacy. Chimpanzees can be infected with HIV-1 but are not practical for research. However, several species of macaques are susceptible to the simian immunodeficiency viruses (SIVs) that cause disease in macaques, which also closely mimic HIV in humans. Thus, macaque-SIV models of HIV infection have become a critical foundation for AIDS vaccine development. Here we examine the multiple variables and considerations that must be ta...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Dynamic quantitative assays of phagosomal function.
Authors: Podinovskaia M, VanderVen BC, Yates RM, Glennie S, Fullerton D, Mwandumba HC, Russell DG
Abstract
Much of the activity of the macrophage as an effector cell is performed within its phagocytic compartment. This ranges from the degradation of tissue debris as part of its homeostatic function to the generation of the superoxide burst as part of its microbicidal response to infection. We have developed a range of real-time readouts of phagosomal function that enable these activities to be rigorously quantified. This unit contains descriptions of several of these assays assessed by different methods of...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
A practical guide to culturing mouse and human bone marrow stromal cells.
Authors: Nemeth K, Mayer B, Sworder BJ, Kuznetsov SA, Mezey E
Abstract
Bone marrow stromal cells (BMSCs, frequently also called MSCs) represent a cell population within the bone marrow, a subset of which contains multipotent stem cells. Their primary role is to produce and maintain both bone tissue and bone marrow microenvironment necessary for hematopoiesis. The latter is achieved by secreting a wide variety of different cytokines and growth factors, many of which also have a regulatory role in immune processes. BMSCs have recently been introduced into the field of immunobiology after their successful cli...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Single-domain antibodies and their utility.
Authors: Baral TN, MacKenzie R, Arbabi Ghahroudi M
Abstract
Engineered monoclonal antibody fragments have gained market attention due to their versatility and tailor-made potential and are now considered to be an important part of future immunobiotherapeutics. Single-domain antibodies (sdAbs), also known as nanobodies, are derived from VHHs [variable domains (V) of heavy-chain-only antibodies (HCAb)] of camelid heavy-chain antibodies. These nature-made sdAbs are well suited for various applications due to their favorable characteristics such as small size, ease of genetic manipulation, high affinity and so...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Immunohistochemistry.
Authors: Hofman FM, Taylor CR
Abstract
This unit describes several methods for localizing specific antigens in various tissue and cell preparations using immunohistochemistry (IHC). Protocols describe preparation of suitable material for IHC including fresh, unfixed, frozen tissue specimens; unfixed cells, either freshly isolated or derived from suspension or adherent cultures; or fixed, paraffin-embedded tissue sections. By careful selection of reagents, it is possible to detect two or even three antigens simultaneously. For antigens that are sensitive to fixative, it may be necessary to unmask the antige...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Schistosomiasis.
Authors: Tucker MS, Karunaratne LB, Lewis FA, Freitas TC, Liang YS
Abstract
Schistosomiasis is the second most important parasitic disease in the world in terms of public health impact. Globally, it is estimated that the disease affects over 200 million people and is responsible for 200,000 deaths each year. The three major schistosomes infecting humans are Schistosoma mansoni, S. japonicum, and S. haematobium. Much immunological research has focused on schistosomiasis because of the pathological effects of the disease, which include liver fibrosis and bladder dysfunction. This unit covers a wide range of ...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
