Isolation of tissue mast cells.
Authors: Kulka M, Metcalfe DD
Abstract
Located primarily in tissues, mast cells are one of the principal effector cells in allergic inflammation. Mast cells derive from mononuclear precursor cells which undergo their final phase of differentiation in the tissues. Mast cells express a unique set of proteases and display functional diversity depending on the tissue in which they differentiate--a phenomenon often referred to as mast cell heterogeneity. Enzymatic digestion and density centrifugation have often been used to isolate human mast cells from tissues such as lung and skin, frequently resulting in cel...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Generation, isolation, and maintenance of human mast cells and mast cell lines derived from peripheral blood or cord blood.
Authors: Rådinger M, Jensen BM, Kuehn HS, Kirshenbaum A, Gilfillan AM
Abstract
Antigen-mediated mast cell activation is a pivotal step in the initiation of allergic disorders including anaphylaxis and atopy. To date, studies aimed at investigating the mechanisms regulating these responses, and studies designed to identify potential ways to prevent them, have primarily been conducted in rodent mast cells. However, to understand how these responses pertain to human disease, and to investigate and develop novel therapies for the treatment of human mast cell-driven disease, human mast cell models may have gre...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Epitope mapping using gram-positive surface display.
Authors: Rockberg J, Löfblom J, Hjelm B, Ståhl S, Uhlén M
Abstract
Antibodies have proven to be invaluable tools for a vast number of applications during the last decades, including protein purification and characterization, medical diagnosis and imaging, and treatment using therapeutic antibiotics. No matter what the aims of the application are, the antibody's binding characteristics will still be the main features determining the assay's reliability. Here, we describe a protocol for determination of antibody-binding epitopes using an antigen-focused, library-based approach where library members are ge...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Efficient in vivo delivery of plasmids and oligonucleotides using hemagglutinating virus of Japan envelope (HVJ-E) vector in immunological disease models.
Authors: Kitani A, Fichtner-Feigl S
Abstract
This unit describes a method for in vivo delivery of oligonucleotides or plasmids using the hemagglutinating virus of Japan envelope (HVJ-E), an inactivated Sendai virus particle, as a delivery system. Viral transfection methods generally show a higher transfection efficiency than nonviral methods for the delivery of genes to cells. However, in using these methods one must bear in mind that the introduction of a virus particle into a host carries a risk for leukemia induction and for creation of disturbances in immune function due to cytotoxicity.
PMID: ...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Measuring TLR function in transfectants.
Authors: Rallabhandi P
Abstract
This unit summarizes a combination of methods that can be optimized for measuring toll-like receptor (TLR) function. TLRs serve as primary innate immune sensors and exhibit high specificity towards evolutionarily conserved microbial and viral structures. The unit focuses specifically on TLR4, the principal Gram-negative lipopolysaccharide (LPS) sensor. Methods described include transient transfections, analyses of activation of various promoters in reporter-gene assays, and induction of IL-8 secretion. Other topics that will be briefly discussed include the necessity for the...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Measurement of myeloid cell immune suppressive activity.
Authors: Dolcetti L, Peranzoni E, Bronte V
Abstract
This unit presents simple methods to assess the immunosuppressive properties of immunoregulatory cells of myeloid origin, such as myeloid-derived suppressor cells (MDSCs), both in vitro and in vivo. These methods are general and could be adapted to test the impact of different suppressive populations on T cell activation, proliferation, and cytotoxic activity; moreover they could be useful to assess the influence exerted on immune suppressive pathways by genetic modifications, chemical inhibitors, and drugs.
PMID: 21053303 [PubMed - indexed for ME...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Cecal ligation and puncture.
Authors: Cuenca AG, Delano MJ, Kelly-Scumpia KM, Moldawer LL, Efron PA
Abstract
The cecum contains a high concentration of microbes, which are a combination of Gram-negative and Gram-positive flora. These bacteria range from anaerobic to facultative aerobic to aerobic organisms. In the procedure described in this unit, the ligation of the cecum produces a source of ischemic tissue as well as polymicrobial infection. This combination of ischemic/necrotic tissue and microbial infection distinguishes this multifactorial model from a number of other bacterial sepsis models, including but not limited to: bacter...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Measuring mast cell mediator release.
Authors: Kuehn HS, Radinger M, Gilfillan AM
Abstract
Mediators released from activated mast cells are responsible for the allergic inflammatory reactions associated with disease states such as anaphylaxis and atopy. These mediators are released as a consequence of immediate degranulation and phospholipid metabolism upon mast cell activation, followed by delayed cytokine gene expression. Thus, techniques that monitor indices of these events in mast cell culture systems, in association with biochemical analysis of parameters of cell signaling, are critical to our understanding of the molecular mechanisms reg...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Evaluation of human natural killer cell activities in whole blood.
Authors: Claus M, Watzl C
Abstract
Natural killer (NK) cells are important effector cells of the innate immune system. Activation of NK cells results in their cytotoxic activity against locally attached target cells and leads to the secretion of cytokines. These activities are usually measured in purified NK cells or isolated peripheral blood mononuclear cells. In this unit, we describe a protocol to measure NK cell cytotoxicity (lysis of (51)Cr labeled target cells), degranulation (externalization of CD107a), and cytokine production (intracellular FACS analysis of IFN-γ) in whole-blood samples. Using the...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Measurement of tumor cytolysis by macrophages.
Authors: Escórcio-Correia M, Hagemann T
Abstract
This unit describes two different protocols for the measurement of tumor cytolysis by macrophages. Traditionally, cytotoxicity assays have relied on the use of radioactive isotopes. In Basic Protocol 1, cytotoxic activity is measured by the release into the culture supernatant of a radioisotope that had been incorporated by the target cell and is released upon cell death. This poses a problem for some cell lines in which spontaneous isotope release occurs in the absence of effector cell cytotoxicity. In Basic Protocol 2, a nonradioactive approach is used to...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
In vivo and ex vivo protocols for measuring the killing of extracellular pathogens by macrophages.
Authors: Medina E, Goldmann O
Abstract
This unit describes a series of in vivo/ex vivo combined protocols for investigating the interactions (adhesion, phagocytosis, and killing) of extracellular bacteria with peritoneal murine macrophages. It includes steps needed for in vivo infection of murine peritoneal macrophages after intraperitoneal inoculation with the pathogen of interest, as well as the measurement of bacteria associated with or truly internalized by these phagocytic cells. Several protocols for the ex vivo measurement of the ability of peritoneal macrophages to kill the microorganisms that have...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Measurement of macrophage growth and differentiation.
Authors: Chitu V, Yeung YG, Yu W, Nandi S, Stanley ER
Abstract
This unit provides protocols for measuring the abundance and growth of macrophage precursors in agar cultures and the proliferation of isolated mature macrophages in vitro, by either direct cell counting or by DNA measurement. Methods for the immunohistochemical identification of macrophages and the determination of their proliferative status in vivo by immunofluorescence are also included. It also describes methods for characterization of macrophage differentiation through the immunofluorescence analysis of cell-surface expression of CSF-1 rec...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Measurement of type I interferon production.
Authors: Seeds RE, Miller JL
Abstract
The Basic Protocol in this unit describes measurement of murine interferon (IFN)α/β by intracellular staining for these cytokines and detection by flow cytometry. Alternate protocols detail an enzyme-linked immunoabsorbent assay (ELISA) for IFNα and a biological assay to measure IFN. The FACS assay allows measurement of IFNα/β production by defined cell populations, while ELISA measures secreted IFNα. The bioassay measures functional antiviral activity and is the most sensitive of the assays discussed. These assays therefore provide complementary methods to asses...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Protein tyrosine phosphatase assays.
Authors: Lorenz U
Abstract
Tyrosine phosphorylation and dephosphorylation of proteins play a critical role in many processes of the immune system, from early development to fully differentiated effector function. Since the opposing actions of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) determine the steady-state level of tyrosine phosphorylation on a given protein, it is often important for mechanistic studies to determine the specific activities of PTKs and PTPs. PTPs are defined by their enzymatic activity that catalyzes the dephosphorylation of phosphotyrosine residues. This...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Quantitative assessment of macrophage functions in repair and fibrosis.
Authors: Wynn TA, Barron L, Thompson RW, Madala SK, Wilson MS, Cheever AW, Ramalingam T
Abstract
Macrophages play key roles in wound repair and fibrosis by regulating extracellular matrix turnover. Macrophages can process matrix components themselves, but also recruit and alter the functions of other cell types that directly build or degrade extracellular matrix. Classically activated macrophages (CAM, also called M1) tend to promote tissue injury while alternatively activated macrophages (AAM, also called M2) are often linked with the mechanisms of wound repair and fibrosis. However, rather than promoting...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
