Experimental and computational tools for analysis of signaling networks in primary cells.
Authors: Schoof EM, Linding R
Abstract
Cellular information processing in signaling networks forms the basis of responses to environmental stimuli. At any given time, cells receive multiple simultaneous input cues, which are processed and integrated to determine cellular responses such as migration, proliferation, apoptosis, or differentiation. Protein phosphorylation events play a major role in this process and are often involved in fundamental biological and cellular processes such as protein-protein interactions, enzyme activity, and immune responses. Determining which kinases phosphorylate specific pho...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Isolation and analysis of mouse microglial cells.
Authors: Garcia JA, Cardona SM, Cardona AE
Abstract
Microglia are mononuclear phagocytes that make up about 10% of the central nervous system (CNS). They are known for their surveillant behavior, which involves continuous monitoring of neural tissue by extending and retracting their processes. Microglial cells are derived from myeloid progenitor cells and play important roles in homeostasis as well as inflammatory and immune responses in the brain. This unit describes several microglial cell isolation protocols that can be easily adapted for projects requiring a rapid and efficient analysis of mouse microg...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Dextran sulfate sodium (DSS)-induced colitis in mice.
Authors: Chassaing B, Aitken JD, Malleshappa M, Vijay-Kumar M
Abstract
Inflammatory bowel diseases (IBD), mainly comprising ulcerative colitis and Crohn's Disease, are complex and multifactorial diseases with unknown etiology. For the past 20 years, to study human IBD mechanistically, a number of murine models of colitis have been developed. These models are indispensable tools to decipher underlying mechanisms of IBD pathogenesis as well as to evaluate a number of potential therapeutics. Among various chemically induced colitis models, the dextran sulfate sodium (DSS)-induced colitis model is widely used ...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Differentiation and characterization of myeloid cells.
Authors: Gupta D, Shah HP, Malu K, Berliner N, Gaines P
Abstract
Ex vivo differentiation of myeloid cells begins with an enriched population of bone marrow-derived hematopoietic stem cells generated by lineage depletion and/or positive selection for CD34(+) antigen (human) or Sca-1(+) (mouse) cells, which are then expanded and subsequently induced in vitro in a process that recapitulates normal myeloid development. Myeloid cell lines include two human leukemic cell lines, NB-4 and HL-60, which have been demonstrated to undergo retinoic acid-induced myeloid development; however, both cell lines exhibit defe...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Immunologic applications of conditional gene modification technology in the mouse.
Authors: Sharma S, Zhu J
Abstract
Since the success of homologous recombination in altering mouse genome and the discovery of Cre-loxP system, the combination of these two breakthroughs has created important applications for studying the immune system in the mouse. Here, we briefly summarize the general principles of this technology and its applications in studying immune cell development and responses; such implications include conditional gene knockout and inducible and/or tissue-specific gene over-expression, as well as lineage fate mapping. We then discuss the pros and cons of a few commonly used Cre-e...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Immunomagnetic isolation of pathogen-containing phagosomes and apoptotic blebs from primary phagocytes.
Authors: Steinhäuser C, Dallenga T, Tchikov V, Schaible UE, Schütze S, Reiling N
Abstract
Macrophages and polymorphonuclear neutrophils are professional phagocytes essential in the initial host response against intracellular pathogens such as Mycobacterium tuberculosis. Phagocytosis is the first step in phagocyte-pathogen interaction, where the pathogen is engulfed into a membrane-enclosed compartment termed a phagosome. Subsequent effector functions of phagocytes result in killing and degradation of the pathogen by promoting phagosome maturation, and, terminally, phago-lysosome fusion. Intracellular pat...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Animal models for candidiasis.
Authors: Conti HR, Huppler AR, Whibley N, Gaffen SL
Abstract
Multiple forms of candidiasis are clinically important in humans. Established murine models of disseminated, oropharyngeal, vaginal, and cutaneous candidiasis caused by Candida albicans are described in this unit. Detailed materials and methods for C. albicans growth and detection are also described. Curr. Protoc. Immunol. 105:19.6.1-19.6.17. © 2014 by John Wiley & Sons, Inc.
PMID: 24700323 [PubMed - in process] (Source: Current Protocols in Immunology)
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Isolation of murine natural killer cells.
Authors: Pak-Wittel MA, Piersma SJ, Plougastel BF, Poursine-Laurent J, Yokoyama WM
Abstract
This unit describes the isolation of natural killer (NK) cells from mouse spleen. The basic protocol describes a method for preparing a highly purified NK cell population from mouse spleen by depletion of contaminating cells with selected monoclonal antibodies (MAbs) and magnetic separation. There are several advantages to this negative selection process. One of these is that the NK cells are not coated with antibody and, therefore, are not at risk of functional perturbation by antibody cross-linking. Additionally, ...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Glomerulonephritis Induced by Heterologous Anti-GBM Globulin as a Planted Foreign Antigen.
Authors: Odobasic D, Ghali JR, O'Sullivan KM, Holdsworth SR, Kitching AR
Abstract
The glomerulonephritides are diseases characterized by immune-mediated glomerular inflammation. Most severe and rapidly progressive forms of glomerulonephritis feature the participation of injurious leukocytes that localize to glomeruli. This unit describes classical models of rapidly progressive glomerulonephritis in mice, induced by injecting heterologous globulin (raised in sheep) that binds to the glomerular basement membrane. These models have been particularly useful in defining the participation of effector leukocytes ...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Oil-based emulsion vaccine adjuvants.
Authors: Schijns VE, Strioga M, Ascarateil S
Abstract
Vaccine adjuvants are critical components in experimental and licensed vaccines used in human and veterinary medicine. When aiming to evoke an immune response to a purified antigen, the administration of antigen alone is often insufficient, unless the antigen contains microbial structures or has a natural particulate structure. In most cases, the rationale to use an adjuvant is obvious to the experimental immunologist or the professional vaccinologist, who is familiar with the nature of the antigen, and the aim of the vaccine to elicit a specific antibo...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Isolation and characterization of mouse innate lymphoid cells.
Authors: Halim TY, Takei F
Abstract
Innate lymphoid cells (ILCs) are rare populations of cytokine-producing lymphocytes and are divided into three groups, namely ILC1, ILC2, and ILC3, based on the cytokines that they produce. They comprise less than 1% of lymphocytes in mucosal tissues and express no unique cell surface markers. Therefore, they can only be identified by combinations of multiple cell surface markers and further characterized by cytokine production in vitro. Thus, multicolor flow cytometry is the only reliable method to purify and characterize ILCs. Here we describe the methods for cell prep...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Measurement of Peptide Binding to MHC Class II Molecules by Fluorescence Polarization.
Authors: Yin L, Stern LJ
Abstract
Peptide binding to major histocompatibility complex class II (MHCII) molecules is a key process in antigen presentation and CD4+ T cell epitope selection. This unit describes a fairly simple but powerful fluorescence polarization-based binding competition assay to measure peptide binding to soluble recombinant MHCII molecules. The binding of a peptide of interest to MHCII molecules is assessed based on its ability to inhibit the binding of a fluorescence-labeled probe peptide, with the strength of binding characterized as IC50 (concentration required for 50% inhibition of ...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
T cell mediated autoimmune glomerular disease in mice.
Authors: Ooi JD, Gan PY, Odobasic D, Holdsworth SR, Kitching AR
Abstract
Many forms of glomerulonephritis are mediated by autoimmunity. While autoantibodies are often pathogenic, cell-mediated immunity plays an important role in a number of forms of rapidly progressive glomerulonephritis. This unit describes the induction of cell-mediated autoimmune glomerular disease in mice. One disease model, experimental anti-glomerular basement membrane (GBM) disease, features autoreactivity to a well-defined component of type IV collagen found in the GBM, α3(IV)NC1. The other models the cell-mediated effector respon...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Isolation and purification of proteasomes from primary cells.
We describe a protocol to purify functionally active proteasomes from human CD4(+) T cells and dendritic cells derived from peripheral blood mononuclear cells. The purification is a three-step process involving ion-exchange chromatography, ammonium sulfate precipitation, and sucrose density gradient ultracentrifugation. This method can be easily adapted to purify proteasomes from cell lines or from organs. Methods to characterize and visualize the purified proteasomes are also described. © 2014 by John Wiley & Sons, Inc.
PMID: 25367127 [PubMed - in process] (Source: Current Protocols in Immunology)
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Isolation of thymic epithelial cells and analysis by flow cytometry.
Authors: Jain R, Gray DH
Abstract
The epithelial cells of the thymus govern the differentiation of hematopoietic precursors into T cells, which are critical for acquired immunity. Thymic epithelial cells (TEC) provide molecular cues that direct precursor recruitment, commitment to the T cell lineage, thymocyte proliferation, and the processes of positive and negative selection. Despite the importance of TEC to the immune system, fundamental questions regarding their differentiation, turnover, and function throughout life remain unanswered. This knowledge gap is largely due to technical difficulties in isol...
Source: Current Protocols in Immunology - November 16, 2014 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
