Gut Microbiome Standardization in Control and Experimental Mice.
Authors: McCoy KD, Geuking MB, Ronchi F
Abstract
Mouse models are used extensively to study human health and to investigate the mechanisms underlying human disease. In the past, most animal studies were performed without taking into consideration the impact of the microbiota. However, the microbiota that colonizes all body surfaces, including the gastrointestinal tract, respiratory tract, genitourinary tract, and skin, heavily impacts nearly every aspect of host physiology. When performing studies utilizing mouse models it is critical to understand that the microbiome is heavily impacted by environmental f...
Source: Current Protocols in Immunology - April 4, 2017 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Quantitation of DNA and RNA with Absorption and Fluorescence Spectroscopy.
Authors: Gallagher SR
Abstract
Quantitation of nucleic acids is a fundamental tool in molecular biology that requires accuracy, reliability, and the use of increasingly smaller sample volumes. This unit describes the traditional absorbance measurement at 260 nm and three more sensitive fluorescence techniques employing Hoechst 33258, ethidium bromide, and PicoGreen. The range of the assays covers 25 pg/ml to 50 µg/ml. Absorbance at 260 nm has an effective range from 1 to 50 µg/ml; Hoechst 33258 from 0.01 to 15 µg/ml; ethidium bromide from 0.1 to 10 µg/ml; and PicoGreen from 25 to 1000 pg/ml. ©...
Source: Current Protocols in Immunology - February 4, 2017 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Isotype Determination of Antibodies.
Authors: Hornbeck P, Fleisher TA, Papadopoulos NM
Abstract
On identifying a new monoclonal antibody, or in characterizing antibodies in sera evoked by disease or immunization, it is particularly informative to determine the serological class of the antibodies. The serological class of the protein is determined by the structure of the antibody constant region. Several methods of class or isotype determination are outlined in this unit: sandwich ELISA, electrophoresis, and immunofixation, or use of a variety of commercially available kits. Different purification schemes and approaches for enzymatic fragmenta...
Source: Current Protocols in Immunology - February 4, 2017 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Double-Immunodiffusion Assay for Detecting Specific Antibodies (Ouchterlony).
Authors: Hornbeck P
Abstract
The method first described by Ouchterlony in 1948 is a classic and simple technique that permits evaluation and comparison of antibodies in animal or human sera directed against protein or complex carbohydrate antigens. It is a low-tech procedure that may provide information on the relative quantity of antibody activity and the nature of the antigenic epitopes in different preparations. © 2017 by John Wiley & Sons, Inc.
PMID: 28150863 [PubMed - in process] (Source: Current Protocols in Immunology)
Source: Current Protocols in Immunology - February 4, 2017 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Web Sites of Interest to Immunologists.
Authors: Lefranc MP
Abstract
This appendix includes a selection of links of interest to immunologists from the international ImMunoGeneTics (IMGT) database, covering a wide variety of subject matter. © 2017 by John Wiley & Sons, Inc.
PMID: 28150864 [PubMed - in process] (Source: Current Protocols in Immunology)
Source: Current Protocols in Immunology - February 4, 2017 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
MHC-Peptide Tetramers to Visualize Antigen-Specific T Cells.
Authors: Altman JD, Davis MM
Abstract
Mature T lymphocytes of the CD8 or CD4 classes bear αβ T cell receptors (TCR) that are specific for a molecular complex consisting of a major histocompatibility complex class I or II (MHC class I or II) molecule bound to a unique self or foreign peptide. Until recently, methods for monitoring antigen-specific T cell immune responses were restricted primarily to functional assays based on limiting dilution analysis, because the lack of specific molecular reagents to identify clonal T cells obviated approaches to identify and enumerate specific T cells. Development of ...
Source: Current Protocols in Immunology - November 3, 2016 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Isolation and Characterization of Exosomes from Cultures of Tissue Explants and Cell Lines.
Authors: Mincheva-Nilsson L, Baranov V, Nagaeva O, Dehlin E
Abstract
Exosomes are specialized, nanometer-sized extracellular vesicles of endosomal origin actively secreted into the extracellular space by a variety of cells under normal and pathological conditions. Exosomes have recently emerged as important intercellular communicators and modulators of diverse mechanisms and cellular responses. Characterization of their composition and function will open possibilities for new diagnostic methods and promising therapeutic approaches based on nanobiology. This unit provides a standard isolation procedure for ...
Source: Current Protocols in Immunology - November 3, 2016 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Induction and Quantification of NETosis.
Authors: Carmona-Rivera C, Kaplan MJ
Abstract
Neutrophil extracellular traps (NET) are web-like structures composed of nuclear material and neutrophil granular proteins that are released to the extracellular space after specific microbial or sterile inflammatory stimuli. NETosis is a specific type of cell death that ensues when neutrophils extrude NETs. It is important to develop validated standards to induce and quantify NETs in order to accurately compare results between laboratories and advance the understanding of this biological process and its implications in health and disease. This unit presents me...
Source: Current Protocols in Immunology - November 3, 2016 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Transduction of Herpesvirus saimiri-Transformed T Cells with Exogenous Genes of Interest.
Authors: Martínez-Barricarte R, de Jong SJ, Markle J, de Paus R, Boisson-Dupuis S, Bustamante J, van de Vosse E, Fleckenstein B, Casanova JL
Abstract
Human T cells can be transformed and expanded with herpesvirus saimiri (HVS). HVS-transformed T cells from patients have facilitated the study of a broad range of primary immunodeficiencies (PID) in which T-cell development or function is altered. However, the utility of HVS-transformed T cells for genetic studies has been limited by technical challenges in the expression of exogenous genes, including wild-type or mutant alleles. A novel, gamma retrovirus-ba...
Source: Current Protocols in Immunology - November 3, 2016 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Analysis and Purification of Mouse Intestinal Dendritic Cell and Macrophage Subsets by Flow Cytometry.
Authors: Koscsó B, Bogunovic M
Abstract
The unit presents a method for analysis of intestinal dendritic cell (DC) and macrophage subsets by flow cytometry in the single cell suspension prepared from the mouse small and large intestine (Basic Protocol). describes a strategy to enrich the hematopoietic cell fraction in the sample by Percoll gradient centrifugation, and describes preparation of single cell suspensions from specific tissue layers of the small intestine, such as the epithelium, villi mucosa, submucosa, and muscularis externa. Finally, Support Protocol explains how to purify specific intestinal...
Source: Current Protocols in Immunology - August 3, 2016 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Assessment of Inflammasome Formation by Flow Cytometry.
Authors: Sester DP, Zamoshnikova A, Thygesen SJ, Vajjhala PR, Cridland SO, Schroder K, Stacey KJ
Abstract
Inflammasomes are large protein complexes formed in response to cellular stresses that are platforms for recruitment and activation of caspase 1. Central to most inflammasome functions is the adapter molecule ASC (apoptosis-associated speck-like protein containing a caspase-recruitment domain) that links the inflammasome initiator protein to the recruited caspases. ASC is normally diffuse within the cell but within minutes of inflammasome activation relocates to a dense speck in the cytosol. The dramat...
Source: Current Protocols in Immunology - August 3, 2016 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
TepiTool: A Pipeline for Computational Prediction of T Cell Epitope Candidates.
Authors: Paul S, Sidney J, Sette A, Peters B
Abstract
Computational prediction of T cell epitope candidates is currently being used in several applications including vaccine discovery studies, development of diagnostics, and removal of unwanted immune responses against protein therapeutics. There have been continuous improvements in the performance of MHC binding prediction tools, but their general adoption by immunologists has been slow due to the lack of user-friendly interfaces and guidelines. Current tools only provide minimal advice on what alleles to include, what lengths to consider, how to deal wi...
Source: Current Protocols in Immunology - August 3, 2016 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Immunoblotting and Immunodetection.
Authors: Ni D, Xu P, Gallagher S
Abstract
Immunoblotting (western blotting) is used to identify specific antigens recognized by polyclonal or monoclonal antibodies. This unit provides protocols for all steps, starting with solubilization of the protein samples, usually by means of SDS and reducing agents. Following solubilization, the material is separated by SDS-PAGE and the antigens are electrophoretically transferred to a membrane, a process that can be monitored by reversible staining with Ponceau S. The transferred proteins are bound to the surface of the membrane, providing access to immunodetection ...
Source: Current Protocols in Immunology - August 3, 2016 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Mouse Models for Immune-Mediated Platelet Destruction or Immune Thrombocytopenia (ITP).
Authors: Neschadim A, Branch DR
Abstract
Immune thrombocytopenia (ITP) is a debilitating, life-threatening autoimmune disorder affecting more than 4 in every 100,000 adults annually, stemming from the production of antiplatelet antibody resulting in accelerated platelet destruction and thrombocytopenia. Numerous animal models of ITP have been developed that contributed to the basic understanding of the underlying mechanisms of ITP onset, progression, and maintenance. Rodent models that develop ITP spontaneously, or by passive transfer of an antiplatelet sera or antibody, play an instrumental role in the in...
Source: Current Protocols in Immunology - April 3, 2016 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
Measuring Bioenergetics in T Cells Using a Seahorse Extracellular Flux Analyzer.
Authors: van der Windt GJ, Chang CH, Pearce EL
Abstract
This unit contains several protocols to determine the energy utilization of T cells in real-time using a Seahorse Extracellular Flux Analyzer (http://www.seahorsebio.com). The advantages to using this machine over traditional metabolic assays include the simultaneous measurement of glycolysis and mitochondrial respiration, in real-time, on relatively small numbers of cells, without any radioactivity. The Basic Protocol describes a standard mitochondrial stress test on the XF(e) 96, which yields information about oxidative phosphorylation and glycolysi...
Source: Current Protocols in Immunology - April 3, 2016 Category: Allergy & Immunology Tags: Curr Protoc Immunol Source Type: research
