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Current Protocols in Immunology

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https://www.readbyqxmd.com/read/28369684/gut-microbiome-standardization-in-control-and-experimental-mice
#1
Kathy D McCoy, Markus B Geuking, Francesca Ronchi
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 factors, including (but not limited to) food, bedding, caging, and temperature...
April 3, 2017: Current Protocols in Immunology
https://www.readbyqxmd.com/read/28369683/basic-multicolor-flow-cytometry
#2
Zofia Maciorowski, Pratip K Chattopadhyay, Paresh Jain
Multicolor flow cytometry is a rapidly evolving technology that uses multiple fluorescent markers to identify and characterize cellular subpopulations of interest, allowing rapid analysis on tens of thousands of cells per second, with the possibility of isolating pure, viable populations by cell sorting for further experimentation. This unit covers the tools needed by the beginning immunologist to plan and run multicolor experiments, with information on fluorochromes and their characteristics, spectral spillover, compensation and spread, instrument and reagent variables, and the basic elements of multicolor panel design...
April 3, 2017: Current Protocols in Immunology
https://www.readbyqxmd.com/read/28369682/characterization-and-functional-analysis-of-mouse-semi-invariant-natural-t-cells
#3
Amrendra Kumar, Jelena S Bezbradica, Aleksandar K Stanic, Sebastian Joyce
Semi-invariant natural killer T (iNKT) cells are CD1d-restricted innate-like lymphocytes that recognize lipid agonists. Activated iNKT cells have immunoregulatory properties. Human and mouse iNKT cell functions elicited by different glycolipid agonists are highly conserved, making the mouse an excellent animal model for understanding iNKT cell biology in vivo. This unit describes basic methods for the characterization and quantification (see Basic Protocol 1) and functional analysis of mouse iNKT cells in vivo or in vitro...
April 3, 2017: Current Protocols in Immunology
https://www.readbyqxmd.com/read/28369681/measurement-of-tumor-necrosis-factor-and-lymphotoxins
#4
M Michele Hogan, Stefanie N Vogel
The tumor necrosis factor (TNF) superfamily of cytokines plays critical roles in all aspects of the immune response. TNF and the lymphotoxins (LT), LTα and LTβ, are particularly important as major effector cytokines and mediators or lymphoid organ development. One of the classical methods for the measurement of TNF and LTα activity is by demonstrating their ability to lyse certain target cells. A detailed protocol for the measurement of this activity using a highly sensitive indicator cell line is presented...
April 3, 2017: Current Protocols in Immunology
https://www.readbyqxmd.com/read/28369680/transfection-by-electroporation
#5
Huntington Potter, Richard Heller
Electroporation-the use of high-voltage electric shocks to introduce DNA into cells-can be used with most cell types, yields a high frequency of both stable transformation and transient gene expression, and, because it requires fewer steps, can be easier than alternative techniques. This unit describes electroporation of mammalian cells, including ES cells, for the preparation of knock-out, knock-in, and transgenic mice. Protocols are described for the use of electroporation in vivo to perform gene therapy for cancer, as well as for DNA vaccination...
April 3, 2017: Current Protocols in Immunology
https://www.readbyqxmd.com/read/28150864/web-sites-of-interest-to-immunologists
#6
Marie-Paule Lefranc
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.
February 2, 2017: Current Protocols in Immunology
https://www.readbyqxmd.com/read/28150863/double-immunodiffusion-assay-for-detecting-specific-antibodies-ouchterlony
#7
Peter Hornbeck
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.
February 2, 2017: Current Protocols in Immunology
https://www.readbyqxmd.com/read/28150862/isotype-determination-of-antibodies
#8
Peter Hornbeck, Thomas A Fleisher, Nicholas M Papadopoulos
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 fragmentation of the antibodies depend on the class or isotype of the antibody, so this information streamlines these processes...
February 2, 2017: Current Protocols in Immunology
https://www.readbyqxmd.com/read/28150861/quantitation-of-dna-and-rna-with-absorption-and-fluorescence-spectroscopy
#9
Sean R Gallagher
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...
February 2, 2017: Current Protocols in Immunology
https://www.readbyqxmd.com/read/27801513/transduction-of-herpesvirus-saimiri-transformed-t-cells-with-exogenous-genes-of-interest
#10
Rubén Martínez-Barricarte, Sarah Jill de Jong, Janet Markle, Roel de Paus, Stephanie Boisson-Dupuis, Jacinta Bustamante, Esther van de Vosse, Bernhard Fleckenstein, Jean-Laurent Casanova
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-based method for the simple and reliable transduction, purification, and study of HVS-transformed T cells is described...
November 1, 2016: Current Protocols in Immunology
https://www.readbyqxmd.com/read/27801512/induction-and-quantification-of-netosis
#11
Carmelo Carmona-Rivera, Mariana J Kaplan
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...
November 1, 2016: Current Protocols in Immunology
https://www.readbyqxmd.com/read/27801511/isolation-and-characterization-of-exosomes-from-cultures-of-tissue-explants-and-cell-lines
#12
Lucia Mincheva-Nilsson, Vladimir Baranov, Olga Nagaeva, Eva Dehlin
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 purification of exosomes based on density gradient ultracentrifugation with sucrose...
November 1, 2016: Current Protocols in Immunology
https://www.readbyqxmd.com/read/27801510/mhc-peptide-tetramers-to-visualize-antigen-specific-t-cells
#13
John D Altman, Mark M Davis
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...
November 1, 2016: Current Protocols in Immunology
https://www.readbyqxmd.com/read/27479659/tepitool-a-pipeline-for-computational-prediction-of-t-cell-epitope-candidates
#14
Sinu Paul, John Sidney, Alessandro Sette, Bjoern Peters
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 with homologous peptides, and what cutoffs should be considered relevant...
August 1, 2016: Current Protocols in Immunology
https://www.readbyqxmd.com/read/27479658/assessment-of-inflammasome-formation-by-flow-cytometry
#15
David P Sester, Alina Zamoshnikova, Sara J Thygesen, Parimala R Vajjhala, Simon O Cridland, Kate Schroder, Katryn J Stacey
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 dramatic redistribution of ASC can be monitored by flow cytometry using parameters of fluorescence peak height and width when immunostained or tagged with a fluorescent protein...
August 1, 2016: Current Protocols in Immunology
https://www.readbyqxmd.com/read/27038464/contact-hypersensitivity
#16
Anthony A Gaspari, Stephen I Katz, Stefan F Martin
Contact hypersensitivity (CHS) is a simple in vivo assay of cell-mediated immune function in which exposure of epidermal and dermal cells to exogenous haptens results in a delayed-type hypersensitivity (DTH) reaction that can be measured and quantified. Epidermal Langerhans cells and dermal dendritic cells are the critical antigen-presenting cells in this reaction which initiate sensitization to haptens by presenting antigens to CD4- and CD8-bearing T lymphocytes which, in turn, secrete cytokines and recruit other cells to the site of the reaction...
April 1, 2016: Current Protocols in Immunology
https://www.readbyqxmd.com/read/27038463/in-vivo-depletion-of-t-lymphocytes
#17
Karen Laky, Ada M Kruisbeek
In vivo depletion of T lymphocytes is a means of studying the role of specific T cell populations during defined phases of in vivo immune responses. In this unit, a protocol is provided for injecting monoclonal antibodies (mAbs) into wild-type adult mice. Depletion of the appropriate subset of cells is verified by flow cytometry analysis of lymph node and spleen cell suspensions in pilot experiments. Once conditions have been established, depleted mice can be used to study the impact of T cell subsets on a variety of in vivo immune responses...
April 1, 2016: Current Protocols in Immunology
https://www.readbyqxmd.com/read/27038462/differentiation-and-characterization-of-tr1-cells
#18
Norio Chihara, Asaf Madi, Katarzyna Karwacz, Amit Awasthi, Vijay K Kuchroo
Regulatory T cell-mediated suppression serves as a pivotal mechanism of negative regulation of immune-mediated inflammation. Type 1 regulatory T cells (Tr1 cells) are an important subset of CD4+ T cells that prevent excessive inflammatory responses and maintain immune tolerance. The anti-inflammatory role of Tr1 cells is mediated in part by their production of interleukin 10 (IL-10), which dampens the function of both antigen-presenting cells and antigen-specific effector T cells. Additionally, Tr1 cells can kill effector and myeloid cells through the perforin-granzyme B pathway...
April 1, 2016: Current Protocols in Immunology
https://www.readbyqxmd.com/read/27038461/measuring-bioenergetics-in-t-cells-using-a-seahorse-extracellular-flux-analyzer
#19
Gerritje J W van der Windt, Chih-Hao Chang, Erika L Pearce
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 glycolysis, two energy-generating pathways...
April 1, 2016: Current Protocols in Immunology
https://www.readbyqxmd.com/read/27038460/mouse-models-for-immune-mediated-platelet-destruction-or-immune-thrombocytopenia-itp
#20
Anton Neschadim, Donald R Branch
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 investigation of ITP mechanisms responsible for the breakdown of tolerance in human ITP, in studies of the immunopathology underlying the progression of platelet destruction, and in elucidation of the mechanisms of therapeutic amelioration of ITP by existing and new therapeutic modalities...
April 1, 2016: Current Protocols in Immunology
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