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Current Protocols in Immunology

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https://www.readbyqxmd.com/read/29091265/mouse-eosinophils-identification-isolation-and-functional-analysis
#1
Hadar Reichman, Perri Rozenberg, Ariel Munitz
Eosinophils are bone marrow-derived cells that differentiate in the bone marrow and migrate into the peripheral blood primarily under the regulation of interleukin (IL)-5. Eosinophil levels in the blood are relatively low. However, under steady-state conditions and in settings of allergic inflammation, parasite infections, or even cancer, they migrate and mainly reside in mucosal tissues where they have key effector and immune-modulating functions. Functional studies using eosinophils are not simple, since these cells are terminally differentiated and rapidly die in vitro...
November 1, 2017: Current Protocols in Immunology
https://www.readbyqxmd.com/read/29091264/analysis-of-cellular-dna-content-by-flow-cytometry
#2
Zbigniew Darzynkiewicz, Xuan Huang, Hong Zhao
Cellular DNA content can be measured by flow cytometry with the aim of : (1) revealing cell distribution within the major phases of the cell cycle, (2) estimating frequency of apoptotic cells with fractional DNA content, and/or (3) disclosing DNA ploidy of the measured cell population. In this unit, simple and universally applicable methods for staining fixed cells are presented, as are methods that utilize detergents and/or proteolytic treatment to permeabilize cells and make DNA accessible to fluorochrome...
November 1, 2017: Current Protocols in Immunology
https://www.readbyqxmd.com/read/29091263/high-dimensional-fluorescence-cytometry
#3
Thomas Myles Ashhurst, Adrian Lloyd Smith, Nicholas Jonathan Cole King
The immune system consists of a complex network of cells, all expressing a wide range of surface and/or intracellular proteins. Using flow cytometry, these cells can be analyzed by labeling with fluorophore-conjugated antibodies. The recent expansion of fluorescence flow cytometry technology, in conjunction with the ever-expanding understanding of the complexity of the immune system, has led to the generation of larger high-dimensional fluorescence flow cytometry panels. However, as panel size and complexity increases, so too does the difficulty involved in constructing high-quality panels, in addition to the challenges of analyzing such high-dimensional datasets...
November 1, 2017: Current Protocols in Immunology
https://www.readbyqxmd.com/read/29091262/isolation-and-functional-use-of-human-nkt-cells
#4
Mark A Exley, S Brian Wilson, Steven P Balk
This unit details methods for the isolation, in vitro expansion, and functional characterization of human iNKT cells. The term 'iNKT' derives from the fact that a large fraction of murine and some human NK marker+ T cells ('NKT') recognize the MHC class I-like CD1d protein and use an identical 'invariant' TCRα chain, which is generated in humans by precise Vα24 and Jα18 rearrangements with either no N-region diversity or subsequent trimming to identical or nearly identical amino acid sequence (hence, 'iNKT' cells)...
November 1, 2017: Current Protocols in Immunology
https://www.readbyqxmd.com/read/29091261/the-mouse-model-of-infection-with-citrobacter-rodentium
#5
Nicolas Bouladoux, Oliver J Harrison, Yasmine Belkaid
Citrobacter rodentium is a murine mucosal pathogen used as a model to elucidate the molecular and cellular pathogenesis of infection with two clinically important human gastrointestinal pathogens, enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC). C. rodentium infection provides an excellent model to study different aspects of host-pathogen interaction in the gut, including intestinal inflammatory responses during bacteria-induced colitis, mucosal healing and epithelial repair, the induction of mucosal immune responses, and the role of the intestinal microbiota in mediating resistance to colonization by enteric pathogens...
November 1, 2017: Current Protocols in Immunology
https://www.readbyqxmd.com/read/28762488/a-real-time-cytotoxicity-assay-as-an-alternative-to-the-standard-chromium-51-release-assay-for-measurement-of-human-nk-and-t-cell-cytotoxic-activity
#6
Julien Fassy, Kyriaki Tsalkitzi, Maria Goncalves-Maia, Véronique M Braud
This unit describes the monitoring and quantification of cellular cytotoxicity using a non-radioactive and real-time cytotoxic assay. The extent of target-cell lysis is monitored over time by imaging and quantifying live fluorescent target cells using a cell-imaging multimode reader. This assay is performed in a 96 well plate in optimized culture conditions at 37°C in the presence of 5% CO2 . The basic protocol describes natural killer cell-mediated cytotoxic assay that can be adapted to include antibodies blocking inhibitory NK receptors or triggering antibody-dependent cell-mediated cytotoxicity (ADCC)...
August 1, 2017: Current Protocols in Immunology
https://www.readbyqxmd.com/read/28762487/nomenclature-and-serology-of-hla-class-i-and-class-ii-alleles
#7
James McCluskey, Carmel Kanaan, Mary Diviney
This overview presents nomenclature and serology information on human leucocyte antigens, or HLA molecules, which are encoded by a cluster of genes linked on the short arm of chromosome 6. This region is known as the major histocompatibility complex and codes for class I and class II molecules, which are distinguished from each other based upon their structure, tissue distribution, and source of peptide antigen, as well as upon their interactions with T cell subsets. © 2017 by John Wiley & Sons, Inc.
August 1, 2017: Current Protocols in Immunology
https://www.readbyqxmd.com/read/28762486/universal-precautions-necessary-safety-procedures-when-handling-human-blood-body-fluids-and-specimens
#8
Barbara E Bierer
Universal precautions are observed whenever handling human blood, body fluids, or specimens as a means of preventing exposure to blood-borne pathogens. This appendix outlines safety procedures to follow whenever undertaking research activities that involve human blood, body fluids, and specimens. © 2017 by John Wiley & Sons, Inc.
August 1, 2017: Current Protocols in Immunology
https://www.readbyqxmd.com/read/28762485/characterization-of-human-blood-monocytes-and-intestinal-macrophages
#9
Evida A Dennis, Tanya O Robinson, Lesley E Smythies, Phillip D Smith
Monocytes and macrophages play fundamental roles in defense against microbes, clearance of senescent and dead cells, and immunoregulation. Although blood monocytes are the source of intestinal macrophages in the developed mucosal immune system, blood monocytes and intestinal macrophages from healthy human subjects display distinct phenotypic and functional differences. Blood monocytes can be induced to polarize into M1 and M2 macrophages, whereas intestinal macrophages appear to be terminally differentiated and are unable to undergo such inducible polarization...
August 1, 2017: Current Protocols in Immunology
https://www.readbyqxmd.com/read/28762484/venipuncture-procedures-sources-of-human-peripheral-blood-cells-serum-and-plasma
#10
Barbara E Bierer, Warren Strober
Peripheral blood is the chief source of mononuclear cells (lymphocytes and monocytes) for immunologic studies of humans. This appendix presents protocols for obtaining blood by simple venipuncture when relatively small amounts of blood (10 to 100 ml) are necessary or by lymphapheresis when large amounts (300 to 5000 ml) are necessary. Cells collected by these procedures can be further separated by techniques described in Chapter 7. © 2017 by John Wiley & Sons, Inc.
August 1, 2017: Current Protocols in Immunology
https://www.readbyqxmd.com/read/28762483/high-dimensional-single-cell-analysis-with-mass-cytometry
#11
Tess Melinda Brodie, Vinko Tosevski
Mass cytometry is an analytical technology that combines the sample preparation workflow typical of flow cytometry and the detection capacity of atomic mass spectroscopy, allowing for highly multiplexed measurements of protein or nucleic acid targets on single cells. In 2014, the mass cytometer was adapted for the acquisition of samples from microscopy slides (termed imaging mass cytometry), greatly increasing the applicability of this technology. By using antibodies (or other probes) labeled with purified metal isotopes, the mass cytometer is able to detect up to 50 different parameters (current practical limit) at the single-cell level, enabling a deep and thorough profiling of individual cells in terms of their cell surface protein phenotype, physiological state, proliferation potential, and many other cell states or features...
August 1, 2017: Current Protocols in Immunology
https://www.readbyqxmd.com/read/28369684/gut-microbiome-standardization-in-control-and-experimental-mice
#12
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
#13
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
#14
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
#15
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
#16
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
#17
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
#18
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
#19
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
#20
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
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