Current Protocols in Immunology最新文献

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. Additionally, supravital cell staining with Hoechst 33342, which is primarily used for sorting live cells based on DNA-content differences for their subsequent culturing, is described. Also presented are methods for staining cell nuclei isolated from paraffin-embedded tissues. Available algorithms are listed for deconvolution of DNA-content-frequency histograms to estimate percentage of cells in major phases of the cell cycle and frequency of apoptotic cells with fractional DNA content. © 2017 by John Wiley & Sons, Inc.

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. As such, this unit seeks to review the key principles involved in building high-dimensional panels, as well as to guide users through the process of building and analyzing quality panels. Here, cytometer configuration, fluorophore brightness, spreading error, antigen density, choosing the best conjugates, titration, optimization, and data analysis will all be addressed. © 2017 by John Wiley & Sons, Inc.

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. Nevertheless, in response to local conditions, monocytes differentiated into intestinal macrophages display phenotypic and functional characteristics that enhance their capacity to provide non-inflammatory host defense and participate in local immunoregulation. Using the protocols described here, this unit presents the key phenotypic and functional differences between human blood monocytes and intestinal macrophages, as well as between mouse and human intestinal macrophages. © 2017 by John Wiley & Sons, Inc.

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. © 2017 by John Wiley & Sons, Inc.

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. Adoptive transfer of in vitro differentiated Tr1 cells can be used to suppress autoimmune tissue inflammation in vivo. This unit describes the in vitro stimulation of naïve murine CD4+ T cells using IL-27 to generate IL-10–producing Tr1 cells. © 2016 by John Wiley & Sons, Inc.

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.

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.

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.

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. This unit provides detailed protocols for growing this bacterium, infecting mice by intragastric inoculation, measuring bacterial loads in feces and organs, and monitoring intestinal pathology induced by infection. Additional protocols describe steps needed to create frozen stocks, establish a growth curve, perform ex vivo organ cultures, isolate immune cells from the large intestine, and measure immune response by flow cytometry. © 2017 by John Wiley & Sons, Inc.

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. Thus, establishing simple methods to characterize, obtain, and functionally assess eosinophil activities is important. In this unit, we describe methodology for identifying tissue eosinophils by flow cytometry. In addition, we provide detailed methods for isolating eosinophils and for differentiating them from bone marrow cells for further functional studies. © 2017 by John Wiley & Sons, Inc.