Current Protocols in Immunology最新文献

Flow cytometry is a technology that provides rapid multi-parametric analysis of single cells in solution. Flow cytometers utilize lasers as light sources to produce both scattered and fluorescent light signals that are read by detectors such as photodiodes or photomultiplier tubes. These light signals are converted into electronic signals that are analyzed by a computer and written to a standardized format (.fcs) data file. Cell populations can be analyzed and/or purified based on their fluorescent or light scattering characteristics. A variety of fluorescent reagents are utilized in flow cytometry. These include fluorescently conjugated antibodies, nucleic acid binding dyes, viability dyes, ion indicator dyes, and fluorescent expression proteins. Flow cytometry is a powerful tool that has applications in immunology, molecular biology, bacteriology, virology, cancer biology, and infectious disease monitoring. It has seen dramatic advances over the last 30 years, allowing unprecedented detail in studies of the immune system and other areas of cell biology. © 2018 by John Wiley & Sons, Inc.

Traumatic brain injury (TBI) is a leading cause of death and disability and is a risk factor for the later development of neuropsychiatric disorders and neurodegenerative diseases. Many models of TBI have been developed, but their further refinement and a more detailed long-term follow-up is needed. We have used the Thy1-YFP-H transgenic mouse line and the parallel rod floor test to produce an unbiased and robust method for the evaluation of the multiple effects of a validated model of controlled cortical injury. This approach reveals short- and long-term progressive changes, including compromised biphasic motor function up to 85 days post-lesion, which correlates with neuronal atrophy, dendrite and spine loss, and long-term axonal pathology evidenced by axon spheroids and fragmentation. Here we present methods for inducing a controlled cortical injury in the Thy1-YFP-H transgenic mouse line and for evaluating the resulting deficits in the parallel rod floor test. This technique constitutes a new, unbiased, and robust method for the evaluation of motor and behavioral alterations after TBI. © 2018 by John Wiley & Sons, Inc.

Innate and adaptive immune interactions within the central nervous system (CNS) and surrounding meninges contribute significantly to neural homeostasis as well as a variety of different neurological disorders. Two-photon laser scanning microscopy is a deep tissue imaging technique that provides a means to image immune cell dynamics and interactions in the living CNS with high spatial and temporal resolution. Optical access to the brain and meninges can be achieved through the creation of thinned skull windows, which can be made without inducing damage and inflammation in the underlying tissue. This protocol provides guidance on how to create a thinned skull window without causing CNS injury. We also describe a highly reproducible method to induce a mild traumatic brain injury using the thinned skull approach. © 2018 by John Wiley & Sons, Inc.

The lungs are continuously exposed to environmental threats, requiring an adequate and stringent immune response of a heterogeneous set of effector cells. Dendritic cells (DCs) form a dense network in the respiratory mucosa and act as the central regulators of the different components of this response, both sensing the nature of the threats and precisely coordinating the effector mechanisms best suited for overcoming it. The DCs are classically subdivided in two main groups, plasmacytoid DCs (pDCs) and conventional DCs (cDCs), the latter being further subdivided into cDC1s and cDC2s based on ontogeny and their distinct non-redundant functions. This protocol provides different enrichment methods and represents an up-to-date, universal framework that uses a minimal set of highly specific lineage markers to discriminate and sort pure cDC subsets from the murine lung but also across tissues and species which is an added value in intra- and interspecies comparative research. © 2018 by John Wiley & Sons, Inc.

Efficient phagocytosis of apoptotic cells (efferocytosis) is essential for immune homeostasis. Phospholipids exposed on the surface of apoptotic cells, such as phosphatidylserine, supply important “eat-me” signals. Liposomes are lipid bilayer vesicles that can be generated from one or several types of phospholipids of interest. Thus, these vesicles offer versatility, flexibility, and, importantly, a three-dimensional structure for studying the interaction between lipids and their receptors as well as the lipid-receptor interaction–mediated signaling events controlling efferocytosis by cells like professional phagocytes. Here, we describe methods to prepare liposomes, perform liposome-based lipid–receptor binding assays, use liposomes to block efferocytosis, and utilize liposome-coated beads as apoptotic cell surrogates for phagocytosis. © 2018 by John Wiley & Sons, Inc.

Autoimmune pancreatitis (AIP) is a chronic fibro-inflammatory disorder of the pancreas. However, extensive clinico-pathological analyses have revealed that AIP is, in reality, a pancreatic manifestation of a newly described systemic disease known as IgG4-related disease (IgG4-RD). IgG4-RD is characterized by enhanced local and systemic IgG4 antibody (Ab) responses as well as inflammation involving multiple organs, including the pancreas, bile ducts, and salivary glands. Although mice lack the IgG4 Ab subtype, autoimmune-prone MRL/Mp mice treated with repeated injection with polyinosinic-polycytidylic acid (poly (I:C)) provide an experimental model of AIP. These mice exhibit massive destruction of pancreatic architecture associated with pancreatic immune cell infiltration and fibrosis. Moreover, this experimental AIP may be accompanied by involvement of multiple organs as well as elevation of serum levels of autoAbs, resembling humans with IgG4-RD. Thus, elucidation of the molecular mechanisms accounting for the development of experimental AIP can potentially provide new insights into the immuno-pathogenesis of human IgG4-related AIP. © 2018 by John Wiley & Sons, Inc.

Cytotoxic T cells (CTLs) are important immune effector cells in the adaptive immune response. It has been well documented that CTLs are important in host immune responses to viral and bacterial intracellular pathogens, tumors, and transplanted tissues. The properties of CTLs have been studied extensively in murine models, and their roles validated in the human setting. Frequently, the presence of these cells correlates well with protective immunity, so the ability to readily measure the activity of these cells is an important immunological measurement. In this unit, several assays are described that are commonly utilized to induce CTLs and to measure CTL activity both in vitro and in vivo. These assays are adaptable to many experimental and/or disease models, and in the case of the in vitro assays can be applied to measure CTL activity in human samples. © 2018 by John Wiley & Sons, Inc.

This unit describes the production of monoclonal antibodies beginning with immunization, cell fusion, and selection. Support protocols are provided for screening primary hybridoma supernatants for antibodies of desired specificity, establishment of stable hybridoma lines, cloning of these B cell lines by limiting dilution to obtain monoclonal lines, and preparation of cloning/expansion medium. An alternate protocol describes cell fusion and one-step selection and cloning of hybridomas utilizing a semi-solid methylcellulose-based medium (ClonaCell-HY from StemCell Technologies). Curr. Protoc. Immunol. 102:2.5.1-2.5.29. © 2013 by John Wiley & Sons, Inc.

The development of HIV vaccines has been hampered by the lack of an animal model that can accurately predict vaccine efficacy. Chimpanzees can be infected with HIV-1 but are not practical for research. However, several species of macaques are susceptible to the simian immunodeficiency viruses (SIVs) that cause disease in macaques, which also closely mimic HIV in humans. Thus, macaque-SIV models of HIV infection have become a critical foundation for AIDS vaccine development. Here we examine the multiple variables and considerations that must be taken into account in order to use this nonhuman primate (NHP) model effectively. These include the species and subspecies of macaques, virus strain, dose and route of administration, and macaque genetics, including the major histocompatibility complex molecules that affect immune responses, and other virus restriction factors. We illustrate how these NHP models can be used to carry out studies of immune responses in mucosal and other tissues that could not easily be performed on human volunteers. Furthermore, macaques are an ideal model system to optimize adjuvants, test vaccine platforms, and identify correlates of protection that can advance the HIV vaccine field. We also illustrate techniques used to identify different macaque lymphocyte populations and review some poxvirus vaccine candidates that are in various stages of clinical trials. Understanding how to effectively use this valuable model will greatly increase the likelihood of finding a successful vaccine for HIV. Curr. Protoc. Immunol. 102:12.14.1-12.14.30. © 2013 by John Wiley & Sons, Inc.