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Current Protocols in Immunology

Soraya Mezouar, Amira Ben Amara, Céline Chartier, Laurent Gorvel, Jean-Louis Mege
Macrophages are specialized cells involved in recognition, uptake, and destruction of microorganisms. Human placental macrophages are poorly investigated because of the lack of a convenient protocol for their isolation. Here, we present a straightforward and reliable method to isolate macrophages from full-term human placentas. After enzymatic digestion of placental tissue and centrifugation of the cell suspension on a Ficoll cushion, placental macrophages are selected using magnetic beads coated with anti-CD14 antibodies...
May 24, 2019: Current Protocols in Immunology
Yuelin Kong, Yi Jing, Maria Bettini
The ability to express and study a single T cell receptor (TCR) in vivo is an important aspect of both basic and translational immunological research. Traditionally, this was achieved by using TCR transgenic mice. In the past decade, a more efficient approach for single TCR expression was developed. This relatively rapid and accessible method utilizes retrovirus-mediated stem cell-based gene transfer and is commonly referred to as the TCR retrogenic approach. In this approach, hematopoietic bone marrow precursors are transduced with retroviral vector carrying both alpha and beta chains of a T cell receptor...
May 15, 2019: Current Protocols in Immunology
Dmitri I Kotov, Marc K Jenkins
Epitope-specific CD4+ T cells can be labeled in complex cell mixtures from secondary lymphoid organs with fluorophore-labeled peptide:major histocompatibility complex class II (p:MHCII) tetramers and then detected by flow cytometry. Magnetic enrichment of tetramer-bound cells before flow cytometry increases the sensitivity of detection to the point where epitope-specific cells can be studied even when very rare at early and late times after the host has been exposed to the epitope. This method is very useful for studying polyclonal epitope-specific CD4+ T cells under physiological conditions...
April 29, 2019: Current Protocols in Immunology
Mónica Romera-Hernández, Laura Mathä, Catherine A Steer, Maryam Ghaedi, Fumio Takei
Innate lymphoid cells (ILCs) are a heterogeneous family of lymphocytes that populate barrier and non-barrier tissues. ILCs regulate immune responses to pathogens and commensals but also sustain metabolic homeostasis, tissue remodeling after injury and establish dialogue with the nervous system. ILCs rapidly become activated in the absence of adaptive antigen receptors by responding to signaling molecules provided by hematopoietic or non-hematopoietic cells. Here we provide protocols designed for processing the lung, liver, small intestine, bone marrow, mediastinal and mesenteric lymph nodes in order to obtain a purified leukocyte fraction of cells, in which ILC2 enrichment is optimized...
April 17, 2019: Current Protocols in Immunology
Ulrike Steffen, Fabian T Andes, Georg Schett
Osteoclasts are the only bone-resorbing cells in the body. Together with bone-forming osteoblasts, they are responsible for bone homeostasis and constant bone remodeling. Aberrant activation of osteoclasts leads to bone loss, as seen in postmenopausal osteoporosis or in autoimmune diseases like rheumatoid arthritis. Although much research has been performed to understand and prevent osteoclast-mediated bone loss, the mechanisms of osteoclast hyperactivation are not completely understood. This unit describes several protocols for ex vivo generation of murine and human osteoclasts, allowing study of the effects of specific cells, cytokines, or chemical substances on osteoclast formation and activity without the need for expensive and time-consuming animal experiments...
April 8, 2019: Current Protocols in Immunology
Kate A Markey, Kate H Gartlan
This unit describes methods for quantifying phagocytosis and imaging the immunological synapse between T cells and antigen-presenting cells (APCs), with both techniques delivering valuable information about APC function. These aspects of APC biology have traditionally been challenging to quantify, and imaging flow cytometry, which harnesses the high-throughput nature of flow cytometry combined with the capacity of microscopy to deliver spatial localization, facilitates analysis of these APC functions in a fashion that was previously not possible...
March 6, 2019: Current Protocols in Immunology
Tej Pratap Singh, Howard H Zhang, Samuel T Hwang, Joshua M Farber
Genome-wide association studies have found that polymorphisms in genes for IL-23 and its receptor are important in psoriasis, and blocking IL-23 is an effective therapy in the disease. The use of Aldara™ , a cream that contains the TLR7 and TLR8 agonist imiquimod (IMQ), was found to exacerbate psoriasis in some patients with pre-existing disease. Intradermal injections of IL-23 and topical application of Aldara/IMQ induce skin inflammation in mice with features similar to psoriasis-including epidermal hyperplasia and accumulation of inflammatory cells in epidermis and dermis-which is mediated by IL-17A, IL-22, and other factors implicated in the human disease...
January 7, 2019: Current Protocols in Immunology
Christopher J Bohlen, F Chris Bennett, Mariko L Bennett
Microglia represent 5-10% of cells in the central nervous system and contribute to the development, homeostasis, injury, and repair of neural tissues. As the tissue-resident macrophages of the central nervous system, microglia execute core innate immune functions such as detection of pathogens/damage, cytokine secretion, and phagocytosis. However, additional properties that are specific to microglia and their neural environment are beginning to be appreciated. This article describes approaches for purification of microglia by fluorescence-activated cell sorting using microglia-specific surface markers and for enrichment of microglia by magnetic sorting and immunopanning...
November 10, 2018: Current Protocols in Immunology
Christian Humpel
Ex vivo cell culture models are of particular interest for neurobiologists, as these allow the study of brain cells in an isolated environment. Organotypic brain slice cultures allow growth of three-dimensional brain tissue that conserves the cellular architecture. This unit describes the preparation and culturing of organotypic brain slices from mice. In three basic protocols, the general procedure, the specific preparation of chopper slices, and slicing of whole-brain vibratome sections are described. Support protocols explain the use of postnatal or adult mice, the preparation of coronal or sagittal slices, the preparation of co-cultures, post-processing of slices, the use of extra filter membranes, and the re-use of membrane inserts...
November 2018: Current Protocols in Immunology
Laure-Anne Ligeon, Monica Loi, Christian Münz
LC3-associated phagocytosis (LAP) is an unconventional form of autophagy that relies on parts of the canonical autophagy machinery for its function. LAP is triggered upon receptor-mediated phagocytosis and is characterized by the formation of a single-membrane vesicle decorated with the autophagy protein LC3. In professional phagocytic cells, such as macrophages, the role of LAP in immune processes has been characterized, although how LAP functions at the molecular level remains poorly defined. It is important to point out that as for all autophagic pathways, the study of LAP is still challenging for the scientific community because it is a dynamic and complex process, requiring interactions among several proteins...
November 2018: Current Protocols in Immunology
Jesús Amo-Aparicio, Anna Martínez-Muriana, Alba Sánchez-Fernández, Rubèn López-Vales
Spinal cord injury (SCI) leads to irreversible devastating neurological disabilities. Accumulated evidence in the literature indicates that the inflammatory response that occurs in the spinal cord following injury contributes importantly to spread tissue damage to healthy regions adjacent to the lesion site, and consequently, to increase neurological deficits. Therefore, targeting inflammation could lead to the development of new therapies to prevent tissue damage and neurological impairments after SCI. Inflammation is regulated, in part, by the expression of pro-inflammatory and anti-inflammatory cytokines synthesized, mainly, by glial cells...
November 2018: Current Protocols in Immunology
Muhammad S Alam
Proximity ligation assay (PLA), also referred to as Duolink® PLA technology, permits detection of protein-protein interactions in situ (at distances <40 nm) at endogenous protein levels. It exploits specific antibodies identifying (either directly or indirectly) the two proteins of interest and utilizes specific DNA primers covalently linked to the antibodies. A hybridization step followed by DNA amplification with fluorescent probes permit visualization of spots of proximity by fluorescence microscopy...
November 2018: Current Protocols in Immunology
Nathalie Laflamme, Paul Préfontaine, Antoine Lampron, Serge Rivest
Bone marrow transplantation is the standard of care for a host of diseases such as leukemia and multiple myeloma, as well as genetically inherited metabolic diseases affecting the central nervous system. In mouse models, bone marrow transplantation has proven a valuable tool for understanding the hematopoietic system and the homing of hematopoietic cells to their target organs. Many techniques have been developed to create chimeric mice, animals with a hematopoietic system derived from a genetic background that differs from the rest of the body...
November 2018: Current Protocols in Immunology
Soyoung A Oh, Akiko Seki, Sascha Rutz
CRISPR/Cas9 has enabled the rapid and efficient generation of gene knockouts across various cell types of several species. T cells are central players in adaptive immune responses. Gene editing in primary T cells not only represents a valuable research tool, but is also critical for next generation immunotherapies, such as CAR T cells. Broad application of CRIPSR/Cas9 for gene editing in primary T cells has been hampered by limitations in transfection efficiency and the requirement for TCR stimulation. In this article, we provide a detailed protocol for Cas9/gRNA ribonucleoprotein (RNP) transfection of primary mouse and human T cells without the need for TCR stimulation that achieves near complete loss of target gene expression at the population level...
October 18, 2018: Current Protocols in Immunology
Bonnie Huang, Kristoffer Haurum Johansen, Pamela L Schwartzberg
The ability to alter gene expression directly in T lymphocytes has provided a powerful tool for understanding T cell biology, signaling, and function. Manipulation of T cell clones and primary T cells has been accomplished primarily through overexpression or gene-silencing studies using cDNAs or shRNAs, respectively, which are often delivered by retroviral or lentiviral transduction or direct transfection methods. The recent development of CRISPR/Cas9-based mutagenesis has revolutionized genomic editing, allowing unprecedented genetic manipulation of many cell types with greater precision and ease...
October 12, 2018: Current Protocols in Immunology
Samantha Solito, Laura Pinton, Francesco De Sanctis, Stefano Ugel, Vincenzo Bronte, Susanna Mandruzzato, Ilaria Marigo
This unit presents methods to assess the immunosuppressive properties of immunoregulatory cells of myeloid origin, such as myeloid-derived suppressor cells (MDSCs), both in vitro and in vivo in mice, as well as in biological samples from cancer patients. These methods could be adapted to test the impact of different suppressive populations on T cell activation, proliferation, and cytotoxic activity; moreover, they could be useful to assess the influence exerted by genetic modifications, chemical inhibitors, and drugs on immune suppressive pathways © 2018 by John Wiley & Sons, Inc...
October 10, 2018: Current Protocols in Immunology
Rebecca L Riding, Jillian M Richmond, John E Harris
Vitiligo is an autoimmune skin disease in which the pigment-producing melanocytes are destroyed by autoreactive CD8+ T cells. As a result, patients develop disfiguring white spots on the skin. This article discusses the first mouse model of vitiligo that develops epidermal depigmentation, similar to disease in human patients. To achieve epidermal depigmentation, mice are genetically engineered to retain melanocytes in the skin epidermis. Induction of disease occurs by adoptive transfer of melanocyte-specific CD8+ T cells into recipient mice and the subsequent activation of these T cells using a viral vector...
September 25, 2018: Current Protocols in Immunology
(no author information available yet)
Statistical tests and graphs are an important part of any immunological research publication or presentation, but not all immunologists have the statistical expertise to choose the best methods to evaluate and represent their experiments. These protocols provide a brief overview of the statistical methods most relevant to immunology researchers, so they can either analyze their own data or better understand their statistician collaborators. Protocols cover the basics of t‐tests, ANOVA, nonparametric tests, linear and nonlinear regression models, categorical response models, statistical design of experiments, and advanced methods like re‐sampling tests, Bayesian statistics, and methods for high‐throughput ‐omics data...
August 2, 2018: Current Protocols in Immunology
Ilija Brizić, Berislav Lisnić, Wolfram Brune, Hartmut Hengel, Stipan Jonjić
This unit describes procedures for infecting newborn and adult mice with murine cytomegalovirus (MCMV). Methods are included for propagating MCMV in cell cultures and for preparing a more virulent form of MCMV from salivary glands of infected mice. A plaque assay is provided for determining MCMV titers of infected tissues or virus stocks. Also, a method is described for preparing the murine embryonic fibroblasts used for propagating MCMV and for the plaque assay. © 2018 by John Wiley & Sons, Inc.
July 25, 2018: Current Protocols in Immunology
Andreas B den Hartigh, Susan L Fink
Pyroptosis is a form of programmed pro-inflammatory cell death that plays a protective role in the host response to infection, but can also promote pathogenic inflammation. Pyroptosis is mediated by the cysteine protease, caspase-1. Caspase-1 cleaves gasdermin D, releasing the N-terminal pore-forming domain, which inserts into the plasma membrane and drives osmotic lysis. Caspase-1 also proteolytically activates the inflammatory cytokines interleukin 1β (IL-1β) and IL-18. This unit describes methods for stimulating pyroptosis and assessing subsequent loss of plasma membrane integrity...
July 20, 2018: Current Protocols in Immunology
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