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Current Protocols in Immunology

John T Schroeder, Anja P Bieneman
Isolating human basophils from blood has long been hampered by the fact that these granulocytes represent just 1% or less of the circulating leukocyte population. We describe herein laboratory protocols that have been refined over the past ∼25 years that now enable investigators to prepare basophils for use in a variety of assays to assess the in vitro biology of these immune cells, both in IgE -dependent and -independent responses.
February 2, 2016: Current Protocols in Immunology
Marjorie E Kanof
This unit describes a procedure for separating T cells from other mononuclear cells by exploiting the unique ability of cells to bind to and form rosettes with sheep red blood cells (SRBC). This isolation method also allows recovery of the nonrosetting cell population (B lymphocytes, monocytes, and macrophages). Neuraminidase- and 2-aminoethylisothiouronium bromide (AET)-treated SRBC are used for rosetting because of enhanced binding to T cells. It should be noted that use of the rosetting technique to obtain purified T cells or purified non-T cells by negative selection has largely been superceded by other techniques such as panning and immunomagnetic beads...
February 2, 2016: Current Protocols in Immunology
Harald Schulze
Megakaryocytes (MKs) are the source of circulating platelets and are readily recognized by their large size and distinctive morphology. Their poor representation in hematopoietic tissues often requires considerable ex vivo expansion to generate cells for biochemical and cell biological studies. These experimental protocols describe the assessment of megakaryocytic potential within hematopoietic precursor cells in the bone marrow by colony-forming assays and expansion and enrichment of MKs from cultured fetal liver or spleen or bone marrow cells...
February 2, 2016: Current Protocols in Immunology
Emilie Hollville, Seamus J Martin
Apoptosis is a mode of programmed cell death that plays an important role during development and in the maintenance of tissue homeostasis. Numerous physiological as well as pathological stimuli trigger apoptosis such as engagement of Fas, TRAIL, or TNF receptors, growth factor deprivation, hypoxia, or exposure to cytotoxic drugs. Apoptosis is coordinated from within by members of the caspase family of cysteine proteases that, upon activation, trigger a series of morphological changes including cell shrinkage, extensive plasma membrane blebbing, chromatin condensation, DNA hydrolysis, and nuclear fragmentation...
February 2, 2016: Current Protocols in Immunology
Duojiao Ni, Peng Xu, Sean Gallagher
Immunoblotting (western blotting) is used to identify specific antigens recognized by polyclonal or monoclonal antibodies. This unit provides protocols for all steps, starting with solubilization of the protein samples, usually by means of SDS and reducing agents. Following solubilization, the material is separated by SDS-PAGE and the antigens are electrophoretically transferred to a membrane, a process that can be monitored by reversible staining with Ponceau S. The transferred proteins are bound to the surface of the membrane, providing access to immunodetection reagents...
2016: Current Protocols in Immunology
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...
2016: Current Protocols in Immunology
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...
2016: Current Protocols in Immunology
Balázs Koscsó, Milena Bogunovic
The unit presents a method for analysis of intestinal dendritic cell (DC) and macrophage subsets by flow cytometry in the single cell suspension prepared from the mouse small and large intestine (Basic Protocol). describes a strategy to enrich the hematopoietic cell fraction in the sample by Percoll gradient centrifugation, and describes preparation of single cell suspensions from specific tissue layers of the small intestine, such as the epithelium, villi mucosa, submucosa, and muscularis externa. Finally, Support Protocol explains how to purify specific intestinal DC and macrophage subsets by flow-cytometry-based cell sorting...
2016: Current Protocols in Immunology
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...
2016: Current Protocols in Immunology
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...
2016: Current Protocols in Immunology
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...
2016: Current Protocols in Immunology
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 ( 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...
2016: Current Protocols in Immunology
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...
2016: Current Protocols in Immunology
Warren Strober
The protocol described in this appendix allows for light microscopic quantitation of cell viability. Cells are suspended in PBS containing trypan blue and then examined to determine the percentage of cells that have clear cytoplasm (viable cells) versus cells that have blue cytoplasm (nonviable cells).
November 2, 2015: Current Protocols in Immunology
Douglas B Kuhns, Debra A Long Priel, Jessica Chu, Kol A Zarember
This unit describes the isolation of human polymorphonuclear neutrophils (PMN) from blood using dextran sedimentation and Percoll or Ficoll-Paque density gradients. Assays of neutrophil functions including respiratory burst activation, phagocytosis, and microbial killing are also described.
November 2, 2015: Current Protocols in Immunology
Yuzhi Yin, Alyssa Mitson-Salazar, Calman Prussin
Intracellular cytokine staining (ICCS), employing fluorescently labeled MAbs detected by flow cytometry, has emerged as the premier technique for studying cytokine expression at the single-cell level. Advances in polychromatic flow cytometry have dramatically enhanced the sophistication of ICCS investigations. ICCS can simultaneously measure multiple cytokines within a single cell, allowing the detection of complex cytokine phenotypes. Additionally, cytokines can be measured with a variety of other analytes, including transcription factors, proliferation dilution dyes, activation markers, and viability dyes...
2015: Current Protocols in Immunology
Muthulekha Swamydas, Yi Luo, Martin E Dorf, Michail S Lionakis
Neutrophils represent the first line of defense against bacterial and fungal pathogens. Indeed, patients with inherited and acquired qualitative and quantitative neutrophil defects are at high risk for developing bacterial and fungal infections and suffering adverse outcomes from these infections. Therefore, research aiming at defining the molecular factors that modulate neutrophil effector function under homeostatic conditions and during infection is essential for devising strategies to augment neutrophil function and improve the outcome of infected individuals...
2015: Current Protocols in Immunology
Peter V Hornbeck
This unit describes six different ELISA systems for the detection of specific antibodies, soluble antigens, or cell-surface antigens. In all six systems, soluble reactants are removed from solution after specifically binding to solid-phase reactants. In the first four protocols, solid-phase reactants are prepared by adsorbing an antigen or antibody onto plastic microtiter plates; in the next two protocols, the solid-phase reactants are cell-associated molecules. In all protocols, the solid-phase reagents are incubated with secondary or tertiary reactants covalently coupled to an enzyme...
2015: Current Protocols in Immunology
Anthony E Zamora, Steven K Grossenbacher, Ethan G Aguilar, William J Murphy
Natural killer (NK) cells are large granular lymphocytes of the innate immune system, responsible for direct targeting and killing of both virally infected and transformed cells. NK cells rapidly recognize and respond to abnormal cells in the absence of prior sensitization due to their wide array of germline-encoded inhibitory and activating receptors, which differs from the receptor diversity found in B and T lymphocytes that is due to the use of recombination-activation gene (RAG) enzymes. Although NK cells have traditionally been described as natural killers that provide a first line of defense prior to the induction of adaptive immunity, a more complex view of NK cells is beginning to emerge, indicating they may also function in various immunoregulatory roles and have the capacity to shape adaptive immune responses...
2015: Current Protocols in Immunology
Sondra L Downey, Bogdan I Florea, Herman S Overkleeft, Alexei F Kisselev
Proteasome inhibitors are indispensable research tools in immunology and cell biology. With numerous proteasome inhibitors available commercially, choosing the appropriate compound for a biological experiment may be challenging, especially for a novice. This unit provides an overview of the proteasome inhibitors commonly used in research. It discusses how to select an appropriate highly specific inhibitor, its concentration, and length of exposure for mammalian cell culture experiments. In addition, assays that can be used to confirm proteasome inhibition are discussed...
2015: Current Protocols in Immunology
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