bluuebunny/crossref_metadata_2025_split huggingface dataset:
Assessment of Lymphocyte Development in Radiation Bone Marrow Chimeras
Authors: Spangrude Gerald J. 
Published: June 2004

AbstractThe utilizes congenic strains of mice that differ at the CD45 (common leukocyte antigen) locus. Bone marrow‐derived cells from these strains of mice can be identified by monoclonal antibodies specific for the two allelic variants of the CD45 molecule. Because this antigen is expressed on all nucleated blood cells, it provides a convenient genetic marker for following the progeny of transplanted bone marrow cells. Transplantation of marrow between mouse strains congenic for CD45 after lethal irradiation establishes hematopoiesis driven by genetically marked cells in recipient animals. The first support protocol can be used to establish appropriate radiation doses for use in the . After several weeks, peripheral blood or primary and secondary lymphoid organs of transplant recipients can be evaluated for the presence of donor‐derived cells using the second support protocol. Two‐ or three‐color flow cytometry can be used to identify the progeny of transplanted cells, to document their cell‐surface phenotypes, and to follow development of T, B, and myeloid lineages in vivo.
Measurement of Intracellular Ions by Flow Cytometry
Authors: June Carl H. , Abe Ryo , Rabinovitch Peter S. 
Published: June 2004

AbstractThe recent development of a number of new fluorescent probes makes it possible to measure the concentrations of various intracellular free ions in single living cells. Among these ions are calcium, magnesium, sodium, potassium, and hydrogen (pH). This unit describes flow cytometric protocols using the dyes Indo‐1 AM, Fluo‐3, and Fura Red AM to measure intracellular calcium concentration. Support protocols detail the use of calcium buffers to calibrate a flow cytometric calcium assay, and methods to facilitate dye loading; an alternate protocol describes the use of a spectrofluorimeter to measure intracellular calcium for those investigators without access to a flow cytometer.
Measurement of Intercellular Conjugates by Flow Cytometry
Authors: Segal David M. 
Published: June 2004

AbstractAnalysis of fluorescently labeled cellular aggregates can be accomplished quickly and accurately with the technique of flow cytometry. With a dual‐laser flow cytometer, the excitation and emission spectra of fluorophores used to label cells are widely separated, and there is no spillover of emission from one fluorophore into the detector measuring emission from the other. Consequently, conjugates can be measured by a dual‐laser instrument when the extent of labeling two cell types with fluorophores are very different. The more commonly available single‐laser cytometers can also be used to measure multicellular conjugates, but due to overlaps in emission spectra, the extent of labeling cells with fluorophores must be controlled much more carefully when the single‐laser machines are used. This unit describes the labeling of cells and analysis of conjugates with either dual‐laser or single laser flow cytometers.
Measurement of Interleukin 3 and Other Hematopoietic Cytokines, such as GM‐CSF, G‐CSF, M‐CSF, Erythropoietin, Steel Factor, and Flt‐3 Ligand
Authors: Cooper Scott H. , Broxmeyer Hal E. 
Published: June 2004

AbstractDetection and quantitation of IL‐3 and other hematopoietic cytokines in serum samples and culture supernatants has played an important role in understanding their function and pleiotropic properties during the process of hematopoiesis. Several methods for detecting and quantitating cytokines—such as the colony‐stimulating factors granulocyte/macrophage colony‐stimulating factor (GM‐CSF), granulocyte colony‐stimulating factor (G‐CSF), macrophage colony‐stimulating factor (M‐CSF, also called CSF‐1), interleukin 3 (IL‐3), and erythropoietin (Epo), as well as two potent costimulating cytokines steel factor (SLF) and Flt3/Flk‐2 ligand (Flt3‐L)—are described in this unit.
Measurement of Mouse and Human Interferon γ
Authors: Schreiber Robert D. 
Published: June 2004

AbstractThis unit describes two protocols that can be used to quantitate interferon γ (IFN‐γ), an important modulatory lymphokine that regulates natural, cell‐mediated, and humoral immunity by eliciting a number of biological responses in many different cell types. Depending upon the specificity of the reagents used, the assays will detect either human or murine IFN‐γ. The is an enzyme‐linked immunosorbent assay (ELISA) based on IFN‐γ's unique antigenic structure. The is a functional activity assay based on IFN‐γ's ability to induce major histocompatibility (MHC) class II antigens (also termed Ia antigens) on responsive cells. The advantage of the ELISA protocol is its specificity, while the advantage of the MHC protocol is its sensitivity.
Isolation of Whole Mononuclear Cells from Peripheral Blood and Cord Blood
Authors: Kanof Marjorie E. , Smith Phillip D. , Zola Heddy 
Published: June 2004

AbstractPeripheral blood is the primary source of lymphoid cells for investigations of the human immune system. Its use is facilitated by the Ficoll‐Hypaque density gradient centrifugation method described here. It is a simple and rapid method of purifying peripheral blood mononuclear cells (PBMC) that takes advantage of the density differences between mononuclear cells and other elements found in the blood sample. The mononuclear cell sample can be purified from monocytes by adherence or by exposure to L‐leucine methyl ester; methods are described for both procedures. Cord blood and peripheral blood from infants contain immature cells, including nucleated red cells, that can result in significant contamination of the mononuclear cell layer, and removal of these cells requires additional steps that are described. The isolation procedures presented here can also be applied to cell populations derived from tissues.
Purification of T Cell Subpopulations
Authors: Kanof Marjorie E. 
Published: June 2004

AbstractThis unit describes a procedure for isolating T cell populations or subpopulations using the method of indirect panning. In this method, cells are selected by their capacity to bind to antibody‐coated plates on the basis of particular cell‐surface markers. It is superior to the antibody/complement lysis method (also presented) because the nonselected cell population can be retrieved.
Isolation of Mononuclear Cells From Tonsillar Tissue
Authors: Ryon Judith J. 
Published: June 2004

AbstractThis unit describes a protocol for the derivation of lymphoid cell populations from tonsillar tissue. The procedure can also be applied with little or no modification to spleen and lymph node tissue. Human tonsils are the most readily available lymphoid organs and are often used as a source of large numbers of cells characteristic of local lymphoid tissue. Mononuclear cells are isolated by mechanical disruption of tonsil tissue followed by Ficoll‐Hypaque gradient centrifugation. Cells may then be separated into subpopulations according to general protocols used for peripheral blood lymphocytes.
Measurement of Immunoglobulin Synthesis Using the ELISPOT Assay
Authors: Lycke Nils Y. , Coico Richard 
Published: June 2004

AbstractThe enzyme‐linked immunospot (ELISPOT) assay for detection of single antibody‐secreting cells has become the best alternative method to the conventional plaque‐forming cell (PFC) assays. Among its several advantages are better antigen stability and specificity as well as fewer limitations in the diversity of antigens that can be used in the assay. In addition, the ELISPOT assay can be used to detect two antigenically different secreted antibodies simultaneously by two‐color analysis and offers the unique possibility of quantifying the number of antibody molecules secreted per cell. Finally, the assay can be used to detect single antibody‐secreting cells in tissues that usually confront the immunologist with difficulties, e.g., gut lamina propria from humans or mice. This unit presents the ELISPOT assay in three steps: coating of antigen to a solid phase, incubation of antibody‐producing cells in appropriate dilution, and detection of the antigen‐antibody complex formed at the site of the active antibody‐secreting cell. The assay can be performed using polystyrene plates or nitrocellulose membrane in microtiter plates or using nitrocellulose membrane in a blotting manifold.
Measurement of Cellular Adhesion Under Static Conditions
Authors: Mobley James L. , Shimizu Yoji 
Published: June 2004

AbstractThis unit describes procedures for measuring the adhesion of lymphoid cells to immobilized ligands or adherent cell monolayers. The basic protocol, which is optimized for human T cells, can be easily modified to study almost any cell type in suspension if the corresponding adhesive ligand is available. Alternate protocols describe modifications to the basic protocol that allow analysis of cell adhesion in the presence of stimulators of lymphocyte activation or monoclonal antibodies (MAbs) that react with adhesion molecules or their ligands, or analysis of cell adhesion to adherent cell monolayers. A support protocol is provided for determining the optimal concentration of adhesion ligands and choosing the appropriate type of microtiter plate for static adhesion assays.
Isolation of Human Eosinophils
Authors: Lim Kaiser G. , Weller Peter F. 
Published: June 2004

AbstractHighly purified eosinophils can be isolated from peripheral blood by negative selection using anti‐CD16 immunomagnetic beads. The basic protocol describes a sequential fractionation of peripheral blood in which eosinophils are separated first from mononuclear cells and then from CD16+ neutrophils. This technique is easy to use, fast, and highly reproducible. A support protocol describes a staining method that can be used to evaluate the purity of eosinphils.
Immunoaffinity Chromatography
Authors: Springer Timothy A. 
Published: June 2004

AbstractThis unit describes the isolation of soluble or membrane‐bound protein antigens from cells or homogenized tissue by immunoaffinity chromatography. The technique involves the elution of a single protein from an immunoaffinity column after prior elution of nonspecifically adsorbed proteins. To elute the bound antigen from the immunoaffinity matrix, the antibody‐antigen interaction is destabilized by brief exposure to high‐pH or low‐pH buffer. The use of batch purification of antigens is an alternate procedure and results in shorter column loading times. The detergent octyl β‐D‐glucoside can be used instead of Triton X‐100 for elution. Because octyl β‐D‐glucoside has a high critical micelle concentration (CMC), it can be readily removed by dialysis, as described.
Electroelution of Proteins from Stained Gels
Authors: Smith John A. 
Published: June 2004

AbstractIn this procedure an SDS‐polyacrylamide gel containing electrophoresed proteins is stained with Coomassie brilliant blue, the gel band of interest is excised, and the stained protein is electrophoretically eluted from the minced gel. After removal of most of the salt and sodium dodecyl sulfate (SDS) by dialysis, the protein is precipitated to remove remaining detergent and salt and then analyzed.
Phage Display Selection and Analysis of Ab‐Binding Epitopes
Authors: Enshell‐Seijffers David , Gershoni Jonathan M. 
Published: June 2004

AbstractThe identification and characterization of B cell epitopes by combinatorial phage display peptide analyses is based on the principle that unique peptides can be affinity‐purified from an enormous collection of random peptides. Moreover, once selected, the peptide sequence can be elucidated; filamentous bacteriophages have been genetically engineered to incorporate the DNA template corresponding to the peptide displayed on its surface. This unit begins with a discussion of some of the factors that distinguish available libraries. Protocols are then provided for affinity selection of antibody‐specific phages, determination of phage titer, confirmation and amplification of positive phages, phage characterization, and construction of custom‐tailored phages. The selection protocol in this unit is specific and designed for libraries that are used in the authors' laboratory and are based on the fth1 or fd‐tet derived vectors. However, information is included for adapting these protocols to the specific requirements of other phage display libraries.
Preparation of Genomic DNA from Mammalian Tissue
Authors: Strauss William.M. 
Published: June 2004

AbstractThere are a number of different procedures for the preparation of genomic DNA. They all start with some form of cell lysis, followed by deproteinization and recovery of DNA. The main differences between various approaches lie in the extent of deproteinization and in molecular weight of the DNA produced. The isolation procedure described here is relatively brief and relies on the powerful proteolytic activity of proteinase K combined with the denaturing ability of the ionic detergent SDS.
Reagents and Radioisotopes Used to Manipulate Nucleic Acids
Authors: Struhl Kevin 
Published: June 2004

AbstractThe application of molecular biological techniques to immunological studies is based in large part on the manipulation of nucleic acids‐‐RNA, DNA, and oligonucleotides. This unit offers very basic procedures that are used in a number of protocols later in the chapter. In particular, preparation of nucleoside triphosphates‐‐the substrates for almost all synthetic reactions using nucleic acids‐‐is described; the relevant radioisotopes, 32P, 35S, and 3H are discussed; and methods for quantitating radioisotope and biotin incorporation into nucleic acids are presented. Finally, column chromatographic (gel filtration) procedures for separating labeled DNA from synthetic precursors are described.
Isolation and Use of Rafts
Authors: Brown Deborah A. 
Published: June 2004

AbstractThis unit describes methods for isolating and analyzing rafts by detergent insolubility. To distinguish these rafts from raft‐like membranes isolated by other methods, they are referred to here as detergent‐resistant membranes (DRMs). DRMs can be isolated by flotation on sucrose density gradients or by pelleting after detergent extraction. DRM proteins can be analyzed by SDS‐PAGE and immunoblotting. Additionally, radiolabeled DRM proteins can be analyzed, and lipids can be quantitated by high‐performance thin layer chromatography. Support protocols needed for the lipid analysis are also provided. Finally, protocols for raft disruption by cholesterol removal and measuring the kinetics of such removal are included together with a method that reverses the cholesterol removal (cholesterol repletion).
Measurement of Fcγ Receptor–Mediated Binding and Phagocytosis
Authors: Politis Alexander D. , Vogel Stefanie N. 
Published: June 2004

AbstractMacrophages are perhaps best recognized for their ability to phagocytose foreign particles. Phagocytosis can be nonspecific, but is often dramatically augmented with the aid of receptors that recognize the Fc portion of antibodies bound to the intruder. Macrophages utilize receptors that recognize IgG (FcγR), which is a major opsonic mediator of phagocytosis. This unit describes two functional assays that quantify macrophage FcγR surface expression. In each assay, IgG‐opsonized sheep red blood cells (SRBC) labeled with Na251CrO4 are used to detect interaction with macrophages. The is used to measure phagocytosis of target cells. The is used to measure FcγR‐mediated binding of target cells to macrophages. A choice of opsonin in both methods allows for some discrimination between subclasses of FcγRs that are responsible for the observed results.
Measurement of Interferon‐Mediated Antiviral Activity of Macrophages
Authors: Falk Lydia A. 
Published: June 2004

AbstractMacrophages have been shown to produce interferons (IFNs) that exert an antiviral effect‐‐resistance to the cytopathic effect (CPE) of viral infection‐‐on sensitive cell types such as fibroblasts. In some cases, IFN levels in macrophage supernatants may be below the limits of detection but still may be sufficient to confer antiviral activity on macrophages. This unit provides an assay for the qualitative demonstration of the presence or absence of endogenous IFN production in murine macrophages by measuring the resistance of macrophages to the CPE of infection with vesicular stomatitis virus (VSV).
Animal Models of Airway Sensitization
Authors: Hamelmann Eckard , Gelfand Erwin W. 
Published: June 2004

AbstractAirway inflammation and hyperresponsiveness (AHR) are hallmarks and distinguishing features of bronchial asthma. This unit contains protocols that employ a mouse model for the study of allergen‐induced AHR. It includes methods for sensitizing mice to allergens via the airways and assessing tracheal smooth muscle reactivity in vitro and in vivo. Protocols to measure and evaluate immunological mechanisms that contribute to the development to allergen‐induced asthma are also provided.
Presenting Exogenous Antigen to T Cells
Authors: Harding Clifford V. 
Published: June 2004

Abstract
Antigen‐processing experiments can be performed with a wide variety of antigen‐presenting cells (APCs). An important technical distinction for some types of experiments is whether the APCs are adherent or nonadherent, since this dictates the procedures that must be used to wash the cells as the medium is changed. This unit describes approaches that can be used for adherent cells (e.g., macrophages or fibroblasts) or nonadherent cells (e.g., B lymphoma cell lines), or both, using similar cell numbers. The protocols utilize T hybridomas to detect expression of peptide‐MHC complexes, since these cells provide the most convenient, consistent, and flexible T cell readout systems for these purposes. If desired, antigen‐specific T cell clones can be used in lieu of T hybridoma cells, but T cell clones often give poorer responses than T hybridomas to fixed APCs due to fixation‐induced loss of costimulator function.
Measurement of Peptide‐MHC Interactions in Solution Using the Spin Column Filtration Assay
Authors: Buus Søren , Lise Lauemøller Sanne , Stryhn Anette , Østergaard Pedersen Lars 
Published: June 2004

AbstractThis unit describes how peptide‐MHC complexes can be generated in vitro using affinity‐purified MHC and synthetic peptide. The unit first describes how the interaction between peptide and MHC interaction can be measured in an accurate, quantitative biochemical assay. This procedure has been optimized for efficient separation of free peptide and MHC‐bound peptide through a novel principle, termed “gradient centrifugation.” The first two support protocols describe how to set up a biochemical fluid‐phase binding reaction between peptide and MHC class I and class II, respectively. Also, an alternative procedure for setting up a biochemical fluid phase binding reaction between β2m and MHC class I is included. Finally a more versatile inhibition assay is described. The assay is simple and robust, and has several advantages compared to the classical gel‐filtration assay, including increased sensitivity and throughput. It also demands fewer resources both in terms of unique reagents and labor, and it generates less hazardous waste. Thus, the spin column gel‐filtration assay is ideal for routine work.
Animal Models for the Analysis of Immune Responses to Leishmaniasis
Authors: Sacks David L. , Melby Peter C. 
Published: June 2004

AbstractThis unit focuses on the murine model of cutaneous leishmaniasis and models of visceral leishmaniasis in mice and hamsters. Each basic protocol describes the methods used to inoculate parasites and to evaluate infections with regard to lesion progression and visceralization, and quantification of parasite load. Five support protocols are provided; two for the growth and isolation of metacyclic promastigotes from in vitro culture, one for isolation of tissue amastigotes from infected animals, one for cryopreservation of parasites, and one for the preparation of blood agar plates for quantitation of parasite numbers in infected tissue.
Animal Model for Infection with Listeria monocytogenes
Authors: Busch Dirk H. , Vijh Sujata , Pamer Eric G. 
Published: June 2004

AbstractThis unit describes methods for infecting mice with L. monocytogenes. Optimal media for growth and methods to maintain bacterial virulence by passage through mice are included. Methods for determining the severity of splenic and hepatic infection are detailed, with strategies for distinguishing innate from specific immune responses following L. monocytogenes infection. This infection induces MHC class I‐restricted CD8+ cytolytic T lymphocytes that clear infection and provide long‐term immunity. This unit describes methods that can be used for in vitro expansion of L. monocytogenes‐specific T cells.
Animal Model of Nippostrongylus brasiliensis and Heligmosomoides polygyrus
Authors: Camberis Mali , Le Gros Graham , Urban Joseph 
Published: June 2004

AbstractAnimal models of Nippostrongylus brasiliensis and Heligmosomoides polygyrus infection are powerful tools for the investigation of the basic biology of immune responses and protective immunity. In particular, they model the induction and maintenance of Th2 type immune responses and exhibit all the requisite hallmarks of CD4 T cell–dependent IgE production, eosinophilia, mastocytosis, and mucus production. This chapter describes simple, cost‐effective techniques for using and maintaining these easy‐to‐work‐with parasites in the context of a modern laboratory.
The P815 Mastocytoma Tumor Model
Authors: Gajewski Thomas F. , Markiewicz Mary A. , Uyttenhove Catherine 
Published: June 2004

AbstractThis unit presents an experimental tumor model which has led to pivotal advances in tumor immunology culminating in the preclinical development of human cancer vaccines for melanoma. The model employs the use of the P815 mastocytoma cell line. Although the P815 cell line belongs to the mast cell lineage, it offers several advantages for in vivo experimentation of the tumor‐host relationship. It grows progressively in the majority of syngeneic DBA/2 mice and can be implanted either intraperitoneally or subcutaneously. Moreover, immunogeneic variants have been created yielding tumors that are spontaneously rejected BB a behavior that has provided a context in which to study the immunologically relevant molecules and cells that dictate a successful anti‐tumor response.
The TRAMP Mouse as a Model for Prostate Cancer
Authors: Hurwitz Arthur A. , Foster Barbara A. , Allison James P. , Greenberg Norman M. , Kwon Eugene D. 
Published: June 2004

AbstractThe transgenic adenocarcinoma of the mouse prostate (TRAMP) model closely mirrors the pathogenesis of human prostate cancer. Male TRAMP mice uniformly and spontaneously develop autochthonous (orthotopic) prostate tumors following the onset of puberty. Prostate cancer occurs consequent to the expression of SV40 T antigen. The versatility of the TRAMP model has been extended by establishment of several TRAMP‐derived prostate tumor cells lines that can be injected into syngeneic male nontransgenic C57BL/6 hosts to induce ectopic prostate tumorigenesis. Subcutaneous tumor induction using the TRAMP‐C cell lines has provided the basis for two additional murine models, the first of which can be used for rapid screening of experimental therapies for the treatment of primary prostate tumors and the second for testing the effectiveness of adjunctive therapies targeting prostate cancer metastases. Detailed descriptions for the harvesting and microdissection of TRAMP prostates and tumors, and the evaluation and scoring of tumors are also provided.