bluuebunny/crossref_metadata_2025_split huggingface dataset:
Measurement of MHC/Peptide Interactions by Gel Filtration
Authors: Sidney John , Southwood Scott , Oseroff Carla , del Guercio Marie‐France , Sette Alessandro , Grey Howard M. 
Published: June 2004

AbstractThis unit describes a technique for the direct and quantitative measurement of the capacity of peptide ligands to bind Class I and Class II MHC molecules. The binding of a peptide of interest to MHC is assessed based on its ability to inhibit the binding of a radiolabeled probe peptide to MHC molecules. The establishment of an MHC/peptide binding assay, and its subsequent use in determining the MHC binding capacities of peptide ligands, requires sufficient stocks of purified MHC and both labeled and unlabeled peptides. Accordingly, this unit includes protocols for the purification of Class I and Class II MHC molecules by affinity chromatography, and for the radiolabeling of peptides using the chloramine T method. A support protocol describes alterations in the basic protocol that are necessary when performing direct binding assays, which are required for (1) selecting appropriate high‐affinity, assay‐specific, radiolabeled ligands and (2) determining the amount of MHC necessary to yield assays with the highest sensitivity. After a 2‐day incubation, the bound and unbound radiolabeled species are separated, and their relative amounts are determined. Two methods for separation by size‐exclusion gel‐filtration chromatography are described, as is data analysis.
Measurement of the Kinetics of Biomolecular Interactions Using the IAsys Resonant Mirror Biosensor
Authors: George Andrew J. T. 
Published: June 2004

AbstractIn this unit, the resonant mirror biosensor or IAsys, produced by Affinity Sensors, is discussed for the measurement of the kinetics of biomolecular interactions. All biosensors operate using a common principle‐‐that binding events occurring on the sensing surface of the biosensor are converted by a transducer into an electronic signal which can then be processed by a computer to yield information on the amount of binding. Where biosensors vary is in their configuration and the nature of the transducer. Both the IAsys and BIACORE series of machines use optical techniques to detect the binding event, but the IAsys uses a resonant mirror device as the transducer, while the BIACORE uses surface plasmon resonance.When using biosensors the experiment can be divided into various phases, which are separately in this unit. The phases involve (1) the immobilization of the ligand onto the sensing surface; (2) addition of the ligate to the sensing surface, and following its association to the ligand, including the kinetics of binding; (3) washing of free ligate from chamber and following the dissociation of the ligand‐ligate complex; (4) regeneration of the surface; repetition of phases 2 and 3; and (5) data analysis. A protocol is also included for optimization of ligand pH for immobilization to the carboxymethylated dextran surfaces described in this unit.
Measuring Protein‐Protein Interactions by Equilibrium Sedimentation
Authors: Schuck Peter , Braswell Emory H. 
Published: June 2004

AbstractThis commentary unit provides a basic introduction to the principles and practice of sedimentaiton equilibrium analytical ultracentrifugation for the study of reversible protein interactions. Equilibrium sedimentation is one of the most effective methods for the detection and characterization of protein interactions and can be applied to the measurement of self association, heterologous association, binding stoichiometry, and the determination of association constants. The unit includes an extensive discussion of the instrumentation required to carry out equilibrium analytical ultracentrifugation and to perform data analysis. A strategic planning section gives the reader several experimental scenarios and conditions for using equilibrium sedimentation.
Binding of Small Peptides to Immobilized Antibodies: Kinetic Analysis by Surface Plasmon Resonance
Authors: Andreu David , Gomes Paula 
Published: June 2004

AbstractThis unit describes a method for screening small viral peptides as specific antigens using a surface plasmon resonance (SPR) biosensor. The basic protocol in this unit is suited for direct single‐step SPR analysis of small ligand‐large receptor interactions, where small peptides are used as analytes (injected in the continuous buffer flow) and monoclonal antibodies (MAbs) are immobilized on the SPR sensor chip surface. An alternate protocol is included for situations where kinetic analysis is not possible and uses a surface competition assay to indirectly measure the kinetics of small analyte binding.
Peptide Induction of Surface Expression of Class I MHC
Authors: Hansen Ted , Myers Nancy 
Published: June 2004

AbstractThis unit describes a method for comparing the relative binding of different peptides to the same MHC class I (MHC‐I) molecule using live cells. Live cells expressing suboptimally loaded MHC‐I proteins are incubated with medium containing diluted amounts of synthetic peptides to be tested for binding to class I. After overnight incubation with peptide, surface class I expression is monitored by flow cytometry using an allele‐specific MAb. Relative binding affinity of peptide reliably correlates with the amount of surface induction of the class I molecule to which it specifically binds. The mechanistic basis of this assay is that surface MHC‐I molecules become conformationally unstable shortly after peptide dissociation. However, the binding of an exogenous peptide can stabilize the surface class I molecule, prevent conformational instability, and thus increase class I surface expression in an allele‐specific manner.
Schistosomiasis
Authors: Lewis Fred 
Published: June 2004

AbstractThe trematode parasites in the family Schistosomatidae (phylum Platyhelminthes) infect a wide range of vertebrates. Three species of the genus Schistosoma are of major medical importance. This unit deals exclusively with the parasite Schistosoma mansoni, which is the species most frequently maintained in the laboratory. Among the far‐ranging investigations in the immunology of schistosomiasis are studies in vaccine development, immunopathology of granulomatous inflammation and fibrosis, eosinophil function, and in vivo regulation of TH1 and TH2 responses. This unit describes maintenance and collection procedures for various stages of the schistosome that have immunologic interest, including infection of mice with cercariae, collection of cercariae, preparation of in vitro‐derived schistosomules and in vivo‐derived schistosomules, and collection of adult worms and eggs. Included also are techniques for preparing soluble egg antigen (SEA), one of the more commonly used schistosome antigenic preparations. A discussion is given of the basic steps that are important in maintaining the snail intermediate host, and infecting the snails with schistosome miracidia. The unit deals exclusively with the parasite Schistosoma mansoni, which is the species most frequently maintained in the laboratory. Since part of the life cycle of all schistosomes involves a snail host, a description of proper maintenance for the snails is provided. Often, problems in experiments can be traced back to improper snail and parasite maintenance, or lack of attention to detail during mammalian exposure to the infective stage (cercaria) of the parasite.
Experimental Asexual Blood Stage Malaria Immunity
Authors: Amante Fiona H. , Good Michael F. 
Published: June 2004

AbstractThis unit describes three methods for investigating the immune response to murine malaria parasites. Immunization protocols using recombinant fragments of the merozoite surface protein‐1 of Plasmodium yoelii, whole blood stage malaria parasites, and live infection with parasitized red blood cells from a Plasmodium‐infected donor are provided. Methods for chemotherapeutic drug care of Plasmodium‐infected mice and for inducing malaria by adoptive transfer of antigen‐specific T cells are included. Finally, support protocols describe methods for growing, maintaining, and cryopreserving murine asexual blood stage malaria parasites and for preparing blood stage antigen for use in ELISAs.
Animal Models for Candidiasis
Authors: Romani Luigina 
Published: June 2004

AbstractAs little is known about the yeast/host interaction in Candida species other than C. albicans, this unit details the materials and experimental procedures required for development of experimental C.albicans infections, such as GI and disseminated infections in infant Swiss White mice and in adult immunocompromised BALB/c mice, primary acute disseminated infection in BALB/c and DBA/2 mice, and vaginal infection in CBA/J mice and in Wistar rats. Detailed materials and methods for C. albicans growth and detection are also described.
A Mouse Model for Cytomegalovirus Infection
Authors: Brune Wolfram , Hengel Hartmut , Koszinowski Ulrich H. 
Published: June 2004

AbstractThis 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‐forming cell (PFC) assay is provided for measuring mCMV titers of infected tissues or virus stocks. In addition, a method is described for preparing the murine embryonic fibroblasts used for propagating mCMV and for the PFC assay.
Animal Models Using Lymphocytic Choriomeningitis Virus
Authors: von Herrath Matthias , Whitton J. Lindsay 
Published: June 2004

AbstractThis unit includes protocols for inducing systemic infection and persistent infection of mice with lymphocytic choriomeningitis virus (LCMV). Methods used to measure T cell responses to LCMV are then described. A protocol to assess anti‐LCMV immunity in vivo is also included. Support protocols for preparing LCMV stocks and measuring LCMV titers using a plaque assay are also included. Finally, a support protocol for detecting anti‐LCMV antibodies by ELISA is presented.
Immunohistochemistry
Authors: Hofman Florence 
Published: June 2004

AbstractThis unit describes several methods for localizing specific antigens in various tissue and cell preparations using immunohistochemistry (IHC). Protocols describe preparation of suitable material for IHC including fresh, unfixed, frozen tissue specimens; unfixed cells, either freshly isolated or from suspension or adherent cultures, or fixed, paraffin‐embedded tissue sections. By careful selection of reagents, it is possible to detect two antigens simultaneously. For antigens that are sensitive to fixative, it may be necessary to unmask the antigen by a new technique called “antigen retrieval”. If there is cross‐reactivity between the secondary antibody and antigens present in the target cells or tissue, the secondary antibody can be preabsorbed. The different IHC protocols are represented schematically and summarized in a table, which also lists advantages and disadvantages of each approach. Causes of background staining and ways to eliminate it are also discussed.
Commonly Used Mouse Strains
Authors: Kruisbeek Ada M. 
Published: June 2004

AbstractThis appendix lists commonly used mouse strains and selected cell‐surface molecules expressed by these strains. H‐2 is the gene locus residing on chromosome 17 of the mouse which encodes major histocompatibility proteins. Only those H‐2 loci involved in immunologic interactions frequently cited in this manual are listed here‐‐i.e., Aa‐, Ab‐, Ea‐, and Eb‐‐region‐encoded MHC class II molecules, and K‐and
D‐region‐encoded MHC class I molecules. In addition to H‐2 loci, others are listed that encode immunologically important proteins. Congenic strains listed in this appendix represent inbred strains that were derived from their origin (BALB/c, C57Bl/10, and C3H) by selective matings, such that they differ from that originating strain at only one independently segregating genetic locus. The locus of choice in these cases was H‐2; thus, these strains differ from their strain of origin only in their H‐2 complex, but not in any other loci. During the production of the congenic lines, several strains were derived that had undergone intra‐H‐2 recombination events. These strains are listed here and have been invaluable in defining the major roles of various MHC glycoproteins.
Commonly Used Detergents
Authors: Coligan John E. 
Published: June 2004

AbstractThis appendix lists reference Epstein‐Barr virus (EBV)‐transformed B lymphoblastoid cell lines (B‐LCL) and the HLA types expressed by these cell lines. These B‐LCL are useful for studies of cellular immunity (e.g., restriction of antigen‐specific responses), biochemical characterization of histocompatibility molecules, and as controls for DNA‐based typing methods.
Murine Models of Immunodeficiency and Autoimmune Disease
Authors: Cohen Philip 
Published: June 2004

AbstractGenetically determined murine immunodeficiency states are useful for understanding the function of specific immune‐system genes and cellpopulations. In addition, certain immunodeficient strains may be exploited as hosts for foreign tumors or immune cells. The more commonly used immunodeficiency models are described in this appendix. Not included are strains better known for primary neurological or neuromuscular abnormalities or for defective osteoclast function. Many of the recently described immune‐deficient “knockout” strains are described, including cytokine and cytokine receptor knockout strains. The most widely studied murine strains for autoimmune disease and experimental autoreactivity are also listed.
HLA Type of EBV‐Transformed Human B Cell Lines
Authors: Hurley Carolyn Katovich , Johnson Armead H. 
Published: June 2004

AbstractThis appendix lists reference Epstein‐Barr virus (EBV)‐transformed B lymphoblastoid cell lines (B‐LCL) and the HLA types expressed by these cell lines. These B‐LCL are useful for studies of cellular immunity (e.g., restriction of antigen‐specific responses), biochemical characterization of histocompatibility molecules, and as controls for DNA‐based typing methods.
Web Sites of Interest to Immunologists
Authors: Lefranc Marie‐Paule 
Published: June 2004

AbstractThe astonishing increase in the amount of biologically relevant data on the World Wide Web (WWW) enables immunologists to immediately access databases; scan literature; use tools and software; find information on collections, educational resources, and research groups; and exploit that information for scientific projects and clinical trials. This appendix presents a selection of the links available at the international ImMunoGeneTics (IMGT) database. Most of the sections such as Databases, Immunology themes, Journals, Societies, and Tools, provide direct access to Web sites. Three other sections (Molecular Biology Servers, Resources, and Universities and Institutes) offer their own lists of useful links.
Listing, Location, Binding Motifs, and Expression of Nonclassical Class I and Related Genes and Molecules
Authors: Forman James , Lindahl Kirsten Fischer 
Published: June 2004

AbstractThe tables presented in this appendix list nonclassical class I or related genes/molecules arranged by the chromosomal region where they are encoded. This includes genes that fall into the Ib region of the murine major histocompatibility complex (MHC) which includes H2‐Q, ‐T, and ‐M, as well as CD1, which lies outside the MHC region. A final table includes genes/molecules that are encoded in diverse regions. They are included in this section because they are either class I related in that their heavy chain is related to classical class I and/or they are associated with ion given is for the C57BL/6 (B6) strain unless otherwise noted.
Receptors Involved in the Regulation of Murine NK Cells
Authors: Washington A. Valance , McVicar Daniel W. 
Published: June 2004

AbstractThis appendix presents a table of receptors found on murine NK cells. Information is given regarding the specific type of cell(s) in which the particular receptor is found, the chromosomal location, and other names and characteristics including cytoplasmic tyrosine motifs. Only documented receptors and receptor groups are presented, focusing on surface markers found on murine NK cells that regulate the balance between status quo and activation. This appendix also highlights a definite need in the field: the identification of the ligands to these receptors. Less than half of the receptors presented in this table have identified ligands. While the receptors represent advances in understanding the natural killer cell, each unidentified ligand indicates that there is still a long road ahead.
Heat‐Shock Proteins
Authors: Li Zihai , Srivastava Pramod 
Published: June 2004

AbstractHeat‐shock proteins (HSPs), or stress proteins, are highly conserved and present in all organisms and in all cells of all organisms. Selected HSPs, also known as chaperones, play crucial roles in folding/unfolding of proteins, assembly of multiprotein complexes, transport/sorting of proteins into correct subcellular compartments, cell‐cycle control and signaling, and protection of cells against stress/apoptosis. More recently, HSPs have been implicated in antigen presentation with the role of chaperoning and transferring antigenic peptides to the class I and class II molecules of the major histocompatibility complexes. In addition, extracellular HSPs can stimulate professional antigen‐presenting cells of the immune system, such as macrophages and dendritic cells. HSPs constitute a large family of proteins that are often classified based on their molecular weight: hsp10, hsp40, hsp60, hsp70, hsp90, etc. This unit contains a table that lists common HSPs and summarizes their characteristics including (a) name, (b) subcellular localization, (c) known function, (d) chromosome assignment, (e) brief comments, and (f) references.
Equipment
Authors: Donovan John , Brown Patricia 
Published: June 2004

AbstractSpecial equipment and utensils are itemized in the materials list of each protocol. Here, no attempt has been made to list all items required for each procedure, but rather only those items that might not be readily available in the laboratory, have specific specifications, or require special preparation. This appendix lists standard items found in the modern immunology laboratory, i.e., items used extensively in this manual and thus generally not included in the individual materials lists. For a full inventory of equipment and utensils used in a modern immunology laboratory, it will be useful to review both the listings here and the materials lists accompanying each unit (or those units of interest).
Wright‐Giemsa and Nonspecific Esterase Staining of Cells
Authors: Strober Warren 
Published: June 2004

AbstractThis appendix provides two protocols for staining cells. The stains used in the Wright‐Giemsa protocol, the Romanowsky stains, are a mixture of methylene blue (and other closely related thiazine dyes) and eosin. The staining protocol is a two‐stage method that allows for a more intense staining of the nuclei than would be possible if the Wright and Giemsa stains were mixed together. In the second protocol, nonspecific esterase stain are used to identify cell types containing esterases that have a characteristic ability to split esters under particular conditions. In the staining method given here, the substrate, a‐naphthyl butyrate, is incubated with cells under conditions in which esterases present in monocytes/macrophages split the substrate to yield an intermediate that can be coupled with a substance, hexazotized pararosaniline, to yield a colored precipitate. Thus, this staining reaction can be used to identify monocytes/macrophages in cell preparations.
Diagnosis and Treatment of Mycoplasma‐Contaminated Cell Cultures
Authors: Fitch Frank W. , Gajewski Thomas F. , Yokoyama Wayne M. 
Published: June 2004

AbstractMycoplasma contamination is a serious and frequent problem in the culture laboratory. Although mycoplasma contamination may be suspected by the failure of cells to thrive, the formal diagnosis rests on the detection of adenosine phosphorylase secretion by infected cell lines. This appendix describes how to test for mycoplasma contamination, and also presents methods for antibiotic treatment of infected cultures.
Quantitation of DNA and RNA with Absorption and Fluorescence Spectroscopy
Authors: Gallagher Sean R. 
Published: June 2004

AbstractReliable quantitation of nanogram and microgram amounts of DNA and RNA in solution is essential to researchers using molecular biology techniques. In addition to the traditional absorbance measurements at 260 nm, two more sensitive fluorescence techniques are presented in this appendix. These three procedures cover a range from 5 to 10 ng DNA/ml to 50mg DNA/ml.
Gram Staining
Authors: Coico Richard 
Published: June 2004

AbstractNamed after Hans Christian Gram who developed the method in 1884, the Gram stain allows one to distinguish between Gram‐positive and Gram‐negative bacteria on the basis of differential staining with a crystal violet‐iodine complex and a safranin counterstain. The cell walls of Gram‐positive organisms retain this complex after treatment with alcohol and appear purple, whereas gram‐negative organisms decolorize following such treatment and appear pink. The method described here is useful for assessing bacterial contamination of tissue culture samples or for examining the Gram stain status and morphological features of bacteria isolated from mixed or isolated bacterial cultures.
Monoclonal Antibodies to Mouse Cell‐Surface Antigens
Authors: Alaverdi Noosheen 
Published: June 2004

AbstractThe cluster of differentiation (CD) nomenclature, originally devised by the International Workshop on Human Leukocyte Differentiation Antigens (HLDA) to classify leukocyte surface antigens, has become more accepted by researchers in nonhuman animal models. The CD homologues from other species are usually identified by multiple studies, including biochemical, functional analysis, cloning, and immunological assays. This unit compiles a complete (as of August, 2001) list of monoclonal antibodies (mAb) to the mouse CD antigens and several non‐CD surface antigens that are commonly used as phenotypic and functional markers in the mouse system. There are many reagents recognizing polymorphic epitopes of the mouse MHC and different subunits of TCR; for simplicity, only a few mAbs to the framework determinants of these molecules are listed here.
Genetic Mapping in the Mouse
Authors: Pavan William J. 
Published: June 2004

AbstractSeveral molecular genetic approaches are available for identifying the location of a conserved human DNA sequence in the mouse genome. The choice of approach depends on the reagents available and the precise information that the investigator would like to determine. This unit presents a method for determining the location of the murine homologue of a human gene using linkage analysis with available mouse interspecific backcrosses. Restriction fragment length polymorphism (RFLP) analysis is used to map human gene fragments in the mouse genome. The approaches described in this unit are analogous to those used for linkage analysis in human pedigrees, except the mouse crosses can and have been designed so only two alleles are segregating for each locus and the phase of every chromosome is known.
Pedigree Selection and Information Content
Authors: Hauser Elizabeth R. , Weeks Daniel E. 
Published: June 2004

AbstractThis unit covers the important criteria and methods for appropriately selecting pedigrees for linkage analysis. It includes methods for determining the power of a dataset to detect genetic loci for both model‐based (lod score) and model‐free (affected sib‐pair, affected‐relative pair) analyses.This unit covers the important criteria and methods for appropriately selecting pedigrees for linkage analysis.