bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification and Concentration of DNA from Aqueous Solutions
Authors: Moore David
Published: June 2004

This unit presents basic procedures for manipulating solutions of single‐ or double‐stranded DNA through purification and concentration steps. The , using phenol extraction and ethanol precipitation, is appropriate for the purification of DNA from small volumes (<0.4 ml) at concentrations <1 mg/ml. Isopropanol may also be used to precipitate DNA, as described in an alternate protocol. Three support protocols outline methods to buffer the phenol used in extractions, concentrate DNA using butanol, and extract residual organic solvents with ether. An alternative to these methods is nucleic acid purification using glass beads, and is described here. These protocol may also be used for purifying RNA. The final protocols provide modifications to the that are used for concentrating RNA and extracting and precipitating DNA from larger volumes and from dilute solutions, and for removing ow‐molecular‐weight oligonucleotides and triphosphates.
Hybridization Analysis of DNA Blots
Authors: Brown Terry
Published: June 2004

The principle of hybridization analysis is that a single‐stranded DNA or RNA molecule of defined sequence (the probe) can base‐pair to a second DNA or RNA molecule that contains a complementary sequence (the target), with the stability of the hybrid depending on the extent of base pairing that occurs. Experimentally, the analysis is usually carried out with a probe that has been labeled and target DNA that has been immobilized on a membrane support. Hybridization analysis is sensitive and permits detection of single‐copy genes in complex genomes. This unit presents a basic procedure for hybridization analysis with a radiolabeled DNA probe. Despite its lack of embellishment, the protocol gives acceptable results with Southern blots on nitrocellulose and nylon (uncharged and charged) membranes. An describes a similar method for probing DNA blots with a radiolabeled RNA probe. A for stripping blots to ready them for reprobing is also provided.
Preparation of RNA from Tissues and Cells
Authors: Ribaudo Randall , Gilman Michael , Kingston Robert E. , Choczynski Piotr , Sacchi Nicoletta
Published: June 2004

Most procedures for isolating RNA from eukaryotic cells involve lysing and denaturing cells to liberate total nucleic acids. Additional steps are then required to remove DNA.

The first basic protocol describes hot phenol extraction of RNA; the method eliminates or minimizes DNA contamination by the shearing of DNA. The second basic protocol allows rapid preparation of total cytoplasmic RNA by using a nonionic detergent to lyse the plasma membrane, leaving the nuclei intact. The nuclei and hence the bulk of the cellular DNA are then removed with a simple brief centrifugation. A guanidinium thiocyanate protocol describes the separation of RNA from other cellular macromolecules in a guanidinium lysate using a CsCl step gradient. A protocol is also provided for isolation of poly(A+) mRNAs from total RNA.
Transfection by Electroporation
Authors: Potter Huntington
Published: June 2004

Electroporation yields a high frequency of permanent transfectants, has a high efficiency of transient gene expression, and can be easier to carry out than alternative techniques. Electroporation makes use of the fact that the cell membrane acts as an electrical capacitor which is unable (except through ion channels) to pass current. Subjecting membranes to a high‐voltage electric field results in their temporary breakdown and the formation of pores that are large enough to allow macromolecules (as well as smaller molecules such as ATP) to enter or leave the cell. The reclosing of the membrane pores is a natural decay process which is delayed at decreased temperatures. This unit presents the procedures which can be used for both transient and stable transfections.
Stable Transfer of Genes into Mammalian Cells
Authors: Kingston Robert E.
Published: June 2004

Analysis of gene function frequently requires the formation of mammalian cell lines that contain the studied gene in a stably integrated form. Approximately one in 104 cells in a transfection will stably integrate DNA, so a dominant selectable marker is used to permit isolation of stable transfectants. Appropriate selection conditions for the parental cell line are first determined, and the gene to be studied is then transfected into that cell line with a gene that expresses a selectable marker. The cells are allowed to grow under selection for approximately 10 doublings before individual colonies are picked and expanded into cell lines. This unit includes conditions for six markers commonly used for selection of mammalian cells. An alternative, specialized procedure for transferring immunoglobulin chains into lymphoid cells is presented in Unit .
Transfection of Lymphoid Cells
Authors: Morrison Sherie L.
Published: June 2004

Using available vectors, a single immunoglobulin chain or both heavy (H) and light (L) chains can be simultaneously transferred to recipient myeloma cells. Several methods can be used to prepare stable transfected lymphoid cells. The in this unit describes protoplast fusion, which is suitable for certain lymphoid cells but tends to be more cumbersome than electroporation or liposome‐mediated transfection, which are described in the two alternate protocols. All three techniques may be adapted for stable transfection of other eukaryotic cell lines.
Rapid Production of Retroviruses for Efficient Gene Delivery to Mammalian Cells Using 293T Cell–Based Systems
Authors: Swift Susan , Lorens James , Achacoso Philip , Nolan Garry P.
Published: June 2004

This unit details the applications of one of the more common retroviral packaging systems, based on the highly transfectable 293T cell. The packaging system employs the use of the Phoenix cell lines. Calcium phosphate‐mediated transfection is described for efficient introduction of retroviral vector plasmid DNA into the cells to generate high yields of virion‐containing supernatant. An alternate protocol describes a method for transfecting retroviruses that contain a vesicular stomatitis virus G (VSV G) protein. Such virions are said to be “pseudotyped” with VSV G glycoprotein. Support protocols provide a simple method for concentrating VSV‐G‐pseudotyped retroviruses, as well as methods for culturing, cryopreserving, thawing, and drug selecting the Phoenix packaging cell lines. Finally, several methods for transfecting adherent or suspension cells with retroviruses are described.
Construction of Immunoglobulin Fusion Proteins
Authors: Hollenbaugh Diane , Aruffo Alejandro
Published: June 2004

Recombinant DNA technology has allowed the preparation of chimeric genes encoding proteins with novel properties. This unit describes the construction and subsequent testing of genes encoding immunoglobulin chimeras. The first protocol details fusion of a protein (or protein fragment) of interest onto an immunoglobulin constant region using a modified version of the expression vector pCDM8. The resulting fusion protein generally retains the functional properties of both the protein of interest and the immunoglobulin constant region; this can be demonstrated as described here.
Phage Display of Single‐Chain Antibody Constructs
Authors: Benhar Itai , Reiter Yoram
Published: June 2004

Phage display is based on expressing recombinant proteins fused to a phage coat protein. The genetic information encoding for the displayed molecule is physically linked to its product via the displaying particle. With phage display, tailor‐made proteins (fused peptides, antibodies, enzymes, DNA‐binding proteins) may be synthesized and selected to acquire the desired catalytic properties or affinity and specificity of binding. This unit provides protocols for the construction of, and selection from antibody phage display libraries, together with useful approaches for the analysis of phage antibodies. This unit focuses on the construction of immune libraries where the sources for antibody genes are spleens of immunized mice and the isolation and initial characterization of single‐chain antibodies (scFvs) from such libraries.
cDNA Amplification Using One‐Sided (Anchored) PCR
Authors: Dorit Robert L. , Ohara Osamu
Published: June 2004

This unit presents a modification of PCR, called anchored PCR, that allows amplification of full‐length mRNA when only a small amount of sequence information lying within the mRNA is available. Both the original and reamplifications use an oligo(dT) primer complementary either to the poly(A) tail of the mature mRNA [when amplifying downstream (3') to the known sequence] or to an enzymatically synthesized homopolymer tail added to the cDNA following first strand synthesis [when amplifying upstream (5') to the known sequence]. The two rounds of PCR amplification result in a single product that can be sequenced directly or cloned into an appropriate vector for further analysis.
Molecular Analysis of Mouse T Cell Receptor Expression Using PCR
Authors: Fox Casey J. , Danska Jayne S.
Published: June 2004

This unit describes the use of the polymerase chain reaction (PCR) to characterize rearranged murine T cell receptor (TCR) transcripts present in primary lymphoid tissues, peripheral lymphoid tissues, and extra‐lymphoid sites of inflammation, and to quantify them either relatively or absolutely. This methodology has been used extensively to characterize TCR variability in murine thymus and liver in T cell clones, and in extralymphoid tissues where the absolute number of T cells is limited. A procedure for cloning and sequencing PCR‐amplified cDNA transcripts is provided to examine the junctional diversity of expressed genes. Also included is a method for reverse transcription of RNA into cDNA that is optimal for analysis of tissue samples where the number of T cells is limited. The detailed protocols are followed by a commentary that discusses strategies and artifacts as well as providing troubleshooting suggestions for PCR amplification and TCR analysis.
Multiprobe Ribonuclease Protection Assay for Simultaneous Measurement of mRNA Expression
Authors: Young Howard A. , Subleski Jeffrey J. , Krebs Stephanie M.
Published: June 2004

The multiprobe RNase protection assay enables investigators to monitor RNA expression of 8‐12 genes with as little as 1 µg of total RNA. The commercial availability of numerous multi‐gene template sets makes this assay practical for all basic research programs.
Immune‐Complex Assays for Tyrosine Protein Kinases
Authors: Burkhardt Anne L. , Bolen Joseph B.
Published: June 2004

Tyrosine protein kinases (TPKs) represent a diverse group of enzymes that contribute to cellular signal transduction. The generally low abundance of TPKs, coupled with their rapid activation and deactivation, usually precludes their purification through conventional biochemical means. Using immune‐complex protein kinase assays, the presence or absence of a given TPK can be established and an estimation of its functional state obtained. In the of this unit, TPKs are immunoprecipitated, allowed to autophosphorylate in the presence of labeled ATP, run out on an SDS‐PAGE gel, and detected by autoradiography. Alternate protocols are provided for the assessment of the functional state of TPKs by providing a potential substrate along with the labeled ATP in the reaction mixture. In the first alternate protocol, the exogenous substrate is a protein, permitting simultaneous assessment of autophosphorylation and exogenous substrate phosphorylation. The second alternate protocol utilizes a peptide substrate, resulting in a rapid, high‐throughput assay that evaluates only exogenous substrate phosphorylation.
Analyzing Mitogen‐Activated Protein Kinase (MAPK) Activities in T Cells
Authors: Whitmarsh Alan J. , Davis Roger J.
Published: June 2004

The recognition of peptide antigens by T cells through their antigen receptors (T cell receptors, or TCR), together with the ligation of additional surface molecules called costimulatory receptors, rapidly induces interactive signaling pathways that lead to transcriptional initiation at genes such as that of the autocrine growth factor interleukin 2 (IL‐2). Activation of the ERK and JNK subfamilies of MAPK mediates some of these signals. This unit presents procedures for a solid‐phase kinase assay and immune‐complex kinase assay to measure JNK and ERK activities, respectively, in T cells that have been appropriately stimulated. Also described is a procedure for preparing GSThyphen;cJun/GSH‐Sepharose beads needed in the solid‐phase JNK protein kinase activity assay.
Key Molecules Involved in Receptor‐Mediated Lymphocyte Activation
Authors: O'Shea John J. , Johnston James A. , Kehrl John , Koretzky Gary , Samelson Lawrence E.
Published: June 2004

This unit, along with Unit 11.9B, provides a summary of our current knowledge about various signaling pathways critical to the function of immune cells. Here, our understanding of T cell receptor (TCR)‐ and B cell receptor (BCR)‐mediated signaling is summarized. A schematic representation of immunologically relevant cytokine receptors and the Janus Family Kinases (JAKs) that is activated through these receptors is provided, along with details about molecules involved in interleukin 2 mediated signal transduction.
Signal Transduction During Natural Killer Cell Activation
Authors: Chini Claudia C.S. , Leibson Paul J.
Published: June 2004

Natural killer (NK) cells are a subpopulation of lymphocytes that can mediate cytotoxicity of certain tumor cells, virus‐infected cells, and normal cells. In addition to their cytotoxic potential, NK cells secrete a variety of cytokines and chemokines that can modulate the function, growth, and differentiation of other immune cells. These different responses are initiated by the interaction of specific NK surface receptors with defined soluble or cell‐associated ligands. There are several different types of receptors on the NK cell surface including “triggering” receptors, adhesion molecules, cytokine receptors, and MHC‐recognizing killer‐cell inhibitory receptors. The functional response of an NK cell is the result of the integration of signals transduced by these different types of receptors. Some of these signaling pathways are similar to other lymphoid cells, but there are also unique features employed by NK cells. This overview focuses on receptor‐initiated signaling pathways that modulate NK functions.
Apoptosis Signaling Pathways
Authors: Siegel Richard M. , Lenardo Michael J.
Published: June 2004

In the past few years much has been learned about the molecular signals governing apoptosis, or programmed cell death, in lymphocytes and other cells of the immune system. Two major pathways, active and passive apoptosis, have been identified and are discussed in this overview. Active apoptosis, also termed propriocidal cell death or antigen‐induced cell death, occurs when cells are stimulated through a family of TNF‐related receptors termed death receptors. These receptors are up‐regulated in activated lymphocytes and trigger apoptosis chiefly in effector cells that have been recently stimulated through the antigen receptor. Passive or lymphokine withdrawal apoptosis occurs when activated lymphocytes are deprived of essential growth cytokines and does not require death receptors. Cell death initiated by either pathway is carried out by a unique family of intracellular cysteine proteases, the caspases. Defects in each of these pathways produce distinct pathologies.
General Guidelines for Experimenting with HIV
Authors: Byers Karen B. , Engelman Alan , Fontes Benjamin
Published: June 2004

Performing experiments using human immunodeficiency virus (HIV)‐infected materials represents a potential biological hazard for the investigator. An intensive training program for laboratory personnel must always precede the actual execution of research involving manipulation of HIV. In addition, appropriate sterile tissue culture techniques are absolute prerequisites for producing meaningful experiments and for achieving safe working conditions. In most circumstances more than one investigator uses the same HIV research facility; therefore, careful training in biosafety is mandatory not only for self‐protection, but for the safety of other investigators as well. The first protocol in this unit establishes a general framework for working safely with HIV and the second describes the proper storage of HIV stocks and test supernatants.
Detection Assays for HIV Proteins
Authors: Ostrowski Mario A. , Chun Tae‐Wook , Stanley Sharilyn K. , Fauci Anthony S.
Published: June 2004

This updated unit describes four assays for detection of HIV proteins including: (1) a quantitative ELISA, (2) a quantitative immunoblotting assay, (3) a qualitative immunofluorescence assay, and (4) a functional assay to measure virus‐associated reverse transcriptase activity as an indicator of viral production.
Detection of HIVDNA and RNA Using PCR
Authors: Moir Susan , Chun Tae‐Wook
Published: June 2004

This extensively updated unit describes the detection of HIV DNA and RNA using PCR using two basic techniques for quantifying the levels of viral DNA and RNA in infected cells. The schemes for both techniques include isolation of nucleic acids, PCR reactions and detection of amplified products using Southern blotting.
Isolation and Functional Assay of the Membrane Complement Inhibitors CD55 (DAF) and CD59 (MIRL)
Authors: Parker Charles J.
Published: June 2004

The complement system is the primary effector of humoral immunity. Because of its enormous destructive capacity, mechanisms for confining the activity of the system to the desired target and elaborate safeguards for protecting self against complement‐mediated injury have evolved. Human cells, particularly those found at sites of inflammation (e.g., hematopoietic and endothelial cells), express highly specialized membrane constituents that act independently or in concert with plasma regulatory proteins to inhibit the functional activity of complement. Decay‐accelerating factor (DAF), or CD55, directly inhibits the formation and stability of the amplification C3 and C5 convertases of both the classical and the alternative pathways. Failure of a cell to regulate the amplification C3 and C5 convertases allows the generation of the potentially cytolytic membrane attack complex (MAC), or C5b‐9 (consisting of the complement components C5b, C6, C7, C8, and C9). The primary cellular regulator of the MAC is the membrane inhibitor of reactive lysis (MIRL), or CD59, which restricts complement‐mediated lysis by blocking assembly of the MAC (primarily at the stage of C9 binding and polymerization). This unit provides a basic protocol for isolating CD55 and CD59, along with two support protocols describing separate functional assays for CD59 and CD55.
Oxidative Metabolism of Murine Macrophages
Authors: Green Shawn J. , Aniagolu Jacinta , Raney Jennifer J.
Published: June 2004

This unit describes two simple and straightforward microassays that can be used to measure the levels of NO2− and O2−, respectively that are generated by a small number of immunologically‐stimulated macrophages. Detection of these products may be used to identify cytokine(s), microbe(s), or microbial products(s) that regulate oxidative metabolism and effector activity. Although a number of other reliable and sensitive methods are available for assaying these two oxidative metabolites, the microassays described here require little time, technical expertise, or materials. It is not clear at present whether human monocytes/macrophages can also produce NO2−. These protocols are therefore restricted to mouse macrophages.
Assay for Macrophage‐Mediated Anti‐Tumor Cytotoxicity
Authors: Cox George W.
Published: June 2004

This unit describes a procedure for determining ability of mouse macrophages to lyse tumor cells in vitro. The outlines a three‐stage assay that includes: (1) culture of macrophages (freshly explanted from mice or grown from a cell line) with suspected activating reagents; (2) extensive washing of the cultured macrophages to remove residual reagents, followed by incubation with [111In]‐labeled tumor cells to allow lysis to occur; and (3) collection of cell‐free culture supernatants and measurement of cytolytic activity as a function of 111In released from tumor cells destroyed by activated macrophages. The outlines a method for radiolabeling tumor cells with [111In]oxine.
Experimental Autoimmune Encephalomyelitis in the Mouse
Authors: Miller Stephen D. , Karpus William J.
Published: June 2004

This unit details the materials and methods required for both active induction and adoptive transfer of experimental autoimmune encephalomyelitis (EAE) in the SJL mouse strain using intact proteins or peptides from the two major myelin proteins: proteolipid protein (PLP) and myelin basic protein (MBP). Detailed materials and methods required for the purification of both PLP and MBP are also described. Modifications of the specified protocols may be necessary for efficient induction of active or adoptive EAE in other mouse strains.
Experimental Autoimmune Encephalomyelitis in the Rat
Authors: Swanborg Robert H. , Stepaniak Jolie A.
Published: June 2004

This unit details the materials and methods required for both active induction and adoptive transfer of experimental autoimmune encephalomyelitis (EAE) in the SJL mouse strain using intact proteins or peptides from the two major myelin proteins: proteolipid protein (PLP) and myelin basic protein (MBP). Detailed materials and methods required for the purification of both PLP and MBP are also described. Modifications of the specified protocols may be necessary for efficient induction of active or adoptive EAE in other mouse strains.
The BB Rat as a Model of Human Insulin‐Dependent Diabetes Mellitus
Authors: Whalen Barbara J. , Mordes John P. , Rossini Aldo A.
Published: June 2004

Use of the BioBreeding (BB) rat to model human insulin‐dependent diabetes mellitus (IDDM) is useful in that characteristics of diabetes in the BB rat closely parallel those observed in human IDDM. Diabetic animals can be biopsied, autopsied, and bred to study the genetic basis of IDDM. The genetic, immunological, and environmental components of the disease can all be investigated under controlled conditions. Two inbred lines of BB have been used in the majority of published studies using this model system; these rats are designated as diabetes prone (DP‐BB/Wor) and diabetes resistant (DR‐BB/Wor). This unit presents two protocols for the diagnosis and prevention of spontaneous IDDM in DP‐BB/Wor rats. Two alternate protocols are given for the induction of autoimmune disease in DR‐BB/Wor rats and for the adoptive transfer of autoimmunity into histocompatible athymic WAG
nu/nu recipient rats. Support protocols are also given for diagnosis of insulitis, treatment of diabetic rats with insulin, and serological analysis of blood samples from sentinel rats to monitor the pathogen status of the colony.
Adjuvant Arthritis in the Rat
Authors: van Eden Willem , Wagenaar‐Hilbers Josee P.A. , Wauben Marca H.M.
Published: June 2004

Adjuvant arthritis (AA) is an induced form of (sub)chronic arthritis. Strains of rats have a varying genetic susceptibility to AA, whereas mice generally are not susceptible. AA is most easily induced with mycobacteria suspended in oil, although in some strains of rats it can be induced with oily adjuvants in the absence of mycobacteria. The disease is a T cell‐mediated autoimmune arthritis that is frequently used to study immunological aspects of rheumatoid arthritis (RA) and other arthritic or inflammatory diseases in humans. Furthermore, it is used as a model for developing and testing antiinflammatory drugs. There are no particularly well‐defined autoantigens in AA; in this respect, the model resembles spontaneous arthritic diseases in humans. In all susceptible rat strains, the inflammatory process of AA is self remitting, although usually the disease is severe and leads to permanent joint malformations, including ankylosis; a time line for AA development is presented. This unit describes the induction and evaluation of AA and the preparation of adjuvant used to induce AA.
Experimental Autoimmune Uveoretinitis in the Rat and Mouse
Authors: Caspi Rachel R.
Published: June 2004

Experimental autoimmune uveoretinitis (EAU) in rats and mice is a prototypic T cell‐mediated autoimmune disease that targets the neural retina and related tissues. The model is used to represent human sight‐threatening inflammatory eye diseases that are believed to have an autoimmune etiology, and to study basic mechanisms of tolerance and autoimmunity to organ‐specific antigens from immunologically privileged sites. In this unit, EAU is induced in rats and mice by immunization with uveitogenic peptide antigens emulsified in complete Freund's adjuvant (CFA). Clinical onset is observed by both external and microscopic examination. A protocol is provided for preparation of tissue sections of affected eyes for microscopic analysis. EAU can also be induced in the rat (as described) by adoptive transfer of lymphocytes from uveitic rats into unimmunized recipients, which obviates the use of CFA. To induce EAU in mice, Bordetella pertussis toxin (PTX) is included to overcome immunological resistance mechanisms.
Experimental Autoimmune Thyroiditis in the Mouse
Authors: Kong Yi‐chi M.
Published: June 2004

Experimental autoimmune thyroiditis (EAT) in mice is an excellent model for Hashimoto's thyroiditis (HT). It is induced with thyroglobulin (Tg), a known thyroid autoantigen that is common to both mouse and human and for which several conserved, thyroiditogenic epitopes have been identified. This unit describes induction and evaluation of EAT using thyroid histology and in vitro proliferative response assays. An ELISA is presented to detect thelevel of antibody to mouse thyroglobulin (MTg). To induce EAT, either bacterial lipopolysaccharide (LPS) or supplemented complete Freund's adjuvant (CFA) can be used as adjuvant. A support protocol for preparing MTg is included. The T cell proliferation assay can be used to examine the antigenicity of synthetic peptides derived from MTg or heterologous Tg. EAT can be adoptively transferred utilizing cells that have been expanded in vitro, as described. A protocol is provided for inducing tolerance using deaggregated MTg; induction of tolerance requires larger amounts of MTg but efficiently suppresses EAT development.
Experimental Autoimmune Myasthenia Gravis in the Mouse
Authors: Wu Bo , Goluszko Elzbieta , Christadoss Premkumar
Published: June 2004

Myasthenia gravis (MG) is a T cell‐dependent antibody‐mediated autoimmune neuromuscular disease. Antibodies to the nicotinic acetylcholine receptor (AChR) destroy the AChR, thus leading to defective neuromuscular transmission of electrical impulse and to muscle weakness. This unit is a practical guide to the induction and evaluation of experimental autoimmune myasthenia gravis (EAMG) in the mouse, the animal model for MG. Protocols are provided for the extraction and purification of AChR from the electric organs of Torpedo californica, or eel. The purified receptor is used as an immunogen to induce autoimmunity to AChR, thus causing EAMG. The defect in neuromuscular transmission can also be measured quantitatively by electromyography, as described here. In addition, EAMG is frequently characterized by the presence of antibodies to AChR, which are measured by radioimmunoassay and by a marked antibody‐mediated reduction in the number of muscle AChRs. AChR extracted from mouse muscle is used in measuring serum antibody levels and for quantifying muscle AChR content.
Streptococcal Cell Wall Arthritis
Authors: Wilder Ronald L.
Published: June 2004

Streptococcal cell wall (SCW) arthritis in rats is an experimentally‐induced inflammatory model with many features that resemble rheumatoid arthritis (RA) in humans. In this unit, Lewis rats are injected with an aqueous suspension of Group A SCW streptococcal cell wall peptidoglycan‐polysaccharide polymers (SCW PG‐PS) and observed for the development of arthritis. The resulting arthritis is biphasic. An acute phase typically develops within 48 hr, followed 10 to 21 days later by a chronic phase which persists for months. Support protocols are included for preparing Group A PG‐PS and measuring PG‐PS concentration.
Induction of Inflammatory Bowel Disease in Immunodeficient Mice by Depletion of Regulatory T Cells
Authors: Read Simon , Powrie Fiona
Published: June 2004

The severe combined immunodeficiency (SCID) model of colitis shares many features of idiopathic inflammatory bowel disease (IBD) in humans. The SCID model is highly reproducible and easily manipulated, and as such provides a useful tool for studying mucosal immune regulation as it relates to the pathogenesis and treatment of IBD in humans. This unit describes a cell transfer system in which SCID mice are reconstituted with CD4+ CD45RBhigh cells to induce IBD. The CD4+ cells are isolated by immunomagnetic negative selection using monoclonal antibodies, and are then separated by fluorescent cell sorting into CD45RBhigh and CD45RBlow cells. The former population reproducibly induces disease in recipients. A support protocol describes methods to monitor disease progression, which is characterized by weight loss, loose stools, and histologically assessed lesions in the colon.
Autoimmune Myocarditis
Authors: Smith Stacy C.
Published: June 2004

Myosin‐induced autoimmune myocarditis (EAM) is a model of inflammatory heart disease initiated by CD4+ T cells. It is a paradigm of the immune‐mediated cardiac damage believed to play a role in the pathogenesis of a subset of postinfectious human cardiomyopathies. Myocarditis is induced in susceptible mice by immunization with purified cardiac myosin or specific peptides derived from cardiac myosin, or by adoptive transfer of myosin‐reactive T cells. Myocarditis is induced in Lewis rats by immunization with purified cardiac myosin or by adoptive transfer of T cells stimulated by specific peptides derived from cardiac myosin. Myocarditis begins 12 to 14 days after the first immunization, and is maximal after 21 days. In addition to the protocols used to induce EAM in mice and rats, support protocols are included for preparing purified cardiac myosin using mouse or rat heart tissue, preparing purified cardiac myosin for injection, and collecting and assessing hearts by histopathological means.
Induction of TNBS Colitis in Mice
Authors: Scheiffele Frank , Fuss Ivan J.
Published: June 2004

The animal model described in this unit is one of several that have been used to study the immunopathogenesis of inflammatory bowel disease. The model employs the use of 2,4,6‐trinitrobenzenesulfonic acid (TNBS), which induces severe colonic inflammation when administered intrarectally in SJL/J mice. The colitis which results from this procedure presents clinical and histopathological findings that resemble those seen in Crohn's disease. The unit describes the critical parameters needed for successful induction of TNBS‐colitis as well methods for monitoring and grading disease levels. A support protocol for isolating lamina propria mononuclear cells from mouse colons is also included.
Monitoring Antigen‐Specific T Cells Using MHC‐Ig Dimers
Authors: Schneck Jonathan P. , Slansky Jill E. , O'Herrin Sean M. , Greten Tim F.
Published: June 2004

The lack of high affinity reagents has made distinguishing T cells on the basis of antigen specificity difficult to accomplish. This unit provides protocols that utilize innovations in molecular design to permit construction of soluble multivalent MHC complexes (MHC‐Ig dimers) with high avidity for cognate T cell receptors. MHC‐Ig dimers display stable binding properties when they interact with antigen‐specific T cells thus allowing their use in the staining of antigen‐specific T cells by flow cytometry. Methods for constructing and detecting these MHC‐Ig dimers are included along with protocols for applying their use for the quantitation of antigen‐specific T cells.
Biotin Labeling and Quantitation of Cell‐Surface Proteins
Authors: Turvy Diane N. , Blum Janice S.
Published: June 2004

Cell surface receptors, such as transferrin receptors and MHC molecules, are internalized into the endocytic pathway and recycled to the plasma membrane. Previous assays used to measure endocytosis and recycling were cumbersome and often required radioactive reagents. This unit describes protocols that employ the combination of a cleavable biotin reagent to label surface molecules and a capture ELISA to detect these molecules allowing for rapid and safe quantitation of cell surface protein expression, endocytosis, and recycling.
Fluorescence Resonance Energy Transfer: Techniques for Measuring Molecular Conformation and Molecular Proximity
Authors: Edidin Michael
Published: June 2004

This overview unit focuses on the basics of fluorescence and of the FRET phenomenon, and on methods for detecting FRET and data interpretation. FRET is very versatile and there are more application of these basics than can be covered in a single overview. However, some examples are given of applications of various FRET techniques.
Animal Models for Toxoplasma gondii Infection
Authors: Subauste Carlos , Remington Jack
Published: June 2004

Toxoplasma gondii is an obligate intracellular protozoan that commonly infects mammals and birds throughout the world. This unit describes murine models of acute T. gondii infection and toxoplasmic encephalitis. T. gondii infection in severe combined immunodeficient (SCID) mice, which lack T and B cells, has allowed for the study of T cell‐independent mechanisms of defense against intracellular organisms, as described here. The establishment of temperature‐sensitive mutant strains of T. gondii has allowed adoptive‐transfer experiments without the concern for the transfer of the parasite at the same time. The temperature‐sensitive mutant ts‐4 strain disappears from tissues of immunocompetent mice without forming tissue cysts and induces protection against challenge with virulent strains of the parasite, and a protocol is provided for infection with this mutant strain. Support protocols present methodology for evaluation of progression of infection and immune response to the parasite, maintenance of T. gondii tissue cysts and tachyzoites, as well as preparation of T. gondii lysate antigens.
B16 as a Mouse Model for Human Melanoma
Authors: Overwijk Willem W. , Restifo Nicholas P.
Published: June 2004

This unit details protocols for in vivo models of subcutaneous growth and pulmonary metastases of B16 melanoma. Therapeutic approaches include the use of B16.GM‐CSF and rVVmTRP‐1 to induce autoimmune vitiligo and tumor protection. The induction and use of gp100‐specific therapeutic cytotoxic T lymphocytes (CTL) are discussed. Methods are also included for CTL induction, isolation and testing, CTL maintenance, and adoptive transfer. Support protocols detail the testing of mouse sera for presence of MDA‐specific antibodies by immunoblotting and ELISA, respectively. Additional sections, including growing B16 melanoma, enumerating pulmonary metastases, and use of recombinant viruses for vaccination, are discussed together with safety concerns.
Mouse SaI Sarcoma Tumor Model
Authors: Ostrand‐Rosenberg Suzanne
Published: June 2004

SaI is an A/J‐derived (H‐2KkDd) transplantable mouse fibrosarcoma that produces a solid tumor when inoculated subcutaneously, intradermally, or intramuscularly, and an ascites tumor when inoculated intraperitoneally into syngeneic mice. This unit describes the establishment of primary solid SaI/N tumor, the establishment of SaI ascites tumor, and the removal for sampling of in vivo‐passaged SaI ascites tumor cells in mice.
Models for Lymphoma
Authors: Biragyn Arya , Kwak Larry W.
Published: June 2004

This unit describes experimental procedures for development of therapeutic vaccines, particularly “second‐generation” recombinant vaccines. Specifically, a general procedure for handling and culturing lymphoma cell lines in vitro and their subsequent challenge into syngeneic mice is described. Several protocols describe the production of various idiotype (Id)‐based or cellular lymphoma vaccine formulations. In particular, the novel approach of rendering nonimmunogenic lymphoma‐derived scFv or Id immunogenic by fusing it with a chemokine moiety is described. As an alternative, a protocol for expression and purification of these chemokine‐fusion proteins from E. coli is included. A general procedure is included for cloning of cytokine genes, for example, murine GM‐CSF, in A20 lymphoma cells. Support protocols are presented for chemically conjugating intact Ig protein with KLH to produce a prototype protein vaccine and using the Helios Gene Gun System to immunize mice with recombinant DNA tumor vaccines.
Immunofluorescence Staining
Authors: Donaldson Julie G.
Published: June 2004

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications (e.g., brightfield, phase‐contrast, differential interference contrast, and fluorescence epi‐illumination), and how it should be maintained as required to obtain maximum image‐forming capacity and resolution. The principles of microscopy and step‐by‐step alignment and adjustment procedures are described in this unit.
Mouse Endogenous Superantigens: Ms and Mls‐like Determinants Encoded by Mouse Retroviruses
Authors: Hodes Richard J. , Abe Ryo
Published: June 2004

Commonly used inbred mouse strains express different combinations of integrated mouse mammary tumor proviruses (MMTV). This appendix summarizes the proviruses that have been detected. The reported functional properties of those MMTV proviral products which have been identified as superantigens are also summarized, including the ability to elicit primary or secondary T cell responses and to induce Vb‐specific clonal deletion during T cell differentiation. In addition, the amino acid sequences of putative ORF gene products of different MMTV are compared.
Commonly Used Rat Strains
Authors: Kunz Heinz W. , Gill Thomas J.
Published: June 2004

It is critical that genetically defined animals be used in immunological studies so that data can be adequately compared both within and between strains to examine the genetic effects on the phenomena being studied. This appendix lists some of the most commonly used rat strains and their immunogenetic properties.
Human MHC Genes and Products
Authors: Trowsdale John , Campbell R. Duncan
Published: June 2004

This appendix lists the human major histocompatibility complex (MHC) genes, their common alternative names and their protein products. In addition, the genomic organization of the MHC gene loci found on human chromosome 6 is illustrated.
Overview of Protein Folds in the Immune System
Authors: Sun Peter D. , Boyington Jeffrey C.
Published: June 2004

The rapid advancement of X‐ray crystallography and nuclear magnetic resonance techniques in recent years has resulted in the solution of macromolecular structures at an unprecedented rate. This review aims at providing a comprehensive description of structures and folds related to the function of the immune system. Focus is placed on immunologically relevant proteins such as immunoreceptors and major histocompatibility complexes. Information is also provided regarding protein structure data banks.
Large‐Scale Cell Culture
Authors: Bleckwenn Nicole A. , Shiloach Joseph
Published: June 2004

Mammalian cells offer a good production system for complex biologic products that require proper post‐translational processing and folding. These products can be endogenous proteins, recombinant proteins, or recombinant antibodies, which are often needed in large quantities for clinical evaluations and structural studies. Many cell lines are available for production purposes and are derived from various sources, therefore, exhibiting a wide variety of growth and production characteristics. In general, cell types can be divided into two major categories: those that are able to grow in suspension (anchorage‐independent) and those that require a physical support for growth (anchorage‐dependent). In either case, the products produced can be secreted into the production medium or can be accumulated inside the cells. The properties of the cells and the location of the produced product dictate the method used for growth and production, and consequently the selection of a suitable type of bioreactor. This unit focuses on the methods used to cultivate large amounts of cells for the purpose of obtaining an endogenous or recombinant product.
Trypan Blue Exclusion Test of Cell Viability
Authors: Strober Warren
Published: June 2004

The dye exclusion test is used to determine the number of viable cells present in a cell suspension. It is based on the principle that live cells possess intact cell membranes that exclude certain dyes, such as trypan blue, Eosin, or propidium, whereas dead cells do not. In this test, a cell suspension is simply mixed with dye and then visually examined to determine whether cells take up or exclude dye. In the protocol presented here, a viable cell will have a clear cytoplasm whereas a nonviable cell will have a blue cytoplasm.
Obtaining Human Peripheral Blood Cells
Authors: Strober Warren
Published: June 2004

Peripheral blood is the chief source of mononuclear cells (lymphocytes and monocytes) for immunologic studies of humans. This appendix presents protocols for obtaining blood by simple venipuncture when relatively small amounts of blood (10 to 100 ml) are necessary or by lymphapheresis when large amounts (300 to 5000 ml) are necessary. Cells collected by these procedures can be further separated by techniques described in Chapter 7.
Dialysis and Concentration of Protein Solutions
Authors: Andrew Sarah M. , Titus Julie A. , Zumstein Louis
Published: June 2004

Conventional dialysis separates small molecules from large molecules by allowing diffusion of only the small molecules through selectively permeable membranes. This appendix describes dialysis of large‐ and small‐volume samples using cellulose membranes with pore sizes designed to exclude molecules above a selected molecular weight. A describes preparation of membranes for dialysis and discusses issues related to the selection of membranes including commercial kits.
Column Chromatography of Antibodies and Fragments
Authors: Smith John A.
Published: June 2004

Since both size‐exclusion (SE) chromatography (also known as gel filtration) and ion‐exchange (IEX) chromatography are the fundamental methods on which antibody and fragment purifications are based, they are dealt with in detail in this appendix. The procedures for preparing the column matrix, for pouring the chromatography column, and for estimating the appropriate size of the column for the amount of antibody and fragment that are to be purified are all described.
Silanizing Glassware
Authors: Seed Brian
Published: June 2004

Glassware is silanized (siliconized) to prevent adsorption of solute to glass surfaces or to increase its hydrophobicity. This is particularly important with low concentrations of particularly “sticky“ solutes such as single‐stranded nucleic acids or proteins. This appendix provides a protocol for treating glassware.
Monoclonal Antibodies to Rat Leukocyte Surface Antigens, MHC Antigens, and Immunoglobulins
Authors: Puklavec Michael J. , Barclay A. Neil
Published: June 2004

The CD nomenclature used for human‐leukocyte surface antigens is now being widely applied to naming their homologs in other species. This appendix catalogs those CD antigens that have been clearly defined in the rat. There are also many other antigens defined in the rat, but only those for which good biochemical data are available, such as amino acid sequences, are given here. The most commonly used antibodies are summarized.
Construction of Reference Genetic Maps
Authors: Buetow Kenneth H.
Published: June 2004

This unit details the specialized resources and procedures used for constructing reference genetic maps. Construction of such maps in humans represents a subset of the linkage analysis problem. Objectives include the addition of a new locus to an established map, development of a detailed map of loci in a localized area, and construction of de novo maps. Conceptually, the procedures for updating a reference map through the addition of a subset of new loci are similar to those used in establishing linkage for a disease locus. However, construction of new maps of multiple loci is most efficiently accomplished using different family resources that permit the use of accumulated typing resources and alternative, highly efficient statistical methods.
Use of LINKAGE Programs for Linkage Analysis
Authors: Speer Marcy C.
Published: June 2004

This unit provides an introduction to human genetic linkage analysis for cases in which the genetic model is known (parametric or lod score linkage analysis). The LINKAGE computer package is used for computer analysis because it remains a training standard. The concepts presented for the LINKAGE programs, however, translate directly to other commonly used programs including the FASTLINK and VITESSE programs. This unit includes seven examples selected to illustrate many of the key concepts involved in performing linkage analysis. In Examples 1 through 4, a large pedigree segregating an autosomal dominant disease with reduced penetrance is studied for linkage to various markers. In Example 5, the effects of misspecification of the marker allele frequency on linkage analysis are demonstrated.
Overview of Linkage Analysis in Complex Traits
Authors: Pericak‐Vance Margaret A.
Published: June 2004

The goal of this unit is to instruct the reader on the general approaches now available for mapping complex disease. The term complex genetic disease refers to a trait phenotype with an unknown mode of inheritance.The goal of this unit is to instruct the reader on the general approaches now available for mapping complex disease
Disease Associations and Family‐Based Tests
Authors: Ewens Warren J. , Spielman Richard S. , Kaplan Norman L. , Martin Eden R.
Published: June 2004

This unit describes the statistical techniques necessary for performing family‐based association studies (e.g., the TDT) for genetic polymorphisms. Such studies have become increasingly important in the identification of genes that confer an increased risk to disease, particularly for common diseases with a complex etiology. The family‐based approach avoids some of the problems often encountered when applying the traditional case‐control design to genetic studies. The unit includes the Sib TDT (S‐TDT) method, which allows application of the principle of the TDT to sibships without parental data. This extension of TDT is potentially valuable for studying late onset diseases.
Human Mapping Databases
Authors: Talbot Conover , Cuticchia A. Jamie
Published: June 2004

This unit concentrates on the data contained within two human genome databasesGDB (Genome Database) and OMIM (Online Mendelian Inheritance in Man)and includes discussion of different methods for submitting and accessing data. An understanding of electronic mail, FTP, and the use of a World Wide Web (WWW) navigational tool such as Netscape or Internet Explorer is a prerequisite for utilizing the information in this unit.