bluuebunny/crossref_metadata_2025_split huggingface dataset:
Animal Identification
Authors: Donovan John , Brown Patricia 
Published: June 2004

AbstractProper identification of animals plays an important role in research, whether animals are being used as experimental subjects, for breeding purposes, or as a source for tissues, cells, or fluids. Identification of animals links animal‐specific information to experimental records, and facilitates communication between investigators, technicians, and animal care personnel. Specific procedures for marking or identifying individual rodents and rabbits are described in this unit.
Survival Surgery: Removal of the Spleen or Thymus
Authors: Reeves J.P. , Reeves P.A. , Chin L. Thomas 
Published: June 2004

AbstractAlthough for most experiments lymphoid tissue is removed from the freshly sacrificed animal, there are several circumstances which require the surgical removal, under anesthesia, of either the thymus or spleen, and the recovery of the animal for further investigation. This unit describes survival surgery for removal of the adult spleen as well as the adult and neonatal thymus of the mouse.
Surgical Removal of Mouse Salivary Glands
Authors: Jonjic Stipan 
Published: June 2004

AbstractSurgical removal of the salivary glands is a simple procedure aimed at providing glandular tissues for studies in histopathology, immunohistology, DNA and RNA analysis, cytokine production, and virus detection and isolation. This unit describes the surgical removal of the salivary glands in mice, but a similar protocol is applicable to rats and other rodents.
Double‐Immunodiffusion Assay for Detecting Specific Antibodies
Authors: Hornbeck Peter 
Published: June 2004

AbstractDouble immunodiffusion owes its success to the unique nature of antibody‐antigen interactions. When polyvalent antibodies with moderate‐to‐high intrinsic affinities are mixed with antigen at the right ratio (called the zone of equivalence) lattices of antibody‐antigen complexes form and precipitate out of solution. When, as described in this unit, gradients of antigen and antibody are established by diffusion from adjacent wells in a bed of agar, a line of practically insoluble precipitation forms at the equivalence zone (precipitin lines).
Production of Monoclonal Antibodies
Authors: Yokoyama Wayne M. 
Published: June 2004

AbstractThis unit describes the production of monoclonal antibodies beginning with immunization and cell fusion and selection. Support protocols are provided for screening primary hybridoma supernatants for antibodies of desired specificity, establishment of stable hybridoma lines, cloning of these B cell lines by limiting dilution to obtain monoclonal lines, and preparation of cloning/expansion medium (thymocyte‐conditioned medium).
Monoclonal Antibody Supernatant and Ascites Fluid Production
Authors: Yokoyama Wayne M. 
Published: June 2004

AbstractProcedures detailing the production of monoclonal antibody supernatants, including the production of larger amounts (liters), are presented here. The protocol for large‐scale production of hybridomas or cells (e.g., for isolation of cellular proteins) involves a similar procedure. A method for producing and obtaining ascites fluid containing the monoclonal antibody is also presented. The unit ends with a set of recommendations developed by the Committee on Methods of Producing Monoclonal Antibodies (Institute of Laboratory Animal Research, National Research Council). The recommendations are designed to foster the judicious use of animals for the production of monoclonal antibodies and to strongly encourage the use of in vitro methods whenever possible.
Purification of Immunoglobulin G
Authors: Andrew Sarah M. , Titus Julie A. 
Published: June 2004

AbstractIgG can be purified, as described here, by ammonium sulfate precipitation followed by size‐exclusion (SE) chromatography. This is the least expensive option available for purification of antibodies. Protein A‐ and protein G‐affinity chromatography are the fastest methods for purifying antibodies, but they are not effective for all subclasses of rat antibody. A protocol for affinity chromatography using anti‐rat antibody is provided for purification of rat antibodies. Ion‐exchange (IEX) chromatography is described for purifying intact monoclonal and polyclonal antibodies and antibody fragments.
Fragmentation of Immunoglobulin M
Authors: Andrew Sarah M. , Titus Julie A. 
Published: June 2004

AbstractFragmentation of IgM antibodies may be necessary because of the large molecular weight of the native molecule (900 kDa). IgMs fragments resemble IgG in size and structure, but they may have a decreased binding affinity. The Fc portion of IgM can have powerful biological effector functions such as complement activation. Because some T cells have receptors for IgM, it may be desirable to produce fragments of IgM for both cytotoxicity studies and for in vivo use. A protocol is presented for digestion of IgM with pepsin to produce F(ab′)2µ. The fragment can be reduced to produce the monovalent F(ab')u, if desired. IgM can also be reduced and alkylated in a single step, as described, using cysteine to produce IgMs, the bivalent monomer or subunit of IgM.
T Cell Enrichment by Cytotoxic Elimination of B Cells and Accessory Cells
Authors: Hathcock Karen 
Published: June 2004

AbstractT cells from mouse spleen and lymph node do not express the cell‐surface glycoproteins encoded for by MHC class II genes, whereas most non‐T cells in these organs do (i.e., B cells and accessory cells). This unique feature of T cells makes it possible to enrich for them using cytotoxic anti‐class II monoclonal antibodies and activating complement. Because there are two isotypes of MHC class II genes (A and E genes, located adjacent to each other in the MHC region originally called the I region), it is necessary to identify which of these two genes are expressed in the mouse strain used. This unit lists suitable antibody‐producing hybridomas available from ATCC, with antigen specificities (I‐A vs. I‐E) and corresponding mouse strains (H‐2 strains a, b, k, etc., indicated as superscripts). Other antibodies can be tested in pilot experiments using the procedure outlined. This unit describes T cell enrichment using cytotoxic antibodies, and UNIT here describes the depletion of T cells and their subpopulations using the same approach. In the latter unit, T cell surface markers (Thy‐1, CD4, and CD8) are targeted by the cytotoxic antibodies.
T Cell Depletion by Cytotoxic Elimination
Authors: Hathcock Karen S. 
Published: June 2004

AbstractThis unit describes the complete removal of T cells from lymphocyte preparations based on the presence of the glycoprotein Thy‐1 on the cell surface of T lymphocytes. As in UNIT here, cytotoxic elimination is employed; however, Thy‐1‐specific antibodies are used rather than MHC class II‐specific antibodies so that T cells are eliminated rather than B cells and accessory cells. Murine T cells can be further fractionated into subpopulations based on their expression of cell differentiation markers CD4 (Leu3, L3T4, T4) and CD8 (Leu2, Lyt2,3). A protocol in this unit describes removal of T cell subsets by cytotoxic elimination using CD‐specific antibodies. An alternate but technically more demanding method of enriching T cell subsets is the panning procedure described in UNIT here.
Isolation of Ly‐1+/CD5+B Cells by Cell Sorting
Authors: Hardy Richard R. 
Published: June 2004

AbstractLy‐1/CD5 is a 68‐kDa glycoprotein that was originally thought to mark the helper subset of T cells. Later it was shown to be present on all T cells and, more recently, on a subset of B cells. Although its function remains the subject of speculation, CD5 expression serves as a useful marker for a functionally distinct population of B cells that has attracted a considerable amount of interest from investigators of both murine and human immune systems. Of critical importance in much of this work is the isolation of pure populations of CD5+ B cells along with appropriate control populations. A flow cytometry technique is presented in this unit, which results in the selection and isolation of two populations of cells from a complex mixture based on physical properties (e.g., size and internal granularity) and correlated expression of several surface molecules.
Assays for B Lymphocyte Function
Authors: Bondada Subbarao , Robertson Darrell A. 
Published: June 2004

AbstractThis unit describes the antigenic stimulation of in vitro antibody production by B cells and the subsequent measurement of secreted antibodies. The first basic protocol is a generalized system for inducing in vitro antibody production and can accommodate various types of antigens under study. Secreted antibodies can then be measured with an enzyme‐linked immunosorbent assay (ELISA) or other soluble‐antibody detection systems. Alternatively, the number of antibody‐producing cells can be quantified by plaque‐forming cell (PFC) assays presented in this unit: the Cunningham‐Szenberg and the Jerne‐Nordin techniques. Both methods employ specially prepared slide chambers, described here, in which the antibody‐producing B cells are mixed with complement and indicator sheep red blood cells (SRBC), or with trinitrophenol‐modified SRBC (TNP‐SRBC), with subsequent lysis and counting of plaques. Because IgM antibodies fix complement efficiently, whereas IgG and IgA antibodies do not, unmodified PFC assays measure only IgM antibodies. The assay can be modified, however, to measure all classes of antibodies or to enumerate total immunoglobulin‐secreting B cells, as described in alternate protocols. Yet another method of measuring the number of antibody‐producing B cells (in a class‐specific fashion) is to use the ELISPOT technique described in UNIT 7.14. The resting B cells used in these procedures are prepared as described in the final support protocols for Percoll gradient centrifugation.
Proliferative Assays for B Cell Function
Authors: Mond James J. , Brunswick Mark 
Published: June 2004

AbstractThis unit describes procedures for measuring the capacity of purified B cells to undergo proliferation. The method centers on the use of polyclonal stimulating agents (mitogens) because these agents stimulate the majority of B cells and because the alternative (measurement of antigen‐induced proliferation) requires the laborious procedures of isolating antigen‐specific B cells (which are otherwise present in too low a concentration in whole B cell populations). Cross‐linking of the B cell antigen receptor, surface immunoglobulin (sIg), by specific antigen stimulates cells to proliferate prior to secreting Ig. For this purpose, monoclonal or heterologous affinity‐purified anti‐Ig antibodies are used. B cells can also be stimulated to proliferate by antigen‐nonspecific reagents (mitogens), and it is also critical to study the role of these mitogens in B cell responses. Both of these systems have the advantage that the majority of B cells will be activated. The first basic protocol describes B cell proliferation induced by two commonly used stimulants–anti‐Ig antibody (either anti‐IgM or anti‐IgD) and lipopolysaccharide (LPS)–as measured by incorporation of [3H]thymidine into dividing cells. Alternate protocols describe other commonly used mitogens as well as other means of measuring cell proliferation.
Proliferative Assays for T Cell Function
Authors: Kruisbeek Ada M. , Shevach Ethan , Thornton Angela M. 
Published: June 2004

AbstractThis addition to UNIT here will describe the assays needed to evaluate CD4·CD25·T cell non‐responsiveness and function. Unlike the conventional T cells described in , CD4·CD25· cells do not proliferate to TCR stimuli alone. The conditions required to induce proliferation are described. This addition will also describe the assay in which CD4·CD25·T cells are co‐cultured with conventional T cells in order to assess their suppressive function. Finally, this addition will describe the culture conditions for the activation and expansion of CD4·CD25· cells.
Production of T Cell Clones
Authors: Fitch Frank W. , Gajewski Thomas F. 
Published: June 2004

AbstractThis unit provides methods for the derivation of specific types of T cell clones: preparation and maintenance of alloreactive murine helper T (TH) lymphocyte and cytotoxic T lymphocyte (CTL) clones using the limiting dilution technique and derivation of TH clones reactive with soluble protein antigens including a method for the selection of either TH1 or TH2 lymphocyte subsets. These two subsets of TH cells exhibit helper function in different ways and can be distinguished by the patterns of lymphokines they synthesize. Support protocols describe preparation of different types of conditioned media that can be used to enhance the growth of desired clones.
Production of Mouse T Cell Hybridomas
Authors: Kruisbeek Ada M. 
Published: June 2004

AbstractT cell hybridomas can be obtained by fusing activated T cells with tumor cells. A heterogeneous population of hybridomas can be cloned by limiting dilution to obtain hybridomas that express specificity to one T cell receptor (TCR). This unit describes cell fusion and selection of T cell hybridomas. A protocol is provided for screening of T cell hybridomas for expression of the CD3‐TCR complex by flow cytometry analysis. Those hybridomas expressing a CD3‐TCR complex are then further tested for expression of antigen specificity by screening for specific antigen‐induced lymphokine production. The procedures for establishment of stable hybridoma lines and cloning of stable lines by limiting dilution are identical to those described for B cell hybridomas and can be found in UNIT 2.5. Protocols for freezing and thawing T cell hybridomas are identical to those described for B cell hybridomas and other cell lines.
Measurement of Apoptosis and Other Forms of Cell Death
Authors: Muppidi Jagan , Porter Melissa , Siegel Richard M. 
Published: June 2004

AbstractAs programmed cell death (PCD) or apoptosis has emerged as an important regulator of development and homeostasis in multicellular organisms, methods to quantify apoptosis and to distinguish it from necrosis have been developed. This unit presents a set of assays for these purposes, many of which are technically very simple and ideally suited to the study of hematopoietic cells. The first basic protocol allows the qualitative and quantitative assessment of apoptosis in lymphocyte cell cultures using light or fluorescent microscopy. Three protocols follow that are designed to detect nuclear DNA fragmentation and support protocols describe methods to radiolabel the DNA and cytoplasm of the cells to be tested. Techniques that quantitate apoptotic cells using flow cytometry are then described and support protocols provide methods for priming T cell clones and freshly isolated lymph node cells, respectively, for T cell receptor (TCR)‐induced apoptosis. Quantitative detection of DNA fragmentation in apoptotic cells is also described. TdT‐mediated dUTP‐biotin nick end‐labeling (TUNEL) methods are provided for the detection of apoptotic cells, along with procedures for the flow cytometric quantitation of apoptotic cells using TUNEL, and TUNEL, staining of tissue sections to identify apoptotic cells. Since much remains incompletely understood about the molecular pathways of programmed death, and it is probably best to perform more than one of the basic protocols to confirm an observation of apoptotic cell death.
Fetal Thymus Organ Culture for T Cell Development Studies
Authors: Ramsdell Fred 
Published: June 2004

AbstractMost T cell development occurs within the thymus and includes a series of selection processes that are, in large part, still poorly understood. Studies of T cell development have been greatly advanced by the description of multiple phenotypic subsets of T cells and their maturational relationships. This unit describes a system for observing and modulating T cell development in vitro via the culture of entire mouse fetal thymic lobes. The basic protocol delineates the isolation of fetal thymi and the culture system used to allow for normal T cell development on transwell plates. It is easier and somewhat more expensive to carry out than the first alternate protocol which describes a more traditional method of culture on Gelfoam sponges (used originally to develop the culture system). A method for depleting hematopoietic cells from thymic lobes using 2′‐deoxyguanosine and subsequent reconstitution with precursor cells is described in the second alternate protocol. This protocol is valuable for the study of tolerance and T cell selection. In the support protocol, methods of altering and monitoring T cell development are outlined.
Isolation of Mouse Neutrophils
Authors: Luo Yi , Dorf Martin E. 
Published: June 2004

AbstractNeutrophils are widely used in research to elucidate the mechanisms of inflammation, including such processes as cell adhesion, chemotaxis, superoxide release, production of reactive nitrogen intermediates, and granule exocytosis. This unit presents a protocol for the induction of an enriched exudate of polymorphonuclear leukocytes (PMN) in the peritoneal cavity of mice. Neutrophils are then isolated using continuous gradient centrifugation. This is followed by a protocol for isolating neutrophils from peripheral blood. An alternate method for the purification of PMN from peritoneal exudate cells or peripheral blood by Histopaque density gradient centrifugation is also provided. The cell purification protocol can be modified to obtain nonelicited neutrophils from peritoneal fluid.
Isolation of Mouse Intrahepatic Lymphocytes
Authors: Crispe I. Nicholas 
Published: June 2004

AbstractThe liver is a distinctive immunological environment in which are found a wide variety of cell types. The absolute number of intrahepatic lymphocytes (IHL) and the frequency of the various components of the cell mixture in the liver are influenced by the age and strain of the mouse, as well as by the presence of hormones, cytokines, and pathogens. The bulk of the liver consists of hepatocytes. In addition to IHL, the liver also contains a macrophage population, the Kupffer cells, and sinusoidal endothelial cells. The protocols described here can be used to deplete the liver tissue of hepatocytes, endothelial cells, and red blood cells, leaving a cell suspension consisting mainly of IHL in which the major contaminant is Kupffer cells (which can be removed by adherence to plastic). This unit provides two protocols that may be used to isolate IHL. One can be used to isolate IHL from multiple livers in parallel, whereas the more elaborate alternate protocol yields more cells per liver but is more appropriately used to recover the IHL from a single liver.
Animal Models of Acute and Chronic Graft‐Versus‐Host Disease
Authors: Hakim Frances , Fowler Daniel H. , Shearer Gene M. , Gress Ronald E. 
Published: June 2004

AbstractGraft‐versus‐host disease (GVHD) represents a special situation in transplantation immunology in which immunocompetent donor cells are engrafted into recipients that are incapable of rejecting them due to tolerance, immaturity, or radiation‐ or chemotherapy‐induced immune deficiency. Donor T cells encountering allogeneic stimulators become activated, secrete cytokines, proliferate, and differentiate into effectors; this in vivo immune response is known as the graft‐versus‐host reaction (GVHR). The systemic effects of this initial donor anti‐host reaction comprise a multiorgan syndrome, graft‐versus‐host disease (GVHD). Murine GVHD experiments have been utilized to model the clinical disorders of acute and chronic GVHD (AGVHD and CGVHD) that occur after allogeneic bone marrow transplantation, and also to study T cell regulation, induction of tolerance, and autoimmune diseases. Presented in this unit are methods for generating and assessing both AGVHD and CGVHD in mice. While the two syndromes differ markedly in immunopathogenesis, both can be induced by the two main methods presented: transfer of allogenic donor lymphocytes and stem cells into irradiated hosts, and transfer of parental strain lymphocytes and stem cells into unirradiated, immune‐competent F1 strain hosts. Several endpoints of AGVHD and CGVHD should be evaluated in experimental mice, with comparisons made to the syngeneic transplant control or the T cell‐depleted allogeneic control. To this end, protocols are provided for the assessment of survival rates, weight loss, chimerism, donor‐host cytotoxicity, and cytokine and proliferative responses to mitogenic or allogeneic stimuli. Histopathology and assays of B cell immune function are also described for evaluation of the pathogenesis of GVHD.
Delayed‐Type Hypersensitivity
Authors: Luo Yi , Dorf Martin E. 
Published: June 2004

AbstractDelayed‐type hypersensitivity (DTH) is an in vivo assay of cell‐mediated immune function. DTH reactions are often divided into two phases: the sensitization phase, referring to the initial immunization with specific antigen, and the efferent or challenge phase of the DTH response, which usually follows 6 to 14 days after sensitization. This unit details the protocol for stimulating DTH responses in mice to the O‐succinimide ester of the hapten 4‐hydroxy‐3‐nitrophenyl acetyl (abbreviated NP‐O‐Su).
Construction of Human‐SCID Chimeric Mice
Authors: Roncarolo Maria Grazia , Carballido José M. 
Published: June 2004

AbstractUntil recently, testing of new therapeutic agents has relied extensively upon the use of mice and nonhuman primates for in vivo preclinical studies. Unfortunately, these animal models do not always mimic the physiological and pathophysiological processes that occur in humans. The finding that C.B‐17 severe combined immunodeficiency (SCID) mice lack a competent immune system, and therefore are unable to mount effective cellular and humoral responses to foreign antigens, has led to their use as recipients for xenografts of human tissues. This unit is focused on the construction of human‐SCID chimeric through the surgical implantation of human fetal hematolymphoid tissues into SCID mice (SCID‐hu mice). The describes the surgical implantation of human fetal thymus and liver under the kidney capsules of SCID mice (SCID‐hu Thy/Liv model). Subcutaneous transplantation of human fetal bone marrow and thymus (SCID‐hu Bm/Thy mice) is described in the . Additional support protocols provide procedures to analyze human lymphocyte populations in the peripheral blood and grafted organs of SCID‐hu mice. The advantages and disadvantages of each protocol and potential applications are discussed in the Commentary.
Use of the Intracellular Fluorescent DyeCFSEto Monitor Lymphocyte Migration and Proliferation
Authors: Parish Christopher R. , Warren Hilary S. 
Published: June 2004

AbstractThe stable incorporation of the intracellular dye CSFE into lymphocytes is a powerful tool to monitor lymphocyte migration in vivo and to quantitatively analyze cell division both in vivo and in vitro. This unit describes methods for labeling high or low numbers of lymphocytes with CSFE. Protocols are provided to use CSFE‐labeled cells in cell transfer studies or as cells to be cultured in vitro. Detailed guidelines for positioning of CSFE‐labeled lymphocytes in lymphoid organs or other tissues are included for those wishing to use this approach to study lymphocyte migration.