bluuebunny/crossref_metadata_2025 huggingface dataset:
Serial Analysis of Gene Expression (SAGE)
Authors: Blackshaw Seth , Kim Jae B. , Croix Brad St. , Polyak Kornelia
Published: June 2004

Serial Analysis of Gene Expression (SAGE) involves the generation of short fragments of DNA, or tags, from a defined point in the sequence of all cDNAs in the sample analyzed. This short tag, because of its defined presence in the sequence, is typically sufficient to uniquely identify every transcript in the sample. A comprehensive profile of gene expression in any sample can thus be generated. Absolute, rather than relative, measurements of RNA abundance levels are made, which will be useful in the reliable comparison of data between laboratories.Serial Analysis of Gene Expression (SAGE) involves the generation of short fragments of DNA, or tags, from a defined point Serial Analysis of Gene Expression (SAGE) involves the generation of short fragments of DNA, or tags, from a defined point
Representational Difference Analysis
Authors: Chang Yuan
Published: June 2004

Representational difference analysis (RDA) couples subtractive hybridization to PCR‐mediated kinetic enrichment for the detection of differences between two complex genomes. In this unit, protocols start with the restriction digestion of two comparison DNA samples. Specific linkers are ligated to fragments from each pool and amplicons are generated by PCR. Linkers are removed from both samples and a new linker is added only to size‐selected tester amplicons. These tester amplicons are mixed with a large excess of driver amplicons lacking linkers. Hybridization results in three species of dsDNA fragments: (1) both strands derived from driver DNA (lacking linkers on either strand), (2) hybrids with one strand from driver (no linker) and one from tester (with linker), and (3) both strands from tester DNA (linkers on both strands). Excess driver removes DNA fragments common to both samples, and only the DNA fragments unique to the tester are amplified with linker‐specific primers.Representational difference analysis (RDA) couples subtractive hybridization to PCR‐mediated kinetic enrichment for the detect Representational difference analysis (RDA) couples subtractive hybridization to PCR‐mediated kinetic enrichment for the detect
Characteristics of Amino Acids
Authors: Ellington Andrew , Cherry J. Michael
Published: June 2004

This appendix presents useful basic information, including common abbreviations, useful measurements and data, characteristics of amino acids and nucleic acids, information on radioactivity and the safe use of radioisotopes and other hazardous chemicals, conversions for centrifuges and rotors, characteristics of common detergents, and common conversion factors.
Safe Use of Hazardous Chemicals
Authors: Lunn George , Lawler Gretchen
Published: June 2004

This appendix presents useful basic information, including common abbreviations, useful measurements and data, characteristics of amino acids and nucleic acids, information on radioactivity and the safe use of radioisotopes and other hazardous chemicals, conversions for centrifuges and rotors, characteristics of common detergents, and common conversion factors.
Silanizing Glassware
Authors: Seed Brian
Published: June 2004

This unit presents a procedure to silanize (siliconize) glassware to prevent adsorption of solute to the glass surface or to increase its hydrophobicity. This is particularly important when dealing with low concentrations of particularly “sticky” solutes such as single‐stranded nucleic acids or proteins.
Quantitation of DNA and RNA with Absorption and Fluorescence Spectroscopy
Authors: Gallagher Sean R.
Published: June 2004

Reliable quantitation of microgram, nanogram, and even picogram 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, three more sensitive fluorescence techniques are presented in this appendix. These three procedures cover a range from 25 pg DNA/ml to 50 mg DNA/ml.
Introduction of Restriction Enzyme Recognition Sequences by Silent Mutation
Authors: Shankarappa Raj
Published: June 2004

This unit describes silent mutations and provides tables of translation sequences for silent mutagenesis based on six‐ and eight‐base restriction enzymes.
Techniques for Mammalian Cell Tissue Culture
Authors: Phelan Mary C.
Published: June 2004

The first section of this unit discusses basic principles of sterile technique, while the second section discusses medium preparation. The describes subculturing of a monolayer culture grown in petri plates or flasks. Support protocols describe freezing of monolayer cells, thawing and recovery of cells, counting cells using a hemacytometer, and preparing cells for transport. An describes freezing suspension cells.
Kinetic Assay Methods
Authors: Allison R. Donald
Published: June 2004

The purpose of this appendix is to provide a brief review of issues important in the design of initial‐rate assay methods.
Vectors
Authors: Albright Lisa M.
Published: June 2004

This unit presents tables of vectors described throughout the manual, organized by particular function. Individual entries in each table provide figure citations, some specific characteristics, and restriction site data for each vector, as well as sources when available. Restriction site information was generated using MAPSORT from sequence information that was compiled in part from published sequence data.
Animal Health Assurance
Authors: Donovan John , Brown Patricia
Published: June 2004

A program to assure the health of laboratory animals and to provide a stable environment for their maintenance is necessary to reduce unwanted variables or complicating factors in experimentation. To assure animal health throughout a research study, the animals involved should be procured in good health, monitored for continued health status, and protected from pathogenic organisms through testing of any biological materials. This unit addresses all three of these health management practices. Essential in support of these practices is a diagnostic laboratory capable of testing for rodent and rabbit pathogens.
Isolation of Murine Natural Killer Cells
Authors: Reichlin Amy , Iizuka Koho , Yokoyama Wayne M.
Published: June 2004

This unit describes the isolation of natural killer (NK) cells from mouse spleen. The basic protocol describes a method for preparing a highly purified NK cell population from mouse spleen by cytotoxic depletion of contaminating cells with selected monoclonal antibodies (MAbs), complement lysis, and density‐gradient centrifugation to eliminate dead cells. The advantage of this negative selection process is that the NK cells are not coated with antibody and, therefore, are not at risk of activation by antibody cross‐linking. Purity can then be assessed by cell surface phenotype.
Production of Antibodies that Recognize Specific Tyrosine‐Phosphorylated Peptides
Authors: DiGiovanna Michael P. , Stern David F. , Roussel Robert R.
Published: June 2004

It is possible to produce anti‐phosphopeptide antibodies (i.e., antibodies recognizing phosphorylated peptides) that recognize a protein only in its phosphorylated state, and that do not cross‐react with either the cognate unphosphorylated protein or other phosphoproteins. This unit describes production of antibodies against tyrosine‐phosphorylated peptides, with which the authors have the most expertise, but the principles discussed here also apply to peptides phosphorylated on serine and threonine. Protocols are presented for the production of polyclonal and monoclonal anti‐phosphopeptide antibodies. Support protocols are provided for the coupling of peptides and phosphotyrosine to an affinity matrix (Affi‐Gel 10); BSA‐agarose affinity matrix is commercially available.
Handling and Restraint
Authors: Donovan John , Brown Patricia
Published: June 2004

For the safety of the handler and the animal, proper methods for handling and restraining laboratory animals should be followed. Animals can be restrained either manually or in a plastic restrainer. This unit describes handling and manual restraint of mice, rats, hamsters, and rabbits. Alternate protocols describe restraint using the plastic restrainer.
Animal Identification
Authors: Donovan John , Brown Patricia
Published: June 2004

Proper 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

Although 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

Surgical 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

Double 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

This 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

Procedures 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

IgG 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

Fragmentation 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

T 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

This 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

Ly‐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

This 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

This 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

This 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

This 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

T 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

As 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

Most 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

Neutrophils 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

The 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

Graft‐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

Delayed‐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

Until 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

The 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.
Overview of Flow Cytometry
Authors: Sharrow Susan O.
Published: June 2004

This introductory unit provides an overview of terminology and echniques unique to flow ytometry and is divided into two sections. The first section discusses technical aspects of flow cytometry which apply primarily to the instrumentation‐oriented flow cytometry phase. The second section discusses electronic cell separation using flow cytometry.
Preparation of Cells and Reagents for Flow Cytometry
Authors: Holmes Kevin , Lantz Larry M. , Fowlkes B.J. , Schmid Ingrid , Giorgi Janis V.
Published: June 2004

Flow cytometry is widely used for analyzing the expression of cell surface and intracellular molecules (on a per cell basis), characterizing and defining different cell types in heterogeneous populations, assessing the purity of isolated subpopulations, and analyzing cell size and volume. This technique is predominantly used to measure fluorescence intensity produced by fluorescent‐labeled antibodies or ligands that bind to specific cell‐associated molecules. A procedure for direct and indirect staining of single‐cell suspensions of lymphoid tissue or peripheral blood lymphocytes to detect cell surface membrane antigens is presented. In addition, support protocols present methods for fluorescence labeling of purified antibodies. A protocol for flow cytometric analysis of intracellular antigens in single‐cell suspensions is also included. Alternate protocols describe intracellular staining of unfixed cells in the presence of a detergent and staining of nonviable cells to facilitate discrimination of dead cells in fixed or permeabilized cell preparations.
Measurement of Lymphokine Receptors
Authors: Lowenthal John W. , Malek Thomas R. , Saragovi Horacio
Published: June 2004

One of the most widely studied lymphokine systems is the T lymphocyte growth factor interleukin 2 (IL‐2). This unit describes two basic methods for the quantitation and biochemical characterization of IL‐2 receptors. The first method employs the radioreceptor assay. Support protocols to this technique describe quantification of data via calculations of association and dissociation rates and the Scatchard plot analysis. The second approach detects cell‐surface lymphokine receptors by covalently cross‐linking IL‐2 to its receptor. This method employs the chemical disuccinimidyl suberate (DSS) to achieve irreversible cross‐linking of IL‐2 to IL‐2R.
Measurement of Soluble and Membrane‐Bound Interleukin 1 Using a Fibroblast Bioassay
Authors: Dinarello Charles A. , Muegge Kathrin , Durum Scott K.
Published: June 2004

Interleukin 1 (IL‐1) is both a major mediator of inflammation and an important signal for activation and differentiation of lymphoid cells. IL‐1 is produced by a large variety of cell types, but in most immunologic systems, activated macrophages are its major cellular source. This unit describes a bioassay for testing biological fluids based on the ability of IL‐1 to induce IL‐8 or IL‐6 in fibroblasts. Also listed are sources of commercially available kits (ELISAs, immunoassays) to measure IL‐1, and antibodies useful in setting up these assays.
Measurement of Interleukin 7
Authors: Namen Anthony E. , Morrissey Philip J.
Published: June 2004

This unit describes an assay system to detect biologically active interleukin 7 (IL‐7). The target or indicator cells are a clonally derived long‐term pre‐B cell line designated IxN/2bx (or, more simply, 2bx). These cells demonstrate an absolute specificity for IL‐7 for continued growth and viability and are used to establish a thymidine‐uptake proliferation assay similar to those established for other interleukins. A describes maintenance of the IxN/2bx cell line.
Measurement of Antiviral Activity Induced by Interferons α, β, and γ
Authors: Vogel Stefanie N. , Friedman Robert M. , Hogan M. Michele
Published: June 2004

The in this unit describes an assay for murine IFN‐induced antiviral activity and employs vesicular stomatitis virus (VSV) and IFN‐sensitive fibroblasts. describes the preparation of VSV cultures and the calculation of multiplicity of infection (MOI; i.e., concentration of viral particles required to infect cells). describes the antibody neutralization assay, which can be used to identify the bioactive species of IFN in a sample, or to test the potency of an antibody preparation against a particular species of IFN. 1 covers measurement of human IFN‐induced antiviral activity; while the steps are quite similar to , different viral and cell cultures are described.
Measurement of Tumor Necrosis Factor α and β
Authors: Hogan M. Michele , Vogel Stefanie N.
Published: June 2004

Tumor necrosis factor (TNF) a and b are multifunctional cytokines elaborated primarily by monocytes and macrophages (TNF‐a) or T cells (TNF‐b). Some cells that bind TNF‐a or ‐b on their surface receptors lyse as a consequence of the binding reaction. This unit presents a protocol that employs TNF‐sensitive, actinomycin D‐treated murine L929 fibroblasts to quantify TNF activity in supernatants derived from cell cultures, serum samples, or cerebral spinal fluid. While the assay can measure picogram concentrations of human, rat, and murine TNF‐ab, it cannot distinguish between the a and b forms of any species. A describes propagation and preparation of L929 fibroblasts.
Measurement of TGF‐β in Biological Fluids
Authors: Hale‐Donze Hollie , Wahl Sharon M. , Jackson Raymond J. , McGhee Jerry R.
Published: June 2004

Transforming growth factor (TGF‐β) is an immunoregulatory cytokine produced by many different cell types, including lymphocytes and monocytes‐macrophages. Two assays are described for the quantitation of TGF‐β activity in supernatant fluids. The bioassay for TGF‐β is based on the potent ability of TGF‐β to suppress lymphocyte proliferation. The radioreceptor assay is based on the ability of TGF‐β‐containing supernatant fluids to inhibit the binding of radiolabeled TGF‐β to its cellular receptor. Two support protocols describe acid‐activation and neutralization of TGF‐β.
Measurement of Human Interleukin 11
Authors: Bennett Frann , Gianotti JoAnn , Celniker Abbie , Turner Katherine J. , Clark Steven C.
Published: June 2004

This unit describes an ELISA and a cell proliferation assay that can be used, respectively, to measure the protein level or biologic activity of human and murine interleukin 11 (IL‐11). The bioassay is based on the ability of IL‐11 to support growth of the B9‐11 cell line, a subline of B9 that has traditionally been used to measure levels of IL‐6. B9‐11 is substantially more responsive to IL‐11 than the T10 line used in older protocols. This new bioassay therefore provides improved sensitivity, with a detection limit of ˜20 pg/ml. An alternate procedure is provided that employs neutralizing antibodies in the cell proliferation bioassay to use to ensure that the activity of the desired molecule (IL‐11) is being measured in samples containing multiple cytokines. A describes maintenance of B9‐11 cells.
Measurement of Interleukin‐13
Authors: Lauder Angus , McKenzie Andrew N.J.
Published: June 2004

This unit describes two protocols that can be used to quantitate interleukin‐13 (IL‐13). The enzyme‐linked immunosorbent assay (ELISA) has the advantage of being highly specific for human IL‐13 and does not recognize other cytokines present in the sample. A bioassay is also presented based on the ability of IL‐13 to stimulate the growth of the B9 plasmacytoma cell line. The bioassay method can be used to detect both mouse and human IL‐13. B9 cells are dependent on IL‐6 for growth and will also respond to IL‐4. Thus, although B9 cells can readily be used to quantify IL‐13 in the absence of other cytokines, neutralizing antibodies must be incorporated into the bioassay when other cytokines are present in the sample.
Immunoenzymetric Assay of Mouse and Human Cytokines Using NIP‐Labeled Anti‐Cytokine Antibodies
Authors: Abrams John S.
Published: June 2004

This unit describes a general method for immunoenzymetric assay of mouse and human cytokines. The technique is based on use of two anti‐cytokine monoclonal antibodies (MAbs), each specific for a spatially distinct determinant on the cytokine. One of these is a coating antibody, and the other is a derivatized detecting antibody. A support protocol describes chemical labeling of anti‐cytokine monoclonal IgG antibodies with the NIP hapten group to produce the detecting antibody. Another support protocol describes a method for producing horseradish peroxidase (HRPO)‐conjugated J4, a rat IgG1 anti‐NIP monoclonal antibody that confers the anti‐NIP specificity to the HRPO detection system used in the immunoenzymetric assay. The method described in this unit has allowed successful measurement of different cytokines in a wide variety of clinical samples including conditioned medium, serum and plasma, ascites, amniotic fluid, and bronchoalveolar lavage fluid.
Measurement of Interleukin 16
Authors: Center David M. , Cruikshank William W. , Parada Nereida A. , Ryan Thomas , Theodore Arthur C. , Viglianti Gregory , Lim Kaiser G. , Weller Peter F.
Published: June 2004

Interleukin 16 (IL‐16) is a chemoattractant immunomodulatory cytokine that initiates its cellular responses through interaction with membrane‐expressed CD4. The protein may be detected by a number of methods; the choice of protocol will depend on the ultimate object of a particular experiment. The first method presented is the use of ELISA to measure IL‐16 in cell culture supernatants or biological fluids. For some applications, such as identification of IL‐16 in an unknown fluid or medium or direct assessment of its bioactivity, functional assays of IL‐16‐induced responses may be more appropriate. The chemotactic effects of IL‐16 on CD4+ T cells and its specific inhibition may be measured using anti‐IL‐16 antibodies; the same approach may also be applied to monocytes or eosinophils. Another effect of IL‐16 is the induction of CD25, which can be assayed using immunological staining. Finally, cell cycle progression in target cells can be measured by the incorporation of radiolabeled thymidine and confirmed by inhibition with neutralizing antibody.
Detection of Intracellular Cytokines by Flow Cytometry
Authors: Foster Barbara , Prussin Calman
Published: June 2004

Intracellular cytokine detection by flow cytometry has emerged as the premier technique for studying cytokine production at the single‐cell level. Multiparameter flow cytometry permits simultaneous detection of two or more cytokines within a single cell, allowing direct TH1 versus TH2 determination. This capability, combined with the high throughput inherent in the instrumentation, gives intracellular cytokine staining an enormous advantage over existing single‐cell techniques such as ELISPOT, limiting dilution, and T cell cloning. The unit describes the techniques required to perform intracellular staining of cells that have already been stimulated in vitro and fixed. The methods necessary for in vitro activation by PMA and ionomycin or antigens, fixation of cell suspensions, and cell‐surface staining are also described. Because of the greater level of nonspecific binding inherent in fixed, permeabilized cells, greater care must be taken in designing specificity controls. A more rigorous method to verify specificity of staining is also described.
Measurement of Human and Mouse Interleukin 18
Authors: Yoshimoto Tomohiro , Tsutsui Hiroko , Okamura Haruki , Nakanishi Kenji
Published: June 2004

IL‐18, originally designated as interferon‐γ (IFN‐γ)‐inducing factor (IGIF), is a pleiotropic cytokine secreted by activated macrophages and Kupffer cells. The major activity associated with this cytokine is induction of IFN‐γ production from T cells, B cells, and NK cells, especially in collaboration with IL‐12. IL‐18 is synthesized without a signal peptide and must be enzymatically cleaved to become active. Therefore, it is important to determine whether the produced IL‐18 is an active or precursor form. This unit describes functional assays for measurement of bioactive human and mouse IL‐18 and ELISAs for measurement of murine and human IL‐18 proteins. The functional assays are based on the induction of IFN‐γ production by IL‐18. The ELISA measures the concentration of human or mouse IL‐18. Using a combination of monoclonal antibodies against human or mouse IL‐18, the proform and/or mature form of IL‐18 can be detected by ELISA.
Induction and Measurement of In Vivo Antibody Responses
Authors: Engler Renata J.M. , Kurman Carole C. , Nelson David L.
Published: June 2004

The capacity of the human immune system to mount an antibody response following in vivo immunization with a protein or polysaccharide antigen is a revealing indication of the overall integrity of both the B and T cell arms of the immune system. As such, in vivo immunization followed by measurement of the antibody response is an appropriate test of immune function in the various acquired and congenital immunodeficiencies and in a host of other conditions affecting the immune system. This unit describes procedures for in vivo immunization and for the measurement of the subsequent immune response using an ELISA technique.
Immunomagnetic Purification of T Cell Subpopulations
Authors: Horgan Kevin , Shaw Stephen
Published: June 2004

This unit describes a general method for physical separation of cell subpopulations from a heterogeneous mixture of cell types. The technique relies on having an appropriate monoclonal antibody or mix of monoclonal antibodies that distinguish between the cell types being separated. Its advantages over other antibody‐mediated selection techniques are purity of resulting cell preparation, reproducibility of separation, and ease of handling small to large numbers of cells (106 to 1010 cells). The basic protocol outlines preparation of purified human T lymphocytes. The method produces good yields of pure T cells prepared by negative selection (i.e., not labeled with antibody). The alternate protocol applies similar principles for purifying CD4+ T cells, and presents refinements of the basic protocol that minimize cost and improve convenience.
Isolation of Monocyte/Macrophage Populations
Authors: Wahl Larry M. , Smith Phillip D.
Published: June 2004

This unit describes the isolation of monocytes from lymphocytes by adherence, gradient sedimentation on colloidal silica particles, and flow cytometry. Because the first two methods can result in cell activation (induction of gene expression or protein secretion), and the third is technically difficult, a fourth protocol is presented which describes counterflow centrifugal elutriation. This latter procedure can be used to isolate large numbers of purified, nonactivated monocytes.
Isolation of Human NK Cells and Generation of LAK Activity
Authors: Whiteside Theresa L.
Published: June 2004

This unit describes the preparation of natural killer (NK) cells from normal human peripheral blood and their incubation in vitro in the presence of interleukin 2 (IL‐2) to yield lymphokine‐activated killer (LAK) cells. A protocol is presented for isolating highly purified NK cell populations from PBMC, and another method presents steps for generating LAK cells from these purified NK cells via IL‐2 stimulation. An alternate protocol describes the generation of LAK cells directly from whole, unseparated PBMC preparations instead of purified NK populations. The caveat with the alternate protocol is that LAK activity generated in this manner represents the total cytotoxic potential of all LAK precursor cells‐‐i.e., all those PBMC that are capable of responding to IL‐2 by up‐regulation of cytotoxicity against NK‐resistant targets. In PBMC, LAK precursor cells are found among subpopulations of both NK cells and T lymphocytes.
Detection of Unseparated Human Lymphocytes by Flow Cytometry
Authors: Fleisher Thomas A. , Marti Gerald E.
Published: June 2004

The protocol for flow cytometry analysis presented here has been specifically developed for studies of human peripheral blood cells. In this protocol, analysis is performed on unseparated cells in whole peripheral blood, rather than on Ficoll‐Hypaque‐purified mononuclear cells. The advantage of this approach is that it requires less time, uses smaller blood volumes, and eliminates possible differential blood loss as a result of cell separation techniques. In this regard, B cell recovery using the whole blood method is significantly greater than that obtained using Ficoll‐Hypaque‐purified mononuclear cells. However, because lymphocytes generally represent a minority of peripheral cells (especially in adults), careful gating of the test samples for lymphocytes is a more critical requirement in this procedure than in other procedures using purified cells.