bluuebunny/crossref_metadata_2025_split huggingface dataset:
Measurement of Interleukin 6
Authors: Nordan Richard P. , Richards C.D. , Gauldie Jack 
Published: June 2004

AbstractInterleukin 6 (IL‐6) is a pluripotent cytokine with multiple effects on many different cell types. It is produced by a variety of cells in response to immunological and other stimuli. This unit describes a simple and sensitive assay for human, rat, rabbit, pig, and mouse IL‐6, based on IL‐6‐dependent proliferation of a murine B cell hybridoma cell line, B9. Support protocols discuss maintenance of B9 cells, preparation of IL‐6 standards, and production of IL‐6‐containing supernatant. In addition, IL‐6 ELISA kits for the measurement of human or mouse IL‐6 are available from a number of companies. These products are reliable, highly sensitive, and specific, and thus should be considered as an excellent (although more expensive) alternative, keeping in mind that bioactivity is not assessed with this approach
Biological Responses to Chemokine Superfamily Members
Authors: Taub Dennis D. , Schaffer Eric 
Published: June 2004

AbstractA detailed summary of all the chemokine subfamily members including alternative names, receptor‐ligand pairs, receptor cellular distribution, and leukocyte responsiveness is presented here along with assays to measure the biological activities of chemokines on each of the major leukocyte subsets (i.e., microchemotaxis chambers, fluorescence‐based assays, use of nitrocellulose filters and membranes coated with extracellular matrix proteins, cellular adhesion assays, mobilization of intracellular free calcium, actin polymerization, degranulation assays, and the use of chemokine receptor‐specific antibodies in flow cytometric analysis and radiolabeled chemokine binding assays).
Measurement of Mouse and Human Interleukin 9
Authors: Renauld Jean‐Christophe , Van Snick Jacques 
Published: June 2004

AbstractThis unit describes two proliferation assays to detect or quantitate human and murine interleukin 9 (IL‐9). The first is based on the ability of IL‐9 to stimulate the proliferation of the TS1h9RA3 cell line, a murine IL‐9‐dependent cell line transfected with the human IL‐9 receptor. An alternate protocol is based on the ability of IL‐9 to stimulate the proliferation of the human megakaryoblastic leukemia cell line, M‐O7e. M‐O7e cells depend on either human IL‐3 or granulocyte/macrophage colony‐stimulating factor (GM‐CSF) for growth, although other cytokines including IL‐2, ‐4, ‐6, and ‐9 and steel factor are also weakly mitogenic (relative to IL‐3 and GM‐CSF). Thus, although M‐O7e cells can readily be used to quantitate levels of IL‐9 in the absence of other cytokines, analysis in the presence of complex mixtures of cytokines (e.g., natural sources) requires the use of specific antibodies against IL‐9 and the other cytokines, as described in this unit.
Measurement of Human and Mouse Interleukin‐12
Authors: Gately Maurice K. , Chizzonite Richard , Presky David H. 
Published: June 2004

AbstractThis unit describes functional assays for measurement of bioactive IL‐12 and ELISAs for measurement of IL‐12 protein. The functional assays are based on the ability of IL‐12 to stimulate proliferation of PHA‐activated T lymphoblasts (“PHA blasts”). The ELISAs are technically simpler to perform than the functional assays, but cannot distinguish bioactive from inactive cytokine.
ELISPOT Assay to Detect Cytokine‐Secreting Murine and Human Cells
Authors: Klinman Dennis M. , Nutman Thomas B. 
Published: June 2004

AbstractThe filter immunoplaque assay, otherwise called the enzyme‐linked immunospot assay (ELISPOT), was initially developed to detect and quantitate individual antibody‐secreting B cells. Recent modifications have improved the sensitivity of the ELISPOT assay such that cells producing as few as 100 molecules of specific protein per second can be detected. The ELISPOT assay utilizes two high‐affinity cytokine‐specific antibodies directed against different epitopes on the same cytokine molecule: either two monoclonal antibodies or a combination of one monoclonal antibody and one polyvalent antiserum. ELISPOT generates spots based on a colorimetric reaction that detects the cytokine secreted by a single cell. The spot represents a “footprint” of the original cytokine‐producing cell. Spots are permanent and can be quantitated visually, microscopically, or electronically.
Detection of Cytokine Receptors by Flow Cytometry
Authors: Zola Heddy 
Published: June 2004

AbstractThis unit describes three protocols for detecting cytokine receptors on human peripheral blood mononuclear cells or murine splenic lymphocytes via immunofluorescence labeling. The three protocols have in common the use of phycoerythrin as a highly effective fluorescent dye, multiple‐layer staining to increase sensitivity, and selection of reagents for maximum sensitivity. The first procedure uses monoclonal antibody (MAb) against receptor protein, detected with biotinylated anti‐Ig (directed against the species in which the MAb is made), which in turn is stained with phycoerythrin‐streptavidin (PE‐SA). The second procedure uses biotinylated cytokines in the first step, followed by PE‐SA. Finally, a third procedure describes multiparameter analysis involving second and third fluorophores introduced as direct antibody conjugates, which is used to study subset expression of receptors. Instructions are also provided for titrating MAbs and cytokines, as well as how to evaluate detection reagents such as anti‐Ig conjugates and fluorophores.
Measurement of Interleukin 15
Authors: Paxton Raymond J. 
Published: June 2004

AbstractInterleukin 15 (IL‐15) is a small helical cytokine that was first characterized by its ability to stimulate proliferation of the murine T cell line CTLL‐2, but is now known to have activity on a variety of cell types. This unit describes two protocols that can be used to measure IL‐15 in culture supernatants and serum samples. The first method uses an enzyme‐linked immunosorbent assay (ELISA) that is specific for human IL‐15, although it can also detect simian IL‐15. The second method relies on the ability of IL‐15 to support proliferation of CTLL‐2 cells. The CTLL‐2 assay is slightly more sensitive than the ELISA, but it is not specific for IL‐15, because CTLL‐2 cells also respond to IL‐2 and IL‐4. The CTLL‐2 assay can be used to measure human, simian, or murine IL‐15.
Measurement of Interleukin 17
Authors: Patera Andriani C. , Cohen Jeffrey I. 
Published: June 2004

AbstractThis unit describes two procedures for measurement of interleukin‐17 (IL‐17). IL‐17 is a pleiotropic proinflammatory cytokine that binds to its ubiquitously expressed receptor, IL‐17R, resulting in the induction of several other cytokines including IL‐6 and IL‐8. The unit guides the reader on the use of individual reagents and commercial kits available for detection of murine and human IL‐17 by ELISA or Western blotting and presents step‐by‐step protocols for each of these approaches. In addition to detection of IL‐17 protein, a functional assay is described which measures the biological activity of IL‐17 by induction of IL‐6 from fibroblasts.
Detection and Isolation of Cytokine Secreting Cells Using the Cytometric Cytokine Secretion Assay
Authors: Assenmacher Mario , Löhning Max , Radbruch Andreas 
Published: June 2004

AbstractThe cell‐surface affinity matrix technology, otherwise called “secretion assay” or “capture assay,” represents a new, innovative method for the analysis and enrichment of viable cells according to secreted molecules such as antibodies or cytokineof T cells to induce cytokine secretion is included. Alternate protocols provide very rapid and simple procedures for the direct detection of antigen‐specific T cells from small numbers of PBMCs or whole blood without magnetic enrichment.
The In Vivo Cytokine Capture Assay for Measurement of Cytokine Production in the Mouse
Authors: Finkelman Fred , Morris Suzanne , Orekhova Tatyana , Sehy David 
Published: June 2004

AbstractBecause most cytokines are utilized, catabolized, or excreted shortly after they are produced, it has been difficult to directly measure in vivo cytokine production. Consequently, it has been necessary to infer in vivo cytokine secretion levels from the results of ex vivo assays of cytokine secretion, assays that measure tissue levels of cytokine mRNA, or assays that stain tissues for cytokine protein levels. Results of these assays provide important and useful information, but do not necessarily reflect in vivo cytokine secretion. To better determine in vivo cytokine production, the in vivo cytokine capture assay (IVCCA) was developed. IVCCA facilitates measurement of cytokines in serum by increasing their in vivo half‐lives. This increases the sensitivity of measurement of in vivo cytokine production 30‐ to 1,000‐fold. The first protocol described in this unit is for luminescence‐based ELISA, while the second is for an absorbance‐based method.
Isolation of T Cells Using Rosetting Procedures
Authors: Kanof Marjorie E. 
Published: June 2004

AbstractThis unit describes a procedure for separating T cells from other mononuclear cells by exploiting the unique ability of cells to bind to and form rosettes with sheep red blood cells (SRBC). This isolation method also allows recovery of the nonrosetting cell population (B lymphocytes, monocytes, and macrophages). Neuraminidase‐ and 2 ‐aminoethylisothiouronium bromide (AET)‐treated SRBC are used for rosetting because of enhanced binding to T cells.
Isolation of B Cell Populations
Authors: Splawski Judy B. , Lipsky Peter E. , Eisenstein Eli M. , Chua Kevin S. 
Published: June 2004

AbstractThis unit describes procedures for the isolation of pure human B cell populations. Two broad approaches to the isolation of pure human B cell populations are available, one based on techniques of negative selection and the other using positive selection. The negative selection approach has the advantage that the purified B cells obtained are less likely to be functionally altered by the isolation procedure. In addition, this approach does not require the use of expensive antibodies, which are usually the key element of a positive selection procedure. On the other hand, negative selection has the disadvantage that cell types not specifically targeted for removal (such as eosinophils) may be present in the final cell population. The positive selection method is an immunomagnetic positive selection using magnetic beads conjugated directly to an antibody specific for the B cell membrane determinant CD19. Also presented is a Support Protocol detailing the preparation of neuraminidase‐treated sheep red blood cells for use in rosetting.
Measurement of Proliferative Responses of Cultured Lymphocytes
Authors: James Stephen P. 
Published: June 2004

AbstractMeasurement of proliferative responses of human lymphocytes is a fundamental technique for the assessment of their biological responses to various stimuli. Most simply, this involves measurement of the number of cells present in a culture before and after the addition of a stimulating agent. This unit contains several different prototype protocols to measure the proliferation response of lymphocytes following exposure to mitogens, antigens, allogeneic or autologous cells, or soluble factors. Each of these protocols can be used in conjunction with an accompanying support protocol which contains methods for pulsing cultures with [3H]thymidine and determining incorporation of [3H]thymidine into DNA. The protocols described here provide an estimate of DNA synthesis and cell proliferation in an entire cell population, but do not provide information on the proliferation of individual cells.
Induction of Polyclonal Immunoglobulin Synthesis
Authors: James Stephen P. 
Published: June 2004

AbstractThis unit is designed to examine the effects of T cells or lymphokines on B cell differentiation in situations where the antigen specificity of the B cells is not of interest. In these cases, antibody production induced by polyclonal stimuli (e.g., mitogens, antibodies, Epstein‐Barr virus (EBV), or lymphokines) can be measured instead of antigen‐specific immunoglobulin production. Because B cells in the circulation and tissues are pleomorphic (containing subpopulations of cells that may be resting, cells that may have had prior antigenic exposure, and cells that may have undergone prior isotype commitment), the antibody responses of these subpopulations to various growth and differentiation factors differ. Therefore, the choice of which lymphocyte subpopulation to culture and which activation signal to use is determined by the particular experimental question.
Measurement of Polyclonal Immunoglobulin Synthesis Using ELISA
Authors: Nutman Thomas B. 
Published: June 2004

AbstractThe enzyme‐linked immunosorbent assay (ELISA) is a simple, specific, highly reproducible, and nonradioactive method for measuring immunoglobulin levels in cell culture supernatants and other biologic fluids. It can be used to measure the common immunoglobulin classes (IgG, IgM, and IgA) as described in the basic protocol, as well as classes and subclasses found in low levels in these fluids (IgG4 and IgE), as detailed in the alternate protocols.
Measurement of Polyclonal Immunoglobulin Synthesis Using the Reverse Plaque Technique
Authors: Tosato Giovanna , Pike Sandra E. , Blaese Michael 
Published: June 2004

AbstractThis unit describes the reverse hemolytic plaque assay, an effective method for measuring the number of immunoglobulin (Ig)‐secreting cells present in a cell population at any particular time. Cell populations that can be assayed using the technique include peripheral blood mononuclear cells or cells from tissues such as the tonsils. The basic protocol is divided into three stages. First, protein A‐sensitized sheep red blood cells (SRBC), guinea pig complement, and anti‐Ig antibody are prepared. Test samples are then combined and incubated with the SRBC, complement, and antibody in appropriate chambers. Finally, the resulting plaques are scored. A support protocol describes the preparation of plaquing chambers.
Isolation and Functional Analysis of Neutrophils
Authors: Clark Robert A. , Nauseef William M. 
Published: June 2004

AbstractThis unit describes the isolation of high‐purity neutrophils (or polymorphonuclear leukocytes; PMN) and the assays that can be performed to assess their function. An assay for the in vitro study of phagocytic capacity is presented, and microbicidal assays using lysostaphin and differential centrifugation are also described. During phagocytosis or on exposure to soluble agonists, neutrophils exhibit a marked increase in oxidative metabolism which is mediated by activation of NADPH oxidase and results in the formation of superoxide anion as well as hydrogen peroxide and other reactive species. The nitroblue tetrazolium reduction slide test is presented, and is a nonquantitative screening test for the presence of reactive oxygen products. The quantitation of superoxide by the kinetic and static assays are detailed, and a “broken cell” assay is also outlined.Finally, measurement of hydrogen peroxide formation is described.
Isolation of Human Basophils
Authors: Metcalfe Dean D. 
Published: June 2004

AbstractHuman basophils are normally the rarest of all circulating leukocytes and thus are among the most difficult of the blood elements to purify. In this unit, basophils are isolated from leukocyte populations, resulting in preparations of cells of which 5% to 50% are basophils and the rest are lymphocytes along with neutrophils and monocytes. This technique is advantageous because of its speed and because it minimizes basophil stimulation and histamine release.
51Cr Release Assay of Antibody‐Dependent Cell‐Mediated Cytotoxicity (ADCC)
Authors: Nelson David L. , Kurman Carole C. , Serbousek Deborah E. 
Published: June 2004

AbstractAntibody‐dependent cell‐mediated cytotoxicity (ADCC) is an immunologic cytotoxic effector mechanism that is dependent on the cooperative interaction of humoral and cellular effector elements. This unit describes an assay of ADCC activity that can be used as a test for immunocompetence in effector cells or to test the activity of a monoclonal antibody to mediate ADCC. In this form of cytotoxicity, effector cells with receptors for the Fc portion of immunoglobulin produce target cell lysis by attachment to the Fc portion of antibodies that are bound to target cells via their antigen‐combining sites. Therefore, an ADCC assay involves three essential components: labeled target cells, antibodies with specificity for target‐cell surface antigens, and effector‐cell populations. The basic protocol describes a method of measuring ADCC effector activity in lymphoid cells (peripheral blood mononuclear cells, or PBMC) that employs
51Cr‐labeled target cells. The three components are mixed in microtiter‐plate wells and lysis of the target cells is detected by measuring the release of radioactivity into the cell supernatant. Support protocols describe procedures for preparing anti‐target cell antiserum and 51Cr‐labeled target cells.
Cell Adhesion Under Hydrodynamic Flow Conditions
Authors: Gopalan Priya K. , Jones David A. , McIntire Larry V. , Smith C. Wayne 
Published: June 2004

AbstractThis unit describes a hydrodynamic assay to study the relative importance of various receptor/ligand interactions in cell‐cell and cell‐substrate adhesion and to quantitate the strength of their binding. The basic protocol describes how to assemble the single‐chamber flow system with the substrate, add the cells in suspension, and record the experiment on videotape. Alternate protocols present assays to determine how monoclonal antibodies and stimulating and inhibiting agents affect the substrate and the perfusing cells in suspension. Another alternate protocol details the use of the double‐chamber flow system. Support protocols describe how to construct the single‐ and double‐chamber flow systems and how to analyze the data from an experiment. Recording and analyzing the flow experiment requires the use of video equipment and, optionally, a computer and imaging software.
Isolation of Human Intestinal Mucosal Mononuclear Cells
Authors: Fiocchi Claudio , Youngman Kenneth R. 
Published: June 2004

AbstractThe aim of this procedure is to obtain large numbers of isolated, viable, and functional mononuclear cells that are representative of the lymphoid population present in the mucosa of the human gastrointestinal tract under physiological and pathological conditions. The basic protocol is based on the use of surgically resected small and large bowel specimens, and consists of two basic stages: (1) a combination of chemical, enzymatic, and mechanical treatments to dissociate intestinal tissue and free the mononuclear cells from the surrounding interstitial framework; and (2) separation, isolation, and purification of viable mucosal lamina propria mononuclear cells from other cellular and amorphous components. The proportion of viable cells obtained can be increased by including extra separation steps using nylon wool columns or Percoll gradients, as described.
Isolation and Generation of Human Dendritic Cells
Authors: Tedder Thomas F. , Jansen Paul J. 
Published: June 2004

AbstractDendritic cells are highly specialized antigen‐presenting cells (APC), which may be isolated or generated from human blood mononuclear cells. Although mature blood dendritic cells normally represent ˜0.2% of human blood mononuclear cells, their frequency can be greatly increased using the cell enrichment methods described in this unit. More highly purified dendritic cell preparations can be obtained from these populations by sorting of fluorescence‐labeled cells. Alternatively, dendritic cells can be generated from monocytes by culture with the appropriate cytokines, as described here. In addition, a negative selection approach is provided that may be employed to generate cell preparations that have been depleted of dendritic cells to be used for comparison in functional studies.
Measurement of Prostaglandins and Other Eicosanoids
Authors: Stenson William F. 
Published: June 2004

AbstractEicosanoids are arachidonic acid metabolites formed through the cyclooxygenase or one of the lipoxygenase metabolic pathways. They include the prostaglandins (PG), leukotrienes (LT), thromboxanes (Tx), peptidoleukotrienes, and 5‐hydroxyeicosatetraenoic acid (5‐HETE). This unit describes procedures for measuring individual eicosanoids using several commercial kits. These include kits for an enzyme immunoasssay (ELISA) and a radioimmunoassay (RIA). A method is also described that can be used to analyze the full range of eicosanoids produced in a biological system, and a support protocol presents an acetone/chloroform extraction method for purifying eicosanoids prior to assay. Another support protocol describes a different extraction protocol in which samples are run over an octadecylsilyl silica column.
Solubilization of Lymphocytes
Authors: Weissman Allan M. 
Published: June 2004

AbstractPurification and study of transmembrane proteins require isolation of these structures from their lipid environment. This isolation is carried out through the use of detergents. In this unit, several approaches to solubilization of membrane proteins are presented. Solubilization of whole lymphocytes, using conditions aimed at minimizing the disruption of protein‐protein interactions, is described with an optional step that may be useful when the disruption of protein interactions is desired as part of a purification protocol. In some situations, it may be desirable to purify membranes prior to their solubilization or to determine the physical relationship between proteins, which can be accomplished by a cross‐linking.
Purification of Organelles from Mammalian Cells
Authors: Castle J. David 
Published: June 2004

AbstractOrganelle purification procedures capitalize on the differences in size, density, and (occasionally) surface charge density of individual types of organelles. Most fractionation procedures that are based on centrifugation involve some combination of procedures that distinguish both size and density. Initially, a homogenate is prepared in isoosmotic (or slightly hyperosmotic) sucrose or some other predominantly nonelectrolyte medium. A wide range of procedures have been used to fractionate tissue homogenates. The protocols in this unit emphasize different fractionation techniques that have been used for rat liver, an abundant tissue that has been a favorite of many investigators and has served as the source of many organelle preparations of excellent purity. For selected procedures, examples have been given using other tissue sources (e.g., glandular tissues that maintain protein storage granules for regulated secretion) or, where particularly favorable, cultured cells.
Diagonal Gel Electrophoresis
Authors: Samelson L.E. 
Published: June 2004

AbstractDiagonal gel electrophoresis is a form of two‐dimensional analysis useful for investigating the subunit composition of multisubunit proteins containing interchain disulfide bonds. Proteins are electrophoresed in the first dimension in a slab or tube gel under nonreducing conditions. The proteins are then reduced in the gel and this piece of gel is layered onto a second gel and electrophoresed. In the second gel, the proteins migrate at right angles to their original, first‐dimension migration. The majority of cellular proteins are not disulfide‐linked and will fall on the “diagonal” in this system; that is, they migrate approximately equal distances in both directions during electrophoresis and lie approximately on the diagonal line connecting opposite corners of the gel. Upon reduction, component subunits of proteins connected by interchain disulfide bonds will resolve below the diagonal because the individual subunits migrate faster than the disulfide‐linked complex during the second electrophoresis.