bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

AbstractFlow 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

AbstractOne 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

AbstractInterleukin 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

AbstractThis 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

AbstractThe 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

AbstractTumor 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

AbstractTransforming 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

AbstractThis 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

AbstractThis 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

AbstractThis 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

AbstractInterleukin 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

AbstractIntracellular 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

AbstractIL‐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

AbstractThe 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

AbstractThis 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

AbstractThis 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

AbstractThis 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

AbstractThe 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.
Measurement of In Vitro Specific‐Antibody Synthesis
Authors: Yarchoan Robert 
Published: June 2004

AbstractIn this unit, a protocol is provided in which antigen‐specific responses by human lymphocytes are elicited and then quantitated by the enzyme‐linked immunosorbent assay (ELISA). Initially, Ficoll‐Hypaque‐isolated mononuclear cells are treated to remove cytophilic antibody. The antibody response being studied is then elicited by the appropriate stimulating antigen (in this case, influenza virus, although other antigens can also be used) or mitogen. Finally, after a 12‐day incubation, the supernatant is harvested for assay.
Measurement of Polyclonal and Antigen‐Specific Cytotoxic T Cell Function
Authors: Biddison William E. , Lichtenfels Rudolf , Adibzadeh Medi , Martin Roland 
Published: June 2004

AbstractMeasurement of in vitro cytotoxic function of human T cells can be accomplished by polyclonal stimulation of T cell effectors using anti‐CD3 antibody, which stimulates all cytolytic effector cells, or with a specific stimulating antigen. Accordingly, two sets of assays of cytolytic T cell function are described in this unit, one for measuring anti‐CD3‐mediated cytotoxicity and the other for measuring antigen‐specific cytotoxicity. Although the calcein release assay (CARE‐LASS) described here is for use with antigen‐activated cytotoxic T lymphocytes (CTL) as well as natural killer (NK) or lymphokine‐activated killer (LAK) cells, minor changes in the protocols that address polyclonal T cell activation are described that make them suitable for use with calcein‐labeled target cells.
Measurement of Cytotoxic Activity of NK/LAK Cells
Authors: Whiteside Theresa L. 
Published: June 2004

AbstractIn this unit, methodology is presented for measuring the capacity of human natural killer (NK) or lymphokine‐activated killer (LAK) cells to lyse tumor cell targets. Cytotoxicity of these effector cells is evaluated in a short‐term 51Cr‐release assay using NK‐sensitive tumor cells as the targets for NK cells or NK‐resistant tumor cells as the targets for LAK cells. In the basic protocol, a generic 51Cr‐release assay is described in which PBMC, purified NK cells or interleukin 2 (IL‐2)‐activated lymphocyte populations (LAK cells) are utilized as effector cells. Support protocols describe preparation of nonadherent tumor cells, cells obtained from malignant effusions, trypsinized tumor cells from adherent monolayer culture, or freshly isolated tumor cells from surgical specimens. All of these cell types can serve as the 51Cr‐labeled target cells in the basic protocol. The procedure for labeling target cells from any of these sources with 51Cr is also provided.
Generation and Maintenance of Cloned Human T Cell Lines
Authors: Yssel Hans , Spits Hergen 
Published: June 2004

AbstractThis unit describes protocols for the generation of human (allo‐) antigen‐specific T cell lines, and T lymphocyte subpopulations with distinct cytokine production profiles from purified peripheral or cord blood CD4+ T cells, respectively. Methods for the cloning and maintenance of these cell lines are given, as well as a protocol for freeze/thaw procedures.
Generation of T Cell Hybridomas
Authors: Mayer Lloyd 
Published: June 2004

AbstractThis unit presents a method of immortalizing T cells by fusing them with a preexisting T cell line (the fusion partner) to form a T cell hybridoma. Support protocols present key elements in successful fusions, including the selection of an appropriate fusion partner and activation of the donor T cells. Other support protocols describe the selection processes used for hybridomas, including measures to ensure long‐term stability and maintenance of cell lines.
Generation of Epstein‐Barr Virus (EBV)–Immortalized B Cell Lines
Authors: Tosato Giovanna 
Published: June 2004

AbstractImmortalization of B lymphocytes by EBV is an effective procedure for inducing long‐term growth of certain human B lymphocytes. The protocol described in this unit to accomplish this can be divided into three stages: preparation of virus, preparation of target cells to be immortalized, and EBV infection and growth of infected cells.
Isolation of Tissue Mast Cells
Authors: Metcalfe Dean D. 
Published: June 2004

AbstractMast cells, unlike other immune effector cells, are not found in the circulation and thus must be obtained from accessible tissues such as gastrointestinal and lung tissue. In this unit, a combination of mechanical fragmentation, enzyme digestion, and centrifugation procedures are used to obtain suspensions enriched for mast cells from gastrointestinal tissue. A support protocol details a procedure for acid toluidine blue staining of mast cells.
Isolation and Characterization of Human Natural Killer Cell Subsets
Authors: Cooper Megan A. , Caligiuri Michael A. 
Published: June 2004

AbstractNatural killer (NK) cells are innate immune lymphocytes that play a critical role in the host defense against pathogens through their production of cytokines and cytotoxic activity. Human NK cells can be divided into two subsets, each with distinct phenotypic and functional properties, based on cell‐surface density expression of CD56 (CD56bright and CD56dim). This unit describes the purification of human NK cell subsets from blood, protocols for the further characterization of NK cell function, and further background information on these cell types.
Subcellular Fractionation of Tissue Culture Cells
Authors: Aniento Fernando , Gruenberg Jean 
Published: June 2004

AbstractDevelopment of cell fractionation techniques over the last few decades has provided the means to analyze the composition and properties of purified cellular elements. In particular, subcellular fractionation is essential for the development of cell‐free assays that reconstitute complicated cellular processes. These assays have provided new and important tools to understand the molecular mechanisms of complex cellular functions, permitting these functions to be studied in the test tube as a series of biochemical reactions. The protocols in this unit describe fractionation of tissue culture cells to immunoisolate early and late endosomes under conditions where these compartments retain their capacity to support membrane transport in vitro.
Immunoprecipitation
Authors: Bonifacino Juan S. , Dell'Angelica Esteban C. , Springer Timothy A. 
Published: June 2004

AbstractImmunoprecipitation consists of multiple ordered steps: lysing the cell with detergent if the antigen (usually a protein) to be precipitated is membrane‐bound; binding of a specific antigen to an antibody; precipitating the antibody‐antigen complex; washing the precipitate; and dissociating the antigen from the immune complex. The dissociated antigen is then analyzed by electrophoretic methods. In this unit, the basic protocol details the immunoprecipitation of a radiolabeled antigen with a specific antibody (polyclonal or monoclonal) covalently linked to Sepharose. Preparation of Ab‐Sepharose is described in the Support Protocol. The first two alternate protocols present methods for precipitating or isolating the soluble immune complexes formed between a specific antibody and a radiolabeled antigen. Immunoprecipitation is achieved with polyclonal anti‐immunoglobulin (Ig) serum, anti‐Ig‐Sepharose, Staphylococcus protein A or Streptococcus protein G bound to Sepharose, or Staphylococcus aureus bacteria which contain protein A on the cell surface. The third alternate protocol should be used for immunoprecipitation of antigens that are nonspecifically associated with other proteins. The fourth alternate protocol describes immunoprecipitation of unlabeled protein antigens with Ab‐Sepharose.