bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cloning, Expression, and Modification of Antibody V Regions
Authors: Morrison Sherie L. 
Published: June 2004

AbstractCloned variable (V) regions of antibodies can be expressed joined to any constant (C) region, from either the same or a different species. The resulting antibodies will have the desired associated effector functions. Chimeric antibodies obtained by joining murine V regions to human C regions should have decreased immunogenicity in humans. The process of complementarity determining region (CDR) grafting, in which the CDRs from an antibody of one species are transferred to the framework regions of another species, constitutes a further modification of this approach. The protocols presented in this unit are designed to permit PCR‐based cloning of heavy and light chain V regions. This is an advanced molecular biology protocol and should be employed only by investigators who are sufficiently skilled and experienced.
Depletion of Accessory Cells by Adherence to Sephadex G‐10
Authors: Hathcock Karen S. 
Published: June 2004

AbstractAccessory cells such as macrophages and other antigen‐presenting cells (APC) in suspensions of mouse spleen and lymph node are removed by means of their preferential adherence to Sephadex G‐10 polymers. Unlike the use of nylon wool columns to remove B and accessory cells (UNIT ), Sephadex G‐10 columns are employed when the main purpose is to yield functional populations of T cells and B cells (by depleting mostly macrophages and other APC). The following technique is simple, fast, and reproducible; however, it results in some retention of B cells on the resin. The basic protocol in this unit involves assembling the column, loading and equilibrating the sterile Sephadex slurry, and passing the cells through the column followed by collection and analysis of eluate. Two support protocols describe preparation of the Sephadex G‐10 slurry for use in the column by fining and sterilization, plus recycling resin from used columns.
Analysis of Flow Cytometry Data
Authors: Sharrow Susan O. 
Published: June 2004

AbstractThis unit provides several approaches to flow cytometry data analysis. Frequency determinations based on analysis of single‐parameter fluorescence histograms and dual‐parameter contour plots are presented. Next, steps are described for calculating values for signal‐to‐noise ratios when logarithmic amplification is used for data collection. This calculation can be used to compare the amounts of antigenic determinants per cell between different cells stained with the same reagent. Finally, a procedure is described for constructing a calibration curve for logarithmic amplifiers. This calibration curve is required for calculation of signal‐to‐noise values and can also be used to determine if the amplifiers are working properly.
Measurement of Human and Murine Interleukin 2 and Interleukin 4
Authors: Davis Laurie S. , Lipsky Peter E. , Bottomly Kim 
Published: June 2004

AbstractThis unit describes protocols employing cell lines or bioassays that can be used for the quantitation of murine total T cell growth factor (TCGF) activity, interleukin 2 (IL‐2), and interleukin 4 (IL‐4), and of human IL‐2 and IL‐4. The ability to distinguish between different growth factors is crucial to understanding the regulation of the immune response. The describes the use of the CTLL‐2 line to detect murine IL‐2 and IL‐4 in supernatants. One alternate protocol describes the detection of IL‐2 in samples of human serum or supernatants using CTLL‐2 cells, while other alternate procedures describe the detection and quantitation of murine IL‐4 using a mutagenized subline of CTLL‐2, CT.4S, and the detection of human IL‐4 using a derivative of the CT.4S mouse cell line, CT.h4S. Support protocols are provided for the quantitation of CTLL‐2, CT4.S, or CT.h4S proliferation using a standard [3H]thymidine incorporation method or by using the 3‐(4,5‐dimenthylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) colorimetric assay (). Support protocols also describe the calculation of cytokine units from samples based on DNA synthesis data and procedures for the maintenance of the CTLL‐2, CT.4S, and CT.h4S cell lines.
Measurement of Mouse and Human Interleukin 10
Authors: Mosmann Tim 
Published: June 2004

AbstractInterleukin 10 (IL‐10) is a multifunctional cytokine produced by T cells and other cell types. It regulates activities of a variety of the cells of the immune system and may play a crucial role in cross‐regulation of different types of T cells. Two methods of quantitating IL‐10 are provided in this unit. The first is an enzyme‐linked immunosorbent assay (ELISA) using a pair of monoclonal antibodies specific for mouse IL‐10. This method has the advantages of high specificity and insensitivity to other cytokines present in test samples. The second method is a bioassay which has the advantages that both human and mouse IL‐10 are detected and that functional protein is measured. Both methods have similar sensitivity.
Measurement of Human and Murine Stem Cell Factor (c‐kit Ligand)
Authors: Smith Kent A. , Zsebo Krisztina M. 
Published: June 2004

AbstractStem cell factor (SCF) is an early‐acting, hematopoietic growth factor that binds to the receptor encoded by the proto‐oncogene c‐kit. It is a potent growth factor for primitive bone marrow cells as well as thymocytes. This unit describes three protocols for detecting human and murine SCF. In the first, human or rodent SCF is measured by its ability to stimulate proliferation of the human megakaryoblastic leukemia cell line, UT‐7. Because rat and mouse SCF bind well to human c‐kit, human and rodent SCF can both be measured using the first basic protocol. In an , rodent SCF is assayed by its ability to stimulate proliferation of the clonal murine mast cell line, MC/9. Human SCF is not very active on rodent cells and thus cannot be measured using this protocol. Both of the cell proliferation assays lack specificity because they are capable of detecting other cytokines in addition to SCF. The third protocol is a radioreceptor assay using the human erythroleukemia cell line, OCIM1; it specifically measures murine or human SCF and not other cytokines. Support protocols describe maintenance of UT‐7 and MC/9 cells and preparation of plasma membranes from OCIM1 cells.
Generation of HTLV‐I‐Transformed T Cell Lines
Authors: Nutman Thomas B. 
Published: June 2004

AbstractViral transformation of T cells is an effective method for obtaining large numbers of T cells that are easily maintained in the laboratory. Human T cell leukemia/lymphoma virus (HTLV‐I), a type C retrovirus clearly implicated as the etiologic agent of adult T cell leukemia, can both infect and transform normal T cells. This unit presents a protocol in which human T cells are activated and then transformed with HTLV‐I. Detection of virally transformed cells is discussed in the commentary.
Measuring Degranulation of Mast Cells
Authors: Hohman Robert J. , Dreskin Stephen C. 
Published: June 2004

AbstractThis unit describes methods for measuring exocytosis of preformed mediators from secretory granules as an indication of IgE receptor‐mediated activation of mast cells. The first basic protocol describes the measurement of biogenic amines (serotonin and histamine) secreted by activated rodent mast cells (for serotonin) or rodent and human mast cells (for histamine). The second basic and alternate protocols detail techniques for measuring the release of β‐glucuronidase, an enzyme that is synthesized by human and rodent mast cells, stored in secretory granules, and released during degranulation. Methods for assaying other enzymes released during degranulation, such as β‐hexosaminidase and tryptase, are discussed in the . These protocols can also be applied to basophils where appropriate.
Two‐Dimensional Gel Electrophoresis Using the O'Farrell System
Authors: Adams Lonnie D. , Gallagher Sean R. 
Published: June 2004

AbstractTwo‐dimensional gel electrophoresis is the combination of two high‐resolution electrophoretic procedures (isoelectric focusing and SDS‐polyacrylamide gel electrophoresis) to provide much greater resolution than either procedure alone. In the first‐dimension gel, solubilized proteins are separated according to their isoelectric point (pI) by isoelectric focusing. This gel is then applied to the top of an SDS‐slab gel and electrophoresed. The proteins in the first‐dimension gel migrate into the second‐dimension gel where they are separated on the basis of their molecular weight. The basic protocols in this unit are based on the type of equipment originally described by O'Farrell in 1975. Such equipment is relatively inexpensive and can produce satisfactory results in situations that do not require running large numbers of two‐dimensional gels. Many variations of the original procedure have been described in the literature and by equipment suppliers. The procedures in this unit are appropriate, with minor modifications, for different equipment available from commercial suppliers or custom‐made equipment similar to that described by O'Farrell; detailed instructions are usually supplied by the manufacturer. For very basic or very acidic proteins, two alternate protocols are provided. A third alternate protocol describes how two‐dimensional electrophoresis can be performed using a minigel system. Protein sample preparation is presented in the Support Protocol.
Synthesis and Application of Peptide Dendrimers As Protein Mimetics
Authors: Tam James P. , Spetzler Jane C. 
Published: June 2004

AbstractThe multiple antigenic peptide (MAP) is a novel approach to preparing peptide immunogens. The MAP consists of an inner core matrix built up of a large layer of Lys residues and a surface of peptide chains attached to the core matrix. Because of its dendrimeric structure, MAP can be very useful as a template for assembling potential peptide surfaces. Two methods for preparing MAP systems are described in this unit. The direct approach for preparing MAP systems is presented in the first two protocols, including the procedure for b‐butyloxycarbonyl (Boc) chemistry and the procedure for 9 ‐fluorenylmethyloxycarbonyl (Fmoc) chemistry. An indirect approach for preparing MAP systems, in which peptide and core matrix are synthesized separately and conjugated by several ligation methods, is then described. The cMAP approach is also executed using either the direct or indirect approach, but requires an additional cyclization step to constrain the peptides after synthesis. The synthesis of cMAP is described, and the preparation of cyclic peptides is illustrated. A support protocol describes the ninhydrin assay to assess the completeness of the coupling reaction. In most cases, MAP systems can be used directly after simple dialysis or desalting. Some immunological studies, however, require purified MAPs. Additional support protocols describe MAP system purification by dialysis and high‐performance gel‐filtration chromatography.
Synthesis of Multiple Peptides on Plastic Pins
Authors: Rodda Stuart J. 
Published: June 2004

AbstractScanning protein sequences by bioassay for smaller bioactive peptide sequences requires a source of many peptides homologous with the parent protein sequence. This unit deals with one of the synthetic methods for making such sets of peptides. The strategy of the multiple peptide approach to biological scanning is discussed, along with the synthetic protocols, and the handling of peptides after synthesis, i.e., cleavage, preliminary purification, storage, and analysis. It is specific for the multipin technique using equipment obtained from Chiron Technologies, although some of the approaches are applicable to other multiple synthesis techniques. Procedures for multipin equipment obtained from other suppliers may differ from the procedures described here, and the manufacturer's literature should be consulted. This unit also includes protocols for preparing Fmoc‐amino acid solutions and for acetylating or biotinylating synthesized peptides.
Deprotection of Oligonucleotides and Purification Using Denaturing PAGE
Authors: Ellington Andrew , Chory Joanne 
Published: June 2004

AbstractThe advantages of purification by denaturing polyacrylamide gel electrophoresis (PAGE) are speed, simplicity, and high resolution. Although yields tend to be low (<50% of applied sample), the amount of material recovered is usually far in excess of that required for most molecular biology applications (e.g., cloning and sequencing). Denaturing PAGE can resolve oligonucleotides from 2 to 300 bases, depending on the percentage of polyacrylamide used. The method presented here is thus useful not only for isolating chemically synthesized deoxyribonucleotides but also small RNAs or other single‐stranded polynucleotides.
Liposome‐Mediated Transfection
Authors: Whitt Michael , Buonocore Linda , Rose John K. 
Published: June 2004

AbstractUsing liposomes to deliver DNA into different eukaryotic cell types results in higher efficiency and greater reproducibility than other transfection methods. In this unit, the describes a transient expression system while an involves stable transformation and expression of DNA integrated into the genome of the transfected cell. In both protocols, plasmid DNA derived from either crude (miniprep) or purified (through CsCl) preparations is mixed with a liposome suspension comprised of cationic lipids and applied to monolayer cell cultures.
Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization
Authors: Kramer Martha F. , Coen Donald M. 
Published: June 2004

AbstractThis unit describes a method for amplifying DNA enzymatically by the polymerase chain reaction (PCR) and for optimizing this reaction for the sequence and primer set of interest. Important variables that can influence the outcome of PCR include the MgCl2 concentration and the cycling temperatures. Additives that promote polymerase stability and processivity or increase hybridization stringency, and strategies that reduce nonspecific primer‐template interactions, especially prior to the critical first cycle, can greatly improve sensitivity, specificity, and yield. This protocol is designed to optimize the reaction components and conditions in one or two stages. The first stage determines the optimal MgCl2 concentration and screens several enhancing additives. To further improve specificity, sensitivity and yield, the second stage compares methods for optimizing initial specific hybridization to prevent polymerization of misprimed sequences prior to thermal cycling. For initial inhibition of polymerase activity, temperature (i.e., cooling reagents), physical separation (“hot start” method), and reversible antibody binding are compared.
Detection of Cytokine mRNA Expression by PCR
Authors: James Stephen P. 
Published: June 2004

AbstractAssessment of cytokine expression during an immune response is a critical requirement for studies of basic immune mechanisms and the pathogenesis of disease. As an alternative to measuring cytokine protein levels, analysis of cytokine mRNA may be appropriate, because production of many cytokines is primarily transcriptionally regulated. Therefore, the level of cytokine mRNA present in a sample may serve as a good estimate of the level of cytokine protein present. This unit outlines the amplification of IL‐2 mRNA from clinical biopsy specimens by the polymerase chain reaction (PCR), but the approach can be readily modified to study any cytokine RNA of interest in biopsies or small samples. In the , cDNA is synthesized from the sample RNA by reverse transcriptase; to control for experimental variation in both the reverse transcription and PCR amplification steps, an internal control RNA is included in the reaction. Together, the reverse‐transcribed cDNAs are amplified by PCR.
Evaluating Human T Cell Receptor Gene Expression by PCR
Authors: Gussoni Emanuela , Panzara Michael A. , Steinman Lawrence 
Published: June 2004

AbstractThis unit describes the use of PCR to characterize and quantify rearranged transcripts from specific T cell receptor variable gene families in human tissue and peripheral blood lymphocytes. The strategy outlined in this unit has been extensively used on different sources of human tissue including brain, spinal cord, and skeletal muscle. A protocol is provided to clone and sequence PCR‐amplified cDNA transcripts to study the junctional diversity of the expressed genes. A support protocol describes a method for reverse transcribing total RNA to make the cDNA required by the other protocols.
Analysis of Inositol Phospholipid Turnover During Lymphocyte Activation
Authors: Imboden John B. , Shoback Dolores M. , Inokuchi Sharon 
Published: June 2004

AbstractReceptor‐mediated activation of phospholipase C (PLC) leads to the hydrolysis of membrane inositol phospholipids, generating diacylglycerol (DAG) and water‐soluble inositol phosphates. This signaling mechanism is used by antigen receptors on T and B cells that have been implicated as mediators of receptor‐induced influx of extracellular Ca2+. This unit provides protocols that describe the resolution of InsP by Dowex anion‐exchange chromatography. This technique provides a reliable means of separating inositol monophosphate, inositol bisphosphate, and inositol trisphosphate, but does not resolve isomers of these. An describes the separation of inositol phosphates by anion‐exchange HPLC. A protocol for resolution of inositol phospholipids by thin‐layer chromatography (TLC) is also provided.
Antiphosphotyrosine Blotting
Authors: Siegel Jeffrey N. 
Published: June 2004

AbstractAntiphosphotyrosine blotting is a technique for detecting tyrosine‐phosphorylated substrates by the use of antibodies that recognize these residues on a wide variety of proteins. This unit describes conditions for cell lysis and immunoprecipitation of proteins with an antiphosphotyrosine antibody, followed by electrophoretic separation, immunoblotting, and color detection of the blotted proteins. This combination of steps provides particularly sensitive conditions for the detection of tyrosine‐phosphorylated substrates, but also gives good results for any protein transferred to nitrocellulose, including whole‐cell lysates or proteins immunoprecipitated with another antibody. Although the alkaline phosphatase color‐detection system has the advantage of providing superior resolution and higher sensitivity without the use of any radioactivity, the 125I‐labeled Staphylococcus protein A detection system is described for use in conjunction with the blotting protocol.
Using Protein Kinase and Protein Phosphatase Inhibitors to Dissect Protein Phosphorylation Pathways
Authors: Karaszkiewicz James W. , Henrich Curtis J. 
Published: June 2004

AbstractIt is often desirable to attempt to measure and to modulate the activity of the protein kinases and phosphatases responsible for a given phenotypic change. The protocols in this unit describe how to assay a variety of protein kinase activities in complex mixtures, such as whole cell lysates, in order to screen compounds as potential inhibitors of these activities. The effects of known or potential inhibitors can be evaluated by monitoring phenotypic changes in intact cells following treatment with the inhibitor. The activities of the major serine/threonine‐specific protein phosphatases in most mammalian cells can be distinguished using a selective inhibitor, okadaic acid. Support protocols describe preparation of cell extracts needed for the kinase assays, and of32P‐labeled phosphorylase used in the inhibition assays.
TRAF‐Mediated TNFR‐Family Signaling
Authors: Arron Joseph R. , Walsh Matthew C. , Choi Yongwon 
Published: June 2004

AbstractThe tumor necrosis factor (TNF) superfamily consists of a wide variety of proteins, some cell‐bound and others secreted, that regulate many cellular processes. In particular, TNF‐family proteins regulate the life and death not only of tumor cells, but of activated cells of the immune system. This overview discusses the six known mammalian TRAF proteins, of which TRAF1, 2, 3, 5, and 6 have been shown to interact directly or indirectly with members of the TNFR superfamily. Structural features of TRAF proteins are described along with a discussion of TRAF‐interacting proteins and the signaling pathways activated by TRAF proteins. Finally, a discussion is provided on the phenotypes of TRAF‐knockout mice.
Alternative Pathway Evaluation
Authors: Giclas Patricia C. 
Published: June 2004

AbstractThe alternative pathway of complement shares its terminal components (C3 and C5 through 9) with the classical pathway, but has several unique components, including factors D, B, and P (properdin). This unit presents methods for assaying total alternative pathway activity and the activity of factors B and D. Radial immunodiffusion (RID) can also be used to measure factor D, B, and P concentrations.
Assays for Membrane Complement Receptors
Authors: O'Rear Lynda D. , Ross Gordon D. 
Published: June 2004

AbstractLeukocytes express several types of receptors for activated products of the complement (C) system. This unit describes assays for a subset of these receptors: those specific for fixed fragments of C3 (CR1, CR2, CR3, and CR4). Although the assays have been designed for tests of human C receptors, they can often be carried out with rodent (e.g., mouse, rat, or guinea pig) cells by substitution of rodent‐specific reagents. Two basic protocols are presented for rosette assay of CR1, CR2, CR3, and CR4 on adherent and nonadherent cells. Isolated leukocytes may be tested for rosette formation in suspension or adherent to a surface. Nonadherent cells such as lymphocytes, NK cells, or cells from an undifferentiated leukocyte cell line must be tested in suspension. The unit also contains three support protocols for formation and quantitation of sheep erythrocyte‐C3 complexes for use in the two basic protocols.
Assays for the Mannan‐Binding Lectin Pathway
Authors: Gadjeva Mihaela , Thiel Steffen , Jensenius Jens C. 
Published: June 2004

AbstractThis unit contains protocols that can be used to measure mannan‐binding lectin (MBL) levels and MBL pathway activity in human plasma or serum. Using a modification of the conventional ELISA, the detection reagent used in the methods described (e.g., an antibody or streptavidin) is labeled with Eu3+ instead of an enzyme. The sensitizing reagent used in these assays employ the use of anti‐MBL antibody or mannan. The unit also includes an MBL/MASP complex activity assay to evaluate MBL activity in serum or plasma samples, which allows for the measurement of the specific activity of the MBL pathway.
Measurement of Bacterial Ingestion and Killing by Macrophages
Authors: Campbell Priscilla A. , Canono Beth P. , Drevets Douglas A. 
Published: June 2004

AbstractThis unit presents fairly simple assays for measuring the binding of bacteria to macrophages, internalization of bacteria (also called ingestion or phagocytosis), and bacterial killing by macrophages. The first basic protocol describes how to measure the ability of macrophages to ingest bacteria. Because it is critical to remove residual extracellular organisms, the protocol presents two alternative steps to accomplish this: a washing procedure and a more stringent method in which cells are sedimented through sucrose. In addition, it is important to distinguish those bacteria truly ingested by a macrophage from those that are bound to, but not internalized by, the cell. A simple but effective way to do this is described in an alternate protocol. The unit also presents two ways to measure the ability of a macrophage to kill bacteria it has internalized. The first is a straightforward assay in which bacterial colonies are enumerated before and after a killing period; a subsequent colony count will indicate whether the bacteria grew within or were killed by the macrophage. The second protocol describes a way to measure bacterial viability based on bacterial metabolism, in which the ability of bacterial dehydrogenases to mediate the reduction of a tetrazolium salt to purple formazan is monitored by measuring absorbance spectrophotometrically.