bluuebunny/crossref_metadata_2025_split huggingface dataset:
RNA Interference in Cultured Drosophila Cells
Authors: Worby Carolyn A. , Dixon Jack E. 
Published: June 2004

AbstractRNA interference (RNAi) can be used to silence genes in a number of species, including plants, nematodes, protozoans, Drosophila melanogaster, mouse embryos, and mammalian and Drosophila cell cultures. Drosophila cell culture provides the opportunity to study signal transduction pathways and protein function in a simple, well defined cell culture paradigm. Furthermore, because Drosophila are RNAi responsive, the results obtained from experiments performed on cultured cells can be confirmed in the whole organism. RNAi takes advantage of the unique ability of double‐stranded RNA (dsRNA) molecules to induce gene silencing in a highly specific manner. This phenomenon is efficacious and long lived, being passed to subsequent generations in Drosophila cell culture. To date, many Drosophila cell lines tested respond to dsRNAs by ablating expression of the target protein. Furthermore, all dsRNAs tested have been efficacious at silencing the target gene. Drosophila cell cultures are simple, easily manipulated model systems that will facilitate loss of function studies applicable to a wide variety of questions.
Identification of RNA Binding Proteins by UV Cross‐Linking
Authors: Luo Ming‐Juan , Reed Robin 
Published: June 2004

AbstractOne of the major focuses of modern biology is to understand the dynamics of RNA‐protein interactions, including factors that interact with mRNA, rRNA, tRNA, snRNA, hnRNA, siRNA, and viral RNA. Identification the direct interactions between proteins and RNA has greatly advanced our knowledge about the function of RNA‐protein machines. UV crosslinking is a straightforward and powerful tool in this endeavor.
Purification of Functional RNA‐Protein Complexes using MS2‐MBP
Authors: Zhou Zhaolan , Reed Robin 
Published: June 2004

AbstractBiological machines composed of RNAs and proteins play essential roles in many biological processes. To better understand the mechanism and function of these machines, it is critical to isolate them in a highly purified and functional form. A method for isolating functional RNA‐protein complexes assembled in vitro is described. The approach combines gel filtration and an affinity‐chromatography strategy using the bacteriophage MS2 coat protein, which binds to a specific RNA‐hairpin structure. Using this method, highly purified and functional human spliceosomes have been isolated. The purified spliceosome preparation is used to determine the protein components of the spliceosome by mass spectrometry and to examine the structure of the spliceosome by electron microscopy.
Compendium of Drugs Commonly Used in Molecular Biology Research
Authors: Cole Nelson B. 
Published: June 2004

AbstractThis 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.
Detection and Quantitation of Radiolabeled Proteins and DNA in Gels and Blots
Authors: Voytas Daniel , Ke Ning 
Published: June 2004

AbstractThis unit presents procedures for visualizing and quantitating radiolabeled proteins or DNA separated by polyacrylamide gel electrophoresis (PAGE) or affixed to filter membranes. Procedures for autoradiography and densitometry are provided, but even greater sensitivity can be achieved with fluorography or by the use of intensifying screens, both of which enhance the radioactive signal. Preflashing the film is described to increase the linear measurement range for weakly radioactive samples. Sensitivity and linear ranges of measurement can also be greatly extended by using a phosphor imaging system to quickly and easily quantify radioactive samples.
Toll Receptors
Authors: Takeda Kiyoshi , Akira Shizuo 
Published: June 2004

AbstractThis has led to the identification of several important roles for Toll receptors in mammals. This unit discusses mammalian Toll receptors (TLR1‐10) that have an essential role in the innate immune recognition of microorganisms. Also discussed are TLR‐mediated signaling pathways and antibodies that are available to detect specific TLRs.
Managing Immunocompromised Animals
Authors: Donovan John , Brown Patricia 
Published: June 2004

AbstractThis unit describes some commonly used methods for maintaining immunocompromised rodents, specifically mice, to prevent infections from adventitious pathogens. The basic protocol describes the microisolation housing unit, while alternate procedures cover individually ventilated isolator housing, laminar‐flow rack housing, flexible‐film isolator housing, and barrier facility housing.
Anesthesia
Authors: Donovan John , Brown Patricia 
Published: June 2004

AbstractAnesthetic agents are used in laboratory animals to prevent pain or distress due to an experimental procedure or for restraint to facilitate a technically difficult procedure. This unit provides three basic protocols: injectable anesthesia for mouse, rat, and hamster; inhalant anesthesia using methoxyflurane for mouse, rat, and hamster; and injectable anesthesia using ketamine/xylazine for rabbit. An describes sedation using butorphanol/acetylpromazine in the rabbit. The further describes and compares these methods of anesthesia for various applications.
Production of Polyclonal Antisera
Authors: Cooper Helen M. , Paterson Yvonne 
Published: June 2004

AbstractAlthough the advances offered by the development of monoclonal antibody techniques have revolutionized the specificity, uniformity, and quantity of antibodies, there remain many circumstances in which polyclonal antibodies are more desirable than monoclonal antibodies. Production of polyclonal antisera takes less time and effort than production of monoclonal antibodies, requires relatively simple and readily available equipment, and produces reagents that can be used for immunoprecipitation, immunoblotting, and enzyme‐linked immunosorbent assays (ELISAs). Theandin this unit describe the production of polyclonal antisera specific for protein antigens in rabbits, rats, mice, and hamsters. Thepresents a method for preparing serum from blood.
Fragmentation of Immunoglobulin G
Authors: Andrew Sarah M. , Titus Julie A. 
Published: June 2004

AbstractThis unit describes procedures for fragmentation of IgG to the monovalent Fab fragment using papain digestion, and to the bivalent F(ab′)2 fragment by pepsin digestion. Alternative methods of fragmentation to F(ab′)2 include use of papain that is preactivated with cysteine and use of the enzyme ficin. These alternate methods are particularly useful for mouse IgG1 antibodies.
Purification of Immunoglobulin M and Immunoglobulin D
Authors: Andrew Sarah M. , Titus Julie A. , Coico Richard , Amin Ashok 
Published: June 2004

AbstractThis unit describes two classical protocols for the purification of IgM ‐ dialysis of ascites fluid, tissue culture medium, or bioreactor supernatants against distilled water to precipitate pure IgM, and ammonium sulfate precipitation. Both protocols can be followed by size‐exclusion chromatography to obtain a highly purified product. Recently an affinity method for purification of IgM has been developed using mannan binding protein, and is described here. The third approach presented is a one‐step IgD purification method, designed specifically for murine derived samples, that uses Sepharose coupled to lectin derived from the seeds of Griffonia simplicifolia‐1. This represents a simple, rapid, and gentle approach to isolating this highly labile immunoglobulin from IgD‐containing ascites or hybridoma sources.
Production of Bispecific Antibodies
Authors: Segal David M. , Bast Bert J.E.G. 
Published: June 2004

AbstractBispecific antibodies (bsAbs) contain two different binding specificities within a single molecule and can specifically bind two different molecules together. BsAbs can be produced by chemically cross‐linking purified antibodies or Fab fragments with reducible disulfide bonds or nonreducible thioether bonds, both of which are described in this unit. Protocols are also presented for producing BsAbs by fusing two antibody‐producing hybridomas that can be selected for based on drug resistance, or by double labeling with fluorochromes and FACS. Support protocols describe screening and purification of bsAbs.
Nucleic Acid Immunizations
Authors: Robinson Harriet L. , Pertmer Tamera M. 
Published: June 2004

AbstractThis unit provides protocols for two broadly used methods of DNA immunization: saline injections and gene gun deliveries of DNA. Saline injections deliver DNA into extracellular spaces; gene gun deliveries bombard DNA directly into cells. Support protocols present methods for preparation of DNA‐coated gold beads, creation of cartridges containing these beads, and optimization of gene gun parameters. Issues relating to plasmid vectors, DNA preparation and doses, and immunization regimens are also discussed. Expression library immunizations, genetic and conventional adjuvants, alternative boosts, neonatal immunizations, and approaches to mucosal delivery of DNA are covered in the commentary.
T Cell Enrichment By Nonadherence to Nylon
Authors: Hathcock Karen S. 
Published: June 2004

AbstractDifferential adherence properties of T cells, B cells, and accessory cells such as macrophages or other APC (antigen‐presenting cells) can be employed to enrich for T cells. While the basis for this adherence is not understood, either nylon wool or Sephadex G‐10 can be used as column matrices to which B cells and accessory cells will more readily adhere than T cells. Such procedures offer a convenient, although imprecise, means of enriching T cells through removal of accessory and B cells; use of nylon wool is preferred if both of the latter subsets are to be removed, while Sephadex is used when the goal is primarily to remove accessory cells.
Isolation of Dendritic Cells
Authors: Inaba Kayo , Swiggard William J. , Steinman Ralph M. , Romani Nikolaus , Schuler Gerold 
Published: June 2004

AbstractThis unit presents two methods for preparing dendritic cells (DCs), a highly specialized type of antigen‐presenting cell (APC). The first method involves the isolation of DCs from mouse spleen, resulting in a cell population that is highly enriched in accessory cell and APC function. A support protocol for collagenase digestion of splenocyte suspensions is described to increase the yield of dendritic cells. The second method involves generating large numbers of DCs from mouse bone marrow progenitor cells. In that technique, bone marrow cells are cultured in the presence of granulocyte/macrophage colony‐stimulating factor (GM‐CSF) to yield 5‐10 106 cells, 60% of which express DC surface markers (e.g., B‐7‐2/CD86). Additional techniques for isolating DCs from mouse spleens or other mouse tissues, as well as from human tissues, are also discussed.
Early Events in B Lymphocyte Activation
Authors: Bondada Subbarao , Troyer Amy , Chelvarajan Ralph L. 
Published: June 2004

AbstractB cell activation can be quantitated indirectly by assaying antibody production or directly by measuring cellular changes that occur immediately after exposure to an activation signal. This unit provides methods for the latter (direct) approach‐‐namely, methods for quantifying early parameters of B cell activation such as increases in intracellular ionized calcium concentration [Ca2+]I, cell size, and MHC class II‐antigen expression. These assays are not so much B cell specific as activation specific; as such they are a useful means of providing information about cellular events that are independent of antibody secretion. While the protocols included in this unit have been specifically designed for use with murine B cells, they can also be used with B cells obtained from other species. In addition, the method described here also applies to T cells.
Quantitation of Functional T Cells by Limiting Dilution
Authors: Miller Richard A. 
Published: June 2004

AbstractCulturing by limiting dilution (LD) can be used to estimate the proportion of T cells in a cell preparation which can respond to an activating stimulus to produce interleukin 2 or to generate cytotoxic T lymphocyte (CTL) activity. In the first basic protocol, IL‐2‐producing murine T cells are measured following stimulation by the mitogen Con A. This protocol can be modified as described in the first alternate protocol to quantitate responses to alloantigens, anti‐CD3 antibody, protein antigens such as keyhole limpet hemocyanin (KLH), and viruses. The second alternate protocol provides a modification for using human responder cells. The
second basic protocol is used for estimating the proportion of cells that can generate a clone of cytotoxic effector cells when stimulated by Con A with the addition of IL‐2.
Granule Enzyme Exocytosis Assay for Cytotoxic T Lymphocyte Activation
Authors: Taffs Rolf , Sitkovsky Michail , Takayama Hajime 
Published: June 2004

AbstractUpon activation by specific target cells, cytotoxic T lymphocytes (CTL) release into the culture medium the content of cytoplasmic granules that contain serine esterases. The amount of enzyme released during CTL activation can be easily quantitated by spectrophotometric measurement of the colored product of the enzymatic degradation of a synthetic substrate. In the primary method presented here, CTL are activated with monoclonal antibodies prepared against the T cell receptor (TCR) complex, then activation is quantitated according to the amount of serine esterase released in the supernatant. Alternate protocols describe the activation of CTL by a combination of protein kinase C and calcium ionophores (a TCR‐independent approach) and by the more conventional approach of target‐cell mediation. In a third approach, β‐glucuronidase rather than esterase activity is measured, as this enzyme is also present in granules released upon CTL activation. This unit therefore includes a colorimetric assay for CTL‐induced β‐glucuronidase activity employing the substrate phenolphthalein glucuronic acid as well as a corresponding automated fluorimetric assay employing the substrate 4‐methylumbelliferyl‐D‐glucuronide. Finally, the quantitation of granule exocytosis resulting from cell damage or death induced by the activating agent, rather than CTL activation, is described.
Isolation of Mouse Small Intestinal Intraepithelial Lymphocytes, Peyer's Patch, and Lamina Propria Cells
Authors: Lefrançois Leo , Lycke Nils 
Published: June 2004

AbstractThe intestinal mucosal immune system is composed of three major lymphoid areas: the lamina propria (LP), which lies just underneath the basement membrane in the intestinal villi; (2) the intraepithelial compartment, which contains the intraepithelial lymphocytes (IEL) and is located just above the basement membrane, between the columnar epithelial cells; and (3) Peyer's patches (PP), lymphoid nodules (akin to lymph nodes) embedded in the gut wall, separated from the LP and IEL. The LP, PP, and IEL lymphoid populations form a complex, interconnected network that responds to immunological insults in the intestine. Therefore, these lymphocyte populations should be analyzed when studying the immunological status of the intestine, for example in oral immunization or in intestinal disease (including infectious disease and tumors). This unit details techniques for isolation of IEL, PP cells, and LP cells from the small intestine of the mouse.
In Vivo Depletion of CD4‐ and CD8‐Specific T Cells
Authors: Kruisbeek Ada M. 
Published: June 2004

AbstractIn vivo depletion of CD4‐ and CD8‐specific T cells is a means of studying the role of these subpopulations in the initiation and effector phases of particular in vivo immune responses. In this unit, a protocol is provided for harvesting anti‐CD4 or anti‐CD8 monoclonal antibody‐ producing ascites fluid or tissue culture supernatant from rat or mouse T cell hybridomas. The antibody (preferably IgG) is then purified and injected intraperitoneally into adult mice. Depletion of the appropriate subset of T cells is verified by flow cytometry analysis of lymph node and spleen cell suspensions in pilot experiments. Once conditions have been established, depleted mice can be used to study the impact of T cell subsets on in vivo immune responses. The depleted condition is maintained by repeated injections of the monoclonal antibody.
Contact Hypersensitivity
Authors: Gaspari Anthony A. , Katz Stephen I. 
Published: June 2004

AbstractContact hypersensitivity is a simple in vivo assay of cell‐mediated immune function in which exposure of epidermal cells to exogenous haptens results in a delayed‐type hypersensitive reaction that can be measured and quantified. The Langerhans cell is the critical antigen‐presenting cell in this reaction which initiates sensitization to haptens by presenting antigens to CD4‐bearing T lymphocytes which, in turn, secrete lymphokines and recruit other cells to the site of the reaction. In the protocol described here, mice are shaved and the skin of their abdomens is exposed to a hapten. After 6 days (the afferent phase), the baseline ear thickness is measured prior to initiation of the efferent phase. Finally, the ear is treated epicutaneously with the hapten solution and ear thickness is measured in ˜24 hr. The change in ear thickness after allergen treatment can be used to calculate the percent suppression of contact hypersensitivity.
Skin Allograft Rejection
Authors: Rosenberg Amy S. 
Published: June 2004

AbstractSkin allograft rejection is a test of the competence of T lymphocytes to mediate in vivo tissue destruction, which in turn reflects their role in critical functions such as anti‐viral and tumor immunity. The tail‐skin graft procedure described here is useful predominantly because of the ease of preparation and resistance to ischemic (nonspecific) necrosis. Additionally, it is not necessary to sacrifice the donor mouse. However, rejection of tail‐skin grafts should not be used to test for genetic homogeneity in breeding experiments or to detect minor histocompatibility (minor‐H) antigens because tail skin is less sensitive than trunk skin in detecting such differences. The trunk‐skin graft procedure detailed here more difficult to perform than the tail‐skin graft. It requires extensive tissue preparation and is more susceptible to ischemic necrosis. Trunk‐skin grafts are especially useful for detecting minimal histocompatibility differences.
Measurement of T and B Cell Turnover with Bromodeoxyuridine
Authors: Tough David F. , Sprent Jonathan 
Published: June 2004

AbstractThe rate of turnover of T and B cells can be measured in terms of the time between two mitoses or between mitosis and death. In vivo, this is accomplished by administering DNA precursors and measuring the rate at which cells become labeled with these precursors.
5‐bromo‐2'‐deoxyuridine (BrdU) is a thymidine analog that is incorporated into the DNA of cells during the S phase of the cell cycle and can be subsequently detected with a specific monoclonal antibody. In this method, BrdU is given to experimental animals either continuously or as a pulse, depending on the goal of the experiment. Subsequently, cells are stained in vitro with an anti‐BrdU antibody and labeled cells detected by flow cytometry. By combining cell surface staining for multiple lymphocyte markers with anti‐BrdU staining, it is possible to determine the lifespan of phenotypically defined subsets.
Flow Cytometry Analysis Using the Becton Dickinson FACS Calibur
Authors: Holmes Kevin L. , Otten Gillis , Yokoyama Wayne M. 
Published: June 2004

AbstractThis unit details the operation of a FACS Calibur flow cytometer for cell analysis. The operation of the Becton Dickinson FACS Calibur and CELLQuest software version 3.0 is described, but the unit is general enough to be helpful for users of all flow cytometers. The FACS Calibur replaces both the FACSCan and FACSort; the information presented here is also applicable to older BD instruments. In this unit, particular emphasis is placed on data acquisition rather than data analysis. Single‐color analysis using fluorescein isothiocyanate (FITC)‐conjugated antibodies is described along with procedures to check instrument performance and sensitivity, single‐color (FITC) analysis with simultaneous live/dead discrimination using propidium iodide (PI), simultaneous two‐color analysis using FITC‐ and phycoerythrin (PE)‐conjugated antibodies, two‐color FITC/PE analysis with simultaneous live/dead discrimination using PI, simultaneous three‐color immunofluorescence with FITC, PE, and the red fluorescent dyes, and finally, simultaneous four‐color immunofluorescence with FITC, PE, red fluorescence dyes, and APC.
Analysis of Cellular DNA Content by Flow Cytometry
Authors: Darzynkiewicz Zbigniew , Huang Xuan 
Published: June 2004

AbstractCellular DNA content can be measured by flow or laser‐scanning cytometry with the aim of (1) revealing cell distribution within the major phases of the cell cycle, (2) estimating the frequency of apoptotic cells with fractional DNA content, and/or (3) disclosing the DNA‐ploidy of the measured cell population. In this unit, simple and universally applicable methods for staining fixed cells are presented, as are methods that utilize detergents and proteolytic treatment to permeabilize cells. Additionally, supravital cell staining with Hoechst 33342, which is primarily used for sorting live cells for subsequent culturing based on DNA‐content differences, is also described. Also presented are methods for staining of cell nuclei isolated from paraffin‐embedded tissues, and deconvolution of DNA‐content‐frequency histograms to estimate the percentage of cells in major phases of the cell cycle and frequency of apoptotic cells with fractional DNA content.
Multiparameter FACS Analysis
Authors: Roederer Mario 
Published: June 2004

AbstractThe purpose of this unit is three‐fold: (1) toprovide a brief historical perspective to illustrate the demands for this technology and to lay the foundation for its application; (2) to explain the hurdles that were surmounted to achieve the current level of multiparametric analysis which serves to alert researchers to potential problems they may encounter when they either bring this technology to their own laboratories, or when they use extant instrumentation in another laboratory; and (3) to illustrate some of the complexities that arise even when all of the major technical hurdles have been overcome. The majority of the unit is focused on “high‐end” multiparameter analysis (termed “polychromatic flow cytometry,” or PFC); i.e., experiments utilizing more than four fluorescence detectors (in addition to scattered light parameters).
Measurement of Mouse and Human Interleukin 5
Authors: Harriman Gregory R. 
Published: June 2004

AbstractTwo fundamentally different types of assays are available for quantitating interleukin 5 in biological samples. One type, a bioassay, is based on the ability of IL‐5 to enhance B cell proliferation and immunoglobulin secretion or eosinophil proliferation and differentiation. The other type of assay, an ELISA, uses antibodies against IL‐5 to capture and quantitate IL‐5 in samples. Advantages and disadvantages of each assay are discussed. The bioassay described in this unit, utilizing BCL1 cells as the indicator line, has been designed primarily to assay murine IL‐5; however, it can also be used to measure human IL‐5. The IL‐5 ELISA, while sensitive and specific, provides no information about biological activity.