bluuebunny/crossref_metadata_2025_split huggingface dataset:
Measurements of Phagocytosis and Phagosomal Maturation
Authors: Chow Chung‐Wai , Downey Gregory P. , Grinstein Sergio 
Published: June 2004

AbstractPhagocytosis is an essential component of the innate immune response. Invading microorganisms are engulfed by neutrophils and macrophages, and subsequently killed by a complex series of reactions that require fusion of the phagocytic vacuole with multiple endomembrane organelles. This vacuolar remodeling process, known as phagosomal maturation, is often the target of intracellular parasites that evade killing by the host immune system. This unit describes detailed protocols for the assessment of phagosome formation and maturation. Particular attention is given to Fc‐ and complement‐receptor mediated phagocytosis, but the protocols provided can be readily adapted to other types of phagocytic processes.
Production of Monoclonal Antibodies
Authors: Yokoyama Wayne M. 
Published: June 2004

AbstractMonoclonal antibodies are particularly specific for a single epitope and can be produced in large quantities. When immune splenic cells are fused with a tumor cell line, they produce hybridomas each secreting one highly specific antibody. These cells are then characterized and cloned to allow production over time. This unit describes the production of monoclonal antibodies beginning with basic protocols for immunization and cell fusion and selection. Support protocols are provided for screening primary hybridoma supernatants for antibodies of desired specificity, establishment of stable hybridoma lines, cloning of these B cell lines by limiting dilution to obtain monoclonal lines, and preparation of cloning/expansion medium (thymocyte‐conditioned medium).
Production of Polyclonal Antisera
Authors: Cooper Helen M. , Paterson Yvonne 
Published: June 2004

AbstractPolyclonal antibody preparations contain multiple antibody molecules recognizing different epitopes on an antigen molecule. Good quality polyclonal antisera are useful for a number of applications–e.g., immunoprecipitation, immunoblotting, and ELISAs–where the binding of more than one antibody molecule per antigen molecule improves the sensitivity of the assay. It is possible to produce polyclonal antibodies in a variety of species, and this make it possible to detect multiple immunoreactions. The protocols in this unit describe the production of polyclonal antisera specific for protein antigens in rabbits, rats, mice, and hamsters using complete Freund's adjuvant (CFA) or other adjuvants. In addition, the unit presents a method for preparing serum from blood. Polyclonal antipeptide antisera can be produced by substituting carrier‐conjugated peptides for the purified protein antigens.
Purification of Immunoglobulin G
Authors: Andrew Sarah M. , Titus Julie A. 
Published: June 2004

AbstractWhile unpurified antibodies are suitable for a number of applications, purified antibodies are required for assays based on known concentration of antibody, for chemical modifications such as radiolabeling or conjugation with fluorochromes, or for structural modifications such as production of F(ab')2 or monvalent Fab fragments. This unit contains protocols for purification of IgG by ammonium sulfate precipitation coupled with size‐exclusion chromatography, Protein A‐ and Protein G‐affinity chromatography, immunoaffinity chromatography, and ion‐exchange chromatography.
Fragmentation of Immunoglobulin G
Authors: Andrew Sarah M. , Titus Julie A. 
Published: June 2004

AbstractFor some purposes, fragments of the IgG molecule are preferred. The Fc portion is useful for studies of biological effect–binding to the Fc receptor, mediating antibody‐dependent cellular cytotoxicity, and complement fixation. The bivalent F(ab')2 produced by digestion with pepsin and the monovalent Fab produced by digestion with papain are useful for studies based on the interaction between antibody binding site(s) with antigen. This unit also describes alternative methods for preparing F(ab')2 fragments.
Antibody Conjugates for Cell Biology
Authors: Haugland Rosaria P. 
Published: June 2004

AbstractAntibody conjugates are extremely useful reagents for probing many biologically and chemically important molecules in vitro or in vivo. This unit presents some basic protocols for conjugating antibodies with fluorescent dyes, with biotin, and with enzymes. These protocols have been selected because they are relatively easy to perform and have high rates of success. The basic approach for conjugating antibodies is to derivatize amino groups on the antibody with the dye or biotin derivative of choice. Conjugation at amine sites is simple to perform, and, with a fluorescent dye, it generally yields brightly fluorescent conjugates. The conjugates retain high levels of biological activity, notwithstanding the fact that the labels react with random amines on the antibody molecule, and they may even react near or at the antigen‐binding site. Labeling of the amines is the most widely used commercial method to tag antibodies. In this unit, suggestions are presented for variations on these basic protocols that are useful when quantities of available antibody are limited. Methods for estimating antibody concentration are also included.
Production of Antibodies That Recognize Specific Tyrosine‐Phosphorylated Peptides
Authors: DiGiovanna Michael P. , Roussel Robert R. , Stern David F. 
Published: June 2004

AbstractPhosphorylation of tyrosine residues of various proteins plays an important role in regulation of the functional activities of proteins within the cell. Antibodies to specific tyrosine‐phosphorylated peptides are a valuable tool for analysis of the localization and function of individual phosphoproteins. This unit describes the methods used to produce and screen monoclonal and polyclonal antibodies for specificity for only the phosphorylated state of a peptide.
Imaging Protein‐Protein Interactions by Fluorescence Resonance Energy Transfer (FRET) Microscopy
Authors: Wouters Fred S. , Bastiaens Philippe I.H. 
Published: June 2004

AbstractFRET microscopy enables the detection of different biochemical states of proteins in cells. The use of fluorescence in the detection of proteins, by chemical modification, by immunofluorescence, or by genetic encoding of a green fluorescent protein fusion protein, provides more information than just the location of the protein in the cell. The properties of the fluorophore can be exploited to extract information on protein‐protein interactions. A straightforward, quantitative imaging approach is presented to measure FRET that is based on internal calibration by acceptor photobleaching.
Interaction Trap/Two‐Hybrid System to Identify Interacting Proteins
Authors: Golemis Erica A. , Serebriiskii Ilya , Finley Russell L. , Kolonin Mikhail G. , Gyuris Jeno , Brent Roger 
Published: June 2004

AbstractThis unit presents protocols designed to detect interacting proteins. Using yeast as a “test tube” and transcriptional activation of a reporter system, interacting proteins can be identified. The system can also be used to test complex formation for proteins for which there exists a reason to expect interaction.
Mapping Protein‐Protein Interactions with Phage‐Displayed Combinatorial Peptide Libraries
Authors: Kay Brian K. , Castagnoli Luisa 
Published: June 2004

AbstractThis unit describes the process and analysis of affinity selecting bacteriophage M13 from libraries displaying combinatorial peptides fused to either a minor or major capsid protein. Direct affinity selection uses target protein bound to a microtiter plate followed by purification of selected phage by ELISA. Alternatively, there is a bead‐based affinity selection method. These methods allow one to readily isolate peptide ligands that bind to a protein target of interest and use the consensus sequence to search proteomic databases for putative interacting proteins.
Protein‐Protein Interactions Identified by Pull‐Down Experiments and Mass Spectrometry
Authors: Brymora Adam , Valova Valentina A. , Robinson Phillip J. 
Published: June 2004

AbstractThe aim of this unit is to provide a method for the identification of new protein‐protein interactions. Pull‐down experiments with GST fusion proteins attached to glutathione beads are a screening technique for identification of protein‐protein interactions. When coupled with mass spectrometry, pull‐downs can be considered as the protein‐based equivalent of a yeast two‐hybrid screen. To improve the isolation of specific binding partners, pull‐down methods are described involving the use of cross‐linking, large‐scale tissue lysates, and spin columns. Alternative techniques are detailed for isolating activation state‐dependent protein interactions with small GTPases. Appropriate methods of sample preparation for mass spectrometry‐based identification of interacting proteins are described, including specialized gel staining techniques, band excision, and in‐gel tryptic digestion. Data interpretation and the most commonly encountered problems are discussed.
Measuring Protein Interactions by Optical Biosensors
Authors: Schuck Peter , Boyd Lisa F. , Andersen Peter S. 
Published: June 2004

AbstractIn recent years, optical evanescent wave biosensors have been used to characterize protein‐protein interactions, including determination of equilibrium binding constants and bimolecular rate constants. This surface binding technique can provide information about chemical on‐rate constants, the lifetimes of complexes formed, and the time course of the signal. This unit provides a thorough, well‐illustrated discussion of the principles of optical biosensors, experimental design, ligand immobilization, experimental data analysis, and common obstacles and possible solutions.
Assessment of Apoptosis and Necrosis by DNA Fragmentation and Morphological Criteria
Authors: Zhivotosky Boris , Orrenius Sten 
Published: June 2004

AbstractApoptotic cells share a number of common features, such as phosphatidylserine (PS) exposure, cell shrinkage, chromatin cleavage, nuclear condensation, and formation of pyknotic bodies of condensed chromatin. Necrotic cells exhibit nuclear swelling, chromatin flocculation, loss of nuclear basophilia, breakdown of cytoplasmic structure and organelle function, and cytolysis by swelling. This unit describes some of the techniques most commonly used to detect cell death. A number of assays are used for characterizing and distinguishing apoptosis and necrosis. Morphological assays include trypan blue exclusion, differential staining, and Hoechst staining. Methods to detect chromatin cleavage include TUNEL assays for whole cells and paraffin sections, DNA fragmentation assays using whole cells, assays of total genomic DNA, analysis of DNA fragmentation by agarose gel electrophoresis, phenol extraction of DNA for analysis of fragmentation, a quantitative assay for DNA fragmentation, and detection of DNA fragmentation by pulsed‐field gel electrophoresis. A protocol is also provided for Cytospin preparations from cell suspensions.
Quantitative Fluorescence In Situ Hybridization (Q‐FISH)
Authors: Poon Steven S.S. , Lansdorp Peter M. 
Published: June 2004

AbstractThis unit describes a quantitative technique for measuring the lengths of telomere repeat sequences in individual chromosomes from single metaphase cells. The technique is based on fluorescence in situ hybridization (FISH) adapted for use with peptide nucleic acid (PNA) probes. PNA is an example of novel synthetic oligonucleotide “mimetic” which has a higher affinity than regular oligonucleotide (RNA or DNA) probes for complementary single‐strand (ss) DNA sequences. PNA oligonucleotides have excellent penetration properties due to their small size (typically 15 to 18‐mers) and can be directly labeled with fluorochromes. These properties have been exploited to develop quantitative fluorescence in situ hybridization (Q‐FISH) onto denatured single‐stranded chromosomal DNA target sequences. The latter can be present in preparations of fixed metaphase cells on slides (Q‐FISH) or in heat‐treated (interphase) cells in suspension (flow‐FISH).
Analysis of Telomeres and Telomerase
Authors: Herbert Brittney‐Shea , Shay Jerry W. , Wright Woodring E. 
Published: June 2004

AbstractThis unit describes techniques to analyze telomeric length and telomerase activity in human cells. Telomere length can be determined by a modification of Southern blotting in which the analysis of chromosome terminal restriction fragments (TRFs) provides the average lengths of all telomeres in a cell population. Telomerase activity can be measured in vitro by a sensitive and efficient polymerase chain reaction (PCR)‐based detection method, also known as telomeric repeat amplification protocol (TRAP). These assays can be used to study the in vitro cellular effects of aging and cancer treatments on telomere biology and telomerase activity.
Flow Cytometry of Apoptosis
Authors: Pozarowski Piotr , Grabarek Jerzy , Darzynkiewicz Zbigniew 
Published: June 2004

AbstractCommon methods applicable to flow cytometry make it possible to: (1) identify and quantify dead or dying cells, (2) reveal a mode of cell death (apoptosis or necrosis), and (3) study mechanisms involved in cell death. Gross changes in cell morphology and chromatin condensation, which occur during apoptosis, can be detected by analysis with laser light beam scattering. Early events of apoptosis, dissipation of the mitochondrial transmembrane potential and caspase activation, can be detected using either fluorochrome reporter groups or appropriate antibodies. Exposure of phosphatidylserine on the exterior surface of the plasma membrane can be detected by the binding of fluoresceinated annexin V. Another apoptotic event, DNA fragmentation based on DNA content of cells with fractional (“sub‐G1”) or DNA strand‐break labeling, TUNEL; or In Situ End Labeling, ISEL;. Still another hallmark of apoptosis is the activation of tissue transglutaminase (TGase), the enzyme that crosslinks protein and thereby makes them less immunogenic. The major advantage of flow cytometry in these applications is that it provides the possibility of multiparametric measurements of cell attributes.
Overview of Metastasis Assays
Authors: Paweletz Cloud P. , Charboneau Lu , Liotta Lance A. 
Published: June 2004

AbstractDuring tumor progression, cells acquire genetic and proteomic changes as they transform from normal to hyperplastic, through dysplasia, to carcinoma in situ, and finally to invasive and metastatic. The time course of progression may extend as far back as 10 years prior to diagnosis. Discerning the mechanism whereby tumor cells execute metastatic dissemination may provide the foundation necessary for successful treatment of the disease. For example, direct genetic evidence has linked in situ breast cancer to invasive carcinoma of the breast supporting the generally accepted assumption that carcinoma in situ of the breast is a clonal expansion of hyperproliferating cells. This in turn may provide a more comprehensive and/or functionally directed target strategy for intervention and prevention of breast cancer. This overview provides a picture of the processes related to metastasis and the experimental approaches used to study these processes.
Microanalysis of Gene Expression in Tissues Using T7‐SAGE: Serial Analysis of Gene Expression After High‐Fidelity T7‐Based RNA Amplification
Authors: Sakai Takayoshi , Larsen Melinda , Yamada Kenneth M. 
Published: June 2004

AbstractIn this unit, the authors describe a new technique, T7‐SAGE, in which a high‐fidelity T7 amplification step is combined with SAGE analysis. In order to avoid extra PCR or other forms of amplification, the authors incorporate only two cycles of T7‐based RNA amplification as the initial step. This T7‐based amplification step has high accuracy. In addition, T7 RNA polymerase has high processivity and functions effectively even when broad stretches of nucleotides are being amplified. Although no protocol that includes an amplification step can claim to permit determination of absolute transcript number, since slight changes in estimated transcript frequency are always possible, T7 procedures appear to be the safest to date. This new T7‐SAGE procedure should facilitate application of SAGE for gene‐expression profiling using minimal quantities of starting material, such as from embryonic tissues and microdissected cells from histological sections of tissues.
The Chick Chorioallantoic Membrane as an In Vivo Angiogenesis Model
Authors: Ponce M. Lourdes , Kleinmann Hynda K. 
Published: June 2004

AbstractThe chick chorioallantoic membrane (CAM) assay for angiogenic activity is a model originally developed to study the angiogenic activity of tumor samples. It is an in vivo assay that can be readily performed in any laboratory setting. The effects of a test compound on angiogenesis are tested by exposing day 10 embryos to the compound and following the patterns of blood vessel development, scoring the appearance of the CAM at day 12 or 13.
Experimental Metastasis Assays in the Chick Embryo
Authors: Wilson Sylvia M. , Chambers Ann F. 
Published: June 2004

AbstractExperimental metastasis assays are used to measure the ability of cancer cells to grow in secondary organs following injection of the cells into the circulation of an experimental animal. The chicken embryo provides an alternative to the more commonly used assays in mice. Details of the experimental metastasis assay in chick embryo are provided, including protocols for maintenance of fertilized chicken eggs, injection of cells into the circulation of 11‐day‐old chick embryos, recovery and quantification of cancer cells from chick liver using the ouabain plating assay, labeling of cells with fluorescent nanospheres, and monitoring of individual steps in the metastatic process in the chick chorioallantoic membrane using intravital videomicroscopy. These assays provide a cost‐effective, readily accessible, and rapid approach for studying the process of cancer metastasis.
Direct Introduction of Molecules into Cells
Authors: McNeil Paul L. 
Published: June 2004

AbstractTechniques for introducing normally impermeant macromolecules into the living cell—referred to as “cell‐loading techniques”—are useful in a variety of settings for the cell biologist. Microinjection is probably the most commonly used technique for introducing fluorescent probes, fluorescently tagged proteins, and antibodies into living cells for short‐term studies of cell physiology and protein location and function. It is, however, not the only technique available, nor the easiest or least expensive to implement. Among the alternatives are several closely related techniques that, like microinjection, rely on the cell's ability to reseal a mechanically induced plasma membrane disruption created in order to gain temporary access to cell cytosol. Four such techniques are described in this unit: scrape loading, scratch loading, bead loading, and syringe loading. Unlike microinjection, these techniques allow one to rapidly load (in a matter of minutes) thousands or even many millions of many types of mammalian cells with normally impermeant molecules, and so to facilitate quantitative analyses of the effect of loading.
Protein Transduction: Generation of Full‐Length Transducible Proteins Using the TAT System
Authors: Becker‐Hapak Michelle , Dowdy Steven F. 
Published: June 2004

AbstractThis unit describes the technology that allows an investigator to transduce full‐length proteins by utilizing a minimal, eleven‐amino acid, HIV‐TAT transduction domain that can be fused to a protein of choice using the pTAT or pTAT‐HA protein expression plasmids. Bacterial expression, followed by solubilization of protein aggregates with a denaturing agent, affords high yields of transducible fusion protein. The fusion protein, once added to the culture medium, can cross the cell membrane and then be degraded or refolded by the cellular machinery. Correct targeting and function of the fusion protein can be easily examined by fluorescent microscopy or immunohistochemistry. This strategy was established and improved to its current state by the purification and transduction of a multitude of fusion proteins. Because the pool of fusion proteins spans many different functions, the protocols cover a wide variety of commonly used protein isolation and characterization methods.
Calcium Phosphate Transfection
Authors: Kingston Robert E. , Chen Claudia A. , Okayama Hiroto 
Published: June 2004

AbstractThis unit presents two methods of calcium phosphate‐based eukaryotic cell transfection that can be used for both transient and stable transfections. In these protocols, plasmid DNA is introduced to monolayer cell cultures via a precipitate that adheres to the cell surface. A HEPES‐buffered solution is used to form a calcium phosphate precipitate that is directly layered onto the cells. For some cells, shocking the cells with glycerol or DMSO improves transfection efficiency. In the alternate high‐efficiency method, a BES‐buffered system is used that allows the precipitate to form gradually in the medium and then drop onto the cells. While the alternate method is particularly efficient for stable transformation of cells with circular plasmid DNA, both protocols yield similar results for transformation with linear plasmid or genomic DNA, or for transient expression.
Transfection Using DEAE‐Dextran
Authors: Gulick Tod 
Published: June 2004

AbstractTransfection of cultured mammalian cells using diethylaminoethyl (DEAE)‐dextran/DNA can be an attractive alternative to other transfection methods in many circumstances. The major advantages of the technique are its relative simplicity and speed, limited expense, and remarkably reproducible interexperimental and intraexperimental transfection efficiency. Disadvantages include inhibition of cell growth and induction of heterogeneous morphological changes in cells. Furthermore, the concentration of serum in the culture medium must be transiently reduced during the transfection. In general, DEAE‐dextran DNA transfection is ideal for transient transfections with promoter/reporter plasmids in analyses of promoter and enhancer functions, and is suitable for overexpression of recombinant protein in transient transfections or for generation of stable cell lines using vectors designed to exist in the cell as episomes. This unit presents a general description of DEAE‐dextran transfection, as well as two more specific protocols for typical experimental applications. The basic protocol is suitable for transfection of anchorage‐dependent (attached) cells. For cells that grow in suspension, electroporation or lipofection is usually preferred, although DEAE‐dextran‐mediated transfection can be used.
Transfection by Electroporation
Authors: Potter Huntington 
Published: June 2004

AbstractElectroporation–the use of high‐voltage electric shocks to introduce DNA into cells–can be used with most cell types, yields a high frequency of both stable transformation and transient gene expression, and, because it requires fewer steps, can be easier than alternate techniques. In this unit, the basic protocol describes the electroporation of mammalian cells and an alternate protocol outlines modifications for preparation and transfection of plant protoplasts.
Transfection of Cultured Eukaryotic Cells Using Cationic Lipid Reagents
Authors: Hawley‐Nelson Pamela , Ciccarone Valentina 
Published: June 2004

AbstractThe development of high‐efficiency methods for the introduction of functional genetic material into eukaryotic cells using cationic lipids has accelerated biological research in the studies of gene expression, control of cell growth, and cell lineage. Transfection mediated by cationic lipids is commonly used in industrial protein production as well as in some clinical gene therapy protocols. Replacing our previous unit on this topic, this new version describes how to perform transfection of adherent and suspension cells, insect cells, and RNA transfection using the cationic lipid system.