bluuebunny/crossref_metadata_2025_split huggingface dataset:
Tyramide Signal Amplification (TSA) Systems for the Enhancement of ISH Signals in Cytogenetics
Authors: Bobrow Mark N. , Moen Philip T. 
Published: June 2004

AbstractThis unit provides a series of techniques that dramatically improve the sensitivity of direct and indirect in situ detection. TSA is a peroxidase‐based signal amplification system which is compatible with all in situ hybridization as well as immunocytochemical detection systems. The assay is performed on a glass slide and combines the use of fluorescent probes in either direct or indirect format using either an enzyme‐labeled streptavidin or antibody. Protocols are provided for brightfield and fluorescent detection of single DNA targets, DNA or RNA in cultured cells, multitarget detection for DNA/RNA and DNA and RNA. In addition a protocol is included for the TSA plus system, a DNP‐based detection system using horseradish peroxidase and alkaline phosphatase.
Molecular Combing
Authors: Conti Chiara , Caburet Sandrine , Schurra Catherine , Bensimon Aaron 
Published: June 2004

AbstractThis unit describes an important advance in fiber‐FISH technology called molecular combing, in which single DNA molecules are bound by one or both ends to a surface and stretched in a uniform and parallel manner by a receding meniscus. This technique is gentle on the molecules, rapid, and easy to perform. Reliable, quantitative information for genome‐wide studies can be obtained without the need for other techniques and a large number of accurate measurements can be made in a single experiment. The authors provide detailed protocols for basic molecular combing, high‐resolution physical mapping, and gene‐dosage approaches as well as support protocols outlining surface preparation, DNA solution preparation, and probe labeling.
Principles and Applications of PRINS in Cytogenetics
Authors: Koch Jørn 
Published: June 2004

AbstractA flexible, low‐cost alternative to FISH, primed in situ labeling (PRINS) has traditionally been used to detect tandemly repeated target sequences in chromosomes and nuclei. The technique is capable of discriminating among closely related DNA sequences in situ and has the advantage of using very small probes which easily penetrate to almost any target. This unit describes basic PRINS and the alternative version, dideoxy‐PRINS, which can increase the sensitivity of the reaction by an order of magnitude. New material on multicolor PRINS and quantitative PRINS has been added. Protocols for detection of single‐copy sequences and for application to the study of in‐vivo activity of DNA‐modifying enzymes are planned.
Comparative Genomic Hybridization (CGH)—Detection of Unbalanced Genetic Aberrations Using Conventional and Micro‐Array Techniques
Authors: Schröck Evelin , Weaver Zoë , Albertson Donna 
Published: June 2004

AbstractThis unit presents comparative genomic hybridization (CGH), a genome‐wide screening technique for genetic aberrations in tumor samples. Specific emphasis is placed on recent applications to the analysis of murine model systems for human cancer. CGH is an invaluable tool for identifying the characteristic genetic rearrangements in these models. The authors discuss an exciting new method currently being developed, array CGH, which results in a tremendous increase in resolution. Oncogene amplifications and deletions of tumor‐suppressor genes are detected on a single‐gene level. Detailed protocols are supplied for CGH analysis of both human and mouse chromosomes. Keywords: comparative genomic hybridization; tumor genetics; unbalanced chromosomal aberrations; DNA copy number changes; gene amplification; oncogenes; tumor suppressor genes; mouse models of human cancer; array technology; array‐CGH This unit presents comparative genomic hybridization (CGH), a genome‐wide screening technique for genetic aberrations in tumor
Analysis of Intracellular Organelles by Flow Cytometry or Microscopy
Authors: Poot Martin 
Published: June 2004

AbstractFunctional analysis of cellular organelles can be accomplished by staining cells with suitable organelle‐specific dyes and analyzing the fluorescence of the stained cells with a flow cytometer. With this methodology it is possible to resolve suspected heterogeneity in organelle function or content within a population of cells. Morphological information can be provided by quantitative microscopy (confocal microscope or video microscope with digital image‐analysis system). The thirteen protocols cover flow cytometry and microscopy for both live and fixed cells.
Measurement of Intracellular Calcium Ions by Flow Cytometry
Authors: June Carl H. , Abe Ryo , Rabinovitch Peter S. 
Published: June 2004

AbstractUsing flow cytometry, single‐cell measurements of calcium can be made on isolated populations identified by one or more phenotypic characteristics. This unit describes the preparation of cells, including labeling with antibodies and with calcium probes, and discusses the principles of data analysis and interpretation.
In Vitro Invasion Assays: Phagocytosis of the Extracellular Matrix
Authors: Bowden Emma T. , Mueller Susette , Coopman Peter J. 
Published: June 2004

AbstractDegradation of the extracellular matrix is an essential component of phagocytosis by tumor cells and can be correlated with their invasive capacity. This unit presents flow cytometry based assays for rapid quantitative assessment of both proteolysis and internalization or internalization alone. These assays provide high‐throughput, low‐cost methods to compare cell lines and test the efficacy of treatments designed to stimulate or inhibit invasion.
Multiparameter Analysis of Physiological Changes in Apoptosis
Authors: Poot Martin 
Published: June 2004

AbstractChanges in mitochondrial parameters leading to cells with compromised mitochondrial function are a hallmark of apoptosis. These changes can be detected and the number of compromised cells quantitated by multicolor flow cytometry utilizing fluorescent dyes that monitor specific features of this organelle. In this way one can trace physiological changes during apoptosis and investigate whether or not certain changes occur simultaneously in the same cell. This unit presents protocols for combined NADH levels and mitochondrial membrane potential, cell cycle stage specific apoptosis, and cellular thiol and mitochondrial cardiolipin levels.
Data Analysis Through Modeling
Authors: Bagwell C. Bruce 
Published: June 2004

AbstractFlow cytometers collect data in an attempt to understand some aspect of a biological system. Mathematical models are developed to extract relevant population features from the data, to help users attain their goals. This unit examines what models are and how they are routinely used.
Keywords: flow cytometry; listmode data; data analysis; modeling
Flow cytometers collect data in an attempt to understand some aspect of a biological system
Image Processing and 2‐D Morphometry
Authors: Turek John 
Published: June 2004

AbstractImage analysis and image processing are becoming commonplace operations in the cytometry environment. This very practical and useful unit describes the basics of image processing, including image enhancement, feature identification, and classification and segmentation. Written by an author with strong background in electron microscopy and image analysis, the unit is well furnished with figures and examples. Anyone needing a start in doing image analysis will find this very easy and useful reading, with suitable references for those wanting to move a step further.
The Application of Data Mining to Flow Cytometry
Authors: Nguyen Andy N.D. 
Published: June 2004

AbstractData mining is the process of automating information discovery to detect useful patterns, correlations, and trends. Existing data must be fitted into a representative model from which useful information can be derived through a variety of algorithms. The routine generation of vast amounts of data make flow cytometry a logical target for the application of data mining. This informative unit discusses the steps of the data‐mining process using the immunophenotyping of hematologic neoplasms to demonstrate the application. The author describes several types of algorithms and provides a useful resource list of commercially available tools.
Overview of Flow Cytometry and Microbiology
Authors: Robinson J. Paul 
Published: June 2004

AbstractAlthough in recent years flow cytometry has become commonplace in hematology and immunology laboratories, application of the technology to microbiology remains largely unrealized. This overview presents the historical background, discusses applications in various areas of the field, and speculates on the directions of future developments. The availability of high‐quality methods should be a prime factor in convincing microbiologists that flow cytometry may have certain advantages over traditional methods and that it does indeed have much to contribute to microbiology.Keywords: flow cytometry; microbiology; environment; food microbiology; overview Although in recent years flow cytometry has become commonplace in hematology and immunology laboratories, application of the t.
Flow Cytometric Detection of Pathogenic E. coli in Food
Authors: Raybourne Richard B. 
Published: June 2004

AbstractE. coli O157:H7 is one of the more important food pathogens, andrapid, quantitative methods to evaluate foods for the presence of this pathogen are needed. This unit provides exactly that: a very much simplified flow cytometric assay for detection of E. coli O157:H7 in a well established vehicle of infection, ground beef. The method uses commercially available FITC‐conjugated specific antibody to this bacterial serotype. Sample preparation and bacterial enrichment procedures are described. Direct and indirect approaches for quantification of the number of bacteria are given. A key feature of the assay is the reduction in time compared with plate‐counting methods; the tradeoff is a slight reduction in sensitivity. Particularly useful is the simultaneous inclusion of a spiked sample to ensure a positive control. In addition, the unit provides hints on sorting the organisms if desired.
Determination of Bacterial Biomass from Flow Cytometric Measurements of Forward Light Scatter Intensity
Authors: Robertson B.R. , Button D.K. 
Published: June 2004

AbstractThis unit presents methods for measuring biomass of bacteria by flow cytometry. Such measurements are an integral part of determining growth rates and kinetics. This unit discusses calibration and standardization methods for bacterial studies using flow cytometry and provides a detailed discussion of the components involved. The serious microbiologist will appreciate the advantages flow cytometry brings to many aspects of microbial study, and this unit is no exception.
Enumeration of Phytoplankton, Bacteria, and Viruses in Marine Samples
Authors: Marie Dominique , Partensky Frédéric , Vaulot Daniel , Brussaard Corina 
Published: June 2004

AbstractFor many years, a small but dedicated group of scientists have been using flow cytometry for the evaluation of marine microorganisms. One of these scientists now provides us with a detailed series of protocols in this area, spelling out the variations in method and instrument operation that are crucial to the successful extraction of quality flow data from marine organisms. In addition, the use of a number of less frequently employed fluorescent probes gives some insight into alternative staining procedures. As our collection of microbiologically oriented techniques increases, this knowledge database becomes invaluable.
Cell Counting
Authors: Phelan Mary C. , Lawler Gretchen 
Published: June 2004

AbstractThis unit presents protocols for counting cells using either a hemacytometer or electronically using a Coulter counter. Cell counting with a hemacytometer permits effective discrimination of live from dead cells using trypan blue exclusion. In addition, the procedure is less subject to errors arising from cell clumping or size heterogeneity. Counting cells is more quickly and easily performed using an electronic counter, but live‐dead discrimination is unreliable. Cell populations containing large numbers of dead cells and/or cell clumps are difficult to count accurately. In addition, electronic counting requires resetting of the instrument for cell populations of different sizes; heterogeneous populations can give rise to inaccurate counts, and resting and activated cells may require counting at separate settings. In general, electronic cell counting is best performed on fresh peripheral blood cells.
Diagnosis and Treatment of Mycoplasma‐Contaminated Cell Cultures
Authors: Fitch Frank W. , Gajewski Thomas F. , Yokoyama (antibiotic treatment) Wayne M. 
Published: June 2004

AbstractMycoplasma contamination is a serious and frequent problem in the culture laboratory. Although mycoplasma contamination may be suspected by the failure of cells to thrive, the formal diagnosis rests on the detection of adenosine phosphorylase secretion by infected cell lines. This appendix describes how to test for mycoplasma contamination, and also presents methods for antibiotic treatment of infected cultures.
Techniques for Bacterial Cell Culture: Media Preparation and Bacteriological Tools
Authors: Lech Karen , Brent Roger 
Published: June 2004

AbstractBasic information for the culture of bacteria is presented in this appendix, using Escherichia coli as a representative organism. The techniques and recipes should be readily transferrable. Specifics are given for liquid and solid media, rich and minimal media, stab agar, as well as a description of the tools required to inoculate and culture bacteria.
Basic Techniques for Mammalian Cell Tissue Culture
Authors: Phelan Mary C. 
Published: June 2004

AbstractCultured mammalian cells are used extensively in cell biology studies; it requires a number of special skills in order to be able to preserve the structure, function, behavior and biology of the cells. This unit describes the basic skills required to maintain and preserve cell cultures: aseptic technique, medium characteristics, passaging, freezing and storage, recovering frozen stocks, and counting viable cells.
Aseptic Technique for Cell Culture
Authors: Coté Rosalie J. 
Published: June 2004

AbstractThis unit describes some of the ways that a laboratory can deal with the constant threat of microbial contamination in cell cultures. A protocol on aseptic technique is described first. This catch‐all term universally appears in any set of instructions pertaining to procedures in which noncontaminating conditions must be maintained. In reality, aseptic technique encompasses all aspects of environmental control, personal hygiene, equipment and media sterilization, and associated quality control procedures needed to ensure that a procedure is, indeed, performed with aseptic, noncontaminating technique. Although cell culture can theoretically be carried out on an open bench in a low‐traffic area, most cell culture work is carried out using a horizontal laminar‐flow clean bench or a vertical laminar‐flow biosafety cabinet. Both are described here.
Establishment of Fibroblast Cultures
Authors: Takashima Akira 
Published: June 2004

AbstractFibroblasts are fairly easily isolated from a variety of tissues, in this case skin. The cells are fast growing and can be rapidly expanded from small samples. They are suitable for behavioral, functional, biochemical, and genomic studies. Skin fibroblast cultures are started from explants in which the epidermis is enzymatically removed to prevent contamination of the fibroblasts with epidermal cells. Alternatively, the dermis can be enzymatically dissociated to provide a suspension of fibroblasts.
Preparation of Endothelial Cells
Authors: Kleinman Hynda K. , Cid Maria C. 
Published: June 2004

AbstractEndothelial cells, which line blood vessels, can be prepared from a variety of tissues. They are frequently prepared from the umbilical vein, which is relatively easy to obtain. The procedure is clearly described and provides a large population of highly purified endothelial cells. There are also methods for obtaining endothelial cells from other tissues such as fat, skin, and mucosa. These methods require special care and generate smaller populations of cells.
Generation of Continuously Growing B Cell Lines by Epstein‐Barr Virus Transformation
Authors: Biddison William E. 
Published: June 2004

AbstractPrimary cultures of cells tend to have a limited life span, which in turn limits the availability of a consistent population of cells to study. In this unit, Epstein‐Barr virus produced by a marmoset cell line is used to transform B cells into a continuously growing cell line that produces no virus. These cell lines can be used as a constant source of cells for further study.
Isolation of Lysosomes from Tissues and Cells by Differential and Density Gradient Centrifugation
Authors: Graham John M. 
Published: June 2004

AbstractThis unit covers the use of Percoll, Nycodenz, and iodixanol for purification of lysosomes from a light mitochondrial pellet or postnuclear supernatant. The first protocol describes isolation of lysosomes from rat liver using a Percoll gradient; it includes some comments on using brain and kidney tissue as source material. Alternatively, this protocol can be modified to enhance the separation of lysosomes by organelle density perturbation. Another alternative uses a discontinuous gradient of Nycodenz to purify lysosomes from a rat liver light mitochondrial pellet. A continuous iodixanol gradient, which can be used to purify other organelles in the light mitochondrial fraction, is yet another alternative for purifying lysosomes. Lysosomes can also be isolated from some of the more commonly used cultured cells. The unit also includes assays for common lysosome marker enzymes.
Proper Alignment and Adjustment of the Light Microscope
Authors: Salmon Edward D. , Canman Julie C. 
Published: June 2004

AbstractThe light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications (e.g., brightfield, phase‐contrast, differential interference contrast, and fluorescence epi‐illumination), and how it should be maintained as required to obtain maximum image‐forming capacity and resolution. The principles of microscopy and step‐by‐step alignment and adjustment procedures are described in this unit.
Fluorescence Microscopy
Authors: Herman Brian 
Published: June 2004

AbstractAbsorption of energy as light by some molecules and emission of energy as fluorescence can occur only at certain wavelengths, which are characteristic for a given molecule (fluorophore). Fluorescence microscopes are equipped to observe the fluorescence of one or more specific fluorophores to localize specific molecules and analyzed cellular structures. This unit discusses the optics and other components of the fluorescence microscope, their functions and adjustments, and the digital darkroom.
Immunofluorescence Staining
Authors: Donaldson Julie G. 
Published: June 2004

AbstractThis unit provides a protocol for indirect immunofluorescence, which is a method that provides information about the locations of specific molecules and the structure of the cell. Antibody molecules for a specific target molecule are exposed to the cell or tissue being investigated. The binding of these molecules is detected by incubating the sample with a secondary antibody specific for immunoglobulin molecules and conjugated to fluorophore. This provides both a visible signal and amplification of the signal and the results are observed with a fluorescent microscope.
Fluorescent Staining of Subcellular Organelles: ER, Golgi Complex, and Mitochondria
Authors: Terasaki Mark , Loew Leslie , Lippincott‐Schwartz Jennifer , Zaal Kristien 
Published: June 2004

AbstractThe ability to distinguish and identify specific subcellular compartments is essential to understanding organelle function, biogenesis, and maintenance within cells and to defining protein trafficking pathways. Fluorescent dyes and/or fluorescently labeled lipid derivatives can be used to identify ER, Golgi complex, and mitochondria. Specific conditions for labeling each of these compartments are described.
Cryo‐Immunogold Electron Microscopy
Authors: Peters Peter J. 
Published: June 2004

AbstractThis unit describes subcellular localization of proteins/antigens using high‐resolution cryo‐immunogold electron microscopy, which allows study of topological biochemistry at the ultrastructural level. This is the most sensitive procedure for immunodetection of antigens on ultrathin sections prepared from chemically fixed cells or tissues, since aldehyde fixation is the only denaturation step. The omission of harsh organic solvents (such as those used for plastic embedding) ensures better preservation of protein antigenicity. Support protocols describe how to embed fixed material in gelatin, cryosection, and mount the sections on Formvar‐coated grids.
Polarization Microscopy
Authors: Inoué Shinya 
Published: June 2004

AbstractThis unit provides an introduction to polarization microscopy, the optics involved, and practical considerations for observing submicroscopic structures. The unit also includes specific examples for visualization of microtubules in the mitotic spindle, chromatin within maturing spermatids, and the biocrystalline skeletal spicules in larval echinoderms.
Two‐Photon Excitation Microscopy for the Study of Living Cells and Tissues
Authors: Rocheleau Jonathan V. , Piston David W. 
Published: June 2004

AbstractTwo‐photon excitation microscopy is an alternative to confocal microscopy that provides advantages in three‐dimensional and deep tissue imaging. This unit will describe the basic physical principles of two‐photon excitation and discuss the advantages and limitations of its use in laser‐scanning microscopy. The advantages of two‐photon microscopy are reduced phototoxicity, increased imaging depth, and the ability to initiate localized photochemistry.
Two‐Dimensional Gel Electrophoresis
Authors: Harper Sandra , Mozdzanowski Jacek , Speicher David 
Published: June 2004

AbstractWhile one‐dimensional SDS‐PAGE separates proteins on the basis of size, two‐dimensional gel electrophoresis separates proteins first on the basis of isoelectric point, then on the basis of size. This method is capable of resolving 1000 to 2000 separate proteins when combined with sensitive detection methods. This unit describes methods for characterizing cell lysates by two‐dimensional gel electrophoresis, including modifications for acidic and basic proteins, the use of immobilized pH gradients, and nonreducing/reducing electrophoretic separations. In addition there are support protocols for determining pH profiles of gels, casting Immobiline gels, preparing cell and tissue samples for isoelectric focusing, preparing molecular weight standards, and using two‐dimensional protein databases.
Staining Proteins in Gels
Authors: Dell'Angelica Esteban C. , Bonifacino Juan S. 
Published: June 2004

AbstractOnce proteins are separated by gel electrophoresis, staining can be used to visualize the proteins. This unit presents protocols for numerous staining methods. The most common method is staining with Coomassie blue, which after washing gives blue bands on a clear background. This technique can also be applied to isoelectric focusing gels. A second, more sensitive but also more technically challenging method is silver staining. Here the proteins are seen as dark brown to black bands on a clear background. If the gel is incubated with SYPRO Ruby, a fluorescent compound that interacts specifically with proteins, the bands fluoresce when illuminated on a standard transilluminator. Finally, proteins can be reversibly stained with zinc, which precipitates the SDS from the gel leaving protein bands as clear spots against an opaque white background.