bluuebunny/crossref_metadata_2025_split huggingface dataset:
Transformation of E. coli by Electroporation
Authors: Morrison Sherie L. 
Published: June 2004

AbstractUsing electroporation to transform Escherichia coli results in transformation efficiencies greater than can be obtained using the best chemical methods. It is easy to obtain transformation efficiencies 108 per milligram DNA and efficiencies of 1010 have been reported. This appendix describes a procedure for electroporation that can be used to transform many different types of bacteria.
Automated Fluorescent Genotyping
Authors: Hall Jeff , Nanthakumar Elizabeth 
Published: June 2004

AbstractWith the publication of a comprehensive human genetic map consisting of over 5000 microsatellite markers, reagents are in hand to undertake large‐scale genotyping projects. This unit describes the basic methodology, optimization of markers, and allele calling.With the publication of a comprehensive human genetic map consisting of over 5000 microsatellite markers, reagents are in hand.
Multicolor Fluorescence In Situ Hybridization (FISH) Approaches for Simultaneous Analysis of the Entire Human Genome
Authors: Lee Charles , Rens Willem , Yang Fengtang 
Published: June 2004

AbstractThe ability to generate chromosome‐specific paints and employ combinatorial labeling strategies has made it possible to differentiate all 24 human chromosomes in a single metaphase spread. Such technology is particularly useful when there is a limited number of metaphase spreads for analyses and when interchromosomal rearrangements are ill‐defined or very complex. There are three systems currently available for simultaneous FISH analysis of all human chromosomes: spectral karyotyping (SKY), Multiplex FISH (M‐FISH), and Rx‐FISH. This overview discusses each of these systems and the recent advances which have made them possible.
Standardization, Calibration, and Control in Flow Cytometry
Authors: Hoffman Robert A. 
Published: June 2004

AbstractBecause flow cytometers are designed to measure particle characteristics, particles are the most common materials used to calibrate, control, and standardize the instruments. Definitions are provided for common terms to alert the reader to critical distinctions in meaning. The unit presents extensive background on particle types and cautions and goes on to describe practical aspects of methods to standardize and calibrate instruments, in terms of optical alignment, fluorescence and light scatter resolution, and sensitivity. Finally, suggestions follow for analyzing particles used for calibration.
Laser Beam Shaping and Spot Size
Authors: Shapiro Howard M. 
Published: June 2004

AbstractOne or more lasers are the source of illumination in the majority of fluorescence flow cytometers now in use. A typical laser emits a beam on the order of 1 mm in diameter. For efficient utilization this beam must be shaped and focused to a smaller size to illuminate the cell stream passing through the interrogation point. This unit discusses spot size requirements and the methods by which optimal spot size is achieved.
Lasers for Flow Cytometry
Authors: Shapiro Howard M. 
Published: June 2004

AbstractLasers are the principle light sources of flow cytometers. Most modern benchtop instruments feature air‐cooled argon‐ion laser emitting 10‐25 mW of light at 488 nm. This unit covers the various types of lasers available and the qualities that make them suitable or unsuitable for use in flow cytometers. Also included is a discussion of future directions, particularly in the development of solid‐state devices that might replace gas lasers.
Flow Cytometers for Characterization of Microorganisms
Authors: Steen Harald B. 
Published: June 2004

AbstractThe measurement of microbes is an increasingly important application in flow cytometry. The small size of these organisms can create special difficulties for instruments designed to analyze much larger eukaryotic cells. This unit describes the problems encountered in making microbiological measurements, the instrument requirements, and some approaches that can be taken to optimize the instrument. This material is essential background for the protocols found in chapter 11, Microbiological Applications.
Principles of Resonance Energy Transfer
Authors: Szöllősi János , Damjanovich Sándor , Mátyus László 
Published: June 2004

AbstractFluorescence Resonance Energy Transfer (FRET) is a radiationless process in which energy is transferred from an excited donor to an acceptor molecule. For many years this principle has been exploited in flow cytometry to excite long wavelength fluorophores with short wavelength excitation; the donor/acceptor molecules are frequently known as tandem conjugates. This unit explains in careful detail the quite complex FRET process. It characterizes available parameters and instruments for FRET measurements, discusses limitations, and shows a few examples for the application of FRET, such as determining occupation of closely related receptors.
Compensation in Flow Cytometry
Authors: Roederer Mario 
Published: June 2004

AbstractMany people using multi‐color flow cytometry do not understand what compensation is, when it is needed, or how it should be applied. The author provides a clear explanation of the nature of compensation, the factors that affect compensation values, and the effect of compensation on data visualization. Both hardware and software compensation are covered. Although the subject is treated from a mathematical perspective, the manner of presentation is very straightforward. The unit discusses appropriate controls and protocols to check compensation settings, as well as introducing the concept of fluorescence‐minus‐one gates. Many example figures are supplied to give the reader a clear indication of how the author believes compensation should be handled. Keywords: flow cytometry; spectral compensation; fluorescence spillover; autofluorescence; fluorescence minus one
Simultaneous Analysis of the Cyan, Green, and Yellow Fluorescent Proteins
Authors: Beavis Andrew J. , Kalejta Robert F. 
Published: June 2004

AbstractFlow cytometric analysis of fluorescent protein expressing cells is of particular interest to researchers in many areas. The detection of fluorescent proteins in cells allows one to monitor gene expression, determine intracellular protein localization, and identify transfected cells. Wild‐type green fluorescent protein has limited utility as its spectral properties are not suitable for standard cytometers. Site‐directed mutations have produced enhanced variants with improved extinction coefficient and quantum yield with standard 488‐nm excitation. Other variants have been constructed with shifted excitation and emission maxima and high quantum yield. It is now possible to monitor multiple processes in a single cell and detect enhanced green, yellow, and cyan fluorescent proteins using a single excitation beam at 458 nm. The authors carefully describe the custom filter setup required to accomplish this and the Boolean gating logic for analysis of the various subpopulations expressing any given combination of fluorescent proteins.
Microscope Objectives
Authors: Abramowitz Mortimer , Friedman Marc M. 
Published: June 2004

AbstractThe objective is the most crucial image‐forming component of a microscope. A knowledge of the many types of objectives available and their characteristics is critical to the selection of appropriate objectives for image cytometry. This unit discusses aberrations in image formation and their correction, construction and types of objectives, and objectives for other microscopy applications, explaining the advantages and limitations of each one.
Scanning Laser Cytometry
Authors: Shapiro Howard M. 
Published: June 2004

AbstractThere are many tasks to which flow cytometers are unsuited, e.g., measurement of attached cells, repeated measurements of a single cell over time, and localization of probes in or on cells. Devices such as confocal microscopes and microscope‐based imaging systems allow high‐resolution analysis and provide flexibility, but typically have low sample throughput and often require a high level of interaction with a technically sophisticated operator. Scanning laser cytometers, which can make low‐resolution, multiparameter optical measurements of light scattering and fluorescence similar to those of flow cytometers, provide reasonably high sample throughput and may, depending on the purpose for which they are designed, either allow for considerable flexibility and operator interaction or permit analysis of large numbers of samples without an operator in attendance. This unit presents an overview of sanning laser cytometers, including such topics as the operating principles, data analysis, and commercially available systems and their applications.
Photobleaching Measurements of Diffusion in Cell Membranes and Aqueous Cell Compartments
Authors: Lustyik György 
Published: June 2004

AbstractThis commentary unit discusses in great detail the theoretical nature of fluorescence recovery after photobleaching (FRAP). This information is crucial to an understanding of how and why FRAP works in a cell system. Further, understanding how to interpret the data sets requires a sound knowledge of the processes involved. Of primary importance are the nature of membrane diffusion and the nature of the multiple compartments into which fluorescent dyes can enter. The unit provides a complete discussion of all aspects of FRAP from the perspective of cellular measurements.
Testing the Efficiency of Aerosol Containment During Cell Sorting
Authors: Schmid Ingrid , Hultin Lance E. , Ferbas John 
Published: June 2004

AbstractProduction of droplets and microdroplets (aerosols) is part of the normal operation of a cell sorter. These aerosols may contain toxic, carcinogenic, or teratogenic fluorophores or known or unknown pathogens from viable biological specimens. Most newer models of commercially available instruments incorporate features designed to reduce the production of aerosols and prevent their release into the room. This unit presents two protocols for assessment of aerosol containment on jet‐in‐air flow sorters. In both procedures, lytic T4 bacteriophage is run through the instrument at high concentrations to tag aerosol droplets. The instrument is tested in normal operating mode and in simulated failure mode. Aerosols are detected by plaque formation on susceptible E. coli lawns. With the continuing increase in the sorting of viable human cells, it is vital for cytometrists to be aware of the potential dangers.
Safe Use of Hazardous Chemicals
Authors: Lunn George , Lawler Gretchen 
Published: June 2004

AbstractMany of the reagents listed in the protocols are hazardous to the user: toxic, oncogenic, mutagenic, teratogenic, allergenic, or just plain irritating. This informative unit discusses the basic minimum safety requirements for handling, storage, and disposal of chemicals in the laboratory. Extensive tables provide detailed information on types of hazards, chemical incompatibilities, chemical resistance of glove types, and biological stains. The authors provide a number of protocols for the decontamination and disposal of some commonly encountered hazardous chemicals, as well as analytical detection procedures for verifying their effectiveness. Also included are an extensive reference bibliography and a list of useful web sites.
Measurement of CD40 Ligand (CD154) Expression on Resting and In Vitro–Activated T Cells
Authors: O'Gorman Maurice R.G. 
Published: June 2004

AbstractMeasurement of the ability of in vitro activated T lymphocytes to express CD40 ligand (CD154) is a valuable tool for diagnosis of the X‐linked hyper‐IgM syndrome (XHIM) and for investigating abnormalities in the costimulatory functions of T lymphocytes. Resting peripheral blood lymphocytes do not normally express any appreciable levels of CD154, necessitating in vitro activation. This unit details the in vitro activation procedure, the three‐color monoclonal antibody panels used to label the appropriate lymphocyte subsets, the controls required to interpret the assay, the setup and acquisition of listmode data files, and the gating and analysis protocols used to interpret the data.
Enumeration of Absolute Cell Counts Using Immunophenotypic Techniques
Authors: Mandy Frank , Brando Bruno 
Published: June 2004

AbstractAbsolute counting of cells or cell subsets has a number of significant clinical applications: monitoring the disease status of HIV‐infected patients, enumerating residual white blood cells in leukoreduced blood products, and assessing immunodeficiency in a variety of situations. The single‐platform method (flow cytometry alone) has emerged as the method of choice for absolute cell enumeration. This technology counts only the cells of interest in a precisely determined blood volume. Exact cell identification is accomplished by a logical electronic gating algorithm capable of identifying lineage‐specific immunofluorescent markers. Exclusion of unwanted cells is automatic. This extensive and detailed unit presents protocols for both volumetric and flow‐rate determination of residual white blood cells and of leukocyte subsets. Keywords: absolute count; volumetric counting; flow rate cytometry; counting microsphere standards; single‐platform absolute count; dual‐platform absolute count; logical gate; lineage‐specific markers; immunophenotyping; absolute residual WBC
Overview of Nucleic Acid Analysis
Authors: Darzynkiewicz Zbigniew , Juan Gloria 
Published: June 2004

AbstractThis unit discusses the general applicability of nucleic‐acid analytical methods in both basic and clinical research. After immunophenotyping, analysis of nucleic acids is perhaps the most common application of flow cytometry. The methodologies are of particular interest for researchers in basic and clinical oncology, as they provide information on cell attributes associated with tumor malignancy, and are widely used in various areas of cell and molecular biology.
Analysis of DNA Content and DNA Strand Breaks for Detection of Apoptotic Cells
Authors: Darzynkiewicz Zbiginew , Juan Gloria 
Published: June 2004

AbstractThis unit describes a method for identifying apoptotic cells based on in situ detection of DNA strand breaks. Correlated analysis of DNA content and DNA strand breaks allows one not only to identify apoptotic cells but also to pinpoint their location in the cell cycle. The basic protocol uses 5‐bromodeoxyuridine triphosphate to label the 3‐OH termini of the breaks, but the procedure is easily adapted to a variety of other conjugates. Given the multiplicity of commercially available reagents it is possible to label DNA strand breaks with a dye of any fluorescence color and excitation wavelength.
Analysis of Nuclear DNA Content and Ploidy in Higher Plants
Authors: Galbraith David W. , Lambert Georgina M. , Macas Jiri , Dolezel Jaroslav 
Published: June 2004

AbstractThis is the first of a series of units discussing the application of cytometry to plant material. Techniques commonly used for mammalian nuclei evaluation need considerable modification to be successful with plant material. David Galbraith and his colleagues bring together many years of knowledge in plant cytometry. Their unit provides detailed protocols on measuring DNA content, ploidy, and cell cycle status of plant tissue using both conventional laser based instruments as well as arc lamp cytometers. This unit provides an excellent starting point for those interested in doing cytometry with plants.
Assessment of Viability, Immunofluorescence, and DNA Content
Authors: Schmid Ingrid 
Published: June 2004

AbstractThis unit addresses the difficult and important area of differentiation between viable and nonviable cells, a question of particular interest for DNA studies. The basic protocol describes the determination of DNA content using pyronin Y combined with dead cell discrimination using 7‐AAD. Alternate protocols present methods for DNA content and viability assessment and simultaneous surface or intracellular antigen expression.
Sperm Chromatin Structure Assay for Fertility Assessment
Authors: Evenson Donald , Jost Lorna 
Published: June 2004

AbstractThe integrity of mammalian sperm DNA is of prime importance for the paternal genetic contribution to normal offspring. Damaged DNA in the single sperm that fertilizes the female egg can have a dramatic negative impact on fetal development. This comprehensive and detailed unit presents a rapid, reliable, practical test for DNA integrity based on staining with acridine orange. SCSA data have been conclusively shown to predict sub/infertility. This assay is ideally suited to human and animal fertility clinics to assess male sperm DNA integrity as related to fertility potential and embryo development. The authors, who have decades of experience in studying sperm viability, provide extensive commentary and methodological tips, making this unit the most detailed method for this test published to date. Keywords: flow cytometry; sperm chromatin structure assay; SCSA; DNA denaturation; acridine orange;animal and human fertility; toxicology The integrity of mammalian sperm DNA is of prime importance for the paternal genetic contribution to normal offspring
Ultraviolet‐Induced Detection of Halogenated Pyrimidines (UVID)
Authors: Hammers Hans‐Joerg , Schlenke Peter 
Published: June 2004

AbstractWhen halogenated pyrimidines such as BrdU are used to identify DNA‐synthesizing cells, their detection with monoclonal antibodies requires DNA denaturation, which can lead to loss of cells and/or cell markers or antigens. The authors present an alternative method employing ultraviolet light to partially photolyze BrdU and induce extensive damage in the nuclei which have incorporated the pyrimidine. Under appropriate conditions the BrdU in the unfolding chromatin is detected by certain monoclonal antibodies. Since no denaturation step or enzymatic treatment is required, this method preserves cellular markers and avoids enzyme‐specific artifacts. Directions are given for both coagulative (ethanol) and crosslinking (formaldehyde) fixation. Keywords: flow cytometry; BrdU; UV; photolysis; hypotonic; cell cycle; proliferation When halogenated pyrimidines such as BrdU are used to identify DNA‐synthesizing cells, their detection with monoclonal antibod
Analysis of Viral Infection and Viral and Cellular DNA and Proteins by Flow Cytometry
Authors: Lehman John M. , Friedrich Thomas D. , Laffin Judith 
Published: June 2004

AbstractViruses are obligate intracellular parasites that require the host cell replication, transcription, and translation machinery for reproduction. Each viral group provides a unique series of viral‐cellular interactions. Studies have provided insight not only into viral replication and control of host functions, but also into cellular functions such as eukaryotic replication, transcription, and translation as well as the regulation of these events. This unit presents a protocol for flow cytometric monitoring of viral infection and quantitating viral‐cellular events. The availability of monoclonal and/or polyclonal antibodies directed to both viral and cellular proteins offers the ability to assay a specific molecule in the intact fixed cell and the opportunity to correlate viral events with cellular processes such as progression through the cell cycle.
Apoptosis Signaling Pathways
Authors: Siegel Richard M. , Lenardo Michael J. 
Published: June 2004

AbstractCell death is often an active and highly choreographed process of cell suicide known as apoptosis. The molecular signals governing this process have been the subject of intense research in recent years. During apoptosis a profusion of pathways either promote cellular survival or lead to certain death. These pathways engage in constant crosstalk with those that control cell proliferation and other activities, in order to maintain the delicate balance between cell births and deaths. This commentary unit discusses two major pathways, active (antigen‐induced) and passive (lymphokine‐withdal) apoptosis, and provides background for the following unit on flow cytometry of apoptosis.
Flow Cytometry of Apoptosis
Authors: Pozarowski Piotr , Grabarek Jerzy , Darzynkiewicz Zbigniew 
Published: June 2004

AbstractApplication of flow cytometry to the study of cell death has three goals: identification and quantification of dead and dying cells; discrimination between apoptotic and necrotic modes of cell death; and elucidation of mechanisms involved in cell death. This massively detailed unit by a pioneer in the field brings together the most common flow cytometric methods for the study of apoptosis, covering a wide variety of apoptotic indices, from loss of membrane potential, caspase activation, and phosphatidyl exposure to DNA fragmentation and tissue transglutaminase activation. The authors also present their recently developed protocol, analogous to the FLICA approach for caspases, for the detection of serine proteases (‘serpases’). The protocols are accompanied by extensive commentary discussion of applicability, strategic planning, problems, and pitfalls, plus a comprehensive list of references.
Measurement of Cytogenetic Damage in Rodent Blood with a Single‐Laser Flow Cytometer
Authors: Dertinger Stephen , Torous Dorthea , Hall Nikki , Tometsko Carol 
Published: June 2004

AbstractThe in vivo rodent micronucleus test is widely utilized to screen chemicals for genotoxic activity. Double‐strand chromosome breaks or dysfunction of the mitotic spindle apparatus can lead to micronuclei formation in dividing cells. Erythrocytes have become the target population of choice, as precursor cells are continuously dividing and micronuclei are readily observable after extrusion of nuclei. The traditional method has been to stain peripheral blood or bone marrow smears and microscopically determine the frequency of micronucleated erythrocytes. Because these events are rare, the process is tedious and time consuming. This unit describes a procedure for fixing and staining rodent peripheral blood for flow cytometric enumeration. The combination of reagents provides for differential labeling and enumeration of four subpopulations: mature erythrocytes, micronucleus‐containing mature erythrocytes, young erythrocytes (reticulocytes), and micronucleus‐containing young erythrocytes. Malaria‐infected rodent erythrocytes, which closely mimic micronucleus‐containing erythrocytes, serve as a biological standard to facilitate rational and consistent equipment calibration. Keywords: flow cytometry; genotoxicity; chromosome damage; micronuclei; reticulocytes; propidium iodide; CD71‐defined antigen The in vivo rodent micronucleus test is widely utilized to screen chemicals for genotoxic activity.
DRAQ5 Labeling of Nuclear DNA in Live and Fixed Cells
Authors: Smith Paul J. , Wiltshire Marie , Errington Rachel J. 
Published: June 2004

AbstractThis unit describes the use of a novel DNA‐detecting far‐red‐fluorescing dye, DRAQ5, a modified anthraquinone, which has a unique combination of properties exploitable by cytometry. These include a high capacity to permeate the cell membrane, a high DNA binding affinity and selectivity, a fluorescence emission spectrum beyond that of fluorescein, phycoerythrin, Texas Red, Cy3, and EGFP, and excitation characteristics separate from those of propidium iodide. In this unit, methods are presented for preparation and analysis of both live and fixed cells stained with DRAQ5. While the focus is on flow cytometric assays, typical imaging applications are also indicated because the staining protocols share the same essential features.
Basic Preparative Techniques for Fluorescence In Situ Hybridization
Authors: Wiegant J. , Raap A.K. 
Published: June 2004

AbstractThis unit presents protocols for preparing human metaphase chromosome slides from peripheral blood lymphocytes, isolating interphase nuclei from lymphocytes and paraffin‐embedded tissues, and preparing DNA fibers. The protocols are designed so that the resulting preparations are amenable to FISH. The methods correspond to a selection of the specimens that can be analyzed with FISH techniques, and the choice of sample preparation method is highly dependent on the molecular cytogenetics question being addressed.