bluuebunny/crossref_metadata_2025 huggingface dataset:
Fenofibrate‐Induced Elevation in Serum Creatinine
Authors: Ritter Janet L. , Nabulsi Shereen
Published: June 2004

Fenofibrate, a fibric acid derivative, is used as adjunctive therapy with diet for treatment of hyperlipidemia. Side effects include gastrointestinal complaints and, rarely, elevations in liver function tests. According to the drug's package insert, elevations in serum creatinine may occur; although, no postmarketing case reports of fenofibrate‐induced elevations in serum creatinine are on file with the manufacturer. Fenofibrate was the probable cause of elevations in serum creatinine concentrations in six patients at our clinic. As the implications of these elevations may be serious, routine serum creatinine monitoring is recommended at baseline and 1–2 months after starting fenofibrate.
Single‐Isomer β‐Agonists
Authors: Handley Dean A.
Published: June 2004

A new generation of bronchodilators is being developed for acute asthma management—single‐isomer β‐agonists. These drugs consist only of the active bronchodilatory isomer (eutomer); they do not have the inactive and potentially harmful isomer (distomer) that is present in marketed racemic β‐agonists. Clinical studies comparing the effectiveness of (R)‐albuterol (levalbuterol) with racemic albuterol established a strong rationale for using single‐isomer β‐agonists in place of the racemic mixture: reduced dosages provide equivalent bronchodilatory effects with fewer β‐mediated side effects. Higher dosages achieve superior bronchodilation in episodes of severe asthma and may reduce costs of emergency department treatment.
Do Antihistamines Have a Role in Asthma Therapy?
Authors: Larsen Julie S.
Published: June 2004

The coexistence of allergic rhinitis with asthma is widely recognized by clinicians. Histamine, a common mediator for both diseases, has a substantial role in the pathophysiology of asthma through its ability to produce smooth muscle contraction and promote vascular permeability. Because antihistamines often are administered to manage allergic rhinitis symptoms, the effects of antihistamines in asthma should be evaluated. The usefulness of first‐generation antihistamines is limited by their side‐effect profile, namely sedation and cognitive impairment. Second‐generation antihistamines have only a modest effect in attenuating bronchospasm induced by histamine, cold air, exercise, and allergen bronchoprovocation, suggesting that second‐generation antihistamines do not have a direct role as a single agent for treating asthma. Studies have shown that controlling allergic rhinitis with antihistamines has a small, indirect effect in improving asthma symptoms. Future work should be directed at improving the potency of antihistamines and defining their antiinflammatory activity.
Leukotriene Receptor Antagonists in the Treatment of Asthma
Authors: Sorkness Christine A.
Published: June 2004

The role of leukotriene receptor antagonists in the treatment of persistent asthma is in evolution. Pivotal 8–12‐week, randomized, controlled trials in both adults and children have shown efficacy, as defined by standard asthma outcomes. Tolerance to therapy did not develop, nor did rebound worsening of asthma symptoms once therapy was withdrawn. In a comparator trial of montelukast versus beclomethasone, the average percentage change from baseline in forced expiratory volume in 1 second was greater with the inhaled corticosteroid preparation; however, improvements in other asthma outcomes were similar. There was considerable heterogeneity of pulmonary response with both treatments, with good and poor responders in both groups. In an open‐label, crossover comparison of montelukast versus cromolyn, both parents and children preferred montelukast, thus regimen adherence was greater with montelukast. Additional long‐term, randomized, controlled trials will define the effectiveness of leukotriene receptor antagonists compared with established controllers, thus determining the leukotriene receptor antagonists' place in asthma management.
Drug Treatment of Airway Inflammation in Asthma
Authors: Blake Kathryn V.
Published: June 2004

Several methods are available for assessing drug effects on airway inflammation and the antiinflammatory effects of drugs for asthma. Cromolyn and theophylline are well‐established drugs for the treatment of asthma, and each has antiinflammatory properties. Drugs in development include those aimed at inhibiting inflammatory mediators and immunoglobulin E function; clinical studies, however, have been conducted largely in patients with moderate to severe asthma.
Imaging of the Paranasal Sinuses
Authors: Boll Daniel T. , Coffey Michael , Lewin Jonathan S.
Published: June 2004

MRI in most instances, can better distinguish between soft tissues when compared with CT. The distinction of soft tissues becomes most clinically relevant when evaluating for possible sinonasal tumors and when inspecting for extension of tumor or inflammation into the orbit or intracranial spaces. It is in these areas that MRI has found its major application in sinus imaging. This unit presents MRI techniques for imaging the sinuses with and without contrast enhancement. The parameters given in this unit are derived from experience at 1.5 T and may need to be slightly altered depending on the field strength and the equipment manufacturer.
Transformation of E. coli by Electroporation
Authors: Morrison Sherie L.
Published: June 2004

Using 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

With 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

The 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.
Traditional Indirect Methods for Estimation of Water Content: Measurement of °Brix
Authors: Reid David S.
Published: June 2004

Refractometric and hydrometric measurements have long been used as means of estimating solution concentrations. The effectiveness of their use to measure equivalent concentration (°Brix) is discussed. The approximations inherent in the use of such traditional methods to estimate water content are highlighted.
Overview of Flow Cytometry Instrumentation
Authors: Dean Phillip N.
Published: June 2004

This unit describes the technology in general, to give a feel for the interplay between the various parts of a flow cytometer. Topics include cell preparation, detectors, analysis, and sorting. A brief chronology of flow instrumentation development illustrates the long history of the field.
Standardization, Calibration, and Control in Flow Cytometry
Authors: Hoffman Robert A.
Published: June 2004

Because 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

One 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

Lasers 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

The 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

Fluorescence 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

Many 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

Flow 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

The 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.
Confocal Microscopy: Principles and Practices
Authors: Dean Phillip N.
Published: June 2004

The author presents a well rounded and complete overview of the topic, covering optics, detectors, image collection, and analysis, and designed to take the uninitiated person through an excellent understanding of this very actively growing technology.
Scanning Laser Cytometry
Authors: Shapiro Howard M.
Published: June 2004

There 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

This 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.
Principles of Quality Control
Authors: Hurley Anne A.
Published: June 2004

This unit presents a brief overview of the principles of quality control in the clinical laboratory.
Components of Quality Control
Authors: Hurley Anne A.
Published: June 2004

The purpose of a control is to aid the operator in deciding whether an analytical system is producing reliable results for a given assay, and ultimately whether to release the results. This unit presents information on the techniques for determining when results are in control or out of control.
Testing the Efficiency of Aerosol Containment During Cell Sorting
Authors: Schmid Ingrid , Hultin Lance E. , Ferbas John
Published: June 2004

Production 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

Many 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.
Spectroscopic Analysis Using DNA and RNA Fluorescent Probes
Authors: Ghallagher Sean R.
Published: June 2004

The unit takes the nature of fluorescent probes one step further and provides a quantitative approach to analysis of DNA and RNA probes using spectroscopic techniques. Two sensitive fluorescence protocols are provided in addition to the traditional absorbance method.
Quality Control in Phenotypic Analysis by Flow Cytometry
Authors: Hurley Anne A.
Published: June 2004

This unit proposes a specific daily quality control program to ensure instrument reliability and accurate results within current constraints.
Measurement of CD40 Ligand (CD154) Expression on Resting and In Vitro–Activated T Cells
Authors: O'Gorman Maurice R.G.
Published: June 2004

Measurement 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

Absolute 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

This 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

This 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

This 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.
Bivariate Analysis of DNA Content and Expression of Cyclin Proteins
Authors: Juan Gloria , Darzynkiewicz Zbigniew
Published: June 2004

Cyclins are key components of the cell cycle machinery. This unit describes techniques associated with measurement of these transiently expressed molecules and focusses on the combination of cell cycle analysis and expression of proliferation‐associated proteins via immunocytochemically attached antibodies.
Assessment of Viability, Immunofluorescence, and DNA Content
Authors: Schmid Ingrid
Published: June 2004

This 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

The 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

When 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

Viruses 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

Cell 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

Application 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

The 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

This 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

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

This 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

This 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

A 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

This 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

Functional 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

Using 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

Degradation 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

Changes 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.
Listmode Data Processing
Authors: Leary James F.
Published: June 2004

Most flow cytometry data are acquired and stored in listmode format, whereby all measurements on each cell are stored separately in a list, rather than in histograms. This unit discusses the desirability of using listmode format and techniques for handling such data.
Data Analysis Through Modeling
Authors: Bagwell C. Bruce
Published: June 2004

Flow 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

Image 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

Data 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

Although 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

E. 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

This 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

For 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

This 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

Mycoplasma 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

Basic 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.
Growing Bacteria in Liquid Media
Authors: Lech Karen , Brent Roger
Published: June 2004

The procedure for inoculating overnight (starter) cultures of E. coli from a single colony is described along with considerations for growing larger cultures. Also included are two methods for monitoring cell growth using a spectrophotometer or a hemacytometer.
Growing Bacteria on Solid Media
Authors: Lech Karen , Brent Roger
Published: June 2004

Detailed protocols are provided for titering and isolating bacterial colonies by serial dilutions, or alternatively by streaking or spreading a plate. Support protocols describe replica plating as well as methods for storing strains as agar stabs or frozen glycerol stocks.
Basic Techniques for Mammalian Cell Tissue Culture
Authors: Phelan Mary C.
Published: June 2004

Cultured 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

This 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.