bluuebunny/crossref_metadata_2025_split huggingface dataset:
Assessment of Surface Markers and Functionality of Dendritic Cells (DCs)
Authors: Nuñez Rafael 
Published: June 2004

AbstractDendritic cells are a complex group of mainly bone marrow derived cells that possess significant ability to take up, process, and present soluble antigens to responder cells in the lymphoid tissues. Characterization of DC surface markers has been a difficult task owing to the lack of appropriate reagents of sufficient specificity. Molecules closely associated with dendritic cells are being identified and investigated and flow cytometric approaches to evaluation of monoclonal antibodies against DC populations are being developed. This informative unit describes assays for determination of the existence of subsets within two DC populations, the kinetics of antigen expression, and the identification of specific markers for DC subsets. A presents an approach for generating dendritic cells and Langerhans cells (a skin‐derived DC) from blood monocytes.
Stem Cell Identification and Sorting Using the Hoechst 33342 Side Population (SP)
Authors: Goodell Margaret A. 
Published: June 2004

AbstractThis unit describes the use of Hoechst 33342 to identify and purify murine hematopoietic stem cells, the so‐called side population. Three properties of the dye contribute to the ability to distinguish stem cells in this way. Hoechst is a DNA‐binding dye. It has at least two binding modes that result in different spectral properties, allowing resolution of multiple populations by viewing fluorescence at two wavelengths simultaneously. The ability to discriminate SP cells is based on the differential efflux of the dye by a multi‐drug‐like transporter. Particular attention is given to the critical aspects of Hoechst concentration, cell concentration, staining time, and staining temperature.
Assessment of Phagocyte Functions by Flow Cytometry
Authors: Bassøe Carl‐Fredrik 
Published: June 2004

AbstractPhagocytes neutrophils, monocytes, and macrophages are crucial in the defense against infection. Their functions include phagocytosis, intra‐ and extracellular digestion of targets, oxidative burst, and chemotaxis. This extensive, detailed unit outlines a four‐part procedure for in‐depth investigation of these four functions that reflect the attacking and processing of pathogenic microorganisms. Written by the original instigator of flow‐based assays in this area, this unit defines an area that is as old as flow itself. Numerous support protocols provide preparative procedures for bacterial targets, opsonins, and antigen‐coated beads.
Flow Cytometric Analysis of Calcium Mobilization in Whole‐Blood Platelets
Authors: Monteiro Maria‐do‐Céu , Gonçalves Maria‐José , Sansonetty Filipe , O'Connor José‐Enrique 
Published: June 2004

AbstractFlow cytometry provides a convenient method to evaluate platelet activation by following the kinetics of intracellular free Ca2+, using sensitive fluorescent indicators that can be loaded into intact cells. Moreover, in the clinical setting, whole‐blood techniques have obvious advantages to avoid artifactual platelet activation and allow the maintenance of near‐physiological conditions. This unit describes a fast and sensitive flow cytometric procedure using the Ca2+‐sensitive dye fluo‐3 AM and the platelet‐specific antibody CD41‐PE to determine the kinetics of intracellular Ca2+ mobilization in whole‐blood platelets with minimal manipulation of the samples. The technique may be applied to reveal fast and transient increases in cytosolic calcium upon platelet stimulation with the agonists ADP and thrombin. This protocol provides a simple and sensitive tool to assess in vitro the time course and intensity of signal‐transduction responses to agonists under near‐physiological conditions, and should be broadly applicable to studies of platelet reactivity.
Flow Cytometric Analysis of Cytokine Responses in Stimulated Whole Blood: Simultaneous Quantitation of TNF‐α‐Secreting Cells and Soluble Cytokines
Authors: Rodríguez‐Caballero Arancha , García‐Montero Andrés , Bueno Clara , Orfao Alberto 
Published: June 2004

AbstractThis unit describes technical protocols aimed at the ex vivo or in vitro evaluation of the functional status of the immune system through the simultaneous identification and enumeration of cytokine‐secreting cells and quantitation of the soluble cytokines produced by these cells.
Data Management
Authors: Parks David R. 
Published: June 2004

AbstractThis unit presents a broad view of flow cytometry data management, emphasizing ways to create and manage data so as to maximize their immediate and long‐term usefulness. The cost in time, money, and effort to create data files in the first place argues for the importance of making full use of files and keeping them from getting lost or becoming uninterpretable. As flow cytometry measurements become increasingly complex, researchers have more and more reasons to reexamine old data. Careful data management is clearly necessary to ensure that stored data contain enough information about an experiment to enable analysis and interpretation by others even years later.
Two‐Dimensional Image Processing and Analysis
Authors: Castleman Kenneth R. 
Published: June 2004

AbstractProcessing and analysis of image data require very different methods than those used in flow cytometry. Image processing is used to correct inaccuracies in an image and make it easier to interpret, whereas image analysis is used to extract quantitative data about a specimen. This unit provides an overview of image processing methods. Keywords: image processing; image analysis; overview Processing and analysis of image data require very different methods than those used in flow cytometry
Multivariate Analysis
Authors: Boland Michael V. , Murphy Robert F. 
Published: June 2004

AbstractOne of the goals of Current Protocols is to provide information from the very basic level to the very advanced. This unit is an excellent example of providing explanation and application for a complex area. Building upon several other units on data analysis, it provides a detailed explanation of how multivariate analytical techniques are applied to flow cytometry data. The unit deals with the covariance matrix, principal component analysis, and cluster analysis and provides sources for software as well as example data files. This material will be particularly valuable to the graduate student trying to obtain a good understanding of statistical methods in flow cytometry, as well as to the experienced investigator who uses these tools on a regular basis without really understanding how they operate. Keywords: principal components analysis; cluster analysis; FCS file format One of the goals of Current Protocols is to provide information from the very basic level to the very advanced
Detection and Location of Hybridization Domains on Chromosomes by Image Cytometry
Authors: Mascio Laura 
Published: June 2004

AbstractBecause of time considerations in performing image analysis for detection of very small amounts of fluorescence probe, there are advantages in using automated detection systems. This unit provides considerable detail on several algorithms used for location of hybridization domains that reduce the tedium and improve objectivity and repeatability. Not only are these algorithms described and discussed in detail, but sources for the programs are also provided. Here is a carefully crafted textual and graphical description of how to go about learning the art of automated image analysis.
Three‐Dimensional Image Visualization and Analysis
Authors: Lockett Stephen J. 
Published: June 2004

AbstractThis unit introduces the concepts of 3D image analysis and visualization as applied in cytometry. The author discusses the nature of 3D data sets and describes the techniques for visualization and analysis of 3D images. Discussions of noise removal, depth attenuation, and correction and segmentation are also included, as is a brief introduction to 3D analysis options and deconvolution prinicples. This commentary unit is a good way to begin an understanding of the application of 3D data sets.
Dial‐In Flow Cytometry Data Analysis
Authors: Battye Francis L. 
Published: June 2004

AbstractAs listmode data files continue to grow larger, access via any kind of network connections becomes more and more trouble because of the enormous traffic generated. The limited speed of transmission via modem makes analysis almost impossible. This unit presents a solution to these problems, one that involves installation at the central storage facility of a small computer program called a Web servlet. Operating in concert with a Web server, the servlet assists the analysis by extracting the display array from the data file and organizing its transmission over the network to a remote client program that creates the data display. The author discusses a recent implementation of this solution and the results for model transmission of two typical data files. The system greatly speeds access to remotely stored data yet retains the flexibility of manipulation expected with local access.
Calibration and Shading Correction for Fluorescence Microscopes
Authors: Model Michael A. 
Published: June 2004

AbstractStandardization in image cytometry involves intensity calibration and shading correction. This unit presents a method using concentrated solutions of fluorophores. A drop of highly concentrated fluorescein or rhodamine placed between a slide and a coverslip produces a spatially uniform fluorescent sample with reproducible quantum yield and resistance to photobleaching. The technique has a number of practical features making it inexpensive, reproducible, and straightforward.
A Software Method for Color Compensation
Authors: Stewart Carleton C. , Stewart Sigrid J. 
Published: June 2004

AbstractWhen two or more fluorochromes are measured simultaneously, every detector sees some fluorescence from every fluorochrome. Spectral compensation is the process of removing the undesired overlap of signal. Although very successful for two and three fluorochromes, the general practice of adjusting instrument compensation becomes increasingly inadequate and unforgiving as the number of fluorochromes increases. When data are collected uncompensated, software compensation provides the flexibility of setting correct compensation every time for every sample. Software methods do have problems. The linearization assumptions made by the software algorithms may be more or less in error. Binning effects become more of a problem with increasing numbers of compensated parameters. This explanatory unit also contains protocols that illustrate the process of software compensation utilizing matrix algebra that provides for elements of all possible PMT detection combinations. Although details are limited to four colors, the principles described can be applied to any desired number. When two or more fluorochromes are measured simultaneously, every detector sees some fluorescence from every fluorochrome.
Flow Cytometry and Environmental Microbiology
Authors: Porter Jonathan 
Published: June 2004

AbstractThis survey unit discusses many of the issues involved for flow cytometry in the field of microbiology, particularly the preparative procedures, which are far more stringent than many other procedures using larger cells. For instance, it is often necessary to filter laboratory agents multiple times to obtain the true particle‐free solutions needed for flow cytometry of microbes. It is difficult enough to recognize bacteria in cell extracts from soil, sediment, or sludge given the background of same‐size particles. This unit provides an excellent overview of a potentially large application area in flow cytometry and is written by one of the most respected scientists in the field.
Estimation of Microbial Viability Using Flow Cytometry
Authors: Davey Hazel M. , Weichart Dieter H. , Kell Douglas B. , Kaprelyants Arseny S. 
Published: June 2004

AbstractFor microorganisms in particular, viability is a term that is difficult to define and a state consequently difficult to measure. The traditional (and gold‐standard) usage equates viability and culturability (i.e., the ability to multiply), but the process of determining culturability is often too slow. Flow cytometry provides the opportunity to make rapid and quantitative measurements of dye uptake in large numbers of cells, and we can therefore exploit the flow cytometric approach to evaluate so‐called viability stains and to develop protocols for more routine assessments of microbial viability. This unit is primarily commentary, but several basic protocols have been included to ensure that users have a firm basis for attempting these reasonably difficult assays on traditional flow cytometer instruments. What is clear is that each assay must be carefully validated with the particular microorganism of interest before being applied in any research, clinical, or service form.
Sorting of Bacteria
Authors: Harkins Kristi R. 
Published: June 2004

AbstractThis unit provides detailed instructions for the sorting of organisms using the cell sorter. These techniques are very valuable for establishing pure cultures of organisms, for example those expressing GFP. The unit provides information on the culture, sorting, reculture, and verification of the sort purity and GFP expression. For those venturing into the area of bacterial sorting, this unit is a must.
Detection of Borreliacidal Antibodies by Flow Cytometry
Authors: Callister Steven M. , Jobe Dean A. , Schell Ronald F. 
Published: June 2004

AbstractLyme disease is a multisystem disorder that usually begins with a skin lesion called erythema migrans and with constitutional symptoms. If the disease is left untreated or treated inappropriately, dissemination of the organism can lead to more severe sequelae, including nervous system disorders or arthritis. Vaccinations with B. burgdorferi or several individual B. burgdorferi proteins induce borreliacidal antibodies that provide protection against infection by inducing a complement cascade that kills the spirochetes without the necessity of scavenging by phagocytic cells. Detection of borreliacidal antibodies is therefore useful for serodiagnosing Lyme disease and monitoring immune status after vaccination. This unit provides a technique for detecting anti‐B. burgdorferi antibodies, as well as for preparing and determining the quality of Barbour‐Stoenner‐Kelly (BSK medium) and complement. In addition, methods are provided for preparation of a B. burgdorferi stock and Mueller‐Hinton agar containing Bacillus subtilis spores.
Mycobacterium tuberculosis Susceptibility Testing by Flow Cytometry
Authors: Schell Ronald F. , Nardelli Dean T. , DeCoster David J. , Kirk Scott M. , Callister Steven M. 
Published: June 2004

AbstractHere is another unit with clinical relevance. Tuberculosis remains a major health problem throughout the world, with approximately one‐quarter of the population being infected. Rapid and accurate susceptibility testing for the tubercle bacillus is essential for control of the disease. Such testing can be accomplished by flow cytometry within twenty‐four hours, instead of the days to weeks required by traditional methods. The use of flow cytometry both improves the quality of susceptibility testing and advances public health measures for the prevention and control of this ancient scourge.
Flow Cytometry of Yeasts
Authors: Lloyd David 
Published: June 2004

AbstractThis unit promotes increased interest in the use of flow cytometry in several new environments. The author provides protocols and descriptive detail on measurements of cell cycle, viability, respiratory activity, and β‐galactosidase activity. Many of these assays are described for other biological systems in CPC, but are now provided in detail for yeasts. Despite the increasing usefulness of digital imaging techniques, flow cytometry remains the method of choice for the resolution of population heterogeneities.
DNA/RNA Analysis of Phytoplankton by Flow Cytometry
Authors: Marie Dominique , Simon Nathalie , Guillou Laure , Partensky Frédéric , Vaulot Daniel 
Published: June 2004

AbstractThe past 10 years or so have seen the combination of molecular and biochemical techniques within the confines of cytometry. The use of flow cytometry in microbiology is finally coming of age. This unit carefully defines the criteria for evaluation of DNA and RNA in phytoplankton. Of course not everyone works with phytoplankton, but the methods outlined are very appropriately representative for other organisms. In addition, the unit discusses the methods for evaluating cell cycle and discriminating specific taxa using fluorescent oligonucleotide probes targeted to 18S rRNA.
Cell Cycle Analysis of Yeasts
Authors: Fortuna Margarida , João Sousa Maria , Côrte‐Real Manuela , Leão Cecília , Salvador Alexandre , Sansonetty Filipe 
Published: June 2004

AbstractStaining protocols generally designed for the flow cytometric analysis of the cell cycle in mammalian cells are frequently not satisfactory for quantification of the various cell‐cycle phases in yeasts. High CVs limit the accuracy of DNA content measurement and estimates of populations in cell‐cycle compartments. This unit describes a staining procedure for yeasts using the sensitive nucleic acid stain SYBR Green I, which binds to double‐stranded DNA with high selectivity and which has a much higher fluorescence quantum yield upon binding than most other commonly used fluorophores. The properties of this dye combined with optimized sample processing provide high‐resolution DNA analysis, with half‐peak CVs around 3 to 4% and clear‐cut identification of the S phase.
Flow Cytometric Assessment of Drug Susceptibility in Leishmania infantum Promastigotes
Authors: Nuñez Rafael , Kamau Sarah , Grimm Felix 
Published: June 2004

AbstractFlow cytometry can be a valuable tool for parasitology in studies of parasite‐drug interaction and cellular toxicity. This unit presents protocols for assessment of Leishmania promastigote proliferation, viability, and cellular protein content. The cytometric approach permits one to detect, differentiate, and quantify cellular changes in these parasites resulting from drug treatment, in this case allopurinol, as well as demonstrate differences in drug susceptibility between promastigote forms. Rapid and relatively simple flow cytometry replaces incorporation of [3H]‐thymidine, hitherto the most common method for determining cell division.
Resolution of Viable and Membrane‐Compromised Free Bacteria in Aquatic Environments by Flow Cytometry
Authors: Grégori Gérald , Denis Michel , Seorbati Sergio , Citterio Sandra 
Published: June 2004

AbstractIn aquatic environments, free heterotrophic bacteria play an extremely important role because of their high biomass, wide panel of metabolisms, and ubiquity, as well as the toxicity of certain species. This unit presents a nucleic‐acid double‐staining protocol (NADS) for flow cytometry that can distinguish the fractions of viable, damaged, or membrane‐compromised cells within the free‐bacterial community. The NADS protocol is based on the simultaneous utilization of two nucleic acid stains, membrane‐permeant SYBR Green and membrane‐impermeant PI. The efficiency of the double staining is magnified by the FRET from SYBR Green to PI when both are bound to the nucleic acids. Full quenching of SYBR Green fluorescence by PI will identify cells with a compromised membrane, partial quenching will indicate cells with a slightly damaged membrane, and lack of quenching will characterize cells with an intact membrane. Samples do not require any pretreatment and this protocol can be performed almost anywhere.
Techniques for Mammalian Cell Tissue Culture
Authors: Phelan Mary C. 
Published: June 2004

AbstractThe first section of this appendix unit discusses basic principles of sterile technique, while the second section discusses medium preparation. A protocol is then provided for subculturing of a monolayer culture grown in petri dishes or flasks. Support protocols describe freezing of monolayer cells, thawing and recovery of cells, counting cells using a hemacytometer, and preparing cells for transport. An describes freezing suspension cells.
Wright‐Giemsa and Nonspecific Esterase Staining of Cells
Authors: Strober Warren 
Published: June 2004

AbstractThis appendix provides two protocols for staining cells. The stains used in the Wright‐Giemsa protocol, the Romanowsky stains, are a mixture of methylene blue (and other closely related thiazine dyes) and eosin. The staining protocol is a two‐stage method that allows for a more intense staining of the nuclei than would be possible if the Wright and Giemsa stains were mixed together. In the second protocol, nonspecific esterase stain are used to identify cell types containing esterases that have a characteris termediate that can be coupled with a substance, hexazotized pararosaniline, to yield a colored precipitate. Thus, this staining reaction can be used to identify monocytes/macrophages in cell preparations.
Importing Biological Materials
Authors: Wolf Paula 
Published: June 2004

AbstractScientific cooperation between laboratories in the United States is no longer confined to those at the same institution, nor even within the boundaries of the country. More than ever, scientists are forming collaborations with their peers outside the U.S., leading not only to the exchange of knowledge and ideas, but also to the exchange of biological reagents. This unit discusses issues involved with international transfer of these reagents, such as packaging and import restrictions.
Analysis of the Association of Proteins with Membranes
Authors: Okamoto T. , Schwab R.B. , Scherer P.E. , Lisanti M.P. 
Published: June 2004

AbstractProteins associate with membranes in a number of ways. Is the membrane an integral membrane protein? A peripheral protein? Does it have a glycosylphosphoinositol (GPI) tail? Is it associated with caveolae‐derived plasma membrane domains? This unit describes methods for determining how a protein of interest associates with the membrane including extraction with alkaline carbonate, urea, high salt, or Triton X‐114. GPI linkage is probed using PI‐PLC cleavage. There is a method for detergent solubilization of Triton‐X 100 insoluble integral and GPI‐linked proteins. Caveolae membranes can be isolated using Triton X‐100 or a detergent‐free method.
Media for Culture of Mammalian Cells
Authors: Sato J. Denry , Kan Mikio 
Published: June 2004

AbstractWhen mammalian cells are cultured in vitro, the investigator is attempting to reproduce the physiological environment in order to maintain and analyze normal functions and responses. The culture medium is an essential component of the in vitro environment and must be selected or designed with care. This unit provides guidelines for design of serum‐containing and serum‐free media, selective andspecialty media, and media for growth under special conditions such as soft‐agar growth.
Media and Culture of Yeast
Authors: Green Simon R. , Moehle Charles M. 
Published: June 2004

AbstractThe yeast, Saccharyomyces cerevisiae, has become an important organism in molecular, biochemical, and genetic analysis. The organism has specific requirements for growth under a variety of conditions. The media, both liquid and solid, simple, define, and complex are describe in this unit. Also included are methods for handling, storing, and shipping stock of yeast.