bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of DNA Denaturation
Authors: Darzynkiewicz Zbigniew , Juan Gloria
Published: June 2004

This unit provides a detailed method for evaluating in situ DNA denaturation by flow cytometry. The principal technique is based upon the metachromatic properties of acridine orange. This technique has a number of advantages over traditional biochemical methods, but requires very precise methodology; the result is excellent clarity and differentiation between single‐stranded and double‐stranded DNA. This unit provides full details on the correct use of acridine orange together with an excellent discussion of the pitfalls and problems.
Flow Cytometric Analysis of Reticulated Platelets
Authors: Matic Goran B. , Rothe Gregor , Schmitz Gerd
Published: June 2004

Flow Cytometric Analysis of Reticulated Platelets (G. Matic, G. Rothe, and G. Schmitz, University of Regensburg, Regensburg, Germany). In the last several years, flow cytometry has emerged as one of the tools of choice in evaluation of platelets. In particular, the distinction between reticulated platelets and mature platelets based on RNA fluorescence has proved to be a vital tool in the clinical hematology laboratory. Further, as is now well understood, it is important to evaluate platelets in whole blood rather than in isolated populations. Platelet quantification in a dual‐color whole‐blood method is of interest for the characterization of thrombocytopoiesis in (immune)‐thrombocytopenia or in the regenerating bone marrow.
Flow Cytometric Analysis of RNA Synthesis by Detection of Bromouridine Incorporation
Authors: Larsen Jørgen K. , Jensen Peter Østrup , Larsen Jacob
Published: June 2004

RNA synthesis has traditionally been investigated by a laborious and time‐consuming radiographic method involving incorporation of tritiated uridine. Now a faster non‐radioactive alternative has emerged, based on immunocytochemical detection. This method utilizes the brominated RNA precursor bromouridine, which is taken into a cell, phosphorylated, and incorporated into nascent RNA. The BrU‐substituted RNA is detected by permeabilizing the cells and staining with certain anti‐BrdU antibodies. This dynamic approach yields information complementing that provided by cellular RNA content analysis at a given time and may be of value in studies of cellular activation and gene expression.
Analysis of Cell Proliferation and Cell Survival by Continuous BrdU Labeling and Multivariate Flow Cytometry
Authors: Poot Martin , Rosato M. , Rabinovitch Peter S.
Published: June 2004

Halogenated deoxyuridines such as BrdU incorporated into DNA in place of thymidine can be detected by virtue of their ability to quench the fluorescence of Hoechst dyes. This property is the basis for a simple and reliable method of enumerating cells which have incorporated BrdU. Counterstaining with another suitable DNA dye permits resolution of cells in G1, S, and G2 phases of three consecutive cell cycles. In addition to this basic technique, the authors present newer protocols for proliferative analysis of GFP‐expressing or cell surface antigen expressing cells versus nonexpressing cells in culture as well as one to determine the absolute number of proliferating and nonproliferating cells by calibration for the volume analyzed. With this technique the proliferative survival of cells can be determined.
Analysis of DNA Content and Green Fluorescent Protein Expression
Authors: Schmid Ingrid , Sakamoto Kathleen M.
Published: June 2004

Green fluorescent protein (GFP) is an intracellular reporter molecule widely utilized for assessment of gene transfer and expression. Enhanced variants have been cloned into various expression vectors suited for many different cell types. To study the effect of a gene of interest on cell cycle progression, it is desirable to measure GFP expression in combination with DNA content. This approach is difficult, as most suitable fluorescent DNA dyes are too large to pass through intact cell membranes, but permeabilization will allow GFP to leak out. The authors present a protocol with a cell preparation technique designed to maintain the delicate balance between retaining GFP fluorescence and obtaining adequate DNA histogram resolution. An describes a combined GFP fluorescence and cell cycle analysis using unpermeabilized cells stained with the vital dye Hoechst 33342.
Analysis of Fine‐Needle Aspirate Biopsies from Solid Tumors by Laser Scanning Cytometry (LSC)
Authors: Gerstner Andreas O.H. , Tàrnok Attila
Published: June 2004

Both flow and image cytometry have routine, reliable methods for ploidy analysis, each with its dbacks. LSC offers a non‐destructive and non‐consumptive method of analysis that allows repeated sample analysis as well as storage for later re‐evaluation. Owing to the extremely small sample requirement, specimens can be obtained through minimally invasive procedures such as fine‐needle aspirate biopsies or simple swabs. Tumors located at sites unsuitable for routine biopsy can be analyzed even on an out‐patient basis.
Analysis of Tissue Imprints by Scanning Laser Cytometry
Authors: Bollmann Reinhard , Méhes Gábor
Published: June 2004

The analysis of DNA content and simultaneous surface or intracellular antigen expression is described for laser scanning cytometry. Measurements can be performed on microscope slides prepared immediately after dissection of native surgical specimens. These protocols will be useful for both diagnostic pathology and cell biology laboratories.
Cell Cycle Analysis of Budding Yeast Using SYTOX Green
Authors: Haase Steven B.
Published: June 2004

Flow cytometric determination of DNA content is a standard tool for cell cycle analysis in the budding yeast, Saccharomyces cerevisiae. Using SYTOX Green instead of propidium iodide (PI) in a standard ethanol fixation protocol increases the accuracy and reproducibility of cell cycle analyses. SYTOX‐stained cells exhibit improved coefficients of variation (CVs), a better correlation between DNA content and fluorescence, and decreased sensitivity to variances in experimental conditions when compared with PI‐stained cells. The SYTOX Green protocol provides researchers with a more accurate and reliable alternative to standard PI‐based protocols for the analysis of the budding yeast cell cycle.
Detection of Mitotic Cells
Authors: Juan Gloria , Darzynkiewicz Zbigniew
Published: June 2004

In preparation for cell division, nuclear chromatin undergoes a vital rearrangement required for the organization of chromosomes and their allocation to daughter cells. This process is initiated during G2 phase with the most remarkable morphological manifestation being chromatin condensation. This unit provides protocols for identification and quantification of mitotic cells based on immunocytochemical detection of histone H3 phosphorylated on Ser 10 (H3‐P), the critical event occurring during the G2 to M transition (essential for chromatin condensation), using anti‐H3‐P, a commercially available antibody to which apoptotic cells are not reactive, concurrently with differential staining of cellular DNA. Additionally an adaptation of this method used to stain cells mounted on microscope slides for analysis by multiparameter laser scanning cytometry is also presented.
Overview of Fluorescence In Situ Hybridization Techniques for Molecular Cytogenetics
Authors: Raap A.K.
Published: June 2004

This unit presents an overview of the FISH methodology. It covers such topics as direct versus indirect methods, sensitivity, multiplicity, resolution, and applications.
Probe Labeling and Fluorescence In Situ Hybridization
Authors: Wiegant J. , Raap A.K.
Published: June 2004

This unit describes in detail basic protocols for probe labeling, denaturing of in situ target DNA, in situ hybridization, and post‐hybridization washes. Support protocols for probe labeling cover probe purification and quality control. Keywords: molecular cytogenetics; FISH; nick translation; random priming; PCR labeling
Immunocytochemical Detection
Authors: Wiegant J.
Published: June 2004

This unit provides detailed descriptions for direct and indirect in situ hybridization methods. Protocols are designed for bright‐field microscopy using a precipitating chromogenic substrate and for immunofluorescence detection using a hapten‐labeled probe. Several alternative procedures are provided together with an excellent discussion of the strategy used for selecting a detection method.
Processing and Staining of Cell and Tissue Material for Interphase Cytogenetics
Authors: Hopman A.H.N. , Ramaekers F.C.S.
Published: June 2004

This unit describes in detail the protocols for preparation of biological specimens (fresh or fixed) for the purposes of establishing in situ hybridization procedures using probes for specific regions of the genome, allowing targeted detection of numerical and structural chromosome aberrations in the interphase nucleus. The authors present a series of comprehensive protocols from the simplest to the most complex.

Keywords: molecular cytogenetics; in situ hybridization; chromosome aberrations; interphase cytogenetics.
Advanced Preparative Techniques to Establish Probes for Molecular Cytogenetics
Authors: Stap Jan , Aten Jacob A. , Lillington D. , Shelling A. , Young B.D.
Published: June 2004

The methods covered in this unit include flow cytometry of metaphase chromosomes, chromosome dissection, and the DOP‐PCR amplification methods for reverse chromosome painting. Successful application in these areas requires care and attention to methodological details, and this unit is particularly comprehensive.

Keywords: molecular cytogenetics; chromosomes; flow cytometry; microdissection; metaphase; cytogenetics.
Single‐Nucleotide Sequence Discrimination In Situ Using Padlock Probes
Authors: Nilsson Mats , Landegren Ulf , Antson Dan‐Oscar
Published: June 2004

Standard fluorescence in situ hybridization (FISH) techniques using cloned probes are limited in their ability to distinguish between closely similar DNA sequences because long hybridization probes are not detectably destabilized by single mismatched base pairs. This problem has been addressed by using short allele‐specific oligonucleotide probes whose hybridization to target sequences is more sensitive to mismatches. This revised and expanded unit presents protocols for discrimination between closely similar DNA sequences in situ. The discussion of probe synthesis has been greatly expanded and an added for enzymatic probe ligation at low probe concentration. A new describes enzymatic probe synthesis.
Combined Immunofluorescence and FISH: New Prospects for Tumor Cell Detection/Identification
Authors: Ambros Peter F. , Méhes Gábor
Published: June 2004

Fluorescence‐microscope based methods are presented that allow automatic selection and quantification of immunophenotyped cells and sequential FISH analysis to facilitate molecular cytogenetic analysis of single cells and very small cell populations. The protocols in this unit are particularly useful for studying the biological and genetic makeup of disseminated tumor cells or other rare cells.
Overview of Functional Cell Assays
Authors: Rabinovitch Peter S. , Robinson J. Paul
Published: June 2004

Flow cytometry can measure a variety of functional parameters of increasing interest to cell biologists and clinicians. The distinguishing feature of these assays is that the information of interest is the functional behavior of the cell itself, rather than the phenotype. This unit discusses cell viability, oxidative metabolism, membrane potential, intracellular ionized calcium, intracellular pH, intracellular organelles, and gene reporter assays.
Assessment of Cell Viability
Authors: Coder David M.
Published: June 2004

Cell viability may be judged by morphological changes or by changes in membrane permeability and/or physiological state inferred from the exclusion of certain dyes of the uptake and retention of others. This unit presents methods based on dye exclusion, esterase activity, and mitochondrial membrane potential, as well as protocols for determining the pre‐fixation viability of fixed cells either before or after fixation. A dye‐exclusion procedure for microscopy is also included.
Flow Cytometric Measurement of Intracellular pH
Authors: Chow Sue , Hedley David
Published: June 2004

A number of fundamentally important biological processes, such as cell signaling and the initiation of mitosis, are accompanied by a change in intracellular pH. Flow cytometric measurement of pH is a generally straightforward procedure that can be done with any instrument equipped with a 488‐nm argon laser. The overall approach is similar to that for calcium: generation of a calibration curve by imparting known changes in pH and interpolation of the test sample pH. This unit presents the traditional calibration method using high‐potassium buffers and the proton ionophore nigericin and a more recently developed technique, the pseudo null method, which involves resuspension of cells in defined mixtures of weak acids and weak bases.
Reporters of Gene Expression: Enzymatic Assays
Authors: Lorincz Matt , Roederer Mario
Published: June 2004

Initially developed for use in bacteria, reporter gene systems are now being utilized in eukaryotic cells.
Estimation of Membrane Potential by Flow Cytometry
Authors: Shapiro Howard M.
Published: June 2004

In most living cells, differences between interior and exterior concentrations of ions (e.g., Na+, K+, Cl‐) generate an electrical potential across the cytoplasmic membrane. Membrane potential, like other cellular parameters, may change early in the course of surface receptor‐mediated cell activation processes related to the development, differentiated function, and pathology of a large number of cell types. Changes in ionic environment may play a role in transmembrane signaling in response to cell surface ligand‐receptor interactions. Thus, it is frequently of scientific interest to estimate membrane potential in individual cells. This unit provides methods for accomplishing this goal by flow or static cytometry using any of a number of cationic or anionic lipophilic fluorescent probes known as distributional dyes. The first of the two protocols is able to estimate the membrane potential of eukaryotic or bacterial cells. The second is more accurate, but can only be used with bacteria.
Oxidative Metabolism
Authors: Robinson J. Paul
Published: June 2004

Peroxide production is detected using DCFH‐DA, superoxide anion by hydroethidine; the methods can be combined for simultaneous detection. A support protocol describes calibration of the flow cytometer to permit calculation of the actual H2O2 production per cell. These protocols are applicable to many different cell types, but provide detailed information on using neutrophils, highly reactive cells that produce vast quantities of ROS.
Intracellular Cytokines
Authors: Babcock George F.
Published: June 2004

The principal technique described in this unit was developed for detecting cytokines produced by T cells, specifically interferon gamma (IFN‐γ) and interleukin 4 (IL‐4). In addition to providing information about which cytokines are being produced, it also helps to phenotypically identify the specific cells producing them. For example, CD4+ or CD8+ cells can be subdivided into TH1/TH2 helper cells or TC1/TC2 suppressor cells, respectively. This procedure can be useful in examining the response of cells to a variety of agonists, the immune function in various disease states, and the level of lymphocyte activation or suppression. In addition, it can be used to detect a variety of cytokines in many cell types, although the methodology must be carefully evaluated for each cytokine or cell type of interest. Additional methods are included for the stimulation of T cells with allogeneic cells and for the performance of controls for labeling specificity.
Flow Cytometric Analysis of Mitochondrial Membrane Potential Using JC‐1
Authors: Cossarizza Andrea , Salvioli Stefano
Published: June 2004

Changes in membrane potential have long been known to represent early activation events. This unit presents very recent developments in both fluorescent probes and functional applications and demonstrates the use of the JC‐1 probe for measuring mitochondrial membrane potential by flow cytometry. A valuable component of this measurement system is the possibility of making quantitative measurements of changes in membrane potential.
Signal Transduction During Natural Killer Cell Activation
Authors: Chini Claudia C.S. , Leibson Paul J.
Published: June 2004

Understanding of transmembrane signaling during NK‐cell activation has greatly expanded during the past few years. The discovery and characterization of novel triggering and inhibitory receptors have revealed the complexity of these processes. This unit focuses on receptor‐initiated signaling pathways that modulate NK functions. Establishing the roles of different signaling pathways in NK cells is a crucial step in the design of therapeutic approaches for selective enhancement or suppression of NK‐cell activation.
Assessment of Surface Markers and Functionality of Dendritic Cells (DCs)
Authors: Nuñez Rafael
Published: June 2004

Dendritic 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

This 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

Phagocytes 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

Flow 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

This 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

This 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.
Data File Standard for Flow Cytometry, FCS 3.0
Authors: Seamer Larry
Published: June 2004

This unit describes the data file format FCS 3.0 developed to permit the sharing of data among laboratories and to provide some uniformity in data formats from various instruments.
Two‐Dimensional Image Processing and Analysis
Authors: Castleman Kenneth R.
Published: June 2004

Processing 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
Data Presentation
Authors: Overton W. Roy
Published: June 2004

The high data rates of flow cytometers make it imperative to use the best data presentation methods available. This unit discusses techniques designed to maximize user understanding of the data.
Multivariate Analysis
Authors: Boland Michael V. , Murphy Robert F.
Published: June 2004

One 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

Because 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

This 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

As 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

Standardization 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

When 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

This 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

For 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

This 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

Lyme 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

Here 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

This 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

The 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

Staining 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

Flow 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

In 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

The 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

This 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

Scientific 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

Proteins 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

When 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.
Sterilization and Filtration
Authors: Coté Rosalie J.
Published: June 2004

This unit on sterility in the tissue culture environment describes methods for sterilization of liquid and dry goods used for tissue culture and filtration of liquids to prevent contamination of cultures.
Media and Culture of Yeast
Authors: Green Simon R. , Moehle Charles M.
Published: June 2004

The 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.
BY‐2 Cells: Culture and Transformation for Live Cell Imaging
Authors: Brandizzi Federica , Irons Sarah , Kearns Anne , Hawes Chris
Published: June 2004

Tobacco Bright Yellow‐2 (BY‐2) suspension cells are a widely used biological material for studying plant cell morphology and physiology. These cells are easy to transform and maintain in culture and tolerate transformation with fluorescent proteins such as the green fluorescent protein and its derivatives. These, by the addition of plant or mammalian targeting sequences, can be directed to specific subcellular locations for the study of cell dynamics in vivo.

This unit describes the production of BY‐2 cell stable transformants via an Agrobacterium based method to permit the visualisation of cellular components in vivo by epifluorescence or confocal microscopy.
Preparation and Culture of Human Lymphocytes
Authors: Biddison William E.
Published: June 2004

This unit presents protocols for preparation of lymphocytes from peripheral (whole) blood or leucopherisis samples, first by differential centrifugation in Ficoll‐Hypaque to separate them from erythrocytes and granulocytes. Repeated centrifugation removes the plasma and platelets. Monocyte/macrophage cells and dendritic cells can be purified from the T and B cell population by exploiting their adhesion to serum‐coated plates in the presence of recombinant IL‐3. Different subpopulations of lymphocytes can be isolated by specific antibodies bound to magnetic beads.
Laser Capture Microdissection
Authors: Charboneau Lu , Paweletz Cloud P. , Liotta Lance A.
Published: June 2004

This unit describes laser capture microdissection (LCM) using the Pixcell II as a technique to provide the scientific community with the opportunity to perform molecular analyses on pure cell populations procured directly from tissues. After identifying specific cells of interest, the cells are captured by firing a near infrared laser through a thermaplastic polymer film that rests on top of the cells. The cells are then ready for molecular analyses.
Overview of Cell Fractionation
Authors: Castle J. David
Published: June 2004

Cell fractionation is a useful preparative and analytical method in cell biology. It is essential for analysis of composition and function of cellular compartments and it is used to prepare materials for in vitro reconstitution studies This overview discusses the basic principles of centrifugation, the instruments available, choice of media, evaluation of fractionation, and procedure optimization.
Isolation of Rat Hepatocyte Plasma Membrane Sheets and Plasma Membrane Domains
Authors: Tuma Pamela L. , Hubbard Ann L.
Published: June 2004

This unit describes a method for isolation of plasma membrane sheets from rat liver. It also includes protocols for preparation of plasma membrane domains isolated from plasma membrane sheets and indirect immunofluorescence localization of marker proteins associated with plasma membrane sheets. The unit has been updated with assays for the marker enzymes alkaline phosphodiesterase I, 5′ nucleotidase, and K+‐stimulated.
Isolation of Mitochondria from Tissues and Cells by Differential Centrifugation
Authors: Graham John M.
Published: June 2004

Differential centrifugation is used to prepare a “heavy” mitochondrial fraction from liver. These mitochondria are relatively pure, highly coupled, and suitable for respiratory studies. This unit presents protocols for isolation of beef heart mitochondria (also suitable for respiratory studies), skeletal muscle mitochondria, and mitochondria from cultured cells can also be isolated from homogenates by differential centrifugation. Differential centrifugation, which separates cellular organelles based on sedimentation velocity, is a rapid method for preparing mitochondria for metabolic studies.
Isolation of Peroxisomes from Tissues and Cells by Differential and Density Gradient Centrifugation
Authors: Graham John M.
Published: June 2004

Peroxisome purification depends on a two‐step procedure: differential centrifugation to prepare a light mitochondrial fraction and fractionation on a density‐gradient medium preferably iodixanol or Nycodenz, to isolate the peroxisome enriched fraction. The iodixanol gradient may be a preformed continuous gradient or a self‐generating gradient. Alternatively a continuous Nycodenz gradient or a simple Nycodenz barrier may be used for the second step. The unit contains protocols for peroxisome isolation from rat liver, tissue culture cells (HepG2 cells), and yeast spheroplasts. The extent of endoplasmic reticulum contamination of the prep can be assessed using an assay for the marker enzyme NADPH‐cytochromecreductase.
Overview of Subcellular Fractionation Procedures for the YeastSaccharomyces cerevisiae
Authors: Rieder Stephanie E. , Emr Scott D.
Published: June 2004

This unit provides an overview of centrifugation‐based fractionation procedures adapted for the yeastSaccharomyces cerevisiae. The goals, merits, limitations, and critical parameters of are discussed in order to facilitate the development of subcellular fractionation strategies. Topics include yeast cell lysate preparation, differential velocity centrifugation, density gradient centrifugation, and the analysis of subcellular fractions.
Isolation of Subcellular Fractions from the YeastSaccharomyces cerevisiae
Authors: Rieder Stephanie E. , Emr Scott D.
Published: June 2004

This unit presents detailed protocols for a range of centrifugation‐based subcellular fractionation procedures for the yeastSaccharomyces cerevisiae. Techniques include spheroplast preparation, glass‐bead lysis, differential centrifugation, and several density gradient procedures using a variety of gradient media. There are analytical procedures that are primarily designed to evaluate the association of proteins with organelles in the exocytic and endocytic pathways. Additionally, there are preparative protocols for isolation of yeast nuclei, vacuoles, mitochondria, peroxisomes, endoplasmic reticulum, plasma membrane, and cytosol. The unit also contains a table, with references, for alternative approaches to isolation of these organelles and fractions.
Isolation of Golgi Membranes from Tissues and Cells by Differential and Density Gradient Centrifugation
Authors: Graham John M.
Published: June 2004

This unit describes the isolation of Golgi membranes on a preparative basis. Methods are provided for rapid isolation of dextran‐treated Golgi stacks from rat liver using a sucrose density barrier, and Golgi isolation by floatation from a light mitochondrial fraction, along with an alternate procedure using a self‐generated iodixinol gradient. If the Golgi tends to vesiculate during homogenization (commonly the case with cultured cells), a primary requirement is to separate these vesicles from other microsomal compartments, so a procedure is described for a discontinuous gradient of sucrose for cultured cells, and an alternate protocol describes a continuous iodixinol gradient that may provide greater resolution. A self‐generated iodixinol gradient is described to prepare Golgi membranes from a microsomal fraction of rat hepatocytes, and a standard Golgi enzyme marker assay is given in a support protocol.