bluuebunny/crossref_metadata_2025_split huggingface dataset:
Immunophenotypic Identification, Enumeration, and Characterization of Human Peripheral Blood Dendritic Cells and Dendritic‐Cell Precursors
Authors: Almeida Julia , Bueno Clara 
Published: June 2004

AbstractStudy of dendritic cells is complicated by their low frequency and the lack of specific markers for sensitive and specific identification. Furthermore, these cells are not a homogeneous population, but contain a variety of subsets. Flow cytometry has well‐established applications for the analysis of cells present at low frequencies; the use of a two‐step acquisition procedure increases the number of cells for analysis. This unit presents protocols for the identification and enumeration of dendritic cells and cell subsets in erythrocyte‐lysed whole peripheral blood. Special attention is paid to the combinations of reagents used for the identification of all dendritic cells as well as each subset, and on the multiparametric gating strategies for specific analysis.
Immunophenotypic Analysis of Platelets
Authors: Krueger Lori A. , Barnard Marc R. , Frelinger A.L. , Furman Mark I. , Michelson Alan D. 
Published: June 2004

AbstractPlatelets are the smallest cellular component in the peripheral circulation. Their primary role is maintenance of hemostasis. The evaluation of platelets by flow cytometry has proven beneficial in the investigation of many disease states, including inherited defects, cardiovascular disease, stroke, and many other inflammatory processes. In the absence of an added exogenous platelet agonist, the activation state of in vivo circulating platelets can be measured. Inclusion of an exogenous agonist in the assay enables analysis of the reactivity of circulating platelets in vitro. This unit describes protocols to evaluate platelet surface glycoproteins, platelet activation status, and platelet responsiveness to agonist. Detailed discussion of appropriate sample handling, reagent preparation, flow cytometric set‐up, and data interpretation are provided for three independent assays.
Immunophenotypic Analysis of PNH Cells
Authors: Richards Stephen J. , Hillmen Peter 
Published: June 2004

AbstractParoxysmal nocturnal hemoglobinuria (PNH) is a rare acquired hematopoietic stem‐cell disorder in which a gene mutation results in cellular inability to synthesize the glycosylphosphatidylinositol (GPI) anchors needed to attach various proteins to the cell membrane. Traditional clinical laboratory PNH screening and diagnosis are based upon demonstration of increased erythrocyte susceptibility to lysis by activated complement. These methods are neither very specific nor very sensitive, and have largely been superseded by the rapid, sensitive, and specific flow cytometric analysis of GPI‐linked antigen expression. The protocols in this unit cover immunophenotypic analysis of red blood cells, peripheral blood granulocytes, and peripheral blood monocytes, as well as a four‐color modification for simultaneous analysis of peripheral blood granulocytes and monocytes.
Quantitative Flow Cytometric Analysis of Membrane Antigen Expression
Authors: D'hautcourt Jean‐Luc 
Published: June 2004

AbstractImmunological analysis for cell antigens has been performed by flow cytometry in a qualitative fashion for over thirty years. During that time it has become increasingly apparent that quantitative measurements such as number of antigens per cell provide unique and useful information. This unit on quantitative flow cytometry (QFCM) describes the most commonly used protocols, both direct and indirect, and the major methods of analysis for the number of antibody binding sites on a cell or particle. Practical applications include detection of antigen under‐ or overexpression in hematological malignancies, distinguishing between B cell lymphoproliferative disorders, and precise diagnosis of certain rare diseases.
Immunophenotyping Using a Laser Scanning Cytometer
Authors: Tárnok Attila , Gerstner Andreas O.H. 
Published: June 2004

AbstractThree‐ and four‐color immunophenotyping is routine in traditional flow cytometry, as is measurement of cell proliferation, but there are drawbacks. The techniques cannot analyze cell morphology or permit restaining of cells of interest. This unit describes a slide‐based method of immunophenotyping using a laser scanning cytometer. In general, many assays originally developed for flow can be adapted to LSC. Although the speed of analysis is comparatively slow, LSC has the advantage that cells are not lost. Considerable additional information can be obtained by morphological examination and/or by further staining because specimens can be repeatedly analyzed and archived. The method has potential to become a powerful tool in clinical diagnosis.
Enzymatic Amplification Staining for Cell Surface Antigens
Authors: Kaplan David 
Published: June 2004

AbstractMany molecules of biological significance function at very low levels and are difficult to detect with the standard staining procedures for flow cytometric analysis. This unit describes a technology for cell staining that enhances the specific fluorescence signal by as much as 100‐fold. The system is based on the enzyme‐catalyzed deposition of a tagged molecule. Enzymatic amplification staining is qualitatively different from the inclusion of additional layers of binding molecules because background fluorescence levels are not increased along with the specific signal. The technique is compatible with multicolor staining. An alternate protocol explains the performance of multiple amplifications on the same cell population by adding a peroxide incubation.
Flow Cytometric Assessment ofHLAAlloantibodies
Authors: Bray Robert A. , Gebel Howard M. , Ellis Thomas M. 
Published: June 2004

AbstractAntibodies against HLA molecules are formed in response to exposure to foreign HLA molecules, which can occur as a result of blood transfusion, pregnancy, or transplant. Blood components, particularly those containing cellular elements, are the most common cause of HLA antibodies. This unit describes technical aspects of the flow cytometric crossmatch (FCXM), flow cytometric microparticle assays, and cell‐based flow cytometric screening assays. The collective goal for these assays is to clearly identify the presence of HLA antibody, determine the titer of antibody, and elucidate the specificities (i.e., HLA antigens) to which they will react. Knowledge of this information is critical for organ allocation and accurate assessment of the immunological risk for a patient at the time of transplantation. In addition, the identification of HLA antibodies in blood components may be useful in planning appropriate transfusion support strategies for selected patients.
Differential Staining of DNA and RNA
Authors: Darzynkiewicz Zbigniew , Juan Gloria 
Published: June 2004

AbstractCell cycle analysis by means of differential staining of RNA and DNA permits determination of RNA content, which in turn allows one to discriminate G0 versus G1 cells and to detect cell differentiation. This unit presents two protocols for differential staining, one using the metachromatic dye acridine orange (AO) and the other a combination of pyronin Y (PY) and Hoechst 33342. Each method has its advantages and limitations, and the Hoechst‐PY method is not applicable to single‐laser instruments.
DNA Content Measurement for DNA Ploidy and Cell Cycle Analysis
Authors: Darzynkiewicz Zbigniew , Juan Gloria 
Published: June 2004

AbstractThis unit presents the most common methods of DNA content analysis. The techniques differ primarily in the mode of cell permeabilization (detergents versus prefixation with alcohol) and composition of the stain solution. Protocols are given for analysis of fixed cells, tissue samples, live cells (for subsequent sorting and culture), archival paraffin‐embedded tissue, and apoptotic cells.
Analysis of DNA Denaturation
Authors: Darzynkiewicz Zbigniew , Juan Gloria 
Published: June 2004

AbstractThis 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

AbstractFlow 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

AbstractRNA 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

AbstractHalogenated 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

AbstractGreen 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

AbstractBoth 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

AbstractThe 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

AbstractFlow 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

AbstractIn 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.
Probe Labeling and Fluorescence In Situ Hybridization
Authors: Wiegant J. , Raap A.K. 
Published: June 2004

AbstractThis 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
Processing and Staining of Cell and Tissue Material for Interphase Cytogenetics
Authors: Hopman A.H.N. , Ramaekers F.C.S. 
Published: June 2004

AbstractThis 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

AbstractThe 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

AbstractStandard 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

AbstractFluorescence‐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

AbstractFlow 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

AbstractCell 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

AbstractA 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.
Estimation of Membrane Potential by Flow Cytometry
Authors: Shapiro Howard M. 
Published: June 2004

AbstractIn 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

AbstractPeroxide 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

AbstractThe 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

AbstractChanges 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

AbstractUnderstanding 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.