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Abstracts from the Proceedings of the XXVII National Conference of CytometryEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
No abstract. (Source: Cytometry Part A)
Source: Cytometry Part A - November 17, 2009 Category: Molecular Biology Source Type: journals

Three-dimensional polar representation for multispectral fluorescence lifetime imaging microscopyEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We present here a new approach that considerably simplifies this analysis avoiding complex fitting algorithm strategies and permitting a fast and visual graphical representation of the fluorescence lifetimes. By transforming the experimental data from time domain to frequency domain for each spectral channel, we calculate the multispectral polar representation and demonstrate its interest on multiply fluorescent labeled sample. We further apply it on Förster resonance energy transfer (FRET) experiments and demonstrate that FRET measurements with a high level of precision can be performed. With addition of emission wavelen...
Source: Cytometry Part A - November 12, 2009 Category: Molecular Biology Authors: A. Leray, C. Spriet, D. Trinel, Laurent Héliot Source Type: journals

Per-channel basis normalization methods for flow cytometry dataEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Between-sample variation in high-throughput flow cytometry data poses a significant challenge for analysis of large-scale data sets, such as those derived from multicenter clinical trials. It is often hard to match biologically relevant cell populations across samples because of technical variation in sample acquisition and instrumentation differences. Thus, normalization of data is a critical step before analysis, particularly in large-scale data sets from clinical trials, where group-specific differences may be subtle and patient-to-patient variation common. We have developed two normalization methods that remove technic...
Source: Cytometry Part A - November 7, 2009 Category: Molecular Biology Authors: Florian Hahne, Alireza Hadj Khodabakhshi, Ali Bashashati, Chao-Jen Wong, Randy D. Gascoyne, Andrew P. Weng, Vicky Seyfert-Margolis, Katarzyna Bourcier, Adam Asare, Thomas Lumley, Robert Gentleman, Ryan R. Brinkman Source Type: journals

Flow immunocytochemistry of marker expression in cells from body cavity fluidsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Diagnostic cytology based on the examination of cells from body cavity fluids misses [sim]50% of patients with a proven malignancy. In an earlier study, we used immunohistochemical detection of epithelial membrane antigen expression with flow cytometric detection of DNA aneuploidy to reduce the number of false negatives. In the present study, we have combined DNA flow cytometry with flow cytometric detection of marker expression to analyze cells from body cavity fluids. Seventy-nine specimens of ascites and pleural fluids were analyzed by diagnostic cytology, DNA flow cytometry, and for the expression of the following mark...
Source: Cytometry Part A - November 6, 2009 Category: Molecular Biology Authors: Awtar Krishan, Parvin Ganjei-Azar, Ronald Hamelik, Deepti Sharma, Isildinha Reis, Mehrdad Nadji Source Type: journals

A quantitative measure for alterations in the actin cytoskeleton investigated with automated high-throughput microscopyEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The actin cytoskeleton modulates a large variety of physiological and disease-related processes in the cell. For example, actin has been shown to be a crucial host factor for successful infection by HIV-1, but the underlying mechanistic details are still unknown. Automated approaches open up the perspective to clarify such an issue by processing many samples in a high-throughput manner. To analyze the alterations in the actin cytoskeleton within an automated setting, large-scale image acquisition and analysis were established for JC-53 cells stained for actin. As a quantitative measure in such an automated approach, we sug...
Source: Cytometry Part A - November 6, 2009 Category: Molecular Biology Authors: Julian Weichsel, Nikolas Herold, Maik J. Lehmann, Hans-Georg Kräusslich, Ulrich S. Schwarz Source Type: journals

BH3-only proteins: The death-puppeteer's wiresEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Most cell death in vertebrates proceeds through the intrinsic pathway of apoptosis and results from unregulated increase of mitochondrial membrane permeability. Bcl2-associated X protein (Bax) and Bcl2-antagonist/killer protein (Bak), the effector proapoptotic members of the Bcl-2 family, are, in their active state, the principal accomplices for this permeabilization process. How exactly Bax and Bak are activated has been a matter of major investigation in the last decade, and suitable tools offered by quantitative cytometric methodologies have significantly contributed to the understanding of the function of Bcl-2 family ...
Source: Cytometry Part A - November 6, 2009 Category: Molecular Biology Authors: Fabio Ghiotto, Franco Fais, Silvia Bruno Source Type: journals

Development of small and inexpensive digital data acquisition systems using a microcontroller-based approachEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Fully digital data acquisition systems for use in flow cytometry provide excellent flexibility and precision. Here, we demonstrate the development of a low cost, small, and low power digital flow cytometry data acquisition system using a single microcontroller chip with an integrated analog to digital converter (ADC). Our demonstration system uses a commercially available evaluation board making the system simple to integrate into a flow cytometer. We have evaluated this system using calibration microspheres analyzed on commercial, slow-flow, and CCD-based flow cytometers. In our evaluations, our demonstration data system ...
Source: Cytometry Part A - October 22, 2009 Category: Molecular Biology Authors: Mark A. Naivar, Mark E. Wilder, Robert C. Habbersett, Travis A. Woods, David S. Sebba, John P. Nolan, Steven W. Graves Source Type: journals

Stromal vascular progenitors in adult human adipose tissueEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The in vivo progenitor of culture-expanded mesenchymal-like adipose-derived stem cells (ADSC) remains elusive, owing in part to the complex organization of stromal cells surrounding the small vessels, and the rapidity with which adipose stromal vascular cells adopt a mesenchymal phenotype in vitro. Immunohistostaining of intact adipose tissue was used to identify three markers (CD31, CD34, and CD146), which together unambiguously discriminate histologically distinct inner and outer rings of vessel-associated stromal cells, as well as capillary and small vessel endothelial cells. These markers were used in multiparameter fl...
Source: Cytometry Part A - October 21, 2009 Category: Molecular Biology Authors: Ludovic Zimmerlin, Vera S. Donnenberg, Melanie E. Pfeifer, E. Michael Meyer, Bruno Péault, J. Peter Rubin, Albert D. Donnenberg Source Type: journals

An efficient high-throughput flow cytometric method for estimating DNA ploidy level in plantsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We present an efficient high-throughput flow cytometric method that builds on previously published methods and permits rapid ploidy discrimination in plants. By using Brassica napus L. microspore-derived plants as an example, we describe how 192 leaf tissue samples may be processed and analyzed comfortably by one operator in 6 h from tissue sampling to ploidy determination. The technique involves placing young leaf samples in two 96-well racks, using a bead-beating procedure to release nuclei into a lysis solution, filtering the samples on 96-well filter plates, staining with propidium iodide, and then rapidly estimating D...
Source: Cytometry Part A - October 21, 2009 Category: Molecular Biology Authors: A. Cousin, K. Heel, W. A. Cowling, M. N. Nelson Source Type: journals

Image segmentation and dynamic lineage analysis in single-cell fluorescence microscopyEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
An increasingly common component of studies in synthetic and systems biology is analysis of dynamics of gene expression at the single-cell level, a context that is heavily dependent on the use of time-lapse movies. Extracting quantitative data on the single-cell temporal dynamics from such movies remains a major challenge. Here, we describe novel methods for automating key steps in the analysis of single-cell, fluorescent images - segmentation and lineage reconstruction - to recognize and track individual cells over time. The automated analysis iteratively combines a set of extended morphological methods for segmentation, ...
Source: Cytometry Part A - October 19, 2009 Category: Molecular Biology Authors: Quanli Wang, Jarad Niemi, Chee-Meng Tan, Lingchong You, Mike West Source Type: journals

The importance of Foxp3 antibody and fixation/permeabilization buffer combinations in identifying CD4+CD25+Foxp3+ regulatory T cellsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We present a comparative study of PCH101, 236A/E7, 3G3, 206D, 150D, and 259D/C7 clones of anti-human-Foxp3 antibodies used in combination with five different fixation/permeabilization buffers. Staining for CD25, CD152, and CD127 was also compared between fixation/permeabilization treatments. Promising antibody/buffer combinations were tested in a panel of peripheral blood mononuclear cells from 10 individuals, and then on fresh versus frozen cells from four individuals. Finally, different fluorochromes coupled to two representative antibodies were compared to optimize separation of Foxp3+ from Foxp3- events. Foxp3 gates we...
Source: Cytometry Part A - October 19, 2009 Category: Molecular Biology Authors: Jacqueline P. Law, Dale F. Hirschkorn, Rachel E. Owen, Hope H. Biswas, Philip J. Norris, Marion C. Lanteri Source Type: journals

A novel imaging-based high-throughput screening approach to anti-angiogenic drug discoveryEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The successful progression to the clinic of angiogenesis inhibitors for cancer treatment has spurred interest in developing new classes of anti-angiogenic compounds. The resulting surge in available candidate therapeutics highlights the need for robust, high-throughput angiogenesis screening systems that adequately capture the complexity of new vessel formation while providing quantitative evaluation of the potency of these agents. Available in vitro angiogenesis assays are either cumbersome, impeding adaptation to high-throughput screening formats, or inadequately model the complex multistep process of new vessel formatio...
Source: Cytometry Part A - October 15, 2009 Category: Molecular Biology Authors: Lasse Evensen, David R. Micklem, Wolfgang Link, James B. Lorens Source Type: journals

NBDT (3-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-3-toluene) - A novel fluorescent dye for studying mechanisms of toluene uptake into vital bacteriaEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In this study, a fluorescently labeled toluene analogue dye (3-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-3-toluene; NBDT), flow cytometry, and shot gun proteome analysis were used to follow toluene uptake into bacteria in more detail. The new dye has excitation peaks at 444 and 475 nm and an emission peak at 537 nm. The toluene-degraders P. putida mt-2 and P. putida F1 as well as P. putida KT2440 and E. coli K12 as negative controls were included. To enable quantification of NBDT uptake, carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was added to inactivate NBDT efflux pumps. The porin inhibitor cadaverine was added to...
Source: Cytometry Part A - October 9, 2009 Category: Molecular Biology Authors: H. Sträuber, T. Hübschmann, N. Jehmlich, F. Schmidt, M. von Bergen, H. Harms, S. Müller Source Type: journals

High content image cytometry in the context of subnuclear organizationEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The organization of proteins in space and time is essential to their function. To accurately quantify subcellular protein characteristics in a population of cells with regard for the stochasticity of events in a natural context, there is a fast-growing need for image-based cytometry. Simultaneously, the massive amount of data that is generated by image-cytometric analyses, calls for tools that enable pattern recognition and automated classification. In this article, we present a general approach for multivariate phenotypic profiling of individual cell nuclei and quantification of subnuclear spots using automated fluorescen...
Source: Cytometry Part A - October 8, 2009 Category: Molecular Biology Authors: W. H. De Vos, L. Van Neste, B. Dieriks, G. H. Joss, P. Van Oostveldt Source Type: journals

A routinely applicable way for using FCM in cell enumeration with CFSE-labeled CellBeads as internal standardEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
No abstract. (Source: Cytometry Part A)
Source: Cytometry Part A - October 8, 2009 Category: Molecular Biology Authors: Fan-Fan Cao, Li-Min Xu, Bin Peng, Qiu-Hua Xie, Georges Uzan, Deng-Hai Zhang Source Type: journals

Automated quality assessment of autonomously acquired microscopic images of fluorescently stained bacteriaEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We present an approach based on an artificial neural network (ANN) to assess the quality of such images. Spatially invariant estimators were extracted as ANN input data from subdivided images by low level image processing. Different ANN designs were compared and >400 ANNs were trained and tested on a set of 25,000 manually classified images. The optimal ANN featured a correct identification rate of 94% (3% false positives, 3% false negatives) and could process about 10 images per second. We compared its performance with the image quality assessment by different humans and discuss the difficulties in assigning images to the...
Source: Cytometry Part A - October 8, 2009 Category: Molecular Biology Authors: M. Zeder, E. Kohler, J. Pernthaler Source Type: journals

Flow cytometry without alignment of collection opticsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
This study describes the performance of a new waveguide flow cell constructed from Teflon AF® (TFC) and the potential use of fiber optic splitters to replace collection objectives and dichroic mirrors. The TFC has the unique optical property that the refractive index of the polymer is lower than water and therefore, water filled TFC behaves and functions as a liquid core waveguide. Thus, as cells flow through the TFC and are illuminated by a laser orthogonal to the flow direction, scattered and fluorescent light is directed down the axis of the TFC to a fiber optic. The total signal in the fiber optic is then split into m...
Source: Cytometry Part A - October 2, 2009 Category: Molecular Biology Authors: Greg Sitton, Friedrich Srienc Source Type: journals

ABCG2-associated resistance to Hoechst 33342 and topotecan in a murine cell model with constitutive expression of side population characteristicsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Drug resistant tumor "side-populations," enriched in cancer stem cells and identified by reduced accumulation of Hoechst 33342 under ABCG2-mediated efflux, may compromise therapeutic outcome. Side-population cells have predicted resistance to minor groove ligands, including the DNA topoisomerase I poison topotecan. We have used a stable Hoechst 33342-resistant murine L cell system (HoeR415) to study resistance patterns, removing the need for SP isolation before microarray analysis of gene expression and the tracking of cell cycle dynamics and cytotoxicity. The majority of HoeR415 cells displayed a side-population phenotype...
Source: Cytometry Part A - October 1, 2009 Category: Molecular Biology Authors: Paul J. Smith, Emeline Furon, Marie Wiltshire, Lee Campbell, Graham P. Feeney, Ronald D. Snyder, Rachel J. Errington Source Type: journals

Profiling of polychromatic flow cytometry data on B-cells reveals patients' clusters in common variable immunodeficiencyEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The aim of this study was to find an objective computational approach for phenotype analysis of common variable immunodeficiency (CVID) patients that describes all differences in the six-color space and to form groups of patients using computational methods. CVID is a heterogeneous primary immunodeficiency disorder where molecular defect is recognized in (Source: Cytometry Part A)
Source: Cytometry Part A - October 1, 2009 Category: Molecular Biology Authors: Tomá[scaron] Kalina, Jan Stuchlý, Ale[scaron] Janda, Ond[rcaron]ej Hru[scaron]ák, [Scaron]árka R[uring][zcaron]i[ccaron]ková, Anna [Scaron]edivá, Ji[rcaron]í Litzman, Marcela Vlková Source Type: journals

Green fiber lasers: An alternative to traditional DPSS green lasers for flow cytometryEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Green and yellow diode-pumped solid-state (DPSS) lasers (532 and 561 nm) have become common fixtures on flow cytometers, due to their efficient excitation of phycoerythrin (PE) and its tandems, and their ability to excite an expanding array of expressible red fluorescent proteins. Nevertheless, they have some disadvantages. DPSS 532-nm lasers emit very close to the fluorescein bandwidth, necessitating optical modifications to permit detection of fluorescein and GFP. DPSS 561-nm lasers likewise emit very close to the PE detection bandwidth and also cause unwanted excitation of APC and its tandems, requiring high levels of c...
Source: Cytometry Part A - September 23, 2009 Category: Molecular Biology Authors: William G. Telford, Sergey A. Babin, Serge V. Khorev, Stephen H. Rowe Source Type: journals

Flow cytometry and the stability of phycoerythrin-tandem dye conjugatesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Routine clinical flow cytometric procedures demand rigorous, simple, and reproducible procedures for spectral compensation. The current, often laborious, spectral compensation procedures are the result of variability in instrument settings, instrument performance, and variability in reagents. In particular, the use of tandem dye conjugates necessitates elaborate spectral compensation procedures that need to be applied frequently. Manufacturer, lot number, and handling procedures are considered the key aspects affecting the fluorescence characteristics of tandem dyes. A better understanding of how specific conditions affect...
Source: Cytometry Part A - September 22, 2009 Category: Molecular Biology Authors: Ruud Hulspas, David Dombkowski, Frederic Preffer, Derick Douglas, Brian Kildew-Shah, John Gilbert Source Type: journals

Rapid flow cytometric method for measuring senescence associated [beta]-galactosidase activity in human fibroblastsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Senescence associated-[beta]-galactosidase (SA-[beta]-gal) activity is a widely used marker for cellular senenescence. SA-[beta]-gal activity is routinely detected cytochemically, manually discriminating negative from positive cells. This method is time-consuming, subjective and therefore prone to operator-error. We aimed to optimize a flow cytometric method described by other workers using endothelial cells to better differentiate between populations of fibroblasts in degrees of SA-[beta]-gal activity. Skin fibroblasts were isolated from young (mean age ± SD: 25.5 ± 1.8) and very old (age 90.2 ± 0.3) subjects. Differen...
Source: Cytometry Part A - September 22, 2009 Category: Molecular Biology Authors: Gerard Noppe, Pim Dekker, Corine de Koning-Treurniet, Joke Blom, Diana van Heemst, Roeland W. Dirks, Hans J. Tanke, Rudi G. J. Westendorp, Andrea B. Maier Source Type: journals

Robust signal detection in 3D fluorescence microscopyEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Robust detection and localization of biomolecules inside cells is of great importance to better understand the functions related to them. Fluorescence microscopy and specific staining methods make biomolecules appear as point-like signals on image data, often acquired in 3D. Visual detection of such point-like signals can be time consuming and problematic if the 3D images are large, containing many, sometimes overlapping, signals. This sets a demand for robust automated methods for accurate detection of signals in 3D fluorescence microscopy. We propose a new 3D point-source signal detection method that is based on Fourier ...
Source: Cytometry Part A - September 16, 2009 Category: Molecular Biology Authors: Amin Allalou, Amalka Pinidiyaarachchi, Carolina Wählby Source Type: journals

Exploration of chromatic aberration for multiplanar imaging: Proof of concept with implications for fast, efficient autofocusEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Image-based autofocus determines focus directly from the specimen (as opposed to reflective surface positioning with an offset), but sequential acquisition of a stack of images to measure resolution/sharpness and find best focus is slower than reflective positioning. Simultaneous imaging of multiple focal planes, which is also useful for 3D imaging of live cells, is faster but requires complicated optics. With color CCD cameras and white light sources commonly available, we asked if axial chromatic aberration can be utilized to acquire multiple focal planes simultaneously, and if it can be controlled through a range suffic...
Source: Cytometry Part A - September 15, 2009 Category: Molecular Biology Authors: Martin Weinigel, Albert L. Kellner, Jeffrey H. Price Source Type: journals

Differentiation of alloreactive versus CD3/CD28 stimulated T-lymphocytes using Raman spectroscopy: A greater specificity for noninvasive acute renal allograft rejection detectionEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In this study we explore its specificity. Seventy-five inactivated, 40 alloantigen-activated, and 75 CD3/CD28-activated T cells were analyzed using RS. CD3/CD28-activated peak magnitudes (PM) were 4.3% to 23.9% lower than inactivated PM at positions: 903, 1031, 1069, 1093, 1155, 1326, and 1449 cm-1, with a difference in peak ratio (PR) observed at the 1182:1195 cm-1 position (0.91 ± 0.06 vs. 1.2 ± 0.01, respectively: P = 0.006). Differences in CD3/CD28- and alloantigen-activated PM were observed at: 903, 1031, 1093, 1155, 1326, and 1449 cm-1, with no PR differences at the 1182:1195 cm-1 position (0.91 ± 0.06 vs. 0.86 ±...
Source: Cytometry Part A - September 14, 2009 Category: Molecular Biology Authors: Kristian L. Brown, Olena Y. Palyvoda, Jagdish S. Thakur, Sandra L. Nehlsen-Cannarella, Omar R. Fagoaga, Scott A. Gruber, Gregory W. Auner Source Type: journals

Virtual-core flow cytometryEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Traditional flow cytometers use a sheath fluid to position particles or cells for cytometric measurements, but the need for sheath fluid greatly complicates flow cytometric instrumentation. A cytometric detector that is free of the requirements of sheath fluid can simplify the design of flow cytometers and can extend their use into a number of areas. We designed a flow cytometer that uses a combination of three photodetectors to sense the position of a particle in sample stream. The position-sensitive detectors create a virtual core in the sample stream that eliminates the need for sheath fluid. In this article, we demonst...
Source: Cytometry Part A - September 13, 2009 Category: Molecular Biology Authors: Jarred E. Swalwell, Timothy W. Petersen, Ger van den Engh Source Type: journals

Efficient framework for automated classification of subcellular patterns in budding yeastEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Fluorescent-tagging and digital imaging are widely used to determine the subcellular location of proteins. An extensive publicly available collection of images for most proteins expressed in the yeast S. cerevisae has provided both an important source of information on protein location but also a testbed for methods designed to automate the assignment of locations to unknown proteins. The first system for automated classification of subcellular patterns in these yeast images utilized a computationally expensive method for segmentation of images into individual cells and achieved an overall accuracy of 81%. The goal of the ...
Source: Cytometry Part A - September 13, 2009 Category: Molecular Biology Authors: Seungil Huh, Donghun Lee, Robert F. Murphy Source Type: journals

Automated multichannel fluorescent whole slide imaging and its application for cytometryEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Slide-based image cytometry (SBC) has several advantages over flow cytometry but it is not widely used because of its low throughput, complicated workflow, and high price. Fully automated microscopes became affordable with the advent of whole slide imaging (WSI) and they can be transformed into a cytometer. A MIRAX MIDI automated whole slide imager was used with metal-halide and light emitting diode (LED)-based fluorescent illumination, filter block changer, and a cooled monochrome charge coupled device camera. The MIRAX control software was further developed for fluorescent sample detection, autofocusing, multichannel dig...
Source: Cytometry Part A - September 10, 2009 Category: Molecular Biology Authors: Viktor Sebestyén Varga, Levente Ficsor, Viktor Kamarás, Viktor Jónás, Tibor Virág, Zsolt Tulassay, Béla Molnár Source Type: journals

Automated organelle-based colocalization in whole-cell imagingEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
This study clearly demonstrated its value for investigating subcellular structures and their constituent proteins. © 2009 International Society for Advancement of Cytometry (Source: Cytometry Part A)
Source: Cytometry Part A - September 9, 2009 Category: Molecular Biology Authors: Ben J. Woodcroft, Luke Hammond, Jennifer L. Stow, Nicholas A. Hamilton Source Type: journals

The identification and enumeration of dendritic cell populations from individual mouse spleen and Peyer's patches using flow cytometric analysisEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Dendritic cell (DC) research currently involves pooling of tissues from multiple animals followed by enrichment techniques to obtain sufficient numbers of DCs for analysis. Enrichment techniques take advantage of DC adherence, buoyant density properties, and/or positive or negative selection of cell populations using monoclonal antibodies. However, enrichment techniques may significantly change the maturation and/or activation status of DCs or selectively eliminate one or more subpopulations of DCs. To overcome these drawbacks, we designed a multicolor flow cytometric technique for simultaneous analysis of DC populations f...
Source: Cytometry Part A - September 9, 2009 Category: Molecular Biology Authors: David M. Duriancik, Kathleen A. Hoag Source Type: journals

Identification of a NCR+/NKG2D+/LFA-1low/CD94- immature human NK cell subsetEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
CD56bright natural killer (NK) cells, generated in vitro from CD34+ hematopoietic progenitor cells, were characterized after a 30-day culture with flt3 ligand plus IL-15. Virtually, all CD56bright cells expressed CD117, CD25, natural cytotoxicity receptors (NCRs), NKG2D, CD161, and CD244, while only a subset expressed CD18-CD11a (LFA-1), and CD94 molecule, defining an immature CD56bright/NCRs+/NKG2D+/LFA-1-/CD94- subset. Another small subset of cells expressing CD94 but not LFA-1 integrin was also identified, suggesting that during NK differentiation LFA-1 might be upregulated later than CD94. To verify this hypothesis in ...
Source: Cytometry Part A - September 8, 2009 Category: Molecular Biology Authors: Loris Zamai, Laura Galeotti, Genny Del Zotto, Barbara Canonico, Prisco Mirandola, Stefano Papa Source Type: journals

Approaching clinical proteomics: Current state and future fields of application in cellular proteomicsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In this report, we discuss the recent advances and applications that fulfill the criteria for clinical proteomics with the focus on cellular proteomics (cytoproteomics) as related to preanalytical and analytical standardization and to quality control measures required for effective implementation of these technologies and analytes into routine laboratory testing to generate novel actionable health information. It will then be crucial to design and carry out clinical studies that can eventually identify novel clinical diagnostic strategies based on these techniques and validate their impact on clinical decision making. © 2...
Source: Cytometry Part A - September 8, 2009 Category: Molecular Biology Authors: Rolf Apweiler, Charalampos Aslanidis, Thomas Deufel, Andreas Gerstner, Jens Hansen, Dennis Hochstrasser, Roland Kellner, Markus Kubicek, Friedrich Lottspeich, Edmund Maser, Hans-Werner Mewes, Helmut E. Meyer, Stefan Müllner, Wolfgang Mutter, Michael Neum Source Type: journals

An optimized flow cytometry protocol for analysis of angiogenic monocytes and endothelial progenitor cells in peripheral bloodEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Circulating adult CD34+VEGFR2+ endothelial progenitor cells (EPCs) have been shown to differentiate into endothelial cells, thus contributing to vascular homeostasis. Furthermore, a subset of circulating CD14+ monocytes coexpresses CD16 together with the angiopoietin receptor Tie2 and has been functionally implicated in tumor angiogenesis. However, clinically applicable protocols for flow cytometric quantification of EPCs and Tie2+ monocytes in peripheral blood and a consensus on reference values remain elusive. The number of Tie2+CD14+CD16mid angiogenic monocytes and CD34+VEGFR2+CD45low/- EPCs was assessed in the peripher...
Source: Cytometry Part A - September 7, 2009 Category: Molecular Biology Authors: Mihail Hristov, Susanne Schmitz, Christoph Schuhmann, Thorsten Leyendecker, Philipp von Hundelshausen, Florian Krötz, Hae-Young Sohn, Frans A. Nauwelaers, Christian Weber Source Type: journals

Flow cytometry APC-tandem dyes are degraded through a cell-dependent mechanismEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
This study demonstrates that the APC-tandem dyes are the target of cell-dependent degradation, which may be antagonized. These findings will allow cytometer users to optimize their multicolor panels. © 2009 International Society for Advancement of Cytometry (Source: Cytometry Part A)
Source: Cytometry Part A - September 7, 2009 Category: Molecular Biology Authors: Christine Le Roy, Nadine Varin-Blank, Florence Ajchenbaum-Cymbalista, Rémi Letestu Source Type: journals

Detection of site-specific glycosylation in proteins using flow cytometryEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We tested the possibility that we may express unique peptide probes on cell surfaces, and detect site-specific glycosylation on these peptides using flow cytometry. Such development can enhance the application of flow cytometry to detect and quantify post-translational modifications in proteins. To this end, the N-terminal section of the human leukocyte glycoprotein PSGL-1 (P-selectin glycoprotein ligand-1) was modified to contain a poly-histidine tag followed by a proteolytic cleavage site. Amino acids preceding the cleavage site have a single O-linked glycosylation site. The recombinant protein called PSGL-1 (HT) was exp...
Source: Cytometry Part A - September 3, 2009 Category: Molecular Biology Authors: Deepak Jayakumar, Dhananjay D. Marathe, Sriram Neelamegham Source Type: journals

Measurement of wheat germ agglutinin binding with a fluorescence microscopeEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Signal intensity in fluorescence microscopy is often measured relative to arbitrary standards. We propose a calibration method based on a solution of the same fluorophore, whose binding to cells needs to be quantified. The method utilizes the low sensitivity of intensity to the object distance in wide-field imaging of uniform materials. Liquid layers of slowly varying depth were prepared by immersing a spherical lens into a drop of a fluorophore placed on a slide. Flatfield-corrected images of the contact and surrounding areas showed linear dependence of the gray level on the depth of fluorescent liquid. This allowed conve...
Source: Cytometry Part A - August 31, 2009 Category: Molecular Biology Authors: Michael A. Model, Jennifer L. Reese, Gail C. Fraizer Source Type: journals

Proximity or no proximity: That is the question - But the answer is more complexEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
No abstract. (Source: Cytometry Part A)
Source: Cytometry Part A - August 5, 2009 Category: Molecular Biology Authors: Peter Nagy, János Szöll[odblac]si Source Type: journals

Click-iTTM assay with improved DNA distribution histogramsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In this report, we have shown that after EdU incorporation, nuclei isolated by lysis can be incubated with the Click-iTTM Assay and stained with propidium iodide for generation of DNA histograms with low CV's. This modified procedure results in better DNA histograms by replacing 7-AAD with propidium iodide and also saves processing time by eliminating the fixation and permeabilization steps. © 2009 International Society for Advancement of Cytometry (Source: Cytometry Part A)
Source: Cytometry Part A - August 4, 2009 Category: Molecular Biology Authors: Ronald M. Hamelik, Awtar Krishan Source Type: journals

Multispot live-image autofocusing for high-throughput microscopy of fluorescently stained bacteriaEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Screening by automated high-throughput microscopy has become a valuable research tool. An essential component of such systems is the autonomous acquisition of focused images. Here we describe the implementation of a high-precision autofocus routine for imaging of fluorescently stained bacteria on a commercially available microscope. We integrated various concepts and strategies that together substantially enhance the performance of autonomous image acquisition. These are (i) nested focusing in bright-field and fluorescence illumination, (ii) autofocusing by continuous life-image acquisition during movement in z-direction r...
Source: Cytometry Part A - August 4, 2009 Category: Molecular Biology Authors: M. Zeder, J. Pernthaler Source Type: journals

DNA damage response induced by tobacco smoke in normal human bronchial epithelial and A549 pulmonary adenocarcinoma cells assessed by laser scanning cytometryEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Cigarette smoke (CS) is a major cause of lung cancer and a contributor to the development of a wide range of other malignancies. There is an acute need to develop a methodology that can rapidly assess the potential carcinogenic properties of the genotoxic agents present in CS. We recently reported that exposure of normal human bronchial epithelial cells (NHBEs) or A549 pulmonary carcinoma cells to CS induces the activation of ATM through its phosphorylation on Ser1981 and phosphorylation of histone H2AX on Ser139 ([gamma]H2AX) most likely in response to the formation of potentially carcinogenic DNA double-strand breaks (DS...
Source: Cytometry Part A - August 4, 2009 Category: Molecular Biology Authors: Hong Zhao, Anthony P. Albino, Ellen Jorgensen, Frank Traganos, Zbigniew Darzynkiewicz Source Type: journals

A simple and optimized length estimator for digitized DNA contoursEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The determination of the contour length of DNA imaged by either electron microscopy or atomic force microscopy is frequently required for investigating the physical properties of nucleic acids. Nevertheless, these measurements are often carried out with methods that are not optimized for the curvilinear shape of DNA or are too complex to be of practical use. The aim of this study is to provide a method for the contour length measurements of DNA that is accurate, practical, and computationally simple. Computer simulated DNA fragments were used as experimental benchmarks in order to compute the coefficients a and b of the (n...
Source: Cytometry Part A - August 4, 2009 Category: Molecular Biology Authors: Claudio Rivetti Source Type: journals

Flow cytometric in situ proximity ligation analyses of protein interactions and post-translational modification of the epidermal growth factor receptor familyEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Interactions between members of the epidermal growth factor receptor (EGFR) family mediates cellular responses to ligand stimulation. Measurement of these interactions could provide important information and may prove useful as prognostic markers in malignancy. Therefore, to develop methods to study these interactions in genetically unmodified cells, such as clinical samples, in a sensitive and selective way, with good statistical accuracy, is important. The in situ proximity ligation assay (in situ PLA) was used to quantify homo- and heteromeric interactions between EGFR and HER2 in cultured cells, using flow cytometry as...
Source: Cytometry Part A - August 1, 2009 Category: Molecular Biology Authors: Karl-Johan Leuchowius, Irene Weibrecht, Ulf Landegren, Lars Gedda, Ola Söderberg Source Type: journals

Hierarchical clustering of monoclonal antibody reactivity patterns in nonhuman speciesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Monoclonal antibodies (Mab) are an important resource for defining molecular expression and probing molecular function. The characterization of Mab reactivity patterns, however, can be costly and inefficient in nonhuman experimental systems. To develop a computational approach to the pattern analysis of Mab reactivity, we analyzed a panel of 128 Mab recognizing sheep antigens. Quantitative single parameter flow cytometry histograms were obtained from five cell types isolated from normal animals. The resulting 640 histograms were smoothed using a Gaussian kernel over a range of bandwidths. Histogram features were selected b...
Source: Cytometry Part A - July 28, 2009 Category: Molecular Biology Authors: Juan Pablo Pratt, Qing Zeng, Dino Ravnic, Harold Huss, James Rawn, Steven J. Mentzer Source Type: journals

Quantitative mechanics of endothelial phagocytosis of silicon microparticlesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Endothelia, once thought of as a barrier to the delivery of therapeutics, is now a major target for tissue-specific drug delivery. Tissue- and disease-specific molecular presentations on endothelial cells provide targets for anchoring or internalizing delivery vectors. Porous silicon delivery vectors are phagocytosed by vascular endothelial cells. The rapidity and efficiency of silicon microparticle uptake lead us to delineate the kinetics of internalization. To discriminate between surface-attached and -internalized microparticles, we developed a double fluorescent/FRET flow cytometric approach. The approach relies on que...
Source: Cytometry Part A - July 16, 2009 Category: Molecular Biology Authors: Rita E. Serda, Jianhua Gu, Jared K. Burks, Kim Ferrari, Chiara Ferrari, Mauro Ferrari Source Type: journals

Watson's analytical approach for immunofluorescence histogram analysisEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In this short note, the key points of the histogram analysis method proposed by Watson (Cytometry 2001;43:55-68) were examined. The complete relationship among the mean values of the distributions on which that method is based was derived analytically. It was shown that the symmetry assumption is not necessary, and that the assumption of equal variances of the unlabeled and labeled cell distributions cannot be verified a posteriori. Moreover, a possible shift between the unlabeled cell distribution in the test and the negative control may greatly influence the estimation of the labeled cell percentage. Finally, by using an...
Source: Cytometry Part A - July 13, 2009 Category: Molecular Biology Authors: Francesco Lampariello Source Type: journals

Evaluation of intensity-based ratiometric FRET in image cytometry - Approaches and a software solutionEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The intensity-based ratiometric FRET (fluorescence resonance energy transfer) method is a powerful technique for following molecular interactions in living cells. Since it is not based on irreversibly destroying the donor or the acceptor fluorophores, the time course of changes in FRET efficiency values can be monitored by this method. ImageJ, a sophisticated software tool for many types of image processing allows users to extend it with programs for various purposes. Implementing intensity-based ratiometric FRET with ImageJ vastly enhances the applicability of the FRET method. We developed an efficient ImageJ plugin, RiFR...
Source: Cytometry Part A - July 9, 2009 Category: Molecular Biology Authors: János Roszik, Duarte Lisboa, János Szöll[odblac]si, György Vereb Source Type: journals

Efficient characterization of the TCR repertoire in lymph nodes by flow cytometryEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Analysis of the T-cell receptor (TCR) repertoire by flow cytometry proved to be relevant for investigating T-cell diversity and detecting reactive cells in blood samples. We used this approach to characterize non-malignant T-lymphocytes in lymph nodes and give insights into their origin. The TCR repertoire of CD4+ and CD8+ T-cells from 81 lymph nodes was analyzed with a four-color flow cytometer using a wide panel of 25 anti-V[beta] monoclonal antibodies. Flow cytometry proved to be a useful and informative technique. We demonstrated a diversified TCR-V[beta] repertoire, and only low level expansions, in 53% of the samples...
Source: Cytometry Part A - July 7, 2009 Category: Molecular Biology Authors: D. Salameire, Y. Le Bris, B. Fabre, J. Fauconnier, F. Solly, M. Pernollet, T. Bonnefoix, D. Leroux, J. Plumas, M. C. Jacob Source Type: journals

The flow cytometry shared resource laboratory: Best practices to assure a high-quality, cost-effective partnership with biomedical research laboratoriesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
No abstract. (Source: Cytometry Part A)
Source: Cytometry Part A - July 5, 2009 Category: Molecular Biology Authors: Jonni Moore, Mario Roederer Source Type: journals

Scalable analysis of flow cytometry data using R/BioconductorEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Flow cytometry is one of the fundamental research tools available to the life scientist. The ability to observe multidimensional changes in protein expression and activity at single-cell resolution for a large number of cells provides a unique perspective on the behavior of cell populations. However, the analysis of complex multidimensional data is one of the obstacles for wider use of polychromatic flow cytometry. Recent enhancements to an open-source platform - R/Bioconductor - enable the graphical and data analysis of flow cytometry data. Prior examples have focused on high-throughput applications. To facilitate wider u...
Source: Cytometry Part A - July 5, 2009 Category: Molecular Biology Authors: David J. Klinke II, Kathleen M. Brundage Source Type: journals

Disease-specific biomarker discovery by aptamersEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
RNA and DNA aptamers developed by an in vitro selection process, Systematic Evolution of Ligands by EXponential enrichment (SELEX), comprise a novel class of high-affinity and specific capture agents, which can be easily modified for cytometry and in vivo applications. A novel application of this technique (Cell SELEX) explores the expression of cell surface epitopes that differ between two given cell types or between healthy and diseased cells. Using whole cells as targets, aptamer libraries can be identified that bind to biomarkers expressed by target cells and not by any other cells. Aptamers have been developed that sp...
Source: Cytometry Part A - June 29, 2009 Category: Molecular Biology Authors: Henning Ulrich, Carsten Wrenger Source Type: journals