MedWorm.com provides a medical RSS filtering service. Over 6000 RSS medical sources are combined and output via different filters. This feed contains the latest items from the 'Cytometry Part A' source. Sat, 20 Mar 2010 16:07:50 +0100 http://www.medworm.com/index.php?rid=3382227&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20891 A finite-difference time-domain (FDTD) method is used to study the multiple scattering from many organelle-size particles distributed in a biological cell. Conventional flow cytometry, where the small-angle forward scatter (FSC) intensity and side scatter (SSC) intensity are used for cell characterizations, may have difficulties to differentiate the organelle distributions in biological cells. Based on the FDTD simulations, a light-scattering methodology is proposed here to overcome such a problem. This method differentiates the dense and sparse distributions of organelle-size particles in a cell, by counting the peak numbers in both large-angle FSC and wide-angle SSC, with the multiple scattering effects being considered. Implemented with a wide-angle microfluidic cytometer, the approach ... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=3382227 Fri, 19 Mar 2010 00:00:00 +0100 3382227 http://www.medworm.com/index.php?rid=3373663&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20890 This study was conducted to generate a genetic based reporter to label cells that transitioned from the G0 to G1/S phases of the cell cycle, hypothesizing that the proximal promoter of the Ki67 (Ki67p) gene, a commonly used cytology marker induced during this transition, would contain the suitable regulatory elements to drive marker gene expression. This study reports the cloning and characterization of the 1.5 kb proximal promoter (Ki67p) of the human Ki67 gene. Ki67p driven GFP expression colocalizes in cells with endogenous Ki67 expression and is correlated with cells transitioning through S/G2/M phases of the cell cycle. Treatment Ki67p-GFP expressing HT1080 cells with mitomycin C, an antineoplastic agent, induces P21 and P27 expression, G1/S/G2M block and attenuates Ki67p activity. At... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=3373663 Wed, 17 Mar 2010 00:00:00 +0100 3373663 http://www.medworm.com/index.php?rid=3373665&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20889 Over a decade has passed since publication of the last review on "Cytometry in cell necrobiology." During these years we have witnessed many substantial developments in the field of cell necrobiology such as remarkable advancements in cytometric technologies and improvements in analytical biochemistry. The latest innovative platforms such as laser scanning cytometry, multispectral imaging cytometry, spectroscopic cytometry, and microfluidic Lab-on-a-Chip solutions rapidly emerge as highly advantageous tools in cell necrobiology studies. Furthermore, we have recently gained substantial knowledge on alternative cell demise modes such as caspase-independent apoptosis-like programmed cell death (PCD), autophagy, necrosis-like PCD, or mitotic catastrophe, all with profound connotations to patho... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=3373665 Tue, 16 Mar 2010 00:00:00 +0100 3373665 http://www.medworm.com/index.php?rid=3373664&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20886 Microparticles, which include exosomes, micro-vesicles, apoptotic bodies and apoptotic microparticles, are small (0.05 - 3 [mu]m in diameter), membranous vesicles that can contain DNA, RNA, miRNA, intracellular proteins and express extracellular surface markers from the parental cells. They can be secreted from intracellular multivesicular bodies or released from the surface of blebbing membranes. Circulating microparticles are abundant in the plasma of normal individuals and can be derived from circulating blood cells such as platelets, red blood cells and leukocytes as well as from tissue sources, such as endothelial and placental tissues. Elevated levels of microparticles are associated with various diseases such as thrombosis (platelet microparticles), congestive heart failure (endothe... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=3373664 Tue, 16 Mar 2010 00:00:00 +0100 3373664 http://www.medworm.com/index.php?rid=3362074&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20887 In this report we describe the development and application of a statistically supported flow cytometry-based assay to quantify cell-mediated cytolysis. The assay depends on the use of the fluorescent dye CFSE to distinguish target from effector cells, the DNA intercalating dye 7AAD to distinguish dead from live cell events, and on the establishment of a cytolysis curve that allows for the derivation of statistically robust data. We demonstrate that the cytolysis curve is well described by a four parameter logistic regression model provided that (i) the range of effector to target (E:T) ratios studied allows for full description of the logistic curve, and (ii) an adequate number of data points are collected to estimate the model parameters. We show that the assay is highly reproducible and ... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=3362074 Sat, 13 Mar 2010 00:00:00 +0100 3362074 http://www.medworm.com/index.php?rid=3353773&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20888 A simple and reliable method for preparation of whole nuclei of a common oomycete, Phytophthora infestans, is described for laser flow cytometry. The ease of preparation, the absence of detectable debris and aggregates, and the precision in determinations of DNA content per nucleus improve interpretation and understanding of the genetics of the organism. Phytophthora infestans is the pathogen that causes potato and tomato late blight. The genetic flexibility of P. infestans and other oomycete pathogens has complicated understanding of the mechanisms of variation contributing to shifts in race structure and virulence profiles on important agricultural crops. Significant phenotypic and genotypic changes are being reported in the apparent absence of sexual recombination in the field. Laser fl... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=3353773 Thu, 11 Mar 2010 00:00:00 +0100 3353773 http://www.medworm.com/index.php?rid=3349748&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20878 Human embryonic stem cell (hESC) cultures are heterogeneous and constituting paracrine signals are required to maintain pluripotency. The cellular interplay and dynamic nature of this heterogeneity is not understood. Here, long-term hESC imaging and tracking revealed that hESC heterogeneity is dynamic and hESC self-renewal is dependent on colony-proximal distributions of paracrine signals. Tracking of hESCs forming colonies revealed that a biologically distinct cell type arises at the colony periphery in the absence of feeders. Higher rates of cell death occur in these hESC-derived cells, leading to clonal selection of colony reestablishing cells. hESC-derived feeders co-transferred during passaging promoted rapid colony recovery and expansion and reduced overall clonal selection of self-r... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=3349748 Wed, 10 Mar 2010 00:00:00 +0100 3349748 http://www.medworm.com/index.php?rid=3314366&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20879 This study investigates the effect of febrile temperature and proinflammatory cytokines on phosphatidylserine (PS) expression on the exofacial surface of pRBCs during parasite maturation. The expression of PS on the pRBCs was determined by flow cytometry using fluorescein-labeled annexin V, which specifically binds to PS and a vital nucleic acid fluorochrome for parasite staining. The results showed that PS expression on the surface of pRBCs increased in association with parasite maturation, especially at the late parasite stage. Furthermore, the growth of P. falciparum also accelerated senescence of the uninfected RBCs in parasite cultures. Exposure to febrile temperature led to significant increases in the expression of PS on the surface of pRBCs, particularly at the late parasite stage ... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=3314366 Sat, 27 Feb 2010 00:00:00 +0100 3314366 http://www.medworm.com/index.php?rid=3306178&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20880 This study demonstrates how single-cell CE, MS, and peptide substrates can be combined to identify and measure enzyme activities in single live cells. © 2010 International Society for Advancement of Cytometry (Source: Cytometry Part A) Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=3306178 Thu, 25 Feb 2010 00:00:00 +0100 3306178 http://www.medworm.com/index.php?rid=3306179&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20884 No abstract. (Source: Cytometry Part A) Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=3306179 Wed, 24 Feb 2010 00:00:00 +0100 3306179 http://www.medworm.com/index.php?rid=3287222&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20876 Analyzing cellular morphologies on a cell-by-cell basis is vital for drug discovery, cell biology, and many other biological studies. Interactions between cells in their culture environments cause cells to touch each other in acquired microscopy images. Because of this phenomenon, cell segmentation is a challenging task, especially when the cells are of similar brightness and of highly variable shapes. The concept of topological dependence and the maximum common boundary (MCB) algorithm are presented in our previous work (Yu et al., Cytometry Part A 2009;75A:289-297). However, the MCB algorithm suffers a few shortcomings, such as low computational efficiency and difficulties in generalizing to higher dimensions. To overcome these limitations, we present the evolving generalized Voronoi dia... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=3287222 Fri, 19 Feb 2010 00:00:00 +0100 3287222 http://www.medworm.com/index.php?rid=3279457&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20877 Research into the genetic basis of nervous system development and neurodegenerative diseases requires counting neurons to find out the extent of neurogenesis or neuronal loss. Drosophila is a widely used model organism for in vivo studies. However, counting neurons throughout the nervous system of the intact animal is humanly unfeasible. Automatic methods for cell counting in intact Drosophila are desirable. Here, we show a method called DeadEasy Neurons to count the number of neurons stained with anti-HB9 antibodies in Drosophila embryos. DeadEasy Neurons employs image filtering and mathematical morphology techniques in 2D and 3D, followed by identification of nuclei in 3D based on minimum volume, to count automatically the number of HB9 neurons in vivo. The resultant method has been vali... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=3279457 Wed, 17 Feb 2010 00:00:00 +0100 3279457 http://www.medworm.com/index.php?rid=3279458&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20875 FOXP3 is a key transcription factor expressed by regulatory T cells (Treg cells). However, differences in staining and analysis protocols have led to conflicting results. Moreover, the transient upregulation of FOXP3 that follows activation in non-Treg cells renders the interpretation of FOXP3 data more difficult in humans than in mice. Human peripheral blood mononuclear cells (PBMCs), isolated CD25- or CD25+CD4+ T cells were stained with three different anti-FOXP3 clones (PCH101, 206D, and 259D) alone or in combination, and using different permeabilization methods. FOXP3 expression was evaluated following T cell activation by several pathways. Gating based on a population that did not express FOXP3 (such as CD3-CD4- T cells) allowed for the optimal characterization of Treg cells. The 206D... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=3279458 Tue, 16 Feb 2010 00:00:00 +0100 3279458 http://www.medworm.com/index.php?rid=3266432&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20873 N-acetylcysteine (NAC) is known as an antioxidant and used for mucus viscosity reduction. However, this drug prevents or induces cell death depending on the cell type. The response of steroidogenic luteal cells to NAC is unknown. Our data shows that NAC can behave as an antioxidant or prooxidant in dependency on the concentration and mitochondrial energization. NAC elevated the flowcytometric-measured portion of hypodiploid (dying) cells. This rise was completely abolished by aurintricarboxylic acid, an inhibitor of topoisomerase II. NAC increased the secretion of nitric oxide and cellular nitrotyrosine. An image analysis indicated that cells pretreated with NAC and loaded with DHR showed a fluorescent structure probably elicited by the oxidative product of DHR, rhodamine 123 that sequeste... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=3266432 Fri, 12 Feb 2010 00:00:00 +0100 3266432 http://www.medworm.com/index.php?rid=3266437&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20855 Effects of some detergents - most frequently used in membrane raft studies - on the polymerization properties of actin were examined under in vitro and in vivo conditions, for protein and cellular investigations, respectively. Under in vitro conditions the polymerization rates were measured with pyrene-labeled actin. We found that polymerization rate depended on the detergent concentration by following either biphasic characteristics or only decreasing tendency. The strongest effects were observed at relatively low detergent concentrations. SDS-PAGE electrophoresis and dynamic light-scattering measurements provided further evidences for the size distribution of actin filaments formed under the influence of detergents. Comparing the polymerization rates measured in the presence of different... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=3266437 Thu, 11 Feb 2010 00:00:00 +0100 3266437 http://www.medworm.com/index.php?rid=3266436&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20861 No abstract. (Source: Cytometry Part A) Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=3266436 Thu, 11 Feb 2010 00:00:00 +0100 3266436 http://www.medworm.com/index.php?rid=3266435&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20863 In recent years, slide-based cytometry has become a key technology for polychromatic cytometric investigations, and many efforts have been made to increase the number of measurable fluorochromes for multiparametric analysis. Sequential photobleaching of fluorochromes next to very photostable dyes is one approach for this technology. As the ALEXA dyes are known to be photostable as compared to the conventional fluorochromes FITC, PE (Riggs et al., Am J Pathol 1958;34:1081-1097), and APC, a differentiation within a fluorochrome pair is possible. Here, we have analyzed the newly available NorthernLightsTM secondary antibodies for use in slide-based cytometry and microscopy. Currently, these fluorochrome-conjugates are now available with three distinct excitation- and emission maxima (NL493, N... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=3266435 Thu, 11 Feb 2010 00:00:00 +0100 3266435 http://www.medworm.com/index.php?rid=3266434&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20865 In this study, we incubated Naegleria and Acanthamoeba amebas with several directly conjugated anti-human leukocyte monoclonal antibodies (mAb) for similarly recognized amebic epitopes. CD marker selection was based upon a recognized role of each mAb in phagocyte activation and/or uptake of bacteria. These included CD14, CD45, and CD206. In FCM, only one CD45 antibody demonstrated strong reactivity with both Naegleria fowleri and Naegleria gruberi that was not expressed in similarly tested Acanthamoeba species. Additional testing of N. gruberi by IHC demonstrated reactivity to a different CD45 antibody. Our results suggest a possible utility of using anti-human leukocyte antibodies to screen amebic cells for similarly expressed protein epitopes. In doing so, several important items must be... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=3266434 Thu, 11 Feb 2010 00:00:00 +0100 3266434 http://www.medworm.com/index.php?rid=3266433&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20868 The prognostic significance of HER2 expression in human breast carcinomas is beyond dispute nowadays. The HER family of receptor tyrosine kinases comprises four members (HER1/ErbB1/EGFR, HER2/ErbB2, HER3/ErbB3, and HER4/ErbB4) that act in concert via transactivation and consequently compose a functional signaling unit. Besides HER2 overexpression, coexpression of other HER receptors has substantial impact on course of disease and potential therapeutic benefit. This observation is substantiated by numerous preclinical studies and retrospective studies done on patients with breast cancer. Against this background, the quantification of all HER receptor expressions at the same time would significantly extend the information content revealed by routine diagnosis of breast cancer tissues. Moreov... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=3266433 Thu, 11 Feb 2010 00:00:00 +0100 3266433 http://www.medworm.com/index.php?rid=3248456&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20872 Traditionally, flow cytometry analysis of dendritic cells (DC) has followed a negative selection procedure, often limiting the characterization of individual DC subsets to enumeration. We demonstrate the development, evaluation, and clinical application of a novel 6 color/8 parameter flow cytometry panel to allow enumeration and monitoring of activation status of circulating human myeloid (MDC1) and plasmacytoid (PDC) dendritic cells in human whole blood. Enumeration showed a trend of greater numbers of MDC1s and PDCs being collected for fresh whole blood than frozen PBMCs, with this difference being statistically significant (P = 0.04) for unstimulated PDC enumeration. Intra-assay variation had a coefficient of variation 0.95). Our results on fresh whole blood showed a significant up regu... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=3248456 Sun, 07 Feb 2010 00:00:00 +0100 3248456 http://www.medworm.com/index.php?rid=3248457&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20864 Research involving bacterial pathogens often requires enumeration of bacteria colonies. Here, we present a low-cost, high-throughput colony counting system consisting of colony counting software and a consumer-grade digital camera or document scanner. We demonstrate that this software, called "NICE" (NIST's Integrated Colony Enumerator), can count bacterial colonies as part of a high-throughput multiplexed opsonophagocytic killing assay used to characterize pneumococcal vaccine efficacy. The results obtained with NICE correlate well with the results obtained from manual counting, with a mean difference of less than 3%. NICE is also rapid; it can count colonies from multiple reaction wells within minutes and export the results to a spreadsheet for data processing. As this program is freely ... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=3248457 Sat, 06 Feb 2010 00:00:00 +0100 3248457 http://www.medworm.com/index.php?rid=3238975&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20867 This study was designed to provide a mechanistic rationale for the kinetic assays using cell permeability markers. Uptake of PI by live cells, effect on the cell cycle, long-term proliferation capacity, DNA damage response, and pharmacologic interactions with anticancer drugs were studied using both laser scanning microscopy and laser scanning cytometry. Exposure of human carcinomic alveolar basal epithelial A549 cells in cultures to 1.5 or 7.5 [mu]M of PI for 24 h had minimal effect on cell cycle progression including DNA replication as measured by incorporation of 5[prime]-ethynyl-2-deoxyuridine (EdU) detected by the "click chemistry" approach and measured by laser scanning cytometry. A modest reduction, from 44 to 40% or 33%, in frequency of DNA replicating cells was seen after 48 h at ... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=3238975 Thu, 04 Feb 2010 00:00:00 +0100 3238975 http://www.medworm.com/index.php?rid=3238977&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20862 This report is presented in a manner that is fully compliant with the Minimum Information about a Flow Cytometry Experiment (MIFlowCyt) standard, which was recently adopted by the International Society for Advancement of Cytometry (ISAC) () and incorporated in the publishing policies of Cytometry and other journals. We demonstrate how a MIFlowCyt-compliant report can be prepared with minimal effort, and yet provide the reader with a much clearer picture of the portrayed FCM experiment and data. © 2010 International Society for Advancement of Cytometry (Source: Cytometry Part A) Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=3238977 Wed, 03 Feb 2010 00:00:00 +0100 3238977 http://www.medworm.com/index.php?rid=3238976&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20866 Fluorescence recovery after photobleaching (FRAP) is a tool widely used in studies of dynamic behavior of fluorescently-tagged proteins in live cells. We have analyzed published data on dynamics of various nuclear proteins and note that FRAP protocols and methods of data analysis vary between laboratories. A question arises if the experimental protocol can influence the recovery times. To establish if the FRAP protocol can influence fluorescence half-recovery times, we used various FRAP protocols and studied the dynamics of a GFP-tagged H1 (linker) histone. We demonstrate that fluorescence half-recovery times depend on the bleaching protocol, including the photon flux of the bleaching light. Thus, we conclude that due to differences between protocols and ways of analyzing data, the existin... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=3238976 Wed, 03 Feb 2010 00:00:00 +0100 3238976 http://www.medworm.com/index.php?rid=3211763&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20857 We describe a gating strategy using Ki-67 and side light scatter, also a marker of blastogenesis, which correlates strongly with data from CFSE dilution. We show that this method is a valid tool for measuring RM antigen-specific cellular proliferation ex vivo and can be used as an alternative to CFSE dilution assays. © 2010 International Society for Advancement of Cytometry (Source: Cytometry Part A) Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=3211763 Wed, 27 Jan 2010 00:00:00 +0100 3211763 http://www.medworm.com/index.php?rid=3211769&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20833 In this study, we compared detection of MPs using either light scatter or SYTO 13 staining, testing the hypothesis that, with fluorescence detection with SYTO 13, problems of "noise" with light scatter are reduced and the range of MP sizes detected is increased. In these experiments, particles were obtained from lymphoid cell lines treated in vitro to undergo apoptosis. As these results showed, STYO 13 allowed the detection of 1.5-2.9 times as many particles as did light scatter. The increased sensitivity was observed with three different cell lines and was independent of inducing stimulus. Treatment of fixed and permeabilized MPs with DNase and RNase showed that SYTO 13 binding resulted from interaction with both DNA and RNA. Together, these findings indicate that the nucleic acid content... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=3211769 Tue, 26 Jan 2010 00:00:00 +0100 3211769 http://www.medworm.com/index.php?rid=3211768&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20846 In conclusion, this study confirmed the cellular uptake of ligand-free gold nanoparticles during coincubation apparently without using endocytic pathways. © 2010 International Society for Advancement of Cytometry (Source: Cytometry Part A) Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=3211768 Tue, 26 Jan 2010 00:00:00 +0100 3211768 http://www.medworm.com/index.php?rid=3211767&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20847 Chemical address tags can be defined as specific structural features shared by a set of bioimaging probes having a predictable influence on cell-associated visual signals obtained from these probes. Here, using a large image dataset acquired with a high content screening instrument, machine vision and cheminformatics analysis have been applied to reveal chemical address tags. With a combinatorial library of fluorescent molecules, fluorescence signal intensity, spectral, and spatial features characterizing each one of the probes' visual signals were extracted from images acquired with the three different excitation and emission channels of the imaging instrument. With multivariate regression, the additive contribution from each one of the different building blocks of the bioimaging probes t... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=3211767 Tue, 26 Jan 2010 00:00:00 +0100 3211767 http://www.medworm.com/index.php?rid=3211766&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20851 Histone H1 is an important constituent of chromatin, which undergoes major structural rearrangements during mitosis. However, the role of H1, multiple H1 subtypes, and H1 phosphorylation is still unclear. In normal human fibroblasts, phosphorylated H1 was found located in nuclei during prophase and in both cytoplasm and condensed chromosomes during metaphase, anaphase, and telophase as detected by immunocytochemistry. Moreover, we detected remarkable differences in the distribution of the histone H1 subtypes H1.2, H1.3, and H1.5 during mitosis. H1.2 was found in chromatin during prophase and almost solely in the cytoplasm of metaphase and early anaphase cells. In late anaphase, it appeared in both chromatin and cytoplasm and again in chromatin during telophase. H1.5 distribution pattern re... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=3211766 Tue, 26 Jan 2010 00:00:00 +0100 3211766 http://www.medworm.com/index.php?rid=3211765&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20852 This study was, therefore, undertaken to investigate NO generation ability and the molecular/biochemical characteristics of NOS isoforms in neutrophil precursor cells. The neutrophil precursors were separated on Percoll density gradient and characterized by Giemsa staining, CD markers, and by their size and granularity at various stages of maturation as Bands 1, 2, and 3. Mature neutrophils were efficient in free radical generation and phagocytosis, whereas immature cells had more mitochondria and myeloperoxidase. Amount of NO augmented from immature to mature neutrophils as assessed by fluorescent probe DAF-2DA and Griess reagent. Measurement of NOS enzyme activity further confirmed the functional status of NOS in these cells. NOS isoforms were differentially expressed during neutrophil m... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=3211765 Tue, 26 Jan 2010 00:00:00 +0100 3211765 http://www.medworm.com/index.php?rid=3211764&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20854 We describe a parametric conditional model of microtubule distribution that can generate a microtubule network in intact cells using a persistent random walk approach. The model parameters are physically meaningful as they directly describe the spatial distribution of microtubules and include the number of microtubules as well as the mean of the length distribution. We also present an indirect method for estimating the parameters of the model from three-dimensional fluorescence microscope images of cells that relies on comparing acquired images with simulated images generated from the model. Our results show that our method can reasonably recover parameters for a given query image, and we present the distributions of parameters estimated by our method for a collection of HeLa cell images. ... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=3211764 Tue, 26 Jan 2010 00:00:00 +0100 3211764 http://www.medworm.com/index.php?rid=3200028&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20860 Pyroglutamyl proline-rich oligopeptides, present in the venom of the pit viper Bothrops jararaca (Bj-PROs), are the first described naturally occurring inhibitors of the angiotensin I-converting enzyme (ACE). The inhibition of ACE by the decapeptide Bj-PRO-10c ( (Source: Cytometry Part A) Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=3200028 Sat, 23 Jan 2010 00:00:00 +0100 3200028 http://www.medworm.com/index.php?rid=3200031&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20853 We describe a computerized method to detect and classify follicular adenoma of the thyroid, follicular carcinoma of the thyroid, and normal thyroid based on the nuclear chromatin distribution from digital images of tissue obtained by routine histological methods. Our method is based on determining whether a set of nuclei, obtained from histological images using automated image segmentation, is most similar to sets of nuclei obtained from normal or diseased tissues. This comparison is performed utilizing numerical features, a support vector machine, and a simple voting strategy. We also describe novel methods to identify unique and defining chromatin patterns pertaining to each class. Unlike previous attempts in detecting and classifying these thyroid lesions using computational imaging, ou... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=3200031 Fri, 22 Jan 2010 00:00:00 +0100 3200031 http://www.medworm.com/index.php?rid=3200030&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20858 Legionella is a pathogenic bacterium that establishes and proliferates well in water storage and distribution systems. Worldwide it is responsible for numerous outbreaks of legionellosis, which can be fatal. Despite recent advances in molecular and immunological methods, the official, internationally accepted detection method for Legionella spp. in water samples (ISO 11371) is still based on cultivation. This method has major disadvantages such as a long assay time of 10 days and the detection of cultivable cells only. Therefore, we developed a cultivation-independent, quantitative, and fast detection method for Legionella pneumophila in water samples. It consists of four steps, starting with (1) a concentrating step, in which cells present in one litre of water are concentrated into 5 ml ... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=3200030 Fri, 22 Jan 2010 00:00:00 +0100 3200030 http://www.medworm.com/index.php?rid=3200029&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20859 Monitoring changes in human immune cell populations such as lymphocytes, monocytes, and dendritic cells (DCs) during infectious diseases like human immunodeficiency virus (HIV) is crucial. However, difficulties to identify rare or heterogeneous cell populations can be limiting. For example, to accurately measure DC subsets, eight flow cytometry parameters are ideal. The aim of this work was to analyze the phenotype of human lymphocyte, monocyte, and DC subsets using a single 12-color flow cytometry panel. After erythrocyte lysis, blood from healthy human volunteers was washed and labeled with a cocktail of 12 antibodies. Samples were analyzed on a Becton-Dickinson FACSAriaTM equipped with three lasers. Data were compared with lineage-specific panels using 5-8 Ab combinations per lineage. A... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=3200029 Fri, 22 Jan 2010 00:00:00 +0100 3200029 http://www.medworm.com/index.php?rid=3188597&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20856 The malaria parasite, Plasmodium falciparum, develops within human erythrocytes, consuming host hemoglobin to support its own growth. Reactive oxygen species (superoxide and hydrogen peroxide) are by-products of hemoglobin digestion and are believed to exert significant oxidative stress on the parasite. We have characterized a cell permeant, far red fluorescent nucleic acid-binding dye, SYTO 61, that can be used to distinguish between uninfected and infected erythrocytes in a flow cytometric format. The spectral properties of SYTO 61 make it suitable for use in combination with the fluorescent reactive oxygen species reporter 5-(and-6)-chloromethyl-2[prime],7[prime]-dichlorodihydro-fluorescein diacetate acetyl ester. We have used this probe combination to measure oxidative stress in differ... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=3188597 Wed, 20 Jan 2010 00:00:00 +0100 3188597 http://www.medworm.com/index.php?rid=3128754&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20836 No abstract. (Source: Cytometry Part A) Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=3128754 Wed, 30 Dec 2009 00:00:00 +0100 3128754 http://www.medworm.com/index.php?rid=3118399&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20842 No abstract. (Source: Cytometry Part A) Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=3118399 Thu, 24 Dec 2009 00:00:00 +0100 3118399 http://www.medworm.com/index.php?rid=3118401&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20840 Co-immunoprecipitation is the classical approach for investigating protein-protein interactions. Analysis is generally conducted using the Western blot approach. We set out to investigate whether flow cytometry was a feasible alternative to Western blotting. Using the TCR-CD3 complex as a model for intermolecular interactions in the MA5.8 cell line, FLAG-tagged CD3[zeta]-scFv fusion proteins could be captured on anti-FLAG coupled beads and associated TCR[beta] molecules could be detected by flow cytometry. This association was abrogated by mutations to the CD3[zeta] transmembrane domain. Using multicolor flow cytometry, TCR[beta], CD3[epsiv], and the scFv region of the CD3[zeta] fusion molecule could all be detected from a single sample. This multicolor analysis was then applied to demonst... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=3118401 Wed, 23 Dec 2009 00:00:00 +0100 3118401 http://www.medworm.com/index.php?rid=3118400&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20841 To push the 100-plex envelope of suspension array technology, we have developed fully automated methods to acquire multispectral images of multiplexed quantum-dot (QD) encoded microspheres, to segment them in the images, to classify them based on their color code, and to quantify the multiplexed assays. Instead of coding microspheres with two colors and n levels, microspheres were coded with n colors and two levels (present or absent), thus transforming the classification problem from analog to digital. Images of multiplexed microspheres, sedimented at the bottom of microwells, were acquired through a tunable filter at the peak luminescence wavelength of each QD coding species in the system and the assay label wavelength. Another image of the light scattered from microspheres was captured ... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=3118400 Wed, 23 Dec 2009 00:00:00 +0100 3118400 http://www.medworm.com/index.php?rid=3088726&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20839 It has been reported that exposure to UV light triggers DNA damage response (DDR) seen as induction of [gamma]H2AX not only in S- but also in G1-phase cells. In the present study, in addition to [gamma]H2AX, we assessed other markers of DDR, namely phosphorylation of ATM on Ser1981, of ATM/ATR substrate on Ser/Thr at SQ/TQ cluster domains and of the tumor suppressor p53 on Ser15, in human pulmonary carcinoma A549 cells irradiated with 50 J/m2 of UV-B. Phosphorylation of these proteins detected with phospho-specific Abs and measured by laser scanning cytometry in relation the cell cycle phase was found to be selective to S-phase cells. The kinetics of phosphorylation of ATM was strikingly similar to that of ATM/ATR substrate, peaking at 30 min after UV irradiation and followed by rapid deph... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=3088726 Tue, 15 Dec 2009 00:00:00 +0100 3088726 http://www.medworm.com/index.php?rid=3088728&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20828 In this study, human macrophages were incubated with two prototypic atherogenic LDL modifications enzymatically degraded LDL (E-LDL) and oxidized LDL (Ox-LDL) prepared from the same donor LDL. To detect differences in macrophage lipid storage, fluorescent high-content imaging was used. Lipid droplets were stained using Bodipy 493/503, and the fluorescent phospholipid probe NBD-PE was used to detect endolysosomal phospholipidosis in high-content imaging assays. The phospholipidosis assay was validated using phospholipidosis-inducing cationic amphiphilic drugs. In addition, neutral lipids and phospholipidosis were determined using LipidTOX. Images of 96-well cell culture microtiter plates were captured with multichannel laser-based high-content confocal microscopy, and subsequently cell- and... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=3088728 Mon, 14 Dec 2009 00:00:00 +0100 3088728 http://www.medworm.com/index.php?rid=3088727&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20837 No abstract. (Source: Cytometry Part A) Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=3088727 Mon, 14 Dec 2009 00:00:00 +0100 3088727 http://www.medworm.com/index.php?rid=3078653&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20843 No abstract. (Source: Cytometry Part A) Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=3078653 Fri, 11 Dec 2009 00:00:00 +0100 3078653 http://www.medworm.com/index.php?rid=3070585&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20835 No abstract. (Source: Cytometry Part A) Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=3070585 Wed, 09 Dec 2009 00:00:00 +0100 3070585 http://www.medworm.com/index.php?rid=3056024&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20834 In conclusion, we identified and characterized precursors committed to four different lineages in adult human liver. We also optimized a flow cytometry approach that will be useful in exploring the contribution of these cells to the pathogenesis of liver disease. © 2009 International Society for Advancement of Cytometry (Source: Cytometry Part A) Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=3056024 Fri, 04 Dec 2009 00:00:00 +0100 3056024 http://www.medworm.com/index.php?rid=3022487&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20825 We report here on the next generation flow cytometry standard data file format. FCS 3.1 is a minor revision based on suggested improvements from the community. The unchanged goal of the standard is to provide a uniform file format that allows files created by one type of acquisition hardware and software to be analyzed by any other type.The FCS 3.1 standard retains the basic FCS file structure and most features of previous versions of the standard. Changes included in FCS 3.1 address potential ambiguities in the previous versions and provide a more robust standard. The major changes include simplified support for international characters and improved support for storing compensation. The major additions are support for preferred display scale, a standardized way of capturing the sample vol... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=3022487 Tue, 24 Nov 2009 00:00:00 +0100 3022487 http://www.medworm.com/index.php?rid=3022486&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20827 Microorganisms are recognized by specific phagocyte surface receptors. Liganded receptors then signal a series of events leading to phagocytosis and destruction of the organism by oxidative, lytic, and associated processes. Some organisms, such as Mycobacterium tuberculosis (Mtb), Cryptococcus neoformans (Cf), and others, evade such destruction, surviving and sometimes multiplying within the phagosome to later cause disease. To study such evasion, we developed protocols which permit simultaneous kinetic measurement of early cytoplasmic signaling and of phagosomal pH (pHp) and oxidative burst, on a cell-by-cell basis, of polymorphonuclear (PMN) leukocytes exposed to fluorescently labeled, nonpathogenic Staphylococcus epidermidis (Se). The availability of a new, highly sensitive pH probe, pH... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=3022486 Tue, 24 Nov 2009 00:00:00 +0100 3022486 http://www.medworm.com/index.php?rid=2999041&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20826 No abstract. (Source: Cytometry Part A) Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2999041 Tue, 17 Nov 2009 00:00:00 +0100 2999041 http://www.medworm.com/index.php?rid=2984383&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20802 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 wavelength as third dimension in the polar representation, autofluorescence emitted by the sample is thus clearly identified. Analysis artifacts induced by the sample or by fitt... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2984383 Thu, 12 Nov 2009 00:00:00 +0100 2984383 http://www.medworm.com/index.php?rid=2969900&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20823 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 technical between-sample variation by aligning prominent features (landmarks) in the raw data on a per-channel basis. These algorithms were tested on two independent flow cytome... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2969900 Sat, 07 Nov 2009 00:00:00 +0100 2969900 http://www.medworm.com/index.php?rid=2969903&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20824 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 markers: Ber-EP4, progesterone (PR), MUC4, and thyroid transcription factor-1 (TTF-1). DNA index of equal to or greater than 1.2 was seen in 33/79 (41.7%) of the samples. Sta... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2969903 Fri, 06 Nov 2009 00:00:00 +0100 2969903 http://www.medworm.com/index.php?rid=2969902&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20818 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 suggest a parameter called image coherency. We successfully benchmarked our analysis by calculating coherency for both a biophysical model of the actin cytoskeleton and for ... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2969902 Fri, 06 Nov 2009 00:00:00 +0100 2969902 http://www.medworm.com/index.php?rid=2969901&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20819 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 members. Here, we review the most relevant findings in this field and highlight one common trait that has emerged from the diverse new theories: a crucial role in the con... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2969901 Fri, 06 Nov 2009 00:00:00 +0100 2969901 http://www.medworm.com/index.php?rid=2919550&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20814 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 clearly resolves all eight peaks of a Rainbow microsphere set on both a slow-flow flow cytometer and a retrofitted BD FACScalibur, which indicates it has the sensitivity ... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2919550 Thu, 22 Oct 2009 23:00:00 +0100 2919550 http://www.medworm.com/index.php?rid=2919551&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20813 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 flow cytometry in conjunction with stem/progenitor markers (CD90 and CD117) to further characterize stromal vascular fraction (SVF) subpopulations. Two mesenchymal and two ... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2919551 Wed, 21 Oct 2009 23:00:00 +0100 2919551 http://www.medworm.com/index.php?rid=2915547&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20816 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 DNA ploidy using a plate loader on a BD FACSCanto II flow cytometer. Throughout the sample preparation process, multichannel pipetting allows faster and less error-prone s... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2915547 Wed, 21 Oct 2009 23:00:00 +0100 2915547 http://www.medworm.com/index.php?rid=2915549&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20812 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, and uses a neighborhood-based scoring method for frame-to-frame lineage linking. Our studies with bacteria, budding yeast and human cells, demonstrate the portability and... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2915549 Mon, 19 Oct 2009 23:00:00 +0100 2915549 http://www.medworm.com/index.php?rid=2915548&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20815 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 were set using two gating strategies based on CD127+CD25- "non-Tregs" or based on isotype controls. For Foxp3 staining, the best conditions for fixation/permeabilization we... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2915548 Mon, 19 Oct 2009 23:00:00 +0100 2915548 http://www.medworm.com/index.php?rid=2898206&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20808 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 formation. We therefore developed an organotypic endothelial-mural cell co-culture assay system that reflects several facets of angiogenesis while remaining compatible with high-... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2898206 Thu, 15 Oct 2009 23:00:00 +0100 2898206 http://www.medworm.com/index.php?rid=2879121&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20811 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 study the porin-mediated influx of toluene. Cadaverine reduced NBDT uptake by toluene-grown P. putida mt-2 and F1 by 25% and 42%, respectively, thus revealing an involve... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2879121 Fri, 09 Oct 2009 23:00:00 +0100 2879121 http://www.medworm.com/index.php?rid=2879124&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20807 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 fluorescence mosaic microscopy, optimized image processing tools, and supervised classification. We demonstrate the efficiency of our analysis by determination of differential DNA ... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2879124 Thu, 08 Oct 2009 23:00:00 +0100 2879124 http://www.medworm.com/index.php?rid=2879123&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20809 No abstract. (Source: Cytometry Part A) Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2879123 Thu, 08 Oct 2009 23:00:00 +0100 2879123 http://www.medworm.com/index.php?rid=2879122&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20810 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 correct quality class. The computer program and the documented source code (VB.NET) are provided under General Public Licence. © 2009 International Society for Advancem... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2879122 Thu, 08 Oct 2009 23:00:00 +0100 2879122 http://www.medworm.com/index.php?rid=2857194&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20806 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 multiple fibers by fiber optic splitters to enable measurement of signal intensities at different wavelengths. Optical filters are placed at the terminus of each fiber bef... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2857194 Fri, 02 Oct 2009 23:00:00 +0100 2857194 http://www.medworm.com/index.php?rid=2857196&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20800 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 comparable with that of the side-population resident in the ABCG2 over-expressing A549 lung cancer cell line. Photo-crosslinking showed direct protection against minor g... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2857196 Thu, 01 Oct 2009 23:00:00 +0100 2857196 http://www.medworm.com/index.php?rid=2857195&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20801 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) Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2857195 Thu, 01 Oct 2009 23:00:00 +0100 2857195 http://www.medworm.com/index.php?rid=2827654&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20790 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 crossbeam compensation to reduce spectral overlap into the PE tandems. In this article, we report the development of a new generation of green fiber lasers that can be eng... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2827654 Wed, 23 Sep 2009 23:00:00 +0100 2827654 http://www.medworm.com/index.php?rid=2827656&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20799 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 the variability in emission spectra of tandem dyes can lead to a considerable increase in reliability of measurements and a potential simplification of setup procedures ... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2827656 Tue, 22 Sep 2009 23:00:00 +0100 2827656 http://www.medworm.com/index.php?rid=2827655&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20796 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. Different pH modulators were tested for toxicity. To induce stress-induced senescence, fibroblasts were exposed to rotenone. Senescence was assessed measuring SA-[beta]-gal activ... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2827655 Tue, 22 Sep 2009 23:00:00 +0100 2827655 http://www.medworm.com/index.php?rid=2803479&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20795 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 series. The method consists of two parts, a detector, which is a cosine filter to enhance the point-like signals, and a verifier, which is a sine filter to validate the r... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2803479 Wed, 16 Sep 2009 23:00:00 +0100 2803479 http://www.medworm.com/index.php?rid=2803480&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20788 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 sufficient for practical use. For proof of concept, we theoretically and experimentally explored the focal differences between three narrow wavelength bands on a 3-chip color C... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2803480 Tue, 15 Sep 2009 23:00:00 +0100 2803480 http://www.medworm.com/index.php?rid=2795982&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20797 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 ± 0.09: P = 0.8). Spectral signature separation of CD3/CD28 - and alloantigen-activated groups was 100% specific and sensitive. We conclude that RS can differentiate T cel... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2795982 Mon, 14 Sep 2009 23:00:00 +0100 2795982 http://www.medworm.com/index.php?rid=2795984&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20792 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 demonstrate the efficacy of a virtual-core flow cytometer (VCFC) using test particles, immunofluorescently labeled thymocytes, and raw seawater. The VCFC performs accurate measu... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2795984 Sun, 13 Sep 2009 23:00:00 +0100 2795984 http://www.medworm.com/index.php?rid=2795983&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20793 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 present study was to improve on both the computational efficiency and accuracy of this task. Numerical features derived from applying Gabor filters to small image patches... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2795983 Sun, 13 Sep 2009 23:00:00 +0100 2795983 http://www.medworm.com/index.php?rid=2785450&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20791 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 digitization, and signal correction due to nonuniform illumination. Fluorescent calibration beads were used to verify the linearity of the system. The HistoQuant software pa... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2785450 Thu, 10 Sep 2009 23:00:00 +0100 2785450 http://www.medworm.com/index.php?rid=2785451&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20786 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) Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2785451 Wed, 09 Sep 2009 23:00:00 +0100 2785451 http://www.medworm.com/index.php?rid=2781441&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20794 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 from tissues of individual mice. The spleens and Peyer's patches were mechanically and enzymatically digested, then incubated with a panel of six monoclonal antibody-fluor... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2781441 Wed, 09 Sep 2009 23:00:00 +0100 2781441 http://www.medworm.com/index.php?rid=2781442&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20789 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 vivo, we evaluated the NK cell expression of LFA-1 in both peripheral and umbilical cord blood samples. Interestingly, in these blood fluids, we have identified a lineage... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2781442 Tue, 08 Sep 2009 23:00:00 +0100 2781442 http://www.medworm.com/index.php?rid=2777822&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20779 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. © 2009 International Society for Advancement of Cytometry (Source: Cytometry Part A) Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2777822 Tue, 08 Sep 2009 23:00:00 +0100 2777822 http://www.medworm.com/index.php?rid=2777824&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20772 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 peripheral venous blood of patients with stable coronary artery disease by three-color flow cytometry using specific monoclonal antibodies conjugated to PerCP, PE, PE-Cy7, APC, a... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2777824 Mon, 07 Sep 2009 23:00:00 +0100 2777824 http://www.medworm.com/index.php?rid=2777823&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20774 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) Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2777823 Mon, 07 Sep 2009 23:00:00 +0100 2777823 http://www.medworm.com/index.php?rid=2765422&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20773 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 expressed on the surface of two mammalian cell lines, CHO and HL-60, using a lentiviral delivery approach. Results demonstrate that the N-terminal portion of PSGL-1 (HT) can... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2765422 Thu, 03 Sep 2009 23:00:00 +0100 2765422 http://www.medworm.com/index.php?rid=2751233&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20787 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 conversion of the measured intensity into the number of molecules per unit area. The method was applied to different cell types stained by WGA-Alexa 488 and WGA-TRITC. Consist... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2751233 Mon, 31 Aug 2009 23:00:00 +0100 2751233 http://www.medworm.com/index.php?rid=2673926&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20782 No abstract. (Source: Cytometry Part A) Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2673926 Wed, 05 Aug 2009 23:00:00 +0100 2673926 http://www.medworm.com/index.php?rid=2673930&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20780 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) Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2673930 Tue, 04 Aug 2009 23:00:00 +0100 2673930 http://www.medworm.com/index.php?rid=2673929&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20770 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 rather than at distinct z-positions, (iii) assessment of the quality and topology of a field of view (FOV) by multispot focus measurements, and (iv) acquisition of z-stack... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2673929 Tue, 04 Aug 2009 23:00:00 +0100 2673929 http://www.medworm.com/index.php?rid=2673928&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20778 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 (DSBs). To obtain a more complete view of the DNA damage response (DDR) we explored the correlation between ATM activation, H2AX phosphorylation, activation of Chk2 through ... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2673928 Tue, 04 Aug 2009 23:00:00 +0100 2673928 http://www.medworm.com/index.php?rid=2673927&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20781 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 (ne, no)-characterization [L(ne,no) = ane + bno] so as to minimize the error of the measurements. The data show that, at variance with straight lines, a DNA length estimato... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2673927 Tue, 04 Aug 2009 23:00:00 +0100 2673927 http://www.medworm.com/index.php?rid=2662330&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20771 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 the readout method. Cells were monitored for changes in dimerization patterns and phosphorylation status upon stimulation. The different cell lines displayed varying amo... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2662330 Sat, 01 Aug 2009 23:00:00 +0100 2662330 http://www.medworm.com/index.php?rid=2648593&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20768 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 by SiZer - an analytic tool that identifies statistically significant features. The extracted histogram features were compared and grouped using hierarchical clustering. T... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2648593 Tue, 28 Jul 2009 23:00:00 +0100 2648593 http://www.medworm.com/index.php?rid=2610448&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20769 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 quenching of antibody-conjugated fluorescein isothiocyanate covalently attached to the microparticle surface by attachment of a secondary antibody labeled with an acceptor f... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2610448 Thu, 16 Jul 2009 23:00:00 +0100 2610448 http://www.medworm.com/index.php?rid=2597750&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20757 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 experimental set of histograms, it was shown that the assumptions underlying Watson's analytical method are likely not to be fulfilled in real immunofluorescence data. Â... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2597750 Mon, 13 Jul 2009 23:00:00 +0100 2597750 http://www.medworm.com/index.php?rid=2587758&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20747 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, RiFRET, which calculates FRET efficiency on a pixel-by-pixel basis from ratiometric FRET images. It allows the user to correct for channel cross-talk (bleed-through) and to c... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2587758 Thu, 09 Jul 2009 23:00:00 +0100 2587758 http://www.medworm.com/index.php?rid=2579798&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20767 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. They involved nearly all V[beta] families, were more frequent in the CD8+ subset of older patients, but were not related to pathology. No evidence could be demonstrated... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2579798 Tue, 07 Jul 2009 23:00:00 +0100 2579798 http://www.medworm.com/index.php?rid=2579800&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20742 No abstract. (Source: Cytometry Part A) Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2579800 Sun, 05 Jul 2009 23:00:00 +0100 2579800 http://www.medworm.com/index.php?rid=2579799&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20746 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 use of this platform for flow cytometry, the analysis of a dataset, obtained following isolation of CD4+CD62L+ T cells from Balb/c splenocytes using magnetic microbeads, i... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2579799 Sun, 05 Jul 2009 23:00:00 +0100 2579799 http://www.medworm.com/index.php?rid=2555561&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20766 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 specifically interact with cell surface epitopes of trypanosomes or distinguish between the differences in molecular signature of somatic and cancer cells. Aside from its u... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2555561 Mon, 29 Jun 2009 23:00:00 +0100 2555561 http://www.medworm.com/index.php?rid=2555563&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20749 The platelet-activating factor (PAF) is a proinflammatory lipid present in the fluid of ovarian Graafian follicle. Ovarian blockage of the PAF receptor (PAFr) reduces ovulations in the rat whereas underlying mechanism is poorly understood. Mural granulosa cells (MGC) were mechanically isolated from the theca interna of bovine periovulatory follicle. The mRNA abundance for PAFr, progesterone receptor and cyclooxygenase-2 were measured by real-time PCR. Cytosolic calcium (Ca2+) concentration was assayed by microscopy using Fura-2 AM as indicator, 8-isoprostaglandin F2[alpha] (8-isoPGF2[alpha]) by an ELISA kit. Fluorescent products arising from intracellular oxidation of hydroethidine (HE) and dihydrorhodamine (DHR) were quantified by flow cytometry. The cells expressed PAFr mRNA and PAFr pro... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2555563 Sun, 28 Jun 2009 23:00:00 +0100 2555563 http://www.medworm.com/index.php?rid=2555562&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20760 In this study, the use of the Accuri C6 flow cytometer for the analysis of plant C-values is described. This work indicates that the unusually large dynamic range of the C6, a design feature, coupled to the linearity of fluorescence emission conferred by staining of nuclei using propidium iodide, allows simultaneous analysis of species whose C-values span that of almost the entire described angiosperms. © 2009 International Society for Advancement of Cytometry (Source: Cytometry Part A) Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2555562 Sun, 28 Jun 2009 23:00:00 +0100 2555562 http://www.medworm.com/index.php?rid=2527051&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20764 Flow cytometry is used extensively in stem cell investigations but there is wide variation in the methods used for data analysis between laboratories. Data analysis can be challenging in stem cell biology as there is often no clear distinction between positive and negative populations. We have undertaken a critical appraisal of factors that affect the accuracy of results in stem cell applications. We used mouse embryonic stem cells and determined the expression of three common antigens in stem cell investigations, namely CD15, CD184, and c-kit. We have compared different cell preparation methods and gating strategies and also evaluated the use of isotype controls and unstained cells as controls for the identification of positive populations. The use of a "doublet discriminator" using a sid... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2527051 Fri, 26 Jun 2009 23:00:00 +0100 2527051 http://www.medworm.com/index.php?rid=2504285&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20765 No abstract. (Source: Cytometry Part A) Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2504285 Wed, 24 Jun 2009 23:00:00 +0100 2504285 http://www.medworm.com/index.php?rid=2504286&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20754 High-content flow cytometric screening (FC-HCS) is a 21st Century technology that combines robotic fluid handling, flow cytometric instrumentation, and bioinformatics software, so that relatively large numbers of flow cytometric samples can be processed and analysed in a short period of time. We revisit a recent application of FC-HCS to the problem of cellular signature definition for acute graft-versus-host-disease. Our focus is on automation of the data processing steps using recent advances in statistical methodology. We demonstrate that effective results, on par with those obtained via manual processing, can be achieved using our automatic techniques. Such automation of FC-HCS has the potential to drastically improve diagnosis and biomarker identification. © 2009 International Society... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2504286 Sun, 21 Jun 2009 23:00:00 +0100 2504286 http://www.medworm.com/index.php?rid=2504292&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20752 No abstract. (Source: Cytometry Part A) Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2504292 Tue, 16 Jun 2009 23:00:00 +0100 2504292 http://www.medworm.com/index.php?rid=2504287&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20755 The problem of the quantitative analysis of immunofluorescence histograms "with weak labeling" is considered. In these distributions, the fluorescence intensity mean of labeled cells is slightly above that of unlabeled cells, and therefore there is a considerable overlap between the two populations. The procedure of selecting "by eye" an intensity level that separates labeled from unlabeled cells is evidently inadequate, leading to subjective and not reproducible results. Threshold and subtraction methods, although objective, quick, and easy to use, are also inaccurate. Till now, correct enough estimates of the percentage of labeled cells have been obtained only by applying mathematical modeling methods, that are, however, not very easy to handle. A new method for estimating the labeled ce... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2504287 Tue, 16 Jun 2009 23:00:00 +0100 2504287 http://www.medworm.com/index.php?rid=2504291&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20748 ADAMTS13 is a secreted metalloprotease that cleaves von Willebrand Factor multimers in order to maintain proper homeostasis. A severe deficiency in ADAMTS13 triggers a disorder known as thrombotic thrombocytopenic purpura. At present, ADAMTS13 expression levels are determined by immunoblotting. We established a flow cytometry methodology to detect intracellular ADAMTS13 in liver and kidney cells using a polyclonal antibody, BL154G, and several monoclonal antibodies previously used to detect ADAMTS13 by immunoblotting. The results were validated using confocal microscopy, immunoblotting, and an activity assay (FRETS-VWF73). We show that labeling ADAMTS13 with specific antibodies and detection by flow cytometry yields results that are comparable with previously established methods for ADAMTS... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2504291 Thu, 11 Jun 2009 23:00:00 +0100 2504291 http://www.medworm.com/index.php?rid=2504290&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20753 Flow cytometry (FCM) is used with haematology analyzers (HAs) to count cells and differentiate leukocytes in cerebrospinal fluid (CSF). To evaluate the FCM techniques of HAs, 10 external DGKL trials with CSF controls were carried out in 2004 to 2008. Eight single platform HAs with and without CSF equipment were evaluated with living blood leukocytes and erythrocytes in CSF like DGKL controls: Coulter (LH750,755), Abbott CD3200TM, CD3500TM, CD3700TM, CD4000TM, SapphireTM, ADVIA 120® CSF assay, and Sysmex XE-2100®. Results were compared with visual counting of native cells in Fuchs-Rosenthal chamber, unstained, and absolute values of leukocyte differentiation, assayed by dual platform analysis with immune-FCM (FACSCaliburTM, CD45, CD14) and the chamber counts. Reference values X were compa... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2504290 Thu, 11 Jun 2009 23:00:00 +0100 2504290 http://www.medworm.com/index.php?rid=2504289&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20756 Although cationic liposome-mediated transfection has become a standard procedure, the mechanistic details of the process are unknown. It has been suggested that endocytic uptake of lipoplexes is efficient, and transfectability is largely determined by later steps. In this article, we stained GM1-enriched membrane microdomains, a subclass of lipid rafts, with subunit B of cholera toxin and correlated transfection efficiency with their density by quantitatively evaluating microscopic images. We found a strong anticorrelation between the density of GM1-enriched membrane microdomains and the efficacy of transfection monitored by measuring the expression level of GFP in different cell lines transfected by lipofection using two different transfection agents. These findings imply that GM1-enriche... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2504289 Thu, 11 Jun 2009 23:00:00 +0100 2504289 http://www.medworm.com/index.php?rid=2504288&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20758 B cells are very heterogeneous, consisting of more than 109 B-cell clones with distinct specificities for antigens in each individual. To identify single B cells with antigen specificity, we have been developing cell microarray technology using microwell array chips whose microwells each capture a single B cell. Using microwell array chips, we detected antigen-specific B cells by monitoring antigen-induced intracellular Ca2+ mobilization with a CCD scanner (MAC-CCD system) or the binding of fluorescence-labeled antigen to cells with a confocal laser scanner. We retrieved target cells from the chip, cloned immunoglobulin genes, and produced antigen-specific antibodies. However, these methods present some difficulties: the former technique could not detect cells whose frequency was less than... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2504288 Thu, 11 Jun 2009 23:00:00 +0100 2504288 http://www.medworm.com/index.php?rid=2462764&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20750 We present robust and efficient algorithms to automate the measurement of nuclear movement and germ tube extension rates in living fungal networks. The aim is to facilitate the understanding of the dynamics and regulation of nuclear migration in growing fungal colonies. The proposed methodology combines a cascade correlation filter to identify nuclear centers from which 2D nuclear velocities are determined and a level set algorithm for centerline extraction to monitor spore (conidial) germling growth. We show how the proposed cascaded filter improves spatial resolution in the presence of noise and is robust when fluorescently labeled nuclei with different intensities are in close proximity to each other. The performance of the filter is evaluated by simulation in comparison to the well kno... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2462764 Mon, 08 Jun 2009 04:00:00 +0100 2462764 http://www.medworm.com/index.php?rid=2462768&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20735 This study demonstrates the influence of different experimental conditions on this nonspecific fluorescence proportion. IGR1 melanoma cells as a model were specifically altered in cell volume and protein content by depolarizing treatments or cell cycle synchronization. Flow cytometry was performed over a wide range of extracellular DiBAC4(3) concentrations. Fixation and increase in protein content led to a nonspecifically enhanced fluorescence, while changes in the amount of free intracellular dye as a result of altered cell volume proved to be negligible. To establish a calibration curve using totally depolarized cells, the pore-forming action of gramicidin should be preferred to fixation. Below 100 nM DiBAC4(3), the logarithmic relation between cell fluorescence and dye concentration tur... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2462768 Fri, 05 Jun 2009 04:00:00 +0100 2462768 http://www.medworm.com/index.php?rid=2462767&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20738 Current methods for monitoring E3 ubiquitin ligase activity in cell culture or in vivo are limited. As a result, the degradation of cellular targets by many E3 ubiquitin ligases in live cells has not yet been examined. For this study, a target of an E3 ubiquitin ligase was expressed as a fluorescently labeled protein in cell culture. If the E3 ubiquitin ligase mediates the degradation of a target protein in cell culture, it is expected that the target will show a reduced fluorescence signal by FCM analysis. We initially used the E3 ubiquitin ligase, herpes simplex virus type 1 (HSV-1) infected cell protein 0 (ICP0) and one of its targets, promyelocytic leukemia (PML) protein, to determine the feasibility of our approach. Cells expressing a PML-GFP fusion protein were selected by cell sorti... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2462767 Fri, 05 Jun 2009 04:00:00 +0100 2462767 http://www.medworm.com/index.php?rid=2462766&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20739 No abstract. (Source: Cytometry Part A) Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2462766 Fri, 05 Jun 2009 04:00:00 +0100 2462766 http://www.medworm.com/index.php?rid=2462765&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20741 Positive selection, sorting, and collection of single cells from within a heterogeneous population are required for many biological studies. We recently demonstrated a miniaturized cell array for this purpose; however, on-chip pre-enrichment and isolation of specific target cells would provide significant value for cell isolation. In the current work, mixed cell samples of fewer than 30,000 cells were used for panning by means of on-array antibody-capture to pre-enrich the target population. The cell surface receptors Fc[epsiv]R1, c-Kit, and ErbB2 were used for positive selection of RBL, RBL, and SK-BR-3 cells, respectively, from the mixed population. The capture efficiency, selectivity, and enrichment for the target cells were calculated and compared with fibronectin-coated controls. As e... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2462765 Fri, 05 Jun 2009 04:00:00 +0100 2462765 http://www.medworm.com/index.php?rid=2446298&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20734 Heterochromatin protein 1 (HP1) was originally identified as a constitutive component of heterochromatin. However it is recognized now that it plays an important role in a number of dynamic processes in the cell nucleus, including transcriptional repression and regulation of euchromatic genes. Recent reports demonstrate that HP1 may be involved in the DNA damage response. Two seemingly contradictory phenomena have been observed - HP1 detachment from chromatin and HP1 recruitment to damaged DNA foci. Based on quantitative FRAP and FLIP studies carefully designed to minimize phototoxicity, we demonstrate that HP1 is recruited to the damaged regions in hetero- as well as euchromatin within a few minutes after damage. © 2009 International Society for Advancement of Cytometry (Source: Cytometr... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2446298 Sat, 30 May 2009 04:00:00 +0100 2446298 http://www.medworm.com/index.php?rid=2427051&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20740 In this study, topologically quantified nuclear patterns of methylated cytosine and global nuclear DNA are utilized as signatures of cellular response to the treatment of cultured cells with the demethylating anti-cancer agents: 5-azacytidine (5-AZA) and octreotide (OCT). Mouse pituitary folliculostellate TtT-GF cells treated with 5-AZA and OCT for 48 hours, and untreated populations, were studied by immunofluorescence with a specific antibody against 5-methylcytosine (MeC), and 4,6-diamidino-2-phenylindole (DAPI) for delineation of methylated sites and global DNA in nuclei (n = 163). Cell images were processed utilizing an automated 3D analysis software that we developed by combining seeded watershed segmentation to extract nuclear shells with measurements of Kullback-Leibler's (K-L) dive... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2427051 Thu, 21 May 2009 04:00:00 +0100 2427051 http://www.medworm.com/index.php?rid=2413418&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20737 We describe a golden fluorescent apoptosis detection tool, which we generated by a fusion of golden fluorescent protein (GdFP) with human annexin A5 (anxA5). GdFP was obtained by replacement of tryptophan at position 66 with 4-aminotryptophan in the chromophore of enhanced cyan fluorescent protein. The GdFP-anxA5 construct combines highly desirable features originating from both fusion partners. These include (i) strong binding to membrane phosphatidylserine patches of apoptotic cells in the presence of Ca2+ which is brought about by anxA5, (ii) the stable and homogeneous monomeric state, (iii) as well as the red-shifted fluorescence maximum at 574 nm originating from GdFP. We found that GdFP-anxA5 is equally well applicable for apoptosis studies as a routinely used fluorescein 5[prime]-is... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2413418 Fri, 15 May 2009 04:00:00 +0100 2413418 http://www.medworm.com/index.php?rid=2413419&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20736 We have proposed a technique for transforming reticulocyte (RET) flow cytometry count into the age distribution allowing for a quantitative characterization of RETs dynamics. This technique was evaluated in homeostatic and erythropoietically stimulated rats. We administered a single intravenous dose (4,050 IU/kg) of recombinant human erythropoietin to Wistar rats, then stained their RETs with thiazole orange, and measured the distribution of the fluorescent signal using flow cytometry. The RET age distribution was determined assuming an exponential decline of RET RNA. The RET distribution over time was characterized by median age, production rate of mature red blood cell in the blood, bone marrow and blood maturation times, and RET loss from the blood. Erythropoietin was found to cause com... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2413419 Tue, 12 May 2009 04:00:00 +0100 2413419 http://www.medworm.com/index.php?rid=2380141&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20733 Nuclear variation in size and shape and genomic instability are hallmarks of dedifferentiated cancer cells. Although micronuclei are a typical long-term consequence of DNA damage, their contribution to chromosomal instability and clonal diversity in cancer disease is unclear. We isolated cancer cells with or without micronuclei to perform genomic analysis. Cell suspensions of HT1080 fibrosarcoma cells from either 2D culture or after isolation from 3D collagen matrix culture were stained with Hoechst 33342 and after classification for presence or absence of a micronucleus via bright-field and epifluorescence microscopy, cells were individually aspirated with a micropipette. Subsequently, whole-genome amplification and single-cell comparative genomic hybridization (CGH) were applied to detec... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2380141 Thu, 30 Apr 2009 04:00:00 +0100 2380141 http://www.medworm.com/index.php?rid=2380142&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20729 Analysis of female mammalian germ cells has been hindered by difficulties in isolating high purity germ cell populations from embryonic and fetal gonads. Meiotic prophase stage oocytes are particularly difficult to isolate due to the lack of suitable surface markers. Oct4 promoter driven GFP expression has been used to distinguish germ cells/oocytes (GFP positive) from somatic cells (GFP negative), however, the requirement for transgenic animals has limited the use of this technique. We analyzed the side- and forward scattering properties of living cell populations obtained from fetal ovaries of Oct4-GFP transgenic and wild-type mice. On the basis of these measurements, we defined criteria that allow the discrimination and identification of germ cells and somatic cells within cell suspensi... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2380142 Wed, 29 Apr 2009 04:00:00 +0100 2380142 http://www.medworm.com/index.php?rid=2340327&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20727 The aim of this study was to assess the potential DNA damage response (DDR) to four supravitally used biomarkers Hoechst 33342 (Ho 42), DRAQ5, DyeCycle Violet (DCV), and SYTO 17. A549 cells were exposed to these biomarkers at concentrations generally applied to live cells and their effect on histone H2AX (Ser 139), p53 (Ser15), ATM (Ser1981), and Chk2 (Thr68) phosphorylation was assessed using phospho-specific Abs. Short-term treatment with Ho 42 led to modest degree of ATM activation with no evidence of H2AX, Chk2, or p53 phosphorylation. However, pronounced ATM, Chk2, and p53 phosphorylation and perturbed G2 progression were seen after 18 h. While short-term treatment with DRAQ5 induced ATM activation with no effect on H2AX, Chk2, and p53, dramatic changes marked by a high degree of H2AX... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2340327 Sat, 18 Apr 2009 04:00:00 +0100 2340327 http://www.medworm.com/index.php?rid=2340329&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20723 No abstract. (Source: Cytometry Part A) Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2340329 Wed, 08 Apr 2009 04:00:00 +0100 2340329 http://www.medworm.com/index.php?rid=2340328&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20726 Flow cytometric analysis provides a rapid screen for abnormalities of polymorphonuclear neutrophils (PMN) function and reflect their behavior in vivo more accurately. This review summarizes the major fluorescent probes used to study PMN oxidative burst and apoptosis using flow cytometry (FCM). We also provide examples of FCM studies in physiological and pathological situations, illustrating the advantages of FCM for assessment of PMN oxidative burst and PMN apoptosis. These data point to the role of FCM in detecting primary immunodeficiencies such as IRAK4 deficiency and support the use of the assessment of the PMN oxidative burst for routine testing in patients with bacterial infections. We also demonstrate the utility of whole-blood analysis using FCM for a better understanding of PMN fu... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2340328 Wed, 08 Apr 2009 04:00:00 +0100 2340328 http://www.medworm.com/index.php?rid=2283631&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20716 Cancer spheroids are a valuable model for screening anticancer strategies. However, studies are published using various numbers of spheroids and sections per spheroid. Here, we establish the sample size requirements for valid screening strategies to treat glioma: how many spheroids per experimental group and how many sections per spheroid are required to detect one-third reduction in an endpoint measurement after treatment? From two glioblastoma patients, 32 untreated organotypic spheroids were cultured and sectioned entirely (14-100 sections per spheroid). The viable fraction was determined as endpoint by automated image analysis in sections and used to establish the minimally-detectable difference between a treatment and reference group. Variance was considerable with a coefficient of va... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2283631 Sun, 22 Mar 2009 04:00:00 +0100 2283631 http://www.medworm.com/index.php?rid=2273565&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20718 Tumor cells in blood of patients with metastatic carcinomas have been associated with poor survival prospects. Further characterization of these cells may provide further insights into the metastatic process. Circulating Tumor Cells (CTC) were enumerated in 7.5 mL of blood with the CellSearchTM system. After enumeration of Cytokeratin+, CD45-, nucleated cells, the cells are fixed in the cartridge while maintaining their original position. Cartridges were hybridized with FISH probes against the centromeric regions of chromosome 1, 7, 8, and 17. Next fluorescence images of the FISH probes of the previous identified CTC were acquired. Leukocytes surrounding the CTC were used as internal controls. The number of copies of chromosome 1, 7, 8, and 17 could be determined in 118 CTC containing bloo... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2273565 Wed, 18 Mar 2009 04:00:00 +0100 2273565 http://www.medworm.com/index.php?rid=2273569&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20708 In conclusion, the peak day of mutant expression occurs only when cells recover from the toxic effects of the mutagen. A fraction of cells originally quantified as mutants are lost over time due to lethal deletions and slower growth. © 2009 International Society for Advancement of Cytometry (Source: Cytometry Part A) Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2273569 Mon, 16 Mar 2009 04:00:00 +0100 2273569 http://www.medworm.com/index.php?rid=2273567&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20714 Image cytometry still faces the problem of the quality of cell image analysis results. Degradations caused by cell preparation, optics, and electronics considerably affect most 2D and 3D cell image data acquired using optical microscopy. That is why image processing algorithms applied to these data typically offer imprecise and unreliable results. As the ground truth for given image data is not available in most experiments, the outputs of different image analysis methods can be neither verified nor compared to each other. Some papers solve this problem partially with estimates of ground truth by experts in the field (biologists or physicians). However, in many cases, such a ground truth estimate is very subjective and strongly varies between different experts. To overcome these difficulti... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2273567 Mon, 16 Mar 2009 04:00:00 +0100 2273567 http://www.medworm.com/index.php?rid=2213672&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20713 With a combinatorial library of bioimaging probes, it is now possible to use machine vision to analyze the contribution of different building blocks of the molecules to their cell-associated visual signals. For this purpose, cell-permeant, fluorescent styryl molecules were synthesized by condensation of 168 aldehyde with 8 pyridinium/quinolinium building blocks. Images of cells incubated with fluorescent molecules were acquired with a high content screening instrument. Chemical and image feature analysis revealed how variation in one or the other building block of the styryl molecules led to variations in the molecules' visual signals. Across each pair of probes in the library, chemical similarity was significantly associated with spectral and total signal intensity similarity. However, ch... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2213672 Thu, 26 Feb 2009 05:00:00 +0100 2213672 http://www.medworm.com/index.php?rid=2203252&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20707 Barrier function and shape changes of endothelial cells (EC) are regulated by phosphorylation/dephosphorylation of key signaling and contractile elements. EC contraction results in intercellular gap formation and loss of the selective vascular barrier to circulating macromolecules. EC dysfunction elicited by thrombin was found to correlate with actin microfilament redistribution. It is known that calcineurin (Cn) is involved in thrombin-induced EC dysfunction because inhibition of Cn potentiates PKC activity and the phosphorylation state of EC myosin light chain is also affected by Cn activity. Immunofluorescent detection of Cn catalytic subunit (CnA) isoforms coexpressed with GFP was visualized on paraformaldehyde (PFA) fixed bovine pulmonary artery endothelial cells (BPAEC). Actin microf... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2203252 Sun, 22 Feb 2009 05:00:00 +0100 2203252 http://www.medworm.com/index.php?rid=2203253&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20712 Using the nucleoside analogue EdU (5-ethynyl-2[prime]-deoxyuridine) for thymidine substitution instead of BrdU (5-bromo-2[prime]-deoxyuridine) in cell proliferation assays has recently been proposed. However, the effect of EdU on cell viability, DNA synthesis, and cell cycle progression and consequently its usability for dynamic cell proliferation analysis in vitro has not been explored. We compared the effect of EdU and BrdU incorporation into SK-BR-3 and BT474 breast cancer cells and the impact on cell cycle kinetics, cell viability, and DNA damage. We found that EdU can be used not only for pulse but also for continuous cell labeling and henceforth in high resolution EdU/Hoechst quenching assays. BrdU and EdU proliferation assays based on click chemistry revealed comparable results. How... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2203253 Fri, 20 Feb 2009 05:00:00 +0100 2203253 http://www.medworm.com/index.php?rid=2165437&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20706 Flow cytometers designed to analyze large particles are enabling new applications in biology and chemistry. Similarly, flow spectroscopy approaches are extending the capabilities of the flow cytometry platform. Here, we report on the adaptation of a commercial large particle analyzer to measure fluorescence and Raman spectra of individual particles at high speeds. We modified a Union Biometrica COPAS Plus instrument to allow red excitation and optical fiber-based light collection and spectral analysis using a spectrograph and CCD array detector. These modifications did not compromise the ability of the instrument to resolve different sized particles based on their extinction and time of flight signals. The modified instrument has the sensitivity and spectral resolution to measure the fluor... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2165437 Sun, 08 Feb 2009 05:00:00 +0100 2165437 http://www.medworm.com/index.php?rid=2148738&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20705 Physical interactions between different cell types are a requirement for the initiation and maintenance of immune responses. The distribution pattern of cells within a tissue may result from specific cell-cell-interactions or random distribution. Tissue architecture, degree of inflammation, frequencies of cells, number of contact partners, cell type, and size as well as cell movement and contact time determine the distribution of cells within tissues. We developed a matrix model to determine the degree of expected random distribution of two cell types (A and B) and cell-cell-contacts within tissue sections. The model predictions were compared with experimental data derived from immunofluorescence microscopy. We implemented a computer algorithm for automatic image analysis to visualize and ... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2148738 Sat, 31 Jan 2009 05:00:00 +0100 2148738 http://www.medworm.com/index.php?rid=2148739&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20703 Flow cytometry is an important drug discovery tool because it permits high-content multiparameter analysis of individual cells. A new method dramatically enhanced screening throughput by multiplexing many discrete fixed cell populations; however, this method is not suited to assays requiring functional cellular responses. HEK293 cells were transfected with unique mutant glycine receptors. Mutant receptor expression was confirmed by coexpression of yellow fluorescent protein (YFP). Commercially available cell-permeant dyes were used to label each glycine receptor expressing mutant with a unique optical code. All encoded cell lines were combined in a single tube and analyzed on a flow cytometer simultaneously before and after the addition of glycine receptor agonist. We decoded multiplexed c... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2148739 Fri, 30 Jan 2009 05:00:00 +0100 2148739 http://www.medworm.com/index.php?rid=2109186&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20704 We report here the use of a phosphatidylcholine (PC) derivative of BODIPY 581/591, which transfers much less rapidly between membranes. This allows the analysis of oxidative stress in individual cells and in different compartments within cells. Quantitative imaging and flow cytometry were used to measure the ratio of fully conjugated to oxidized probe in model systems and in Plasmodium falciparum-infected erythrocytes. We observed an increase in the oxidation of the parasite-associated BODIPY 581/591-PC as the intraerythrocytic parasite matures. By contrast, BODIPY 581/591-PC associated with the erythrocyte membrane experiences a low level of oxidation even in the later stages of parasite development. Treatment with a pro-oxidant compound caused increased oxidation of the probe in the para... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2109186 Fri, 16 Jan 2009 05:00:00 +0100 2109186 http://www.medworm.com/index.php?rid=2109187&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20697 No abstract. (Source: Cytometry Part A) Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2109187 Thu, 15 Jan 2009 05:00:00 +0100 2109187 http://www.medworm.com/index.php?rid=2088535&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20701 Rapid movements of live tissues during the acquisition of 3D image stacks can result in misalignments between successive image slices. The remodeling of the muscles in Drosophila metamorphosis is an example where sporadic motion during image acquisition impede image analysis and volume visualization. Most of the image stack registration algorithms applied in microscopy are aimed at the linear alignment of fixed histological sections. However, live muscles are nonrigid objects and their contractions and relaxations represent nonlinear transformations that cannot be properly rectified by applying purely linear registration methods. We developed a fully automated area-based nonrigid stack registration (NSR) method that minimizes the mean square error of intensities between successive image sl... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2088535 Thu, 08 Jan 2009 05:00:00 +0100 2088535 http://www.medworm.com/index.php?rid=2063802&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20700 Quantification of cardiomyocyte contraction is usually obtained by measuring globally cell shortening from the displacement of cell extremities. We developed a correlation-based optical flow method, which correlates the whole-cell temporal pattern with a precise quantification of the intracellular strain wave at the sarcomeres level. A two-dimensional image correlation analysis of cardiomyocytes phase-contrast images was developed to extract local cell deformations from videomicroscopy time-lapse sequences. Test images, synthesized from known intensity displacement fields, were first used to validate the method. Intracellular strain fields were then computed from videomicroscopy time-lapse sequences of single adult and neonatal cardiomyocytes. The propagation of the sarcomeres contraction-... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2063802 Thu, 25 Dec 2008 05:00:00 +0100 2063802 http://www.medworm.com/index.php?rid=2049897&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20699 Telomeres are complex end structures that confer functional integrity and positional stability to human chromosomes. Despite their critical importance, there is no clear view on telomere organization in cycling human cells and their dynamic behavior throughout the cell cycle. We investigated spatiotemporal organization of telomeres in living human ECV-304 cells stably expressing telomere binding proteins TRF1 and TRF2 fused to mCitrine using four dimensional microscopy. We thereby made use of controlled light exposure microscopy (CLEM), a novel technology that strongly reduces photodamage by limiting excitation in parts of the image where full exposure is not needed. We found that telomeres share small territories where they dynamically associate. These territories are preferentially posit... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2049897 Fri, 19 Dec 2008 05:00:00 +0100 2049897 http://www.medworm.com/index.php?rid=2049899&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20696 No abstract. (Source: Cytometry Part A) Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2049899 Thu, 18 Dec 2008 05:00:00 +0100 2049899 http://www.medworm.com/index.php?rid=2049898&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20698 A caspase sensor based on Förster resonance energy transfer between fluorescent proteins is reported. Enhanced cyan fluorescent protein anchored to the inner leaflet of the plasma membrane of living cells is optically excited by an evanescent electromagnetic field and transfers its excitation energy via a spacer (DEVD) to an enhanced yellow fluorescent protein. Upon apoptosis, DEVD is cleaved and energy transfer is disrupted, as proven by pronounced changes in fluorescence spectra and decay times. Fluorescence spectroscopy and lifetime imaging (FLIM) is combined with total internal reflection fluorescence microscopy (TIRFM) for selective detection of this membrane-bound caspase sensor. Fluorophores of the cytoplasm are thus excluded, and the signal-to-background ratio is increased conside... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2049898 Thu, 18 Dec 2008 05:00:00 +0100 2049898 http://www.medworm.com/index.php?rid=2034968&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20694 No abstract. (Source: Cytometry Part A) Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2034968 Sun, 14 Dec 2008 05:00:00 +0100 2034968 http://www.medworm.com/index.php?rid=2034970&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20661 The ability of brewing yeast to flocculate is an important feature for brewing of qualitatively good beer. Flocculation involves two main cell wall structures, which are the flocculation proteins (flocculins) and mannans, to which these flocculins bind. Unfortunately, in practice, the flocculation ability may get lost after several repitches. Flow cytometry was employed to analyze glucose and mannose structures of the cell surface by application of fluorescent lectins. Validation of the expression of the flocculin genes Lg-FLO1, FLO1, FLO5, and FLO9 was carried out using microarray techniques. SDS-PAGE, western blot, and ESI-MS/MS analyses served to isolate and determine yeast cell flocculins. Mannose and glucose labeling with fluorescent lectins allowed differentiating powdery and floccul... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2034970 Fri, 12 Dec 2008 05:00:00 +0100 2034970 http://www.medworm.com/index.php?rid=2034969&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20687 In this report, we have used a supercontinuum (SC) white light laser as an excitation source for flow cytometry. By selectively filtering the wavelength of interest, almost any laser wavelength in the visible spectrum can be separated and used for flow cytometric analysis. The white light lasers used in this study were integrated into a commercial flow cytometry platform, and a series of high-transmission bandpass filters used to select wavelength ranges from the blue ([sim]480 nm) to the long red (>700 nm). Cells labeled with a variety of fluorescent probes or expressing fluorescent proteins were then analyzed, in comparison with traditional lasers emitting at wavelengths similar to the filtered SC source. Based on a standard sensitivity metric, the white light laser bandwidths produced s... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2034969 Fri, 12 Dec 2008 05:00:00 +0100 2034969 http://www.medworm.com/index.php?rid=2023958&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20689 Model organisms and in particular the budding yeast Saccharomyces cerevisiae have been instrumental in advancing our understanding of cell cycle progression. The asymmetric division of the budding yeast and the tight coupling between cell growth and division have challenged the theoretical understanding of the cell size structure of growing yeast populations. Past efforts have centered on modeling the steady-state theoretical age distribution for asymmetric division from which a cell size distribution can be derived assuming dispersion of cell size within each age class. Different developments, especially in the field of flow cytometry, allowed the determination of a number of cellular properties and their joint distributions for the entire population and the different subpopulations as we... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2023958 Wed, 10 Dec 2008 05:00:00 +0100 2023958 http://www.medworm.com/index.php?rid=2014902&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20693 Human red blood cells (RBCs) have a normal life span of 120 days in vivo and might be primed in vitro to die in response to apoptotic stimuli through a caspase-independent pathway. It is well known that, in vivo, aging RBCs externalize phosphatidylserine residues but is unknown whether these cells express active caspases at this stage. We isolated RBCs expressing phosphatidylserine on their surface from human blood by applying an original method of affinity chromatography using annexin-V fixed on gelatin or on magnetic beads. The isolated RBCs were then analyzed by flow cytometry for morphological changes (dot-plot forward scatter versus side scatter), phosphatidylserine externalization (annexin-V test), cell viability (calcein-AM test), and caspase activities using fluorescent substrates ... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2014902 Sat, 06 Dec 2008 05:00:00 +0100 2014902 http://www.medworm.com/index.php?rid=2014903&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20692 Current methods to screen for bacterial contamination involve using costly reagents such as antibodies or PCR reagents or time-costly growth in cultures. There is need for portable, real-time, multiplex pathogen detection technology that can predict the safety of food. Surface plasmon resonance (SPR) imaging is a sensitive, label-free method that can detect the binding of an analyte to a surface by the changes in refractive index that occur upon binding. We have designed a hybrid microfluidic biochip to perform multiplexed detection of single-celled pathogens using a combination of SPR and fluorescence imaging. The device consists of an array of gold spots, each functionalized with a capture biomolecule targeting a specific pathogen. This biosensor array is enclosed by a polydimethylsiloxa... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2014903 Fri, 05 Dec 2008 05:00:00 +0100 2014903 http://www.medworm.com/index.php?rid=2009186&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20686 ELF97 phosphate (ELF-P) is a useful compound for assessing the phosphorus-related status of planktonic aquatic populations. The technique has been successfully applied to phytoplankton and more recently to heterotrophic prokaryotes in both freshwater and marine samples. We have used a recently developed protocol that enables the detection by flow cytometry of ELF alcohol (ELFA), the product of ELF-P hydrolysis. This protocol allows for identification of the fraction of cells able to express phosphatase activity (i.e., ELFA-labeled). This protocol is also very valuable in the study of time kinetics in this ELFA-labeling. The percentage of ELFA-labeled cells, the relative median ELFA fluorescence per cell, and the absolute ELFA fluorescence were determined in both freshwater (lake) and marin... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2009186 Thu, 04 Dec 2008 05:00:00 +0100 2009186 http://www.medworm.com/index.php?rid=2009193&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20679 Trogocytosis is a recently discovered phenomenon whereby lymphocytes capture fragments of the plasma membrane from antigen presenting cells (APCs). Using APCs labeled with widely used fluorescent lipophilic probes, we previously described a trogocytosis analysis protocol (TRAP) useful to understand the mechanisms and biological consequences of this process and to identify lymphocytes reacting specifically with antigen-bearing APCs. We have compared the suitability of 22 different fluorescent lipophilic probes for use in TRAP assays with cytotoxic T lymphocytes (CTL). The criteria we used were: simple and efficient incorporation in APC membranes, minimal passive diffusion among cells but efficient transfer onto T cells during trogocytosis. Sphingosin-based probes were found to incorporate i... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2009193 Tue, 02 Dec 2008 05:00:00 +0100 2009193 http://www.medworm.com/index.php?rid=2009192&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20683 Microbiology is important to industry therefore rapid and statistically representative measurements of cell physiological state, proliferation, and viability are essential if informed decisions about fermentation bioprocess optimization or control are to be made, because process performance will depend largely on the number of metabolically active viable cells. Samples of recombinant Escherichia coli W3110, containing the gene for the D1.3 anti-lysozyme Fab fragment under the control of the lac-based expression system, were taken at various stages from fed-batch fermentation processes and stained with a mixture of bis-(1,3-dibutylbarbituric acid) trimethine oxonol and propidium iodide (PI/BOX). Where appropriate, measurements of dissolved oxygen tension (DOT), OD600nm and Fab concentration... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2009192 Tue, 02 Dec 2008 05:00:00 +0100 2009192 http://www.medworm.com/index.php?rid=2009191&cid=s_33764_67_f&fid=33764&url=http%3A%2F%2Fdx.doi.org%2F10.1002%252Fcyto.a.20691 Bone marrow derived endothelial progenitor cells (EPCs) are early precursors of mature endothelial cells which replenish aging and damaged endothelial cells. The authors studied a diabetic swine model to determine if induction of DM adversely affects either bone marrow or circulating EPCs and whether a HMG-CoA reductase inhibitor (statin) improves development and recruitment of EPCs in the absence of cholesterol lowering. Streptozotocin was administered to Yorkshire pigs to induce DM. One month after induction, diabetic pigs were treated with atorvastatin (statin, n = 10), ezetimibe (n = 10) or untreated (n = 10) and evaluated for number of bone marrow and circulating EPCs and femoral artery endothelial function. There was no effect of either medication on cholesterol level. One month afte... Cytometry Part A journals http://www.medworm.com/rss/comments.php?id=2009191 Tue, 02 Dec 2008 05:00:00 +0100 2009191