Web-based analysis and publication of flow cytometry experiments.

PubMed

Kotecha, Nikesh; Krutzik, Peter O; Irish, Jonathan M

2010-07-01

Cytobank is a Web-based application for storage, analysis, and sharing of flow cytometry experiments. Researchers use a Web browser to log in and use a wide range of tools developed for basic and advanced flow cytometry. In addition to providing access to standard cytometry tools from any computer, Cytobank creates a platform and community for developing new analysis and publication tools. Figure layouts created on Cytobank are designed to allow transparent access to the underlying experiment annotation and data processing steps. Since all flow cytometry files and analysis data are stored on a central server, experiments and figures can be viewed or edited by anyone with the proper permission, from any computer with Internet access. Once a primary researcher has performed the initial analysis of the data, collaborators can engage in experiment analysis and make their own figure layouts using the gated, compensated experiment files. Cytobank is available to the scientific community at http://www.cytobank.org. (c) 2010 by John Wiley & Sons, Inc.

Web-Based Analysis and Publication of Flow Cytometry Experiments

PubMed Central

Kotecha, Nikesh; Krutzik, Peter O.; Irish, Jonathan M.

2014-01-01

Cytobank is a web-based application for storage, analysis, and sharing of flow cytometry experiments. Researchers use a web browser to log in and use a wide range of tools developed for basic and advanced flow cytometry. In addition to providing access to standard cytometry tools from any computer, Cytobank creates a platform and community for developing new analysis and publication tools. Figure layouts created on Cytobank are designed to allow transparent access to the underlying experiment annotation and data processing steps. Since all flow cytometry files and analysis data are stored on a central server, experiments and figures can be viewed or edited by anyone with the proper permissions from any computer with Internet access. Once a primary researcher has performed the initial analysis of the data, collaborators can engage in experiment analysis and make their own figure layouts using the gated, compensated experiment files. Cytobank is available to the scientific community at www.cytobank.org PMID:20578106

Effect of halloysite nanotubes on the structure and function of important multiple blood components.

PubMed

Wu, Keke; Feng, Ru; Jiao, Yanpeng; Zhou, Changren

2017-06-01

Many researchers have investigated the application of halloysite nanotubes (HNTs) in biomedicine, because of their special nanoscale hollow tubular structure. Although the cytocompatibility of HNTs has been studied, their blood compatibility has not been systematically investigated. In this work, the effect of HNTs on the structure and function of different blood components has been studied, including the morphology and hemolysis of red blood cells (RBCs). Based on scanning electron microscopy (SEM) observations, optical density test and flow cytometry analysis, we found that HNTs can affect the morphology and membrane integrity of RBCs in phosphate buffered saline (PBS) in a content-dependent way. In particular, based on UV-vis absorption spectra, fluorescence spectra and circular dichroism (CD) spectra, HNTs can alter the secondary structure and conformation of human fibrinogen and γ-globulins. In addition, the detection of biomarker molecules C3a and C5a in plasma suggests that HNTs can trigger complement activation. In the blood clotting assay, HNTs were found to significantly prolong the activated partial thromboplastin time (APTT), shorten the prothrombin time (PT) of platelet-poor plasma (PPP), and change the thromboelastography (TEG) parameters of whole blood coagulation. Furthermore, confocal laser scanning microscopy and flow cytometry analysis were used to test intracellular uptake by macrophages, and the cellular uptake of HNTs in the RAW 264.7 was found to be content-dependent, but not time-dependent. These findings provide insight for the potential use of HNTs as biofriendly nanocontainers for biomaterials in vivo. Copyright © 2017 Elsevier B.V. All rights reserved.

Femtosecond laser fabrication of fiber based optofluidic platform for flow cytometry applications

NASA Astrophysics Data System (ADS)

Serhatlioglu, Murat; Elbuken, Caglar; Ortac, Bulend; Solmaz, Mehmet E.

2017-02-01

Miniaturized optofluidic platforms play an important role in bio-analysis, detection and diagnostic applications. The advantages of such miniaturized devices are extremely low sample requirement, low cost development and rapid analysis capabilities. Fused silica is advantageous for optofluidic systems due to properties such as being chemically inert, mechanically stable, and optically transparent to a wide spectrum of light. As a three dimensional manufacturing method, femtosecond laser scanning followed by chemical etching shows great potential to fabricate glass based optofluidic chips. In this study, we demonstrate fabrication of all-fiber based, optofluidic flow cytometer in fused silica glass by femtosecond laser machining. 3D particle focusing was achieved through a straightforward planar chip design with two separately fabricated fused silica glass slides thermally bonded together. Bioparticles in a fluid stream encounter with optical interrogation region specifically designed to allocate 405nm single mode fiber laser source and two multi-mode collection fibers for forward scattering (FSC) and side scattering (SSC) signals detection. Detected signal data collected with oscilloscope and post processed with MATLAB script file. We were able to count number of events over 4000events/sec, and achieve size distribution for 5.95μm monodisperse polystyrene beads using FSC and SSC signals. Our platform shows promise for optical and fluidic miniaturization of flow cytometry systems.

Simultaneous cathodoluminescence and electron microscopy cytometry of cellular vesicles labeled with fluorescent nanodiamonds.

PubMed

Nagarajan, Sounderya; Pioche-Durieu, Catherine; Tizei, Luiz H G; Fang, Chia-Yi; Bertrand, Jean-Rémi; Le Cam, Eric; Chang, Huan-Cheng; Treussart, François; Kociak, Mathieu

2016-06-02

Light and Transmission Electron Microscopies (LM and TEM) hold potential in bioimaging owing to the advantages of fast imaging of multiple cells with LM and ultrastructure resolution offered by TEM. Integrated or correlated LM and TEM are the current approaches to combine the advantages of both techniques. Here we propose an alternative in which the electron beam of a scanning TEM (STEM) is used to excite concomitantly the luminescence of nanoparticle labels (a process known as cathodoluminescence, CL), and image the cell ultrastructure. This CL-STEM imaging allows obtaining luminescence spectra and imaging ultrastructure simultaneously. We present a proof of principle experiment, showing the potential of this technique in image cytometry of cell vesicular components. To label the vesicles we used fluorescent diamond nanocrystals (nanodiamonds, NDs) of size ≈150 nm coated with different cationic polymers, known to trigger different internalization pathways. Each polymer was associated with a type of ND with a different emission spectrum. With CL-STEM, for each individual vesicle, we were able to measure (i) their size with nanometric resolution, (ii) their content in different ND labels, and realize intracellular component cytometry. In contrast to the recently reported organelle flow cytometry technique that requires cell sonication, CL-STEM-based image cytometry preserves the cell integrity and provides a much higher resolution in size. Although this novel approach is still limited by a low throughput, the automatization of data acquisition and image analysis, combined with improved intracellular targeting, should facilitate applications in cell biology at the subcellular level.

In vivo plant flow cytometry: A first proof-of-concept

PubMed Central

Nedosekin, Dmitry A.; Khodakovskaya, Mariya V.; Biris, Alexandru S.; Wang, Daoyuan; Xu, Yang; Villagarcia, Hector; Galanzha, Ekaterina I.; Zharov, Vladimir P.

2011-01-01

In vivo flow cytometry has facilitated advances in the ultrasensitive detection of tumor cells, bacteria, nanoparticles, dyes, and other normal and abnormal objects directly in blood and lymph circulatory systems. Here, we propose in vivo plant flow cytometry for the real-time noninvasive study of nanomaterial transport in xylem and phloem plant vascular systems. As a proof of this concept, we demonstrate in vivo real-time photoacoustic monitoring of quantum dot-carbon nanotube conjugate uptake and uptake by roots and spreading through stem to leaves in a tomato plant. In addition, in vivo scanning cytometry using multimodal photoacoustic, photothermal, and fluorescent detection schematics provided multiplex detection and identification of nanoparticles accumulated in plant leaves in the presence of intensive absorption, scattering, and autofluorescent backgrounds. The use of a portable fiber-based photoacoustic flow cytometer for studies of plant vasculature was demonstrated. These integrated cytometry modalities using both endogenous and exogenous contrast agents have a potential to open new avenues of in vivo study of the nutrients, products of photosynthesis and metabolism, nanoparticles, infectious agents, and other objects transported through plant vasculature. PMID:21905208

Guidelines for improving the reproducibility of quantitative multiparameter immunofluorescence measurements by laser scanning cytometry on fixed cell suspensions from human solid tumors.

PubMed

Shackney, Stanley; Emlet, David R; Pollice, Agnese; Smith, Charles; Brown, Kathryn; Kociban, Deborah

2006-01-01

Laser scanning Cytometry (LSC) is a versatile technology that makes it possible to perform multiple measurements on individual cells and correlate them cell by cell with other cellular features. It would be highly desirable to be able to perform reproducible, quantitative, correlated cell-based immunofluorescence studies on individual cells from human solid tumors. However, such studies can be challenging because of the presence of large numbers of cell aggregates and other confounding factors. Techniques have been developed to deal with cell aggregates in data sets collected by LSC. Experience has also been gained in addressing other key technical and methodological issues that can affect the reproducibility of such cell-based immunofluorescence measurements. We describe practical aspects of cell sample collection, cell fixation and staining, protocols for performing multiparameter immunofluorescence measurements by LSC, use of controls and reference samples, and approaches to data analysis that we have found useful in improving the accuracy and reproducibility of LSC data obtained in human tumor samples. We provide examples of the potential advantages of LSC in examining quantitative aspects of cell-based analysis. Improvements in the quality of cell-based multiparameter immunofluorescence measurements make it possible to extract useful information from relatively small numbers of cells. This, in turn, permits the performance of multiple multicolor panels on each tumor sample. With links among the different panels that are provided by overlapping measurements, it is possible to develop increasingly more extensive profiles of intracellular expression of multiple proteins in clinical samples of human solid tumors. Examples of such linked panels of measurements are provided. Advances in methodology can improve cell-based multiparameter immunofluorescence measurements on cell suspensions from human solid tumors by LSC for use in prognostic and predictive clinical applications. Copyright (c) 2005 Wiley-Liss, Inc.

Technical advances in flow cytometry-based diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria

PubMed Central

Correia, Rodolfo Patussi; Bento, Laiz Cameirão; Bortolucci, Ana Carolina Apelle; Alexandre, Anderson Marega; Vaz, Andressa da Costa; Schimidell, Daniela; Pedro, Eduardo de Carvalho; Perin, Fabricio Simões; Nozawa, Sonia Tsukasa; Mendes, Cláudio Ernesto Albers; Barroso, Rodrigo de Souza; Bacal, Nydia Strachman

2016-01-01

ABSTRACT Objective: To discuss the implementation of technical advances in laboratory diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria for validation of high-sensitivity flow cytometry protocols. Methods: A retrospective study based on analysis of laboratory data from 745 patient samples submitted to flow cytometry for diagnosis and/or monitoring of paroxysmal nocturnal hemoglobinuria. Results: Implementation of technical advances reduced test costs and improved flow cytometry resolution for paroxysmal nocturnal hemoglobinuria clone detection. Conclusion: High-sensitivity flow cytometry allowed more sensitive determination of paroxysmal nocturnal hemoglobinuria clone type and size, particularly in samples with small clones. PMID:27759825

In vivo flow cytometry for blood cell analysis using differential epi-detection of forward scattered light

NASA Astrophysics Data System (ADS)

Paudel, Hari P.; Jung, Yookyung; Raphael, Anthony; Alt, Clemens; Wu, Juwell; Runnels, Judith; Lin, Charles P.

2018-02-01

The present standard of blood cell analysis is an invasive procedure requiring the extraction of patient's blood, followed by ex-vivo analysis using a flow cytometer or a hemocytometer. We are developing a noninvasive optical technique that alleviates the need for blood extraction. For in-vivo blood analysis we need a high speed, high resolution and high contrast label-free imaging technique. In this proceeding report, we reported a label-free method based on differential epi-detection of forward scattered light, a method inspired by Jerome Mertz's oblique back-illumination microscopy (OBM) (Ford et al, Nat. Meth. 9(12) 2012). The differential epi-detection of forward light gives phase contrast image at diffraction-limited resolution. Unlike reflection confocal microscopy (RCM), which detects only sharp refractive index variation and suffers from speckle noise, this technique is suitable for detection of subtle variation of refractive index in biological tissue and it provides the shape and the size of cells. A custom built high speed electronic detection circuit board produces a real-time differential signal which yields image contrast based on phase gradient in the sample. We recorded blood flow in-vivo at 17.2k lines per second in line scan mode, or 30 frames per second (full frame), or 120 frame per second (quarter frame) in frame scan mode. The image contrast and speed of line scan data recording show the potential of the system for noninvasive blood cell analysis.

In Vivo Myeloperoxidase Imaging and Flow Cytometry Analysis of Intestinal Myeloid Cells.

PubMed

Hülsdünker, Jan; Zeiser, Robert

2016-01-01

Myeloperoxidase (MPO) imaging is a non-invasive method to detect cells that produce the enzyme MPO that is most abundant in neutrophils, macrophages, and inflammatory monocytes. While lacking specificity for any of these three cell types, MPO imaging can provide guidance for further flow cytometry-based analysis of tissues where these cell types reside. Isolation of leukocytes from the intestinal tract is an error-prone procedure. Here, we describe a protocol for intestinal leukocyte isolation that works reliable in our hands and allows for flow cytometry-based analysis, in particular of neutrophils.

Laser scanning cytometry for automation of the micronucleus assay

PubMed Central

Darzynkiewicz, Zbigniew; Smolewski, Piotr; Holden, Elena; Luther, Ed; Henriksen, Mel; François, Maxime; Leifert, Wayne; Fenech, Michael

2011-01-01

Laser scanning cytometry (LSC) provides a novel approach for automated scoring of micronuclei (MN) in different types of mammalian cells, serving as a biomarker of genotoxicity and mutagenicity. In this review, we discuss the advances to date in measuring MN in cell lines, buccal cells and erythrocytes, describe the advantages and outline potential challenges of this distinctive approach of analysis of nuclear anomalies. The use of multiple laser wavelengths in LSC and the high dynamic range of fluorescence and absorption detection allow simultaneous measurement of multiple cellular and nuclear features such as cytoplasmic area, nuclear area, DNA content and density of nuclei and MN, protein content and density of cytoplasm as well as other features using molecular probes. This high-content analysis approach allows the cells of interest to be identified (e.g. binucleated cells in cytokinesis-blocked cultures) and MN scored specifically in them. MN assays in cell lines (e.g. the CHO cell MN assay) using LSC are increasingly used in routine toxicology screening. More high-content MN assays and the expansion of MN analysis by LSC to other models (i.e. exfoliated cells, dermal cell models, etc.) hold great promise for robust and exciting developments in MN assay automation as a high-content high-throughput analysis procedure. PMID:21164197

Cytometric analysis of shape and DNA content in mammalian sperm

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gledhill, B.L.

1983-10-10

Male germ cells respond dramatically to a variety of insults and are important reproductive dosimeters. Semen analyses are very useful in studies on the effects of drugs, chemicals, and environmental hazards on testicular function, male fertility and heritable germinal mutations. Sperm were analyzed by flow cytometry and slit-scan flow analysis for injury following the exposure of testes to mutagens. The utility of flow cytometry in genotoxin screening and monitoring of occupational exposure was evaluated. The technique proved valuable in separation of X- and Y-chromosome bearing sperm and the potential applicability of this technique in artificial insemination and a solution, ofmore » accurately assessing the DNA content of sperm were evaluated-with reference to determination of X- and Y-chromosome bearing sperm.« less

Alternatives to current flow cytometry data analysis for clinical and research studies.

PubMed

Gondhalekar, Carmen; Rajwa, Bartek; Patsekin, Valery; Ragheb, Kathy; Sturgis, Jennifer; Robinson, J Paul

2018-02-01

Flow cytometry has well-established methods for data analysis based on traditional data collection techniques. These techniques typically involved manual insertion of tube samples into an instrument that, historically, could only measure 1-3 colors. The field has since evolved to incorporate new technologies for faster and highly automated sample preparation and data collection. For example, the use of microwell plates on benchtop instruments is now a standard on virtually every new instrument, and so users can easily accumulate multiple data sets quickly. Further, because the user must carefully define the layout of the plate, this information is already defined when considering the analytical process, expanding the opportunities for automated analysis. Advances in multi-parametric data collection, as demonstrated by the development of hyperspectral flow-cytometry, 20-40 color polychromatic flow cytometry, and mass cytometry (CyTOF), are game-changing. As data and assay complexity increase, so too does the complexity of data analysis. Complex data analysis is already a challenge to traditional flow cytometry software. New methods for reviewing large and complex data sets can provide rapid insight into processes difficult to define without more advanced analytical tools. In settings such as clinical labs where rapid and accurate data analysis is a priority, rapid, efficient and intuitive software is needed. This paper outlines opportunities for analysis of complex data sets using examples of multiplexed bead-based assays, drug screens and cell cycle analysis - any of which could become integrated into the clinical environment. Copyright © 2017. Published by Elsevier Inc.

Microfluidic Impedance Flow Cytometry Enabling High-Throughput Single-Cell Electrical Property Characterization

PubMed Central

Chen, Jian; Xue, Chengcheng; Zhao, Yang; Chen, Deyong; Wu, Min-Hsien; Wang, Junbo

2015-01-01

This article reviews recent developments in microfluidic impedance flow cytometry for high-throughput electrical property characterization of single cells. Four major perspectives of microfluidic impedance flow cytometry for single-cell characterization are included in this review: (1) early developments of microfluidic impedance flow cytometry for single-cell electrical property characterization; (2) microfluidic impedance flow cytometry with enhanced sensitivity; (3) microfluidic impedance and optical flow cytometry for single-cell analysis and (4) integrated point of care system based on microfluidic impedance flow cytometry. We examine the advantages and limitations of each technique and discuss future research opportunities from the perspectives of both technical innovation and clinical applications. PMID:25938973

Flow Cytometry Data Preparation Guidelines for Improved Automated Phenotypic Analysis.

PubMed

Jimenez-Carretero, Daniel; Ligos, José M; Martínez-López, María; Sancho, David; Montoya, María C

2018-05-15

Advances in flow cytometry (FCM) increasingly demand adoption of computational analysis tools to tackle the ever-growing data dimensionality. In this study, we tested different data input modes to evaluate how cytometry acquisition configuration and data compensation procedures affect the performance of unsupervised phenotyping tools. An analysis workflow was set up and tested for the detection of changes in reference bead subsets and in a rare subpopulation of murine lymph node CD103 + dendritic cells acquired by conventional or spectral cytometry. Raw spectral data or pseudospectral data acquired with the full set of available detectors by conventional cytometry consistently outperformed datasets acquired and compensated according to FCM standards. Our results thus challenge the paradigm of one-fluorochrome/one-parameter acquisition in FCM for unsupervised cluster-based analysis. Instead, we propose to configure instrument acquisition to use all available fluorescence detectors and to avoid integration and compensation procedures, thereby using raw spectral or pseudospectral data for improved automated phenotypic analysis. Copyright © 2018 by The American Association of Immunologists, Inc.

Hyperspectral cytometry.

PubMed

Grégori, Gérald; Rajwa, Bartek; Patsekin, Valery; Jones, James; Furuki, Motohiro; Yamamoto, Masanobu; Paul Robinson, J

2014-01-01

Hyperspectral cytometry is an emerging technology for single-cell analysis that combines ultrafast optical spectroscopy and flow cytometry. Spectral cytometry systems utilize diffraction gratings or prism-based monochromators to disperse fluorescence signals from multiple labels (organic dyes, nanoparticles, or fluorescent proteins) present in each analyzed bioparticle onto linear detector arrays such as multianode photomultipliers or charge-coupled device sensors. The resultant data, consisting of a series of characterizing every analyzed cell, are not compensated by employing the traditional cytometry approach, but rather are spectrally unmixed utilizing algorithms such as constrained Poisson regression or non-negative matrix factorization. Although implementations of spectral cytometry were envisioned as early as the 1980s, only recently has the development of highly sensitive photomultiplier tube arrays led to design and construction of functional prototypes and subsequently to introduction of commercially available systems. This chapter summarizes the historical efforts and work in the field of spectral cytometry performed at Purdue University Cytometry Laboratories and describes the technology developed by Sony Corporation that resulted in release of the first commercial spectral cytometry system-the Sony SP6800. A brief introduction to spectral data analysis is also provided, with emphasis on the differences between traditional polychromatic and spectral cytometry approaches.

Integration of lyoplate based flow cytometry and computational analysis for standardized immunological biomarker discovery.

PubMed

Villanova, Federica; Di Meglio, Paola; Inokuma, Margaret; Aghaeepour, Nima; Perucha, Esperanza; Mollon, Jennifer; Nomura, Laurel; Hernandez-Fuentes, Maria; Cope, Andrew; Prevost, A Toby; Heck, Susanne; Maino, Vernon; Lord, Graham; Brinkman, Ryan R; Nestle, Frank O

2013-01-01

Discovery of novel immune biomarkers for monitoring of disease prognosis and response to therapy in immune-mediated inflammatory diseases is an important unmet clinical need. Here, we establish a novel framework for immunological biomarker discovery, comparing a conventional (liquid) flow cytometry platform (CFP) and a unique lyoplate-based flow cytometry platform (LFP) in combination with advanced computational data analysis. We demonstrate that LFP had higher sensitivity compared to CFP, with increased detection of cytokines (IFN-γ and IL-10) and activation markers (Foxp3 and CD25). Fluorescent intensity of cells stained with lyophilized antibodies was increased compared to cells stained with liquid antibodies. LFP, using a plate loader, allowed medium-throughput processing of samples with comparable intra- and inter-assay variability between platforms. Automated computational analysis identified novel immunophenotypes that were not detected with manual analysis. Our results establish a new flow cytometry platform for standardized and rapid immunological biomarker discovery with wide application to immune-mediated diseases.

Integration of Lyoplate Based Flow Cytometry and Computational Analysis for Standardized Immunological Biomarker Discovery

PubMed Central

Villanova, Federica; Di Meglio, Paola; Inokuma, Margaret; Aghaeepour, Nima; Perucha, Esperanza; Mollon, Jennifer; Nomura, Laurel; Hernandez-Fuentes, Maria; Cope, Andrew; Prevost, A. Toby; Heck, Susanne; Maino, Vernon; Lord, Graham; Brinkman, Ryan R.; Nestle, Frank O.

2013-01-01

Discovery of novel immune biomarkers for monitoring of disease prognosis and response to therapy in immune-mediated inflammatory diseases is an important unmet clinical need. Here, we establish a novel framework for immunological biomarker discovery, comparing a conventional (liquid) flow cytometry platform (CFP) and a unique lyoplate-based flow cytometry platform (LFP) in combination with advanced computational data analysis. We demonstrate that LFP had higher sensitivity compared to CFP, with increased detection of cytokines (IFN-γ and IL-10) and activation markers (Foxp3 and CD25). Fluorescent intensity of cells stained with lyophilized antibodies was increased compared to cells stained with liquid antibodies. LFP, using a plate loader, allowed medium-throughput processing of samples with comparable intra- and inter-assay variability between platforms. Automated computational analysis identified novel immunophenotypes that were not detected with manual analysis. Our results establish a new flow cytometry platform for standardized and rapid immunological biomarker discovery with wide application to immune-mediated diseases. PMID:23843942

Photothermal and photoacoustic Raman cytometry in vitro and in vivo

PubMed Central

Shashkov, Evgeny V.; Galanzha, Ekaterina I.; Zharov, Vladimir P.

2010-01-01

An integrated Raman-based cytometry was developed with photothermal (PT) and photoacoustic (PA) detection of Raman-induced thermal and acoustic signals in biological samples with Raman-active vibrational modes. The two-frequency, spatially and temporally overlapping pump–Stokes excitation in counterpropagating geometry was provided by a nanosecond tunable (420–2300 nm) optical parametric oscillator and a Raman shifter (639 nm) pumped by a double-pulsed Q-switched Nd:YAG laser using microscopic and fiberoptic delivery of laser radiation. The PA and PT Raman detection and imaging technique was tested in vitro with benzene, acetone, olive oil, carbon nanotubes, chylomicron phantom, and cancer cells, and in vivo in single adipocytes in mouse mesentery model. The integration of linear and nonlinear PA and PT Raman scanning and flow cytometry has the potential to enhance its chemical specificity and sensitivity including nanobubble-based amplification (up to 10- fold) for detection of absorbing and nonabsorbing targets that are important for both basic and clinically relevant studies of lymph and blood biochemistry, cancer, and fat distribution at the single-cell level. PMID:20389713

CytoSPADE: high-performance analysis and visualization of high-dimensional cytometry data

PubMed Central

Linderman, Michael D.; Simonds, Erin F.; Qiu, Peng; Bruggner, Robert V.; Sheode, Ketaki; Meng, Teresa H.; Plevritis, Sylvia K.; Nolan, Garry P.

2012-01-01

Motivation: Recent advances in flow cytometry enable simultaneous single-cell measurement of 30+ surface and intracellular proteins. CytoSPADE is a high-performance implementation of an interface for the Spanning-tree Progression Analysis of Density-normalized Events algorithm for tree-based analysis and visualization of this high-dimensional cytometry data. Availability: Source code and binaries are freely available at http://cytospade.org and via Bioconductor version 2.10 onwards for Linux, OSX and Windows. CytoSPADE is implemented in R, C++ and Java. Contact: michael.linderman@mssm.edu Supplementary Information: Additional documentation available at http://cytospade.org. PMID:22782546

Mass cytometry: a highly multiplexed single-cell technology for advancing drug development.

PubMed

Atkuri, Kondala R; Stevens, Jeffrey C; Neubert, Hendrik

2015-02-01

Advanced single-cell analysis technologies (e.g., mass cytometry) that help in multiplexing cellular measurements in limited-volume primary samples are critical in bridging discovery efforts to successful drug approval. Mass cytometry is the state-of-the-art technology in multiparametric single-cell analysis. Mass cytometers (also known as cytometry by time-of-flight or CyTOF) combine the cellular analysis principles of traditional fluorescence-based flow cytometry with the selectivity and quantitative power of inductively coupled plasma-mass spectrometry. Standard flow cytometry is limited in the number of parameters that can be measured owing to the overlap in signal when detecting fluorescently labeled antibodies. Mass cytometry uses antibodies tagged to stable isotopes of rare earth metals, which requires minimal signal compensation between the different metal tags. This unique feature enables researchers to seamlessly multiplex up to 40 independent measurements on single cells. In this overview we first present an overview of mass cytometry and compare it with traditional flow cytometry. We then discuss the emerging and potential applications of CyTOF technology in the pharmaceutical industry, including quantitative and qualitative deep profiling of immune cells and their applications in assessing drug immunogenicity, extensive mapping of signaling networks in single cells, cell surface receptor quantification and multiplexed internalization kinetics, multiplexing sample analysis by barcoding, and establishing cell ontologies on the basis of phenotype and/or function. We end with a discussion of the anticipated impact of this technology on drug development lifecycle with special emphasis on the utility of mass cytometry in deciphering a drug's pharmacokinetics and pharmacodynamics relationship. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

Attenuation of Replication Stress–Induced Premature Cellular Senescence to Assess Anti-Aging Modalities

PubMed Central

Zhao, Hong; Darzynkiewicz, Zbigniew

2014-01-01

Described is an in vitro model of premature senescence in pulmonary adenocarcinoma A549 cells induced by persistent DNA replication stress in response to treatment with the DNA damaging drug mitoxantrone (Mxt). The degree of cellular senescence, based on characteristic changes in cell morphology, is measured by laser scanning cytometry. Specifically, the flattening of cells grown on slides (considered the hallmark of cellular senescence) is measured as the decline in local intensity of DNA-associated DAPI fluorescence (represented by maximal pixels). This change is paralleled by an increase in nuclear area. Thus, the ratio of mean intensity of maximal pixels to nuclear area provides a very sensitive morphometric biomarker for the degree of senescence. This analysis is combined with immunocytochemical detection of senescence markers, such as overexpression of cyclin kinase inhibitors (e.g., p21WAF1) and phosphorylation of ribosomal protein S6 (rpS6), a key marker associated with aging/senescence that is detected using a phospho-specific antibody. These biomarker indices are presented in quantitative terms defined as a senescence index (SI), which is the fraction of the marker in test cultures relative to the same marker in exponentially growing control cultures. This system can be used to evaluate the anti-aging potential of test agents by assessing attenuation of maximal senescence. As an example, the inclusion of berberine, a natural alkaloid with reported anti-aging properties and a long history of use in traditional Chinese medicine, is shown to markedly attenuate the Mxt-induced SI and phosphorylation of rpS6. The multivariate analysis of senescence markers by laser scanning cytometry offers a promising tool to explore the potential anti-aging properties of a variety agents. PMID:24984966

A CLIPS expert system for clinical flow cytometry data analysis

NASA Technical Reports Server (NTRS)

Salzman, G. C.; Duque, R. E.; Braylan, R. C.; Stewart, C. C.

1990-01-01

An expert system is being developed using CLIPS to assist clinicians in the analysis of multivariate flow cytometry data from cancer patients. Cluster analysis is used to find subpopulations representing various cell types in multiple datasets each consisting of four to five measurements on each of 5000 cells. CLIPS facts are derived from results of the clustering. CLIPS rules are based on the expertise of Drs. Stewart, Duque, and Braylan. The rules incorporate certainty factors based on case histories.

Development of wide-angle 2D light scattering static cytometry

NASA Astrophysics Data System (ADS)

Xie, Linyan; Liu, Qiao; Shao, Changshun; Su, Xuantao

2016-10-01

We have recently developed a 2D light scattering static cytometer for cellular analysis in a label-free manner, which measures side scatter (SSC) light in the polar angular range from 79 to 101 degrees. Compared with conventional flow cytometry, our cytometric technique requires no fluorescent labeling of the cells, and static cytometry measurements can be performed without flow control. In this paper we present an improved label-free static cytometer that can obtain 2D light scattering patterns in a wider angular range. By illuminating the static microspheres on chip with a scanning optical fiber, wide-angle 2D light scattering patterns of single standard microspheres with a mean diameter of 3.87 μm are obtained. The 2D patterns of 3.87 μm microspheres contain both large-angle forward scatter (FSC) and SSC light in the polar angular range from 40 to 100 degrees, approximately. Experimental 2D patterns of 3.87 μm microspheres are in good agreement with Mie theory simulated ones. The wide-angle light scattering measurements may provide a better resolution for particle analysis as compared with the SSC measurements. Two dimensional light scattering patterns of HL-60 human acute leukemia cells are obtained by using our static cytometer. Compared with SSC 2D light scattering patterns, wide-angle 2D patterns contain richer information of the HL-60 cells. The obtaining of 2D light scattering patterns in a wide angular range could help to enhance the capabilities of our label-free static cytometry for cell analysis.

qFlow Cytometry-Based Receptoromic Screening: A High-Throughput Quantification Approach Informing Biomarker Selection and Nanosensor Development.

PubMed

Chen, Si; Weddell, Jared; Gupta, Pavan; Conard, Grace; Parkin, James; Imoukhuede, Princess I

2017-01-01

Nanosensor-based detection of biomarkers can improve medical diagnosis; however, a critical factor in nanosensor development is deciding which biomarker to target, as most diseases present several biomarkers. Biomarker-targeting decisions can be informed via an understanding of biomarker expression. Currently, immunohistochemistry (IHC) is the accepted standard for profiling biomarker expression. While IHC provides a relative mapping of biomarker expression, it does not provide cell-by-cell readouts of biomarker expression or absolute biomarker quantification. Flow cytometry overcomes both these IHC challenges by offering biomarker expression on a cell-by-cell basis, and when combined with calibration standards, providing quantitation of biomarker concentrations: this is known as qFlow cytometry. Here, we outline the key components for applying qFlow cytometry to detect biomarkers within the angiogenic vascular endothelial growth factor receptor family. The key aspects of the qFlow cytometry methodology include: antibody specificity testing, immunofluorescent cell labeling, saturation analysis, fluorescent microsphere calibration, and quantitative analysis of both ensemble and cell-by-cell data. Together, these methods enable high-throughput quantification of biomarker expression.

A Novel Antibody Humanization Method Based on Epitopes Scanning and Molecular Dynamics Simulation

PubMed Central

Zhao, Bin-Bin; Gong, Lu-Lu; Jin, Wen-Jing; Liu, Jing-Jun; Wang, Jing-Fei; Wang, Tian-Tian; Yuan, Xiao-Hui; He, You-Wen

2013-01-01

1-17-2 is a rat anti-human DEC-205 monoclonal antibody that induces internalization and delivers antigen to dendritic cells (DCs). The potentially clinical application of this antibody is limited by its murine origin. Traditional humanization method such as complementarity determining regions (CDRs) graft often leads to a decreased or even lost affinity. Here we have developed a novel antibody humanization method based on computer modeling and bioinformatics analysis. First, we used homology modeling technology to build the precise model of Fab. A novel epitope scanning algorithm was designed to identify antigenic residues in the framework regions (FRs) that need to be mutated to human counterpart in the humanization process. Then virtual mutation and molecular dynamics (MD) simulation were used to assess the conformational impact imposed by all the mutations. By comparing the root-mean-square deviations (RMSDs) of CDRs, we found five key residues whose mutations would destroy the original conformation of CDRs. These residues need to be back-mutated to rescue the antibody binding affinity. Finally we constructed the antibodies in vitro and compared their binding affinity by flow cytometry and surface plasmon resonance (SPR) assay. The binding affinity of the refined humanized antibody was similar to that of the original rat antibody. Our results have established a novel method based on epitopes scanning and MD simulation for antibody humanization. PMID:24278299

Mesenchymal Stem Cells for Vascular Target Discovery in Breast Cancer-Associated Angiogenesis

DTIC Science & Technology

2004-09-01

Matrigel plug and sorted by flow cytometry . Sorting of these retrieved cells based on co-expression of the GFP marker and cell- surface endothelial...express the green fluorescent protein (GFP) and clonal MSC populations can be isolated and phenotypically and genotypically analyzed by flow cytometry ...monoclonal populations of these GFP+ murine MSCs and conducted flow cytometry analysis to determine their phenotype. Specifically, we determined if

Visual analysis of mass cytometry data by hierarchical stochastic neighbour embedding reveals rare cell types.

PubMed

van Unen, Vincent; Höllt, Thomas; Pezzotti, Nicola; Li, Na; Reinders, Marcel J T; Eisemann, Elmar; Koning, Frits; Vilanova, Anna; Lelieveldt, Boudewijn P F

2017-11-23

Mass cytometry allows high-resolution dissection of the cellular composition of the immune system. However, the high-dimensionality, large size, and non-linear structure of the data poses considerable challenges for the data analysis. In particular, dimensionality reduction-based techniques like t-SNE offer single-cell resolution but are limited in the number of cells that can be analyzed. Here we introduce Hierarchical Stochastic Neighbor Embedding (HSNE) for the analysis of mass cytometry data sets. HSNE constructs a hierarchy of non-linear similarities that can be interactively explored with a stepwise increase in detail up to the single-cell level. We apply HSNE to a study on gastrointestinal disorders and three other available mass cytometry data sets. We find that HSNE efficiently replicates previous observations and identifies rare cell populations that were previously missed due to downsampling. Thus, HSNE removes the scalability limit of conventional t-SNE analysis, a feature that makes it highly suitable for the analysis of massive high-dimensional data sets.

Annexin-V/quantum dot probes for multimodal apoptosis monitoring in living cells: improving bioanalysis using electrochemistry

NASA Astrophysics Data System (ADS)

Montón, Helena; Parolo, Claudio; Aranda-Ramos, Antonio; Merkoçi, Arben; Nogués, Carme

2015-02-01

There is a great demand to develop novel techniques that allow useful and complete monitoring of apoptosis, which is a key factor of several diseases and a target for drug development. Here, we present the use of a novel dual electrochemical/optical label for the detection and study of apoptosis. We combined the specificity of Annexin-V for phosphatidylserine, a phospholipid expressed in the outer membrane of apoptotic cells, with the optical and electrochemical properties of quantum dots to create a more efficient label. Using this conjugate we addressed three important issues: (i) we made the labeling of apoptotic cells faster (30 min) and easier; (ii) we fully characterized the samples by common cell biological techniques (confocal laser scanning microscopy, scanning electron microscopy and flow cytometry); and (iii) we developed a fast, cheap and quantitative electrochemical detection method for apoptotic cells with results in full agreement with those obtained by flow cytometry.There is a great demand to develop novel techniques that allow useful and complete monitoring of apoptosis, which is a key factor of several diseases and a target for drug development. Here, we present the use of a novel dual electrochemical/optical label for the detection and study of apoptosis. We combined the specificity of Annexin-V for phosphatidylserine, a phospholipid expressed in the outer membrane of apoptotic cells, with the optical and electrochemical properties of quantum dots to create a more efficient label. Using this conjugate we addressed three important issues: (i) we made the labeling of apoptotic cells faster (30 min) and easier; (ii) we fully characterized the samples by common cell biological techniques (confocal laser scanning microscopy, scanning electron microscopy and flow cytometry); and (iii) we developed a fast, cheap and quantitative electrochemical detection method for apoptotic cells with results in full agreement with those obtained by flow cytometry. Electronic supplementary information (ESI) available: Optical microscopy images of apoptotic induced cell cultures at different times and negative control of flow cytometry. See DOI: 10.1039/c4nr07191c

Critical Role of CD8 T Cells in Mediating Sex-Based Differences in a Murine Model of Lupus

DTIC Science & Technology

2009-08-21

into  female  transfers  (fF)  mice  that  was  reduced  in  CD8  depleted  fF  mice.  Flow   cytometry   analysis  showed increased numbers of splenic...splenocytes were first analyzed by flow cytometry for CD4 and CD8 T cells and F1 mice received either: a) unfractionated splenocytes (CD8 intactF1...using magnetic beads purchased from Invitrogen (Carlsbad, CA) according to the manufacturer’s instructions. Flow cytometry analysis before cell

Mesenchymal Stem Cells for Vascular Target Discovery in Breast Cancer-Associated Angiogenesis

DTIC Science & Technology

2005-09-01

demonstrating this marker as demonstrated by flow cytometry . These GFP+ MSCs were subsequently analyzed for expression of commonly reported markers of...phenotypically and genotypically analyzed by flow cytometry and gene chip analysis, respectively. We have also shown that MSCs can then be stimulated to...positive MSCs retrieved by collagenase digestion of the Matrigel plug and sorted by flow cytometry . Sorting of these retrieved cells based on co-expression

Fisetin as a promising antifungal agent against Cryptocococcus neoformans species complex.

PubMed

Reis, M P C; Carvalho, C R C; Andrade, F A; Fernandes, O F L; Arruda, W; Silva, M R R

2016-08-01

The aim of this study was to investigate the mechanisms of action of fisetin, a flavonol with antifungal activity previously evaluated against the Cryptococcus neoformans species complex. Ergosterol content and flow cytometry analysis were determined for the C. neoformans species complex in the presence of fisetin and ultrastructural analysis of morphology was performed on Cryptococcus gattii and C. neoformans. Decrease in the total cellular ergosterol content after exposure to fisetin ranged from 25·4% after exposure to 128 μg ml(-1) to 21·6% after exposure to 64 μg ml(-1) of fisetin compared with the control (without fisetin). The fisetin effects obtained with flow cytometry showed metabolic impairment, and alterations in its normal morphology caused by fisetin in C. neoformans cells were verified using scanning electron microscopy. Fisetin is a compound that acts in the biosynthesis of ergosterol. Flow cytometry showed that fisetin reduced viability of the metabolically active cells of C. gattii, while morphological changes explain the action of fisetin in inhibiting growth of these fungi. This study supports the idea that fisetin may represent a good starting point for the development of future therapeutic substances for cryptococcosis. © 2016 The Society for Applied Microbiology.

OpenCyto: An Open Source Infrastructure for Scalable, Robust, Reproducible, and Automated, End-to-End Flow Cytometry Data Analysis

PubMed Central

Finak, Greg; Frelinger, Jacob; Jiang, Wenxin; Newell, Evan W.; Ramey, John; Davis, Mark M.; Kalams, Spyros A.; De Rosa, Stephen C.; Gottardo, Raphael

2014-01-01

Flow cytometry is used increasingly in clinical research for cancer, immunology and vaccines. Technological advances in cytometry instrumentation are increasing the size and dimensionality of data sets, posing a challenge for traditional data management and analysis. Automated analysis methods, despite a general consensus of their importance to the future of the field, have been slow to gain widespread adoption. Here we present OpenCyto, a new BioConductor infrastructure and data analysis framework designed to lower the barrier of entry to automated flow data analysis algorithms by addressing key areas that we believe have held back wider adoption of automated approaches. OpenCyto supports end-to-end data analysis that is robust and reproducible while generating results that are easy to interpret. We have improved the existing, widely used core BioConductor flow cytometry infrastructure by allowing analysis to scale in a memory efficient manner to the large flow data sets that arise in clinical trials, and integrating domain-specific knowledge as part of the pipeline through the hierarchical relationships among cell populations. Pipelines are defined through a text-based csv file, limiting the need to write data-specific code, and are data agnostic to simplify repetitive analysis for core facilities. We demonstrate how to analyze two large cytometry data sets: an intracellular cytokine staining (ICS) data set from a published HIV vaccine trial focused on detecting rare, antigen-specific T-cell populations, where we identify a new subset of CD8 T-cells with a vaccine-regimen specific response that could not be identified through manual analysis, and a CyTOF T-cell phenotyping data set where a large staining panel and many cell populations are a challenge for traditional analysis. The substantial improvements to the core BioConductor flow cytometry packages give OpenCyto the potential for wide adoption. It can rapidly leverage new developments in computational cytometry and facilitate reproducible analysis in a unified environment. PMID:25167361

OpenCyto: an open source infrastructure for scalable, robust, reproducible, and automated, end-to-end flow cytometry data analysis.

PubMed

Finak, Greg; Frelinger, Jacob; Jiang, Wenxin; Newell, Evan W; Ramey, John; Davis, Mark M; Kalams, Spyros A; De Rosa, Stephen C; Gottardo, Raphael

2014-08-01

Flow cytometry is used increasingly in clinical research for cancer, immunology and vaccines. Technological advances in cytometry instrumentation are increasing the size and dimensionality of data sets, posing a challenge for traditional data management and analysis. Automated analysis methods, despite a general consensus of their importance to the future of the field, have been slow to gain widespread adoption. Here we present OpenCyto, a new BioConductor infrastructure and data analysis framework designed to lower the barrier of entry to automated flow data analysis algorithms by addressing key areas that we believe have held back wider adoption of automated approaches. OpenCyto supports end-to-end data analysis that is robust and reproducible while generating results that are easy to interpret. We have improved the existing, widely used core BioConductor flow cytometry infrastructure by allowing analysis to scale in a memory efficient manner to the large flow data sets that arise in clinical trials, and integrating domain-specific knowledge as part of the pipeline through the hierarchical relationships among cell populations. Pipelines are defined through a text-based csv file, limiting the need to write data-specific code, and are data agnostic to simplify repetitive analysis for core facilities. We demonstrate how to analyze two large cytometry data sets: an intracellular cytokine staining (ICS) data set from a published HIV vaccine trial focused on detecting rare, antigen-specific T-cell populations, where we identify a new subset of CD8 T-cells with a vaccine-regimen specific response that could not be identified through manual analysis, and a CyTOF T-cell phenotyping data set where a large staining panel and many cell populations are a challenge for traditional analysis. The substantial improvements to the core BioConductor flow cytometry packages give OpenCyto the potential for wide adoption. It can rapidly leverage new developments in computational cytometry and facilitate reproducible analysis in a unified environment.

Development of a two-parameter slit-scan flow cytometer for screening of normal and aberrant chromosomes: application to a karyotype of Sus scrofa domestica (pig)

NASA Astrophysics Data System (ADS)

Hausmann, Michael; Doelle, Juergen; Arnold, Armin; Stepanow, Boris; Wickert, Burkhard; Boscher, Jeannine; Popescu, Paul C.; Cremer, Christoph

1992-07-01

Laser fluorescence activated slit-scan flow cytometry offers an approach to a fast, quantitative characterization of chromosomes due to morphological features. It can be applied for screening of chromosomal abnormalities. We give a preliminary report on the development of the Heidelberg slit-scan flow cytometer. Time-resolved measurement of the fluorescence intensity along the chromosome axis can be registered simultaneously for two parameters when the chromosome axis can be registered simultaneously for two parameters when the chromosome passes perpendicularly through a narrowly focused laser beam combined by a detection slit in the image plane. So far automated data analysis has been performed off-line on a PC. In its final performance, the Heidelberg slit-scan flow cytometer will achieve on-line data analysis that allows an electro-acoustical sorting of chromosomes of interest. Interest is high in the agriculture field to study chromosome aberrations that influence the size of litters in pig (Sus scrofa domestica) breeding. Slit-scan measurements have been performed to characterize chromosomes of pigs; we present results for chromosome 1 and a translocation chromosome 6/15.

A cell-penetrating peptide analogue, P7, exerts antimicrobial activity against Escherichia coli ATCC25922 via penetrating cell membrane and targeting intracellular DNA.

PubMed

Li, Lirong; Shi, Yonghui; Cheng, Xiangrong; Xia, Shufang; Cheserek, Maureen Jepkorir; Le, Guowei

2015-01-01

The antibacterial activities and mechanism of a new P7 were investigated in this study. P7 showed antimicrobial activities against five harmful microorganisms which contaminate and spoil food (MIC=4-32 μM). Flow cytometry and scanning electron microscopy analyses demonstrated that P7 induced pore-formation on the cell surface and led to morphological changes but did not lyse cell. Confocal fluorescence microscopic observations and flow cytometry analysis expressed that P7 could penetrate the Escherichia coli cell membrane and accumulate in the cytoplasm. Moreover, P7 possessed a strong DNA binding affinity. Further cell cycle analysis and change in gene expression analysis suggested that P7 induced a decreased expression in the genes involved in DNA replication. Up-regulated expression genes encoding DNA damage repair. This study suggests that P7 could be applied as a candidate for the development of new food preservatives as it exerts its antibacterial activities by penetrating cell membranes and targets intracellular DNA. Copyright © 2014 Elsevier Ltd. All rights reserved.

Enumerating Pathogenic Microorganism Surrogates for Groundwater Experiments Using Solid-Phase Cytometry

NASA Astrophysics Data System (ADS)

Stevenson, M. E.; Blaschke, A. P.; Kirschner, A.

2010-12-01

Regulators need a dependable method that would enable them to calculate with confidence the setback distance of a drinking water well from a potential point of contamination. Since it is not permissible to perform field tests using pathogenic microorganisms, it is necessary to predict the transport of dangerous microbes in a different way, using surrogates. One such surrogate method involves using bacteriophages, which are viruses that are pathogenic to bacteria, but are not dangerous to humans. Another possible surrogate to model the potential travel time of microbial contamination is the use of synthetic microspheres; we will test microspheres ranging in size from 0.025 to 1 µm. The constraining factor for comparing the transport of microspheres and bacteriophages is the detection limit of the measuring apparatus. Appropriate measuring techniques are mandatory for a comparison. Traditionally, bacteriophages are measured using plaque forming analysis, the detection limit being one plaque forming unit per petri dish. In our study, the use of solid-phase cytometry for enumerating microspheres for wellhead protection projects is being investigated, as the detection limit using this technology is one cell per filter. To the best of our knowledge, there is no other technique available that enables a comparable detection limit. The solid-phase cytometer used for this study is a ChemScan RDI (Chemunex, France). For comparison, epifluorescence microscopy will also be used. The ChemScan RDI device automatically drives an epifluorescent microscope to the site of each cell detected, in order to confirm the validity of the reading. In this way, it is possible to observe whether clumping together of microspheres is a problem or if non-target cells were labelled. Keywords: Microspheres, Solid-phase cytometry, ChemScan, Drinking water protection Acknowledgements: We would like to thank the Austrian Science Fund (FWF) for financial support as part of the Doctoral Program on Water Resource Systems (DK Plus W1219-N22) and the Vienna Waterworks (MA 31) as part of the GWRS-Vienna project.

[Clinical usefulness of urine-formed elements' information obtained from bacteria detection by flow cytometry method that uses nucleic acid staining].

PubMed

Nakagawa, Hiroko; Yuno, Tomoji; Itho, Kiichi

2009-03-01

Recently, specific detection method for Bacteria, by flow cytometry method using nucleic acid staining, was developed as a function of automated urine formed elements analyzer for routine urine testing. Here, we performed a basic study on this bacteria analysis method. In addition, we also have a comparison among urine sediment analysis, urine Gram staining and urine quantitative cultivation, the conventional methods performed up to now. As a result, the bacteria analysis with flow cytometry method that uses nucleic acid staining was excellent in reproducibility, and higher sensitivity compared with microscopic urinary sediment analysis. Based on the ROC curve analysis, which settled urine culture method as standard, cut-off level of 120/microL was defined and its sensitivity = 85.7%, specificity = 88.2%. In the analysis of scattergram, accompanied with urine culture method, among 90% of rod positive samples, 80% of dots were appeared in the area of 30 degrees from axis X. In addition, one case even indicated that analysis of bacteria by flow cytometry and scattergram of time series analysis might be helpful to trace the progress of causative bacteria therefore the information supposed to be clinically significant. Reporting bacteria information with nucleic acid staining flow cytometry method is expected to contribute to a rapid diagnostics and treatment of urinary tract infections. Besides, the contribution to screening examination of microbiology and clinical chemistry, will deliver a more efficient solution to urine analysis.

Multiplexed and Microparticle-based Analyses: Quantitative Tools for the Large-Scale Analysis of Biological Systems

PubMed Central

Nolan, John P.; Mandy, Francis

2008-01-01

While the term flow cytometry refers to the measurement of cells, the approach of making sensitive multiparameter optical measurements in a flowing sample stream is a very general analytical approach. The past few years have seen an explosion in the application of flow cytometry technology for molecular analysis and measurements using micro-particles as solid supports. While microsphere-based molecular analyses using flow cytometry date back three decades, the need for highly parallel quantitative molecular measurements that has arisen from various genomic and proteomic advances has driven the development in particle encoding technology to enable highly multiplexed assays. Multiplexed particle-based immunoassays are now common place, and new assays to study genes, protein function, and molecular assembly. Numerous efforts are underway to extend the multiplexing capabilities of microparticle-based assays through new approaches to particle encoding and analyte reporting. The impact of these developments will be seen in the basic research and clinical laboratories, as well as in drug development. PMID:16604537

Pumpless Microflow Cytometry Enabled by Viscosity Modulation and Immunobead Labeling.

PubMed

Kim, Byeongyeon; Oh, Sein; Shin, Suyeon; Yim, Sang-Gu; Yang, Seung Yun; Hahn, Young Ki; Choi, Sungyoung

2018-06-19

Major challenges of miniaturizing flow cytometry include obviating the need for bulky, expensive, and complex pump-based fluidic and laser-based optical systems while retaining the ability to detect target cells based on their unique surface receptors. We addressed these critical challenges by (i) using a viscous liquid additive to control flow rate passively, without external pumping equipment, and (ii) adopting an immunobead assay that can be quantified with a portable fluorescence cell counter based on a blue light-emitting diode. Such novel features enable pumpless microflow cytometry (pFC) analysis by simply dropping a sample solution onto the inlet reservoir of a disposable cell-counting chamber. With our pFC platform, we achieved reliable cell counting over a dynamic range of 9-298 cells/μL. We demonstrated the practical utility of the platform by identifying a type of cancer cell based on CD326, the epithelial cell adhesion molecule. This portable microflow cytometry platform can be applied generally to a range of cell types using immunobeads labeled with specific antibodies, thus making it valuable for cell-based and point-of-care diagnostics.

Single Cell Mass Cytometry for Analysis of Immune System Functional States

PubMed Central

Bjornson, Zach B.; Nolan, Garry P.; Fantl, Wendy J.

2013-01-01

Single cell mass cytometry facilitates high-dimensional, quantitative analysis of the effects of bioactive molecules on cell populations at single-cell resolution. Datasets are generated with antibody panels (upwards of 40) in which each antibody is conjugated to a polymer chelated with a stable metal isotope, usually in the Lanthanide series of the periodic table. Isotope labelled antibodies recognize surface markers to delineate cell types and intracellular signaling molecules to provide a measure of the network state—and thereby demarcating multiple cell state functions such as apoptosis, DNA damage and cell cycle. By measuring all these parameters simultaneously, the signaling state of an individual cell can be measured at its network state. This review will cover the basics of mass cytometry as well as outline steps already taken to allow it to stand aside traditional fluorescence based cytometry in the immunologist’s analytical arsenal in their study of immune states during infection. PMID:23999316

Biomarkers of Cell Senescence Assessed by Imaging Cytometry

PubMed Central

Zhao, Hong; Darzynkiewicz, Zbigniew

2012-01-01

The characteristic features of senescent cells such as their “flattened” appearance, enlarged nuclei and low saturation density at the plateau phase of cell growth, can be conveniently measured by image-assisted d cytometry such as provided by the laser scanning cytometry (LSC). The “flattening” of senescent cells is reflected by the decline in local density of staining (intensity of maximal pixel) of DNA-associated fluorescence [4,6-diamidino-2- phenylindole (DAPI)] paralleled by an increase in nuclear size (area). Thus, the ratio of the maximal pixel of DAPI fluorescence per nucleus to the nuclear area provides a very sensitive morphometric biomarker of “depth” of senescence, which progressively declines during induction of senescence. Also recorded is cellular DNA content revealing cell cycle phase, as well as the saturation cell density at plateau phase of growth, which is dramatically decreased in cultures of senescent cells. Concurrent immunocytochemical analysis of expression of p21WAF1, p16INK4a or p27KIP1 cyclin kinase inhibitor provides additional markers of senescence. These biomarker indices can be expressed in quantitative terms (“senescence indices”) as a fraction of the same markers of the exponentially growing cells in control cultures. PMID:23296652

Noninvasive High-Throughput Single-Cell Analysis of HIV Protease Activity Using Ratiometric Flow Cytometry

PubMed Central

Gaber, Rok; Majerle, Andreja; Jerala, Roman; Benčina, Mojca

2013-01-01

To effectively fight against the human immunodeficiency virus infection/acquired immunodeficiency syndrome (HIV/AIDS) epidemic, ongoing development of novel HIV protease inhibitors is required. Inexpensive high-throughput screening assays are needed to quickly scan large sets of chemicals for potential inhibitors. We have developed a Förster resonance energy transfer (FRET)-based, HIV protease-sensitive sensor using a combination of a fluorescent protein pair, namely mCerulean and mCitrine. Through extensive in vitro characterization, we show that the FRET-HIV sensor can be used in HIV protease screening assays. Furthermore, we have used the FRET-HIV sensor for intracellular quantitative detection of HIV protease activity in living cells, which more closely resembles an actual viral infection than an in vitro assay. We have developed a high-throughput method that employs a ratiometric flow cytometry for analyzing large populations of cells that express the FRET-HIV sensor. The method enables FRET measurement of single cells with high sensitivity and speed and should be used when subpopulation-specific intracellular activity of HIV protease needs to be estimated. In addition, we have used a confocal microscopy sensitized emission FRET technique to evaluate the usefulness of the FRET-HIV sensor for spatiotemporal detection of intracellular HIV protease activity. PMID:24287545

Single cell analysis using surface enhanced Raman scattering (SERS) tags

PubMed Central

Nolan, John P.; Duggan, Erika; Liu, Er; Condello, Danilo; Dave, Isha; Stoner, Samuel A.

2013-01-01

Fluorescence is a mainstay of bioanalytical methods, offering sensitive and quantitative reporting, often in multiplexed or multiparameter assays. Perhaps the best example of the latter is flow cytometry, where instruments equipped with multiple lasers and detectors allow measurement of 15 or more different fluorophores simultaneously, but increases beyond this number are limited by the relatively broad emission spectra. Surface enhanced Raman scattering (SERS) from metal nanoparticles can produce signal intensities that rival fluorescence, but with narrower spectral features that allow a greater degree of multiplexing. We are developing nanoparticle SERS tags as well as Raman flow cytometers for multiparameter single cell analysis of suspension or adherent cells. SERS tags are based on plasmonically active nanoparticles (gold nanorods) whose plasmon resonance can be tuned to give optimal SERS signals at a desired excitation wavelength. Raman resonant compounds are adsorbed on the nanoparticles to confer a unique spectral fingerprint on each SERS tag, which are then encapsulated in a polymer coating for conjugation to antibodies or other targeting molecules. Raman flow cytometry employs a high resolution spectral flow cytometer capable of measuring the complete SERS spectra, as well as conventional flow cytometry measurements, from thousands of individual cells per minute. Automated spectral unmixing algorithms extract the contributions of each SERS tag from each cell to generate high content, multiparameter single cell population data. SERS-based cytometry is a powerful complement to conventional fluorescence-based cytometry. The narrow spectral features of the SERS signal enables more distinct probes to be measured in a smaller region of the optical spectrum with a single laser and detector, allowing for higher levels of multiplexing and multiparameter analysis. PMID:22498143

Clinical-scale laser-based scanning and processing of live cells: selective photothermal killing of fluorescent tumor targets for autologous stem cell transplantation

NASA Astrophysics Data System (ADS)

Koller, Manfred R.; Hanania, Elie G.; Eisfeld, Timothy; O'Neal, Robert A.; Khovananth, Kevin M.; Palsson, Bernhard O.

2001-04-01

High-dose chemotherapy, followed by autologous hematopoietic stem cell (HSC) transplantation, is widely used for the treatment of cancer. However, contaminating tumor cells within HSC harvests continue to be of major concern since re-infused tumor cells have proven to contribute to disease relapse. Many tumor purging methods have been evaluated, but all leave detectable tumor cells in the transplant and result in significant loss of HSCs. These shortcomings cause engraftment delays and compromise the therapeutic value of purging. A novel approach integrating automated scanning cytometry, image analysis, and selective laser-induced killing of labeled cells within a cell mixture is described here. Non-Hodgkin's lymphoma (NHL) cells were spiked into cell mixtures, and fluorochrome-conjugated antibodies were used to label tumor cells within the mixture. Cells were then allowed to settle on a surface, and as the surface was scanned with a fluorescence excitation source, a laser pulse was fired at every detected tumor cell using high-speed beam steering mirrors. Tumor cells were selectively killed with little effect on adjacent non-target cells, demonstrating the feasibility of this automated cell processing approach. This technology has many potential research and clinical applications, one example of which is tumor cell purging for autologous HSC transplantation.

Preparation of isolated nuclei from K 562 haemopoietic cell line for high resolution scanning electron microscopy.

PubMed

Reipert, S; Reipert, B M; Allen, T D

1994-09-01

The aim of the work is to visualise nuclear pore complexes (NPCs) in mammalian cells by high resolution scanning electron microscopy. A detergent-free isolation protocol was employed to obtain clean nuclei from the haemopoietic cell line K 562. Nuclear isolation was performed by mechanical homogenisation under hypotonic conditions followed by purification of the nuclear fraction. The isolated nuclei were attached to silicon chips, fixed, critical point dried, and sputter coated with a thin film (3-4 nm) of tantalum. Analysis of the nuclear surface by scanning electron microscopy (SEM) revealed a strong sensitivity of the outer nuclear membrane (ONM) to disruption during the isolation procedure. A significant reduction of the characteristic pattern of damage to the ONM was achieved by means of an isopicnic centrifugation on an isoosmolar balanced Percoll gradient. Analysis of the population of isolated nuclei by flow cytometry showed no signs of cell cycle specific losses of nuclei during isolation. The SEM investigations of the morphology of the nuclear envelope (NE) and of substructural details of NPCs and polyribosomes were performed using an in-lens field emission scanning electron microscope.

Novel Methods of Determining Urinary Calculi Composition: Petrographic Thin Sectioning of Calculi and Nanoscale Flow Cytometry Urinalysis

PubMed Central

Gavin, Carson T; Ali, Sohrab N; Tailly, Thomas; Olvera-Posada, Daniel; Alenezi, Husain; Power, Nicholas E; Hou, Jinqiang; St. Amant, Andre H; Luyt, Leonard G; Wood, Stephen; Wu, Charles; Razvi, Hassan; Leong, Hon S

2016-01-01

Accurate determination of urinary stone composition has significant bearing on understanding pathophysiology, choosing treatment modalities and preventing recurrence. A need exists for improved methods to determine stone composition. Urine of 31 patients with known renal calculi was examined with nanoscale flow cytometry and the calculi collected during surgery subsequently underwent petrographic thin sectioning with polarized and fluorescent microscopy. Fluorescently labeled bisphosphonate probes (Alendronate-fluorescein/Alendronate-Cy5) were developed for nanoscale flow cytometry to enumerate nanocrystals that bound the fluorescent probes. Petrographic sections of stones were also imaged by fluorescent and polarized light microscopy with composition analysis correlated to alendronate +ve nanocrystal counts in corresponding urine samples. Urine samples from patients with Ca2+ and Mg2+ based calculi exhibited the highest alendronate +ve nanocrystal counts, ranging from 100–1000 nm in diameter. This novel urine based assay was in agreement with composition determined by petrographic thin sections with Alendronate probes. In some cases, high alendronate +ve nanocrystal counts indicated a Ca2+ or Mg2+ composition, as confirmed by petrographic analysis, overturning initial spectrophotometric diagnosis of stone composition. The combination of nanoscale flow cytometry and petrographic thin sections offer an alternative means for determining stone composition. Nanoscale flow cytometry of alendronate +ve nanocrystals alone may provide a high-throughput means of evaluating stone burden. PMID:26771074

CytometryML binary data standards

NASA Astrophysics Data System (ADS)

Leif, Robert C.

2005-03-01

CytometryML is a proposed new Analytical Cytology (Cytomics) data standard, which is based on a common set of XML schemas for encoding flow cytometry and digital microscopy text based data types (metadata). CytometryML schemas reference both DICOM (Digital Imaging and Communications in Medicine) codes and FCS keywords. Flow Cytometry Standard (FCS) list-mode has been mapped to the DICOM Waveform Information Object. The separation of the large binary data objects (list mode and image data) from the XML description of the metadata permits the metadata to be directly displayed, analyzed, and reported with standard commercial software packages; the direct use of XML languages; and direct interfacing with clinical information systems. The separation of the binary data into its own files simplifies parsing because all extraneous header data has been eliminated. The storage of images as two-dimensional arrays without any extraneous data, such as in the Adobe Photoshop RAW format, facilitates the development by scientists of their own analysis and visualization software. Adobe Photoshop provided the display infrastructure and the translation facility to interconvert between the image data from commercial formats and RAW format. Similarly, the storage and parsing of list mode binary data type with a group of parameters that are specified at compilation time is straight forward. However when the user is permitted at run-time to select a subset of the parameters and/or specify results of mathematical manipulations, the development of special software was required. The use of CytometryML will permit investigators to be able to create their own interoperable data analysis software and to employ commercially available software to disseminate their data.

Platelet compatibility of magnesium alloys.

PubMed

Yahata, Chie; Mochizuki, Akira

2017-09-01

Lately, Mg alloys have been investigated as a new class of biomaterials owing to their excellent biodegradability and biocompatibility. It has previously been reported that the in vitro compatibility of a Mg alloy containing aluminum and zinc (AZ) alloy with the blood coagulation system is excellent due to Mg 2+ ions eluting from the alloy. In this study, the compatibility of the AZ alloy with platelets was evaluated by scanning electron microscopy (SEM) and flow cytometry. In the flow cytometry analysis, the platelets were stained using PAC-1 and P-selectin antibodies. SEM images and PAC-1 analyses showed no negative effects on the platelets, whereas P-selectin analysis showed marked platelet activation. To understand these contradictory results, the amount of β-thromboglobulin (β-TG) released from the platelets was investigated. From that investigation, it was concluded that platelets are markedly activated by the alloys. In addition to clarifying divergent results depending on the analysis method used, the effects of Mg 2+ ions and pH on platelet activation were studied. These results show that platelet activation is caused by an increase in pH at the alloy surface owing to the erosion of the alloy. Copyright © 2017 Elsevier B.V. All rights reserved.

SCENERY: a web application for (causal) network reconstruction from cytometry data

PubMed Central

Papoutsoglou, Georgios; Athineou, Giorgos; Lagani, Vincenzo; Xanthopoulos, Iordanis; Schmidt, Angelika; Éliás, Szabolcs; Tegnér, Jesper

2017-01-01

Abstract Flow and mass cytometry technologies can probe proteins as biological markers in thousands of individual cells simultaneously, providing unprecedented opportunities for reconstructing networks of protein interactions through machine learning algorithms. The network reconstruction (NR) problem has been well-studied by the machine learning community. However, the potentials of available methods remain largely unknown to the cytometry community, mainly due to their intrinsic complexity and the lack of comprehensive, powerful and easy-to-use NR software implementations specific for cytometry data. To bridge this gap, we present Single CEll NEtwork Reconstruction sYstem (SCENERY), a web server featuring several standard and advanced cytometry data analysis methods coupled with NR algorithms in a user-friendly, on-line environment. In SCENERY, users may upload their data and set their own study design. The server offers several data analysis options categorized into three classes of methods: data (pre)processing, statistical analysis and NR. The server also provides interactive visualization and download of results as ready-to-publish images or multimedia reports. Its core is modular and based on the widely-used and robust R platform allowing power users to extend its functionalities by submitting their own NR methods. SCENERY is available at scenery.csd.uoc.gr or http://mensxmachina.org/en/software/. PMID:28525568

Opto-fluidics based microscopy and flow cytometry on a cell phone for blood analysis.

PubMed

Zhu, Hongying; Ozcan, Aydogan

2015-01-01

Blood analysis is one of the most important clinical tests for medical diagnosis. Flow cytometry and optical microscopy are widely used techniques to perform blood analysis and therefore cost-effective translation of these technologies to resource limited settings is critical for various global health as well as telemedicine applications. In this chapter, we review our recent progress on the integration of imaging flow cytometry and fluorescent microscopy on a cell phone using compact, light-weight and cost-effective opto-fluidic attachments integrated onto the camera module of a smartphone. In our cell-phone based opto-fluidic imaging cytometry design, fluorescently labeled cells are delivered into the imaging area using a disposable micro-fluidic chip that is positioned above the existing camera unit of the cell phone. Battery powered light-emitting diodes (LEDs) are butt-coupled to the sides of this micro-fluidic chip without any lenses, which effectively acts as a multimode slab waveguide, where the excitation light is guided to excite the fluorescent targets within the micro-fluidic chip. Since the excitation light propagates perpendicular to the detection path, an inexpensive plastic absorption filter is able to reject most of the scattered light and create a decent dark-field background for fluorescent imaging. With this excitation geometry, the cell-phone camera can record fluorescent movies of the particles/cells as they are flowing through the microchannel. The digital frames of these fluorescent movies are then rapidly processed to quantify the count and the density of the labeled particles/cells within the solution under test. With a similar opto-fluidic design, we have recently demonstrated imaging and automated counting of stationary blood cells (e.g., labeled white blood cells or unlabeled red blood cells) loaded within a disposable cell counting chamber. We tested the performance of this cell-phone based imaging cytometry and blood analysis platform by measuring the density of red and white blood cells as well as hemoglobin concentration in human blood samples, which showed a good match to our measurement results obtained using a commercially available hematology analyzer. Such a cell-phone enabled opto-fluidics microscopy, flow cytometry, and blood analysis platform could be especially useful for various telemedicine applications in remote and resource-limited settings.

Co-delivery of doxorubicin and siRNA using octreotide-conjugated gold nanorods for targeted neuroendocrine cancer therapy

NASA Astrophysics Data System (ADS)

Xiao, Yuling; Jaskula-Sztul, Renata; Javadi, Alireza; Xu, Wenjin; Eide, Jacob; Dammalapati, Ajitha; Kunnimalaiyaan, Muthusamy; Chen, Herbert; Gong, Shaoqin

2012-10-01

A multifunctional gold (Au) nanorod (NR)-based nanocarrier capable of co-delivering small interfering RNA (siRNA) against achaete-scute complex-like 1 (ASCL1) and an anticancer drug (doxorubicin (DOX)) specifically to neuroendocrine (NE) cancer cells was developed and characterized for combined chemotherapy and siRNA-mediated gene silencing. The Au NR was conjugated with (1) DOX, an anticancer drug, via a pH-labile hydrazone linkage to enable pH-controlled drug release, (2) polyarginine, a cationic polymer for complexing siRNA, and (3) octreotide (OCT), a tumor-targeting ligand, to specifically target NE cancer cells with overexpressed somatostatin receptors. The Au NR-based nanocarriers exhibited a uniform size distribution as well as pH-sensitive drug release. The OCT-conjugated Au NR-based nanocarriers (Au-DOX-OCT, targeted) exhibited a much higher cellular uptake in a human carcinoid cell line (BON cells) than non-targeted Au NR-based nanocarriers (Au-DOX) as measured by both flow cytometry and confocal laser scanning microscopy (CLSM). Moreover, Au-DOX-OCT-ASCL1 siRNA (Au-DOX-OCT complexed with ASCL1 siRNA) resulted in significantly higher gene silencing in NE cancer cells than Au-DOX-ASCL1 siRNA (non-targeted Au-DOX complexed with ASCL1 siRNA) as measured by an immunoblot analysis. Additionally, Au-DOX-OCT-ASCL1 siRNA was the most efficient nanocarrier at altering the NE phenotype of NE cancer cells and showed the strongest anti-proliferative effect. Thus, combined chemotherapy and RNA silencing using NE tumor-targeting Au NR-based nanocarriers could potentially enhance the therapeutic outcomes in treating NE cancers.A multifunctional gold (Au) nanorod (NR)-based nanocarrier capable of co-delivering small interfering RNA (siRNA) against achaete-scute complex-like 1 (ASCL1) and an anticancer drug (doxorubicin (DOX)) specifically to neuroendocrine (NE) cancer cells was developed and characterized for combined chemotherapy and siRNA-mediated gene silencing. The Au NR was conjugated with (1) DOX, an anticancer drug, via a pH-labile hydrazone linkage to enable pH-controlled drug release, (2) polyarginine, a cationic polymer for complexing siRNA, and (3) octreotide (OCT), a tumor-targeting ligand, to specifically target NE cancer cells with overexpressed somatostatin receptors. The Au NR-based nanocarriers exhibited a uniform size distribution as well as pH-sensitive drug release. The OCT-conjugated Au NR-based nanocarriers (Au-DOX-OCT, targeted) exhibited a much higher cellular uptake in a human carcinoid cell line (BON cells) than non-targeted Au NR-based nanocarriers (Au-DOX) as measured by both flow cytometry and confocal laser scanning microscopy (CLSM). Moreover, Au-DOX-OCT-ASCL1 siRNA (Au-DOX-OCT complexed with ASCL1 siRNA) resulted in significantly higher gene silencing in NE cancer cells than Au-DOX-ASCL1 siRNA (non-targeted Au-DOX complexed with ASCL1 siRNA) as measured by an immunoblot analysis. Additionally, Au-DOX-OCT-ASCL1 siRNA was the most efficient nanocarrier at altering the NE phenotype of NE cancer cells and showed the strongest anti-proliferative effect. Thus, combined chemotherapy and RNA silencing using NE tumor-targeting Au NR-based nanocarriers could potentially enhance the therapeutic outcomes in treating NE cancers. Electronic supplementary information (ESI) available: Additional flow cytometry histogram profiles of DOX fluorescence and ASCL1 knockdown results. See DOI: 10.1039/c2nr31853a

Super-resolved calibration-free flow cytometric characterization of platelets and cell-derived microparticles in platelet-rich plasma.

PubMed

Konokhova, Anastasiya I; Chernova, Darya N; Moskalensky, Alexander E; Strokotov, Dmitry I; Yurkin, Maxim A; Chernyshev, Andrei V; Maltsev, Valeri P

2016-02-01

Importance of microparticles (MPs), also regarded as extracellular vesicles, in many physiological processes and clinical conditions motivates one to use the most informative and precise methods for their characterization. Methods based on individual particle analysis provide statistically reliable distributions of MP population over characteristics. Although flow cytometry is one of the most powerful technologies of this type, the standard forward-versus-side-scattering plots of MPs and platelets (PLTs) overlap considerably because of similarity of their morphological characteristics. Moreover, ordinary flow cytometry is not capable of measurement of size and refractive index (RI) of MPs. In this study, we 1) employed the potential of the scanning flow cytometer (SFC) for identification and characterization of MPs from light scattering; 2) suggested the reference method to characterize MP morphology (size and RI) with high precision; and 3) determined the lowest size of a MP that can be characterized from light scattering with the SFC. We equipped the SFC with 405 and 488 nm lasers to measure the light-scattering profiles and side scattering from MPs, respectively. The developed two-stage method allowed accurate separation of PLTs and MPs in platelet-rich plasma. We used two optical models for MPs, a sphere and a bisphere, in the solution of the inverse light-scattering problem. This solution provides unprecedented precision in determination of size and RI of individual spherical MPs-median uncertainties (standard deviations) were 6 nm and 0.003, respectively. The developed method provides instrument-independent quantitative information on MPs, which can be used in studies of various factors affecting MP population. © 2015 International Society for Advancement of Cytometry.

Systems biology and clinical cytomics: The 10th Leipziger Workshop and the 3rd International Workshop on Slide-Based Cytometry, Leipzig, Germany, April 2005.

PubMed

Tárnok, Attila; Valet, Günther K; Emmrich, Frank

2006-01-01

Despite very significant technical and software improvements in flow cytometry (FCM) since the 1980's, the demand for a cytometric technology combining both quantitative cell analysis and morphological documentation in Cytomics became evident. Improvements in microtechnology and computing permit nowadays similar quantitative and stoichiometric single cell-based high-throughput analyses by microscopic instruments, like Slide-Based Cytometry (SBC). SBC and related techniques offer unique tools to perform complex immunophenotyping, thereby enabling diagnostic procedures during early disease stages. Multicolor or polychromatic analysis of cells by SBC is of special importance not only as a cytomics technology platform but also because of low quantities of required reagents and biological material. The exact knowledge of the location of each cell on the slide permits repetitive restaining and reanalysis of specimens. Various separate measurements of the same specimen can be ultimately fused to one database increasing the information obtained per cell. Relocation and optical evaluation of cells as typical SBC feature, can be of integral importance for cytometric analysis, since artifacts can be excluded and morphology of measured cells can be documented. Progress in cell analytic: In the SBC, new horizons can be opened by the new techniques of structural and functional analysis with the high resolution from intracellular and membrane (confocal microscopy, nanoscopy, total internal fluorescence microscopy (TIRFM), and tissue level (tissomics), to organ and organism level (in vivo cytometry, optical whole body imaging). Predictive medicine aims at the detection of changes in patient's state prior to the manifestation of the disease or the complication. Such instances concern immune consequences of surgeries or noninfectious posttraumatic shock in intensive care patients or the pretherapeutic identification of high risk patients in cancer cytostatic therapy. Preventive anti-infectious or anti-shock therapy as well as curative chemotherapy in combination with stem cell transplantation may provide better survival chances for patient at concomitant cost containment. Predictive medicine-guided optimization of therapy could lead to individualized medicine that gives significant therapeutic effect and may lower or abrogate potential therapeutic side effects. The 10th Leipziger Workshop combined with the 3rd International Workshop on SBC aimed to offer new methods in Image- and Slide-Based Cytometry for solutions in clinical research. It moved towards practical applications in the clinics and the clinical laboratory. This development will be continued in 2006 at the upcoming Leipziger Workshop and the International Workshop on Slide-Based Cytometry.

A simple and efficient method for poly-3-hydroxybutyrate quantification in diazotrophic bacteria within 5 minutes using flow cytometry.

PubMed

Alves, L P S; Almeida, A T; Cruz, L M; Pedrosa, F O; de Souza, E M; Chubatsu, L S; Müller-Santos, M; Valdameri, G

2017-01-16

The conventional method for quantification of polyhydroxyalkanoates based on whole-cell methanolysis and gas chromatography (GC) is laborious and time-consuming. In this work, a method based on flow cytometry of Nile red stained bacterial cells was established to quantify poly-3-hydroxybutyrate (PHB) production by the diazotrophic and plant-associated bacteria, Herbaspirillum seropedicae and Azospirillum brasilense. The method consists of three steps: i) cell permeabilization, ii) Nile red staining, and iii) analysis by flow cytometry. The method was optimized step-by-step and can be carried out in less than 5 min. The final results indicated a high correlation coefficient (R2=0.99) compared to a standard method based on methanolysis and GC. This method was successfully applied to the quantification of PHB in epiphytic bacteria isolated from rice roots.

Flow cytometric HyPer-based assay for hydrogen peroxide.

PubMed

Lyublinskaya, O G; Antonov, S A; Gorokhovtsev, S G; Pugovkina, N A; Kornienko, Ju S; Ivanova, Ju S; Shatrova, A N; Aksenov, N D; Zenin, V V; Nikolsky, N N

2018-05-30

HyPer is a genetically encoded fluorogenic sensor for hydrogen peroxide which is generally used for the ratiometric imaging of H 2 O 2 fluxes in living cells. Here, we demonstrate the advantages of HyPer-based ratiometric flow cytometry assay for H 2 O 2 , by using K562 and human mesenchymal stem cell lines expressing HyPer. We show that flow cytometry analysis is suitable to detect HyPer response to submicromolar concentrations of extracellularly added H 2 O 2 that is much lower than concentrations addressed previously in the other HyPer-based assays (such as cell imaging or fluorimetry). Suggested technique is also much more sensitive to hydrogen peroxide than the widespread flow cytometry assay exploiting H 2 O 2 -reactive dye H 2 DCFDA and, contrary to the H 2 DCFDA-based assay, can be employed for the kinetic studies of H 2 O 2 utilization by cells, including measurements of the rate constants of H 2 O 2 removal. In addition, flow cytometry multi-parameter ratiometric measurements enable rapid and high-throughput detection of endogenously generated H 2 O 2 in different subpopulations of HyPer-expressing cells. To sum up, HyPer can be used in multi-parameter flow cytometry studies as a highly sensitive indicator of intracellular H 2 O 2 . Copyright © 2018. Published by Elsevier Inc.

Morphological observation and analysis using automated image cytometry for the comparison of trypan blue and fluorescence-based viability detection method.

PubMed

Chan, Leo Li-Ying; Kuksin, Dmitry; Laverty, Daniel J; Saldi, Stephanie; Qiu, Jean

2015-05-01

The ability to accurately determine cell viability is essential to performing a well-controlled biological experiment. Typical experiments range from standard cell culturing to advanced cell-based assays that may require cell viability measurement for downstream experiments. The traditional cell viability measurement method has been the trypan blue (TB) exclusion assay. However, since the introduction of fluorescence-based dyes for cell viability measurement using flow or image-based cytometry systems, there have been numerous publications comparing the two detection methods. Although previous studies have shown discrepancies between TB exclusion and fluorescence-based viability measurements, image-based morphological analysis was not performed in order to examine the viability discrepancies. In this work, we compared TB exclusion and fluorescence-based viability detection methods using image cytometry to observe morphological changes due to the effect of TB on dead cells. Imaging results showed that as the viability of a naturally-dying Jurkat cell sample decreased below 70 %, many TB-stained cells began to exhibit non-uniform morphological characteristics. Dead cells with these characteristics may be difficult to count under light microscopy, thus generating an artificially higher viability measurement compared to fluorescence-based method. These morphological observations can potentially explain the differences in viability measurement between the two methods.

Carboxymethyl chitosan based nanocomposites containing chemically bonded quantum dots and magnetic nanoparticles

NASA Astrophysics Data System (ADS)

Ding, Yongling; Yin, Hong; Chen, Rui; Bai, Ru; Chen, Chunying; Hao, Xiaojuan; Shen, Shirley; Sun, Kangning; Liu, Futian

2018-03-01

A biocompatible nanocomposite consisting of fluorescent quantum dots (QDs) and magnetic nanoparticles (MNPs) has been constructed via carboxymethyl chitosan (CMCS), resulting in magnetic-fluorescent nanoparticles (MFNPs). In these MFNPs, QDs and MNPs are successfully conjugated via covalent bonds onto the surface of CMCS. The composite retains favorable magnetic and fluorescent properties and shows a good colloidal stability in physiological environments. Folate (FA) as a specific targeting ligand was further incorporated into the nanocomposites to form a delivery vehicle with a targeting function. The therapeutic activity was achieved by loading chemotherapeutic drug doxorubicin (DOX) through electrostatic and hydrophobic interactions. The cumulative DOX release profile shows pH-sensitive. Both flow cytometry analysis and confocal laser scanning microscopic observation suggested that these nanocomposites were uptaken by cancer cells via FA receptor-mediated endocytosis pathway. In summary, the CMCS based nanocomposites developed in this work have a great potential for effective cancer-targeting and drug delivery, as well as in situ cellular imaging.

Microfluidic Imaging Flow Cytometry by Asymmetric-detection Time-stretch Optical Microscopy (ATOM).

PubMed

Tang, Anson H L; Lai, Queenie T K; Chung, Bob M F; Lee, Kelvin C M; Mok, Aaron T Y; Yip, G K; Shum, Anderson H C; Wong, Kenneth K Y; Tsia, Kevin K

2017-06-28

Scaling the number of measurable parameters, which allows for multidimensional data analysis and thus higher-confidence statistical results, has been the main trend in the advanced development of flow cytometry. Notably, adding high-resolution imaging capabilities allows for the complex morphological analysis of cellular/sub-cellular structures. This is not possible with standard flow cytometers. However, it is valuable for advancing our knowledge of cellular functions and can benefit life science research, clinical diagnostics, and environmental monitoring. Incorporating imaging capabilities into flow cytometry compromises the assay throughput, primarily due to the limitations on speed and sensitivity in the camera technologies. To overcome this speed or throughput challenge facing imaging flow cytometry while preserving the image quality, asymmetric-detection time-stretch optical microscopy (ATOM) has been demonstrated to enable high-contrast, single-cell imaging with sub-cellular resolution, at an imaging throughput as high as 100,000 cells/s. Based on the imaging concept of conventional time-stretch imaging, which relies on all-optical image encoding and retrieval through the use of ultrafast broadband laser pulses, ATOM further advances imaging performance by enhancing the image contrast of unlabeled/unstained cells. This is achieved by accessing the phase-gradient information of the cells, which is spectrally encoded into single-shot broadband pulses. Hence, ATOM is particularly advantageous in high-throughput measurements of single-cell morphology and texture - information indicative of cell types, states, and even functions. Ultimately, this could become a powerful imaging flow cytometry platform for the biophysical phenotyping of cells, complementing the current state-of-the-art biochemical-marker-based cellular assay. This work describes a protocol to establish the key modules of an ATOM system (from optical frontend to data processing and visualization backend), as well as the workflow of imaging flow cytometry based on ATOM, using human cells and micro-algae as the examples.

SCENERY: a web application for (causal) network reconstruction from cytometry data.

PubMed

Papoutsoglou, Georgios; Athineou, Giorgos; Lagani, Vincenzo; Xanthopoulos, Iordanis; Schmidt, Angelika; Éliás, Szabolcs; Tegnér, Jesper; Tsamardinos, Ioannis

2017-07-03

Flow and mass cytometry technologies can probe proteins as biological markers in thousands of individual cells simultaneously, providing unprecedented opportunities for reconstructing networks of protein interactions through machine learning algorithms. The network reconstruction (NR) problem has been well-studied by the machine learning community. However, the potentials of available methods remain largely unknown to the cytometry community, mainly due to their intrinsic complexity and the lack of comprehensive, powerful and easy-to-use NR software implementations specific for cytometry data. To bridge this gap, we present Single CEll NEtwork Reconstruction sYstem (SCENERY), a web server featuring several standard and advanced cytometry data analysis methods coupled with NR algorithms in a user-friendly, on-line environment. In SCENERY, users may upload their data and set their own study design. The server offers several data analysis options categorized into three classes of methods: data (pre)processing, statistical analysis and NR. The server also provides interactive visualization and download of results as ready-to-publish images or multimedia reports. Its core is modular and based on the widely-used and robust R platform allowing power users to extend its functionalities by submitting their own NR methods. SCENERY is available at scenery.csd.uoc.gr or http://mensxmachina.org/en/software/. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Detecting endotoxin with a flow cytometry-based magnetic aptasensor.

PubMed

Zuo, Ming-Yan; Chen, Li-Juan; Jiang, Hao; Tan, Lin; Luo, Zhao-Feng; Wang, Yan-Mei

2014-12-01

Endotoxin, which is also known as lipopolysaccharide (LPS), is a marker for intruding gram-negative pathogens. It is essential to detect endotoxin quickly and sensitively in a complex milieu. A new flow cytometry (FCM)-based magnetic aptasensor assay that employs two endotoxin-binding aptamers and magnetic beads has been developed to detect endotoxin. The endotoxin-conjugated sandwich complex on magnetic beads was observed by scanning confocal laser microscopy. The resulting magnetic aptasensor rapidly detected (<1 min) endotoxin within a broad dynamic detection range of 10(-8) to 10(0)mg/ml in the presence of bovine serum albumin (BSA), RNA, sucrose, and glucose, which are most likely to coexist with endotoxin in the majority of biological liquids. Only 2 μl of magnetic aptasensor was required to quantify the endotoxin solution. Furthermore, the magnetic aptasensor could be regenerated seven times and still presented an outstanding response to the endotoxin solution. Therefore, the magnetic aptasensor exhibited high sensitivity, selectivity, and reproducibility, thereby serving as a powerful tool for the quality control and high-throughput detection of endotoxin in the food and pharmaceutical industries. Copyright © 2014 Elsevier Inc. All rights reserved.

A simple and efficient method for poly-3-hydroxybutyrate quantification in diazotrophic bacteria within 5 minutes using flow cytometry

PubMed Central

Alves, L.P.S.; Almeida, A.T.; Cruz, L.M.; Pedrosa, F.O.; de Souza, E.M.; Chubatsu, L.S.; Müller-Santos, M.; Valdameri, G.

2017-01-01

The conventional method for quantification of polyhydroxyalkanoates based on whole-cell methanolysis and gas chromatography (GC) is laborious and time-consuming. In this work, a method based on flow cytometry of Nile red stained bacterial cells was established to quantify poly-3-hydroxybutyrate (PHB) production by the diazotrophic and plant-associated bacteria, Herbaspirillum seropedicae and Azospirillum brasilense. The method consists of three steps: i) cell permeabilization, ii) Nile red staining, and iii) analysis by flow cytometry. The method was optimized step-by-step and can be carried out in less than 5 min. The final results indicated a high correlation coefficient (R2=0.99) compared to a standard method based on methanolysis and GC. This method was successfully applied to the quantification of PHB in epiphytic bacteria isolated from rice roots. PMID:28099582

Highly multiparametric analysis by mass cytometry.

PubMed

Ornatsky, Olga; Bandura, Dmitry; Baranov, Vladimir; Nitz, Mark; Winnik, Mitchell A; Tanner, Scott

2010-09-30

This review paper describes a new technology, mass cytometry, that addresses applications typically run by flow cytometer analyzers, but extends the capability to highly multiparametric analysis. The detection technology is based on atomic mass spectrometry. It offers quantitation, specificity and dynamic range of mass spectrometry in a format that is familiar to flow cytometry practitioners. The mass cytometer does not require compensation, allowing the application of statistical techniques; this has been impossible given the constraints of fluorescence noise with traditional cytometry instruments. Instead of "colors" the mass cytometer "reads" the stable isotope tags attached to antibodies using metal-chelating labeling reagents. Because there are many available stable isotopes, and the mass spectrometer provides exquisite resolution between detection channels, many parameters can be measured as easily as one. For example, in a single tube the technique allows for the ready detection and characterization of the major cell subsets in blood or bone marrow. Here we describe mass cytometric immunophenotyping of human leukemia cell lines and leukemia patient samples, differential cell analysis of normal peripheral and umbilical cord blood; intracellular protein identification and metal-encoded bead arrays. Copyright © 2010 Elsevier B.V. All rights reserved.

Flow cytometry as an improved method for the titration of Chlamydiaceae and other intracellular bacteria.

PubMed

Käser, T; Pasternak, J A; Hamonic, G; Rieder, M; Lai, K; Delgado-Ortega, M; Gerdts, V; Meurens, F

2016-05-01

Chlamydiaceae is a family of intracellular bacteria causing a range of diverse pathological outcomes. The most devastating human diseases are ocular infections with C. trachomatis leading to blindness and genital infections causing pelvic inflammatory disease with long-term sequelae including infertility and chronic pelvic pain. In order to enable the comparison of experiments between laboratories investigating host-chlamydia interactions, the infectious titer has to be determined. Titer determination of chlamydia is most commonly performed via microscopy of host cells infected with a serial dilution of chlamydia. However, other methods including fluorescent ELISpot (Fluorospot) and DNA Chip Scanning Technology have also been proposed to enumerate chlamydia-infected cells. For viruses, flow cytometry has been suggested as a superior alternative to standard titration methods. In this study we compared the use of flow cytometry with microscopy and Fluorospot for the titration of C. suis as a representative of other intracellular bacteria. Titer determination via Fluorospot was unreliable, while titration via microscopy led to a linear read-out range of 16 - 64 dilutions and moderate reproducibility with acceptable standard deviations within and between investigators. In contrast, flow cytometry had a vast linear read-out range of 1,024 dilutions and the lowest standard deviations given a basic training in these methods. In addition, flow cytometry was faster and material costs were lower compared to microscopy. Flow cytometry offers a fast, cheap, precise, and reproducible alternative for the titration of intracellular bacteria like C. suis. © 2016 International Society for Advancement of Cytometry. © 2016 International Society for Advancement of Cytometry.

Minimal Residual Disease Evaluation in Childhood Acute Lymphoblastic Leukemia: An Economic Analysis

PubMed Central

Gajic-Veljanoski, O.; Pham, B.; Pechlivanoglou, P.; Krahn, M.; Higgins, Caroline; Bielecki, Joanna

2016-01-01

Background Minimal residual disease (MRD) testing by higher performance techniques such as flow cytometry and polymerase chain reaction (PCR) can be used to detect the proportion of remaining leukemic cells in bone marrow or peripheral blood during and after the first phases of chemotherapy in children with acute lymphoblastic leukemia (ALL). The results of MRD testing are used to reclassify these patients and guide changes in treatment according to their future risk of relapse. We conducted a systematic review of the economic literature, cost-effectiveness analysis, and budget-impact analysis to ascertain the cost-effectiveness and economic impact of MRD testing by flow cytometry for management of childhood precursor B-cell ALL in Ontario. Methods A systematic literature search (1998–2014) identified studies that examined the incremental cost-effectiveness of MRD testing by either flow cytometry or PCR. We developed a lifetime state-transition (Markov) microsimulation model to quantify the cost-effectiveness of MRD testing followed by risk-directed therapy to no MRD testing and to estimate its marginal effect on health outcomes and on costs. Model input parameters were based on the literature, expert opinion, and data from the Pediatric Oncology Group of Ontario Networked Information System. Using predictions from our Markov model, we estimated the 1-year cost burden of MRD testing versus no testing and forecasted its economic impact over 3 and 5 years. Results In a base-case cost-effectiveness analysis, compared with no testing, MRD testing by flow cytometry at the end of induction and consolidation was associated with an increased discounted survival of 0.0958 quality-adjusted life-years (QALYs) and increased discounted costs of $4,180, yielding an incremental cost-effectiveness ratio (ICER) of $43,613/QALY gained. After accounting for parameter uncertainty, incremental cost-effectiveness of MRD testing was associated with an ICER of $50,249/QALY gained. In the budget-impact analysis, the 1-year cost expenditure for MRD testing by flow cytometry in newly diagnosed patients with precursor B-cell ALL was estimated at $340,760. We forecasted that the province would have to pay approximately $1.3 million over 3 years and $2.4 million over 5 years for MRD testing by flow cytometry in this population. Conclusions Compared with no testing, MRD testing by flow cytometry in newly diagnosed patients with precursor B-cell ALL represents good value for money at commonly used willingness-to-pay thresholds of $50,000/QALY and $100,000/QALY. PMID:27099644

Minimal Residual Disease Evaluation in Childhood Acute Lymphoblastic Leukemia: An Economic Analysis.

PubMed

2016-01-01

Minimal residual disease (MRD) testing by higher performance techniques such as flow cytometry and polymerase chain reaction (PCR) can be used to detect the proportion of remaining leukemic cells in bone marrow or peripheral blood during and after the first phases of chemotherapy in children with acute lymphoblastic leukemia (ALL). The results of MRD testing are used to reclassify these patients and guide changes in treatment according to their future risk of relapse. We conducted a systematic review of the economic literature, cost-effectiveness analysis, and budget-impact analysis to ascertain the cost-effectiveness and economic impact of MRD testing by flow cytometry for management of childhood precursor B-cell ALL in Ontario. A systematic literature search (1998-2014) identified studies that examined the incremental cost-effectiveness of MRD testing by either flow cytometry or PCR. We developed a lifetime state-transition (Markov) microsimulation model to quantify the cost-effectiveness of MRD testing followed by risk-directed therapy to no MRD testing and to estimate its marginal effect on health outcomes and on costs. Model input parameters were based on the literature, expert opinion, and data from the Pediatric Oncology Group of Ontario Networked Information System. Using predictions from our Markov model, we estimated the 1-year cost burden of MRD testing versus no testing and forecasted its economic impact over 3 and 5 years. In a base-case cost-effectiveness analysis, compared with no testing, MRD testing by flow cytometry at the end of induction and consolidation was associated with an increased discounted survival of 0.0958 quality-adjusted life-years (QALYs) and increased discounted costs of $4,180, yielding an incremental cost-effectiveness ratio (ICER) of $43,613/QALY gained. After accounting for parameter uncertainty, incremental cost-effectiveness of MRD testing was associated with an ICER of $50,249/QALY gained. In the budget-impact analysis, the 1-year cost expenditure for MRD testing by flow cytometry in newly diagnosed patients with precursor B-cell ALL was estimated at $340,760. We forecasted that the province would have to pay approximately $1.3 million over 3 years and $2.4 million over 5 years for MRD testing by flow cytometry in this population. Compared with no testing, MRD testing by flow cytometry in newly diagnosed patients with precursor B-cell ALL represents good value for money at commonly used willingness-to-pay thresholds of $50,000/QALY and $100,000/QALY.

ggCyto: Next Generation Open-Source Visualization Software for Cytometry.

PubMed

Van, Phu; Jiang, Wenxin; Gottardo, Raphael; Finak, Greg

2018-06-01

Open source software for computational cytometry has gained in popularity over the past few years. Efforts such as FlowCAP, the Lyoplate and Euroflow projects have highlighted the importance of efforts to standardize both experimental and computational aspects of cytometry data analysis. The R/BioConductor platform hosts the largest collection of open source cytometry software covering all aspects of data analysis and providing infrastructure to represent and analyze cytometry data with all relevant experimental, gating, and cell population annotations enabling fully reproducible data analysis. Data visualization frameworks to support this infrastructure have lagged behind. ggCyto is a new open-source BioConductor software package for cytometry data visualization built on ggplot2 that enables ggplot-like functionality with the core BioConductor flow cytometry data structures. Amongst its features are the ability to transform data and axes on-the-fly using cytometry-specific transformations, plot faceting by experimental meta-data variables, and partial matching of channel, marker and cell populations names to the contents of the BioConductor cytometry data structures. We demonstrate the salient features of the package using publicly available cytometry data with complete reproducible examples in a supplementary material vignette. https://bioconductor.org/packages/devel/bioc/html/ggcyto.html. gfinak@fredhutch.org. Supplementary data are available at Bioinformatics online and at http://rglab.org/ggcyto/.

Dynamic autofocus for continuous-scanning time-delay-and-integration image acquisition in automated microscopy.

PubMed

Bravo-Zanoguera, Miguel E; Laris, Casey A; Nguyen, Lam K; Oliva, Mike; Price, Jeffrey H

2007-01-01

Efficient image cytometry of a conventional microscope slide means rapid acquisition and analysis of 20 gigapixels of image data (at 0.3-microm sampling). The voluminous data motivate increased acquisition speed to enable many biomedical applications. Continuous-motion time-delay-and-integrate (TDI) scanning has the potential to speed image acquisition while retaining sensitivity, but the challenge of implementing high-resolution autofocus operating simultaneously with acquisition has limited its adoption. We develop a dynamic autofocus system for this need using: 1. a "volume camera," consisting of nine fiber optic imaging conduits to charge-coupled device (CCD) sensors, that acquires images in parallel from different focal planes, 2. an array of mixed analog-digital processing circuits that measure the high spatial frequencies of the multiple image streams to create focus indices, and 3. a software system that reads and analyzes the focus data streams and calculates best focus for closed feedback loop control. Our system updates autofocus at 56 Hz (or once every 21 microm of stage travel) to collect sharply focused images sampled at 0.3x0.3 microm(2)/pixel at a stage speed of 2.3 mms. The system, tested by focusing in phase contrast and imaging long fluorescence strips, achieves high-performance closed-loop image-content-based autofocus in continuous scanning for the first time.

Dissecting the Role of IGFBP-2 in Development of Acute Myeloid Leukemia

DTIC Science & Technology

2011-06-01

surface proteins on freshly isolated and cultured cells, as determined by flow cytometry ... Surface Immune Molecules on Phenotypic HSCs during Culture (A and B) A summary of the result of flow cytometry analysis of surface expression of indicated...from the distant implanted tumor were counted by flow cytometry analysis. The flow cytometry result was confirmed by counting GFP+ surface foci of

High-throughput autofluorescence flow cytometry of breast cancer metabolism (Conference Presentation)

NASA Astrophysics Data System (ADS)

Shah, Amy T.; Cannon, Taylor M.; Higginbotham, Jim N.; Skala, Melissa C.

2016-02-01

Tumor heterogeneity poses challenges for devising optimal treatment regimens for cancer patients. In particular, subpopulations of cells can escape treatment and cause relapse. There is a need for methods to characterize tumor heterogeneity of treatment response. Cell metabolism is altered in cancer (Warburg effect), and cells use the autofluorescent cofactor NADH in numerous metabolic reactions. Previous studies have shown that microscopy measurements of NADH autofluorescence are sensitive to treatment response in breast cancer, and these techniques typically assess hundreds of cells per group. An alternative approach is flow cytometry, which measures fluorescence on a single-cell level and is attractive for characterizing tumor heterogeneity because it achieves high-throughput analysis and cell sorting in millions of cells per group. Current applications for flow cytometry rely on staining with fluorophores. This study characterizes flow cytometry measurements of NADH autofluorescence in breast cancer cells. Preliminary results indicate flow cytometry of NADH is sensitive to cyanide perturbation, which inhibits oxidative phosphorylation, in nonmalignant MCF10A cells. Additionally, flow cytometry is sensitive to higher NADH intensity for HER2-positive SKBr3 cells compared with triple-negative MDA-MB-231 cells. These results agree with previous microscopy studies. Finally, a mixture of SKBr3 and MDA-MB-231 cells were sorted into each cell type using NADH intensity. Sorted cells were cultured, and microscopy validation showed the expected morphology for each cell type. Ultimately, flow cytometry could be applied to characterize tumor heterogeneity based on treatment response and sort cell subpopulations based on metabolic profile. These achievements could enable individualized treatment strategies and improved patient outcomes.

[Effects of infrasound therapy on proliferation, apoptosis and ultrastructure of human B lymphoma Raji cells].

PubMed

Bao, Yong; Fan, Jian-Zhong; Li, Ke; Li, Chuan; Yang, Jun-Feng

2008-06-01

To investigate the effect of infrasound therapy on the proliferation, apoptosis and ultrastructure of human B lymphoma Raji cells. Human B lymphoma Raji cells were exposed to infrasound treatment for 15, 30, 60, 90 and 120 min and cultured subsequently for 24 or 48 h. MTT assay, flow cytometry analysis, and electron microscopy were performed to examine the proliferative status, cell apoptosis and ultrastructural changes of the exposed cells, respectively. MTT assay revealed no significant changes in the proliferation of the cells exposed to infrasound treatment (P>0.05), nor did flow cytometry analysis identified significant variation in the cell apoptosis (P>0.05). Scanning electron microscopy, however, identified shortened or reduced cell processes and microvilli on the surface of the cells with infrasound exposure and a subsequent 24-hour culture, and the cell membrane surface became smooth. Under transmission electron microscope, the cells with infrasound treatment presented with significantly reduced microvilli, and the cell nuclei appeared homogeneous, with cytoplasmic budding and losses after a 48-hour culture. Infrasound less than 90 dB does not obviously affect the proliferation and apoptosis of Raji cells, but may directly cause cell ultrastructural changes such as reduction of the cell processes.

Automation of sample preparation for mass cytometry barcoding in support of clinical research: protocol optimization.

PubMed

Nassar, Ala F; Wisnewski, Adam V; Raddassi, Khadir

2017-03-01

Analysis of multiplexed assays is highly important for clinical diagnostics and other analytical applications. Mass cytometry enables multi-dimensional, single-cell analysis of cell type and state. In mass cytometry, the rare earth metals used as reporters on antibodies allow determination of marker expression in individual cells. Barcode-based bioassays for CyTOF are able to encode and decode for different experimental conditions or samples within the same experiment, facilitating progress in producing straightforward and consistent results. Herein, an integrated protocol for automated sample preparation for barcoding used in conjunction with mass cytometry for clinical bioanalysis samples is described; we offer results of our work with barcoding protocol optimization. In addition, we present some points to be considered in order to minimize the variability of quantitative mass cytometry measurements. For example, we discuss the importance of having multiple populations during titration of the antibodies and effect of storage and shipping of labelled samples on the stability of staining for purposes of CyTOF analysis. Data quality is not affected when labelled samples are stored either frozen or at 4 °C and used within 10 days; we observed that cell loss is greater if cells are washed with deionized water prior to shipment or are shipped in lower concentration. Once the labelled samples for CyTOF are suspended in deionized water, the analysis should be performed expeditiously, preferably within the first hour. Damage can be minimized if the cells are resuspended in phosphate-buffered saline (PBS) rather than deionized water while waiting for data acquisition.

Assessment of DNA replication in central nervous system by Laser Scanning Cytometry

NASA Astrophysics Data System (ADS)

Lenz, Dominik; Mosch, Birgit; Bocsi, Jozsef; Arendt, Thomas; Tárnok, Attila

2004-07-01

μIn neurons of patients with Alzheimers's disease (AD) signs of cell cycle re-entry as well as polyploidy have been reported1, 2, indicating that the entire or a part of the genome of the neurons is duplicated before its death but mitosis is not initiated so that the cellular DNA content remains tetraploid. It was concluded, that this imbalance is the direct cause of the neuronal loss in AD3. Manual counting of polyploidal cells is possible but time consuming and possibly statistically insufficient. The aim of this study was to develop an automated method that detects the neuronal DNA content abnormalities with Laser Scanning Cytometry (LSC).Frozen sections of formalin-fixed brain tissue of AD patients and control subjects were labelled with anti-cyclin B and anti-NeuN antibodies. Immunolabelling was performed using Cy5- and Cy2-conjugated secondary antibodies and biotin streptavidin or tyramid signal amplification. In the end sections of 20m thickness were incubated with propidium iodide (PI) (50μg/ml) and covered on slides. For analysis by the LSC PI was used as trigger. Cells identified as neurons by NeuN expression were analyzed for cyclin B expression. Per specimen data of at least 10,000 neurons were acquired. In the frozen brain sections an automated quantification of the amount of nuclear DNA is possible with LSC. The DNA ploidy as well as the cell cycle distribution can be analyzed. A high number of neurons can be scanned and the duration of measuring is shorter than a manual examination. The amount of DNA is sufficiently represented by the PI fluorescence to be able to distinguish between eu- and polyploid neurons.

Comparative exploration of multidimensional flow cytometry software: a model approach evaluating T cell polyfunctional behavior.

PubMed

Spear, Timothy T; Nishimura, Michael I; Simms, Patricia E

2017-08-01

Advancement in flow cytometry reagents and instrumentation has allowed for simultaneous analysis of large numbers of lineage/functional immune cell markers. Highly complex datasets generated by polychromatic flow cytometry require proper analytical software to answer investigators' questions. A problem among many investigators and flow cytometry Shared Resource Laboratories (SRLs), including our own, is a lack of access to a flow cytometry-knowledgeable bioinformatics team, making it difficult to learn and choose appropriate analysis tool(s). Here, we comparatively assess various multidimensional flow cytometry software packages for their ability to answer a specific biologic question and provide graphical representation output suitable for publication, as well as their ease of use and cost. We assessed polyfunctional potential of TCR-transduced T cells, serving as a model evaluation, using multidimensional flow cytometry to analyze 6 intracellular cytokines and degranulation on a per-cell basis. Analysis of 7 parameters resulted in 128 possible combinations of positivity/negativity, far too complex for basic flow cytometry software to analyze fully. Various software packages were used, analysis methods used in each described, and representative output displayed. Of the tools investigated, automated classification of cellular expression by nonlinear stochastic embedding (ACCENSE) and coupled analysis in Pestle/simplified presentation of incredibly complex evaluations (SPICE) provided the most user-friendly manipulations and readable output, evaluating effects of altered antigen-specific stimulation on T cell polyfunctionality. This detailed approach may serve as a model for other investigators/SRLs in selecting the most appropriate software to analyze complex flow cytometry datasets. Further development and awareness of available tools will help guide proper data analysis to answer difficult biologic questions arising from incredibly complex datasets. © Society for Leukocyte Biology.

SURVIVAL OF SALMONELLA SPECIES IN RIVER WATER

EPA Science Inventory

The survival of four Salmonella strains in river water microcosms was monitored by culturing techniques, direct counts, whole-cell hybridization, scanning electron microscopy, and resuscitation techniques via the direct viable count method and flow cytometry. Plate counts of bact...

Using laser scanning cytometry to measure PPAR-mediated peroxisome proliferation and beta oxidation.

PubMed

Pruimboom-Brees, Ingrid M; Brees, Dominique J J E; Shen, Amy C; Keener, Mary; Francone, Omar; Amacher, David E; Loy, James K; Kerlin, Roy L

2005-01-01

Laser scanning cytometry (LSC) is a new technology that combines the properties and advantages of flow cytometry (FC) and immunohistochemistry (IHC), thus providing qualitative and quantitative information on protein expression with the additional perspective provided by cell and tissue localization. Formalin-fixed, paraffin embedded liver sections from rats exposed to a Peroxisome Proliferator Activated Receptor (PPAR) agonist were stained with antibodies against peroxisomal targeting signal-1 (PTS-1) (a highly conserved tripeptide contained within all peroxisomal enzymes), Acyl CoA oxidase (AOX) (the rate limiting enzyme of peroxisomal beta oxidation), and catalase (an inducible peroxisomal antioxidant enzyme) to evaluate peroxisomal beta oxidation, oxidative stress, and peroxisome proliferation. The LSC showed increased AOX, catalase, and PTS-1 expression in centrilobular hepatocytes that correlated favorably with the microscopic observation of centrilobular hepatocellular hypertrophy and with the palmitoyl CoA biochemical assay for peroxisomal beta oxidation, and provided additional morphologic information about peroxisome proliferation and tissue patterns of activation. Therefore, the LSC provides qualitative and quantitative evaluation of peroxisome activity with similar sensitivity but higher throughput than the traditional biochemical methods. The additional benefits of the LSC include the direct correlation between histopathologic observations and peroxisomal alterations and the potential utilization of archived formalin-fixed tissues from a variety of organs and species.

Label-free cell-cycle analysis by high-throughput quantitative phase time-stretch imaging flow cytometry

NASA Astrophysics Data System (ADS)

Mok, Aaron T. Y.; Lee, Kelvin C. M.; Wong, Kenneth K. Y.; Tsia, Kevin K.

2018-02-01

Biophysical properties of cells could complement and correlate biochemical markers to characterize a multitude of cellular states. Changes in cell size, dry mass and subcellular morphology, for instance, are relevant to cell-cycle progression which is prevalently evaluated by DNA-targeted fluorescence measurements. Quantitative-phase microscopy (QPM) is among the effective biophysical phenotyping tools that can quantify cell sizes and sub-cellular dry mass density distribution of single cells at high spatial resolution. However, limited camera frame rate and thus imaging throughput makes QPM incompatible with high-throughput flow cytometry - a gold standard in multiparametric cell-based assay. Here we present a high-throughput approach for label-free analysis of cell cycle based on quantitative-phase time-stretch imaging flow cytometry at a throughput of > 10,000 cells/s. Our time-stretch QPM system enables sub-cellular resolution even at high speed, allowing us to extract a multitude (at least 24) of single-cell biophysical phenotypes (from both amplitude and phase images). Those phenotypes can be combined to track cell-cycle progression based on a t-distributed stochastic neighbor embedding (t-SNE) algorithm. Using multivariate analysis of variance (MANOVA) discriminant analysis, cell-cycle phases can also be predicted label-free with high accuracy at >90% in G1 and G2 phase, and >80% in S phase. We anticipate that high throughput label-free cell cycle characterization could open new approaches for large-scale single-cell analysis, bringing new mechanistic insights into complex biological processes including diseases pathogenesis.

Individual Cell Based Traits Obtained by Scanning Flow-Cytometry Show Selection by Biotic and Abiotic Environmental Factors during a Phytoplankton Spring Bloom

PubMed Central

Pomati, Francesco; Kraft, Nathan J. B.; Posch, Thomas; Eugster, Bettina; Jokela, Jukka; Ibelings, Bas W.

2013-01-01

In ecology and evolution, the primary challenge in understanding the processes that shape biodiversity is to assess the relationship between the phenotypic traits of organisms and the environment. Here we tested for selection on physio-morphological traits measured by scanning flow-cytometry at the individual level in phytoplankton communities under a temporally changing biotic and abiotic environment. Our aim was to study how high-frequency temporal changes in the environment influence biodiversity dynamics in a natural community. We focused on a spring bloom in Lake Zurich (Switzerland), characterized by rapid changes in phytoplankton, water conditions, nutrients and grazing (mainly mediated by herbivore ciliates). We described bloom dynamics in terms of taxonomic and trait-based diversity and found that diversity dynamics of trait-based groups were more pronounced than those of identified phytoplankton taxa. We characterized the linkage between measured phytoplankton traits, abiotic environmental factors and abundance of the main grazers and observed weak but significant correlations between changing abiotic and biotic conditions and measured size-related and fluorescence-related traits. We tested for deviations in observed community-wide distributions of focal traits from random patterns and found evidence for both clustering and even spacing of traits, occurring sporadically over the time series. Patterns were consistent with environmental filtering and phenotypic divergence under herbivore pressure, respectively. Size-related traits showed significant even spacing during the peak of herbivore abundance, suggesting that morphology-related traits were under selection from grazing. Pigment distribution within cells and colonies appeared instead to be associated with acclimation to temperature and water chemistry. We found support for trade-offs among grazing resistance and environmental tolerance traits, as well as for substantial periods of dynamics in which our measured traits were not under selection. PMID:23951218

Individual cell based traits obtained by scanning flow-cytometry show selection by biotic and abiotic environmental factors during a phytoplankton spring bloom.

PubMed

Pomati, Francesco; Kraft, Nathan J B; Posch, Thomas; Eugster, Bettina; Jokela, Jukka; Ibelings, Bas W

2013-01-01

In ecology and evolution, the primary challenge in understanding the processes that shape biodiversity is to assess the relationship between the phenotypic traits of organisms and the environment. Here we tested for selection on physio-morphological traits measured by scanning flow-cytometry at the individual level in phytoplankton communities under a temporally changing biotic and abiotic environment. Our aim was to study how high-frequency temporal changes in the environment influence biodiversity dynamics in a natural community. We focused on a spring bloom in Lake Zurich (Switzerland), characterized by rapid changes in phytoplankton, water conditions, nutrients and grazing (mainly mediated by herbivore ciliates). We described bloom dynamics in terms of taxonomic and trait-based diversity and found that diversity dynamics of trait-based groups were more pronounced than those of identified phytoplankton taxa. We characterized the linkage between measured phytoplankton traits, abiotic environmental factors and abundance of the main grazers and observed weak but significant correlations between changing abiotic and biotic conditions and measured size-related and fluorescence-related traits. We tested for deviations in observed community-wide distributions of focal traits from random patterns and found evidence for both clustering and even spacing of traits, occurring sporadically over the time series. Patterns were consistent with environmental filtering and phenotypic divergence under herbivore pressure, respectively. Size-related traits showed significant even spacing during the peak of herbivore abundance, suggesting that morphology-related traits were under selection from grazing. Pigment distribution within cells and colonies appeared instead to be associated with acclimation to temperature and water chemistry. We found support for trade-offs among grazing resistance and environmental tolerance traits, as well as for substantial periods of dynamics in which our measured traits were not under selection.

A pilot study for the integration of cytometry reports in digital cytology telemedicine applications.

PubMed

Giansanti, Daniele; Cerroni, Fabio; Amodeo, Rachele; Filoni, Marco; Giovagnoli, Maria Rosaria

2010-01-01

Up to date, tele-pathology in the three different forms of application, "dynamic", "static" and "virtual microscopy" has been mainly based on tele-hystology remote consulting. Today the diffusion of specialized WAN connections is guiding the research of new applications of tele-pathology. A specific analysis has been conducted, focused on digital cytology, in the biomedical laboratory of Sant'Andrea Hospital to investigate the technologies potentially useful to integrate in the LAN/WAN for telemedicine applications. Among the possible tools useful to be integrated in the LAN/WAN for telemedicine applications, the cytometry equipment available in the technical unity of cytometry has been considered important. The study finally provides a proposal for a tele-consulting architecture for the integration of cytometry reports both in the hospital LAN and the WAN for possible cooperative diagnosis and second opinion support.

Ferroptosis and Cell Death Analysis by Flow Cytometry.

PubMed

Chen, Daishi; Eyupoglu, Ilker Y; Savaskan, Nicolai

2017-01-01

Cell death and its recently discovered regulated form ferroptosis are characterized by distinct morphological, electrophysiological, and pharmacological features. In particular ferroptosis can be induced by experimental compounds and clinical drugs (i.e., erastin, sulfasalazine, sorafenib, and artesunate) in various cell types and cancer cells. Pharmacologically, this cell death process can be inhibited by iron chelators and lipid peroxidation inhibitors. Relevance of this specific cell death form has been found in different pathological conditions such as cancer, neurotoxicity, neurodegeneration, and ischemia. Distinguishing cell viability and cell death is essential for experimental and clinical applications and a key component in flow cytometry experiments. Dead cells can compromise the integrity of the data by nonspecific binding of antibodies and dyes. Therefore it is essential that dead cells are robustly and reproducibly identified and characterized by means of cytometry application. Here we describe a procedure to detect and quantify cell death and its specific form ferroptosis based on standard flow cytometry techniques.

Tracking protein aggregation and mislocalization in cells with flow cytometry.

PubMed

Ramdzan, Yasmin M; Polling, Saskia; Chia, Cheryl P Z; Ng, Ivan H W; Ormsby, Angelique R; Croft, Nathan P; Purcell, Anthony W; Bogoyevitch, Marie A; Ng, Dominic C H; Gleeson, Paul A; Hatters, Danny M

2012-03-18

We applied pulse-shape analysis (PulSA) to monitor protein localization changes in mammalian cells by flow cytometry. PulSA enabled high-throughput tracking of protein aggregation, translocation from the cytoplasm to the nucleus and trafficking from the plasma membrane to the Golgi as well as stress-granule formation. Combining PulSA with tetracysteine-based oligomer sensors in a cell model of Huntington's disease enabled further separation of cells enriched with monomers, oligomers and inclusion bodies.

CytometryML and other data formats

NASA Astrophysics Data System (ADS)

Leif, Robert C.

2006-02-01

Cytology automation and research will be enhanced by the creation of a common data format. This data format would provide the pathology and research communities with a uniform way for annotating and exchanging images, flow cytometry, and associated data. This specification and/or standard will include descriptions of the acquisition device, staining, the binary representations of the image and list-mode data, the measurements derived from the image and/or the list-mode data, and descriptors for clinical/pathology and research. An international, vendor-supported, non-proprietary specification will allow pathologists, researchers, and companies to develop and use image capture/analysis software, as well as list-mode analysis software, without worrying about incompatibilities between proprietary vendor formats. Presently, efforts to create specifications and/or descriptions of these formats include the Laboratory Digital Imaging Project (LDIP) Data Exchange Specification; extensions to the Digital Imaging and Communications in Medicine (DICOM); Open Microscopy Environment (OME); Flowcyt, an extension to the present Flow Cytometry Standard (FCS); and CytometryML. The feasibility of creating a common data specification for digital microscopy and flow cytometry in a manner consistent with its use for medical devices and interoperability with both hospital information and picture archiving systems has been demonstrated by the creation of the CytometryML schemas. The feasibility of creating a software system for digital microscopy has been demonstrated by the OME. CytometryML consists of schemas that describe instruments and their measurements. These instruments include digital microscopes and flow cytometers. Optical components including the instruments' excitation and emission parts are described. The description of the measurements made by these instruments includes the tagged molecule, data acquisition subsystem, and the format of the list-mode and/or image data. Many of the CytometryML data-types are based on the Digital Imaging and Communications in Medicine (DICOM). Binary files for images and list-mode data have been created and read.

Validation of cell-based fluorescence assays: practice guidelines from the ICSH and ICCS - part II - preanalytical issues.

PubMed

Davis, Bruce H; Dasgupta, Amar; Kussick, Steven; Han, Jin-Yeong; Estrellado, Annalee

2013-01-01

Flow cytometry and other technologies of cell-based fluorescence assays are as a matter of good laboratory practice required to validate all assays, which when in clinical practice may pass through regulatory review processes using criteria often defined with a soluble analyte in plasma or serum samples in mind. Recently the U.S. Food and Drug Administration (FDA) has entered into a public dialogue in the U.S. regarding their regulatory interest in laboratory developed tests (LDTs) or so-called "home brew" assays performed in clinical laboratories. The absence of well-defined guidelines for validation of cell-based assays using fluorescence detection has thus become a subject of concern for the International Council for Standardization of Haematology (ICSH) and International Clinical Cytometry Society (ICCS). Accordingly, a group of over 40 international experts in the areas of test development, test validation, and clinical practice of a variety of assay types using flow cytometry and/or morphologic image analysis were invited to develop a set of practical guidelines useful to in vitro diagnostic (IVD) innovators, clinical laboratories, regulatory scientists, and laboratory inspectors. The focus of the group was restricted to fluorescence reporter reagents, although some common principles are shared by immunohistochemistry or immunocytochemistry techniques and noted where appropriate. The work product of this two year effort is the content of this special issue of this journal, which is published as 5 separate articles, this being Validation of Cell-based Fluorescence Assays: Practice Guidelines from the ICSH and ICCS - Part II - Preanalytical issues. © 2013 International Clinical Cytometry Society. © 2013 International Clinical Cytometry Society.

Rapid Flow Cytometry-Based Test for the Diagnosis of Lipopolysaccharide Responsive Beige-Like Anchor (LRBA) Deficiency.

PubMed

Gámez-Díaz, Laura; Sigmund, Elena C; Reiser, Veronika; Vach, Werner; Jung, Sophie; Grimbacher, Bodo

2018-01-01

The diagnosis of lipopolysaccharide-responsive beige-like-anchor-protein (LRBA) deficiency currently relies on gene sequencing approaches that do not support a timely diagnosis and clinical management. We developed a rapid and sensitive test for clinical implementation based on the detection of LRBA protein by flow cytometry in peripheral blood cells after stimulation. LRBA protein was assessed in a prospective cohort of 54 healthy donors and 57 patients suspected of LRBA deficiency. Receiver operating characteristics analysis suggested an LRBA:MFI ratio cutoff point of 2.6 to identify LRBA-deficient patients by FACS with 94% sensitivity and 80% specificity and to discriminate them from patients with a similar clinical picture but other disease-causing mutations. This easy flow cytometry-based assay allows a fast screening of patients with suspicion of LRBA deficiency reducing therefore the number of patients requiring LRBA sequencing and accelerating the treatment implementation. Detection of biallelic mutations in LRBA is however required for a definitive diagnosis.

Identifying the Presence of Prostate Cancer in Individuals with PSA Levels <20 ng ml-1 Using Computational Data Extraction Analysis of High Dimensional Peripheral Blood Flow Cytometric Phenotyping Data.

PubMed

Cosma, Georgina; McArdle, Stéphanie E; Reeder, Stephen; Foulds, Gemma A; Hood, Simon; Khan, Masood; Pockley, A Graham

2017-01-01

Determining whether an asymptomatic individual with Prostate-Specific Antigen (PSA) levels below 20 ng ml -1 has prostate cancer in the absence of definitive, biopsy-based evidence continues to present a significant challenge to clinicians who must decide whether such individuals with low PSA values have prostate cancer. Herein, we present an advanced computational data extraction approach which can identify the presence of prostate cancer in men with PSA levels <20 ng ml -1 on the basis of peripheral blood immune cell profiles that have been generated using multi-parameter flow cytometry. Statistical analysis of immune phenotyping datasets relating to the presence and prevalence of key leukocyte populations in the peripheral blood, as generated from individuals undergoing routine tests for prostate cancer (including tissue biopsy) using multi-parametric flow cytometric analysis, was unable to identify significant relationships between leukocyte population profiles and the presence of benign disease (no prostate cancer) or prostate cancer. By contrast, a Genetic Algorithm computational approach identified a subset of five flow cytometry features ( CD 8 + CD 45 RA - CD 27 - CD 28 - ( CD 8 + Effector Memory cells); CD 4 + CD 45 RA - CD 27 - CD 28 - ( CD 4 + Terminally Differentiated Effector Memory Cells re-expressing CD45RA); CD 3 - CD 19 + (B cells); CD 3 + CD 56 + CD 8 + CD 4 + (NKT cells)) from a set of twenty features, which could potentially discriminate between benign disease and prostate cancer. These features were used to construct a prostate cancer prediction model using the k-Nearest-Neighbor classification algorithm. The proposed model, which takes as input the set of flow cytometry features, outperformed the predictive model which takes PSA values as input. Specifically, the flow cytometry-based model achieved Accuracy = 83.33%, AUC = 83.40%, and optimal ROC points of FPR = 16.13%, TPR = 82.93%, whereas the PSA-based model achieved Accuracy = 77.78%, AUC = 76.95%, and optimal ROC points of FPR = 29.03%, TPR = 82.93%. Combining PSA and flow cytometry predictors achieved Accuracy = 79.17%, AUC = 78.17% and optimal ROC points of FPR = 29.03%, TPR = 85.37%. The results demonstrate the value of computational intelligence-based approaches for interrogating immunophenotyping datasets and that combining peripheral blood phenotypic profiling with PSA levels improves diagnostic accuracy compared to using PSA test alone. These studies also demonstrate that the presence of cancer is reflected in changes in the peripheral blood immune phenotype profile which can be identified using computational analysis and interpretation of complex flow cytometry datasets.

Cytobank: providing an analytics platform for community cytometry data analysis and collaboration.

PubMed

Chen, Tiffany J; Kotecha, Nikesh

2014-01-01

Cytometry is used extensively in clinical and laboratory settings to diagnose and track cell subsets in blood and tissue. High-throughput, single-cell approaches leveraging cytometry are developed and applied in the computational and systems biology communities by researchers, who seek to improve the diagnosis of human diseases, map the structures of cell signaling networks, and identify new cell types. Data analysis and management present a bottleneck in the flow of knowledge from bench to clinic. Multi-parameter flow and mass cytometry enable identification of signaling profiles of patient cell samples. Currently, this process is manual, requiring hours of work to summarize multi-dimensional data and translate these data for input into other analysis programs. In addition, the increase in the number and size of collaborative cytometry studies as well as the computational complexity of analytical tools require the ability to assemble sufficient and appropriately configured computing capacity on demand. There is a critical need for platforms that can be used by both clinical and basic researchers who routinely rely on cytometry. Recent advances provide a unique opportunity to facilitate collaboration and analysis and management of cytometry data. Specifically, advances in cloud computing and virtualization are enabling efficient use of large computing resources for analysis and backup. An example is Cytobank, a platform that allows researchers to annotate, analyze, and share results along with the underlying single-cell data.

Organizing the Cellular and Molecular Heterogeneity in High-Grade Serous Ovarian Cancer by Mass Cytometry

DTIC Science & Technology

2014-10-01

Bendall SC, Sung P, Nolan GP, Arvin AM. Single-cell mass cytometry analysis of human tonsil T cell remodeling by varicella zoster virus. Cell Rep...Perspectives on Flow Cytometry 2013, September 20, 2013, Mass Cytometry and Cell Cycle, Mexico City, Mexico (by Web Conference) Nolan: Nuclear

CytometryML: a markup language for analytical cytology

NASA Astrophysics Data System (ADS)

Leif, Robert C.; Leif, Stephanie H.; Leif, Suzanne B.

2003-06-01

Cytometry Markup Language, CytometryML, is a proposed new analytical cytology data standard. CytometryML is a set of XML schemas for encoding both flow cytometry and digital microscopy text based data types. CytometryML schemas reference both DICOM (Digital Imaging and Communications in Medicine) codes and FCS keywords. These schemas provide representations for the keywords in FCS 3.0 and will soon include DICOM microscopic image data. Flow Cytometry Standard (FCS) list-mode has been mapped to the DICOM Waveform Information Object. A preliminary version of a list mode binary data type, which does not presently exist in DICOM, has been designed. This binary type is required to enhance the storage and transmission of flow cytometry and digital microscopy data. Index files based on Waveform indices will be used to rapidly locate the cells present in individual subsets. DICOM has the advantage of employing standard file types, TIF and JPEG, for Digital Microscopy. Using an XML schema based representation means that standard commercial software packages such as Excel and MathCad can be used to analyze, display, and store analytical cytometry data. Furthermore, by providing one standard for both DICOM data and analytical cytology data, it eliminates the need to create and maintain special purpose interfaces for analytical cytology data thereby integrating the data into the larger DICOM and other clinical communities. A draft version of CytometryML is available at www.newportinstruments.com.

Real-time Image Processing for Microscopy-based Label-free Imaging Flow Cytometry in a Microfluidic Chip.

PubMed

Heo, Young Jin; Lee, Donghyeon; Kang, Junsu; Lee, Keondo; Chung, Wan Kyun

2017-09-14

Imaging flow cytometry (IFC) is an emerging technology that acquires single-cell images at high-throughput for analysis of a cell population. Rich information that comes from high sensitivity and spatial resolution of a single-cell microscopic image is beneficial for single-cell analysis in various biological applications. In this paper, we present a fast image-processing pipeline (R-MOD: Real-time Moving Object Detector) based on deep learning for high-throughput microscopy-based label-free IFC in a microfluidic chip. The R-MOD pipeline acquires all single-cell images of cells in flow, and identifies the acquired images as a real-time process with minimum hardware that consists of a microscope and a high-speed camera. Experiments show that R-MOD has the fast and reliable accuracy (500 fps and 93.3% mAP), and is expected to be used as a powerful tool for biomedical and clinical applications.

Validation of cell-based fluorescence assays: practice guidelines from the ICSH and ICCS - part I - rationale and aims.

PubMed

Davis, Bruce H; Wood, Brent; Oldaker, Teri; Barnett, David

2013-01-01

Flow cytometry and other technologies of cell-based fluorescence assays are as a matter of good laboratory practice required to validate all assays, which when in clinical practice may pass through regulatory review processes using criteria often defined with a soluble analyte in plasma or serum samples in mind. Recently the U.S. Food and Drug Administration (FDA) has entered into a public dialogue in the U.S. regarding their regulatory interest in laboratory developed tests (LDTs) or so-called "home brew" assays performed in clinical laboratories. The absence of well-defined guidelines for validation of cell-based assays using fluorescence detection has thus become a subject of concern for the International Council for Standardization of Haematology (ICSH) and International Clinical Cytometry Society (ICCS). Accordingly, a group of over 40 international experts in the areas of test development, test validation, and clinical practice of a variety of assay types using flow cytometry and/or morphologic image analysis were invited to develop a set of practical guidelines useful to in vitro diagnostic (IVD) innovators, clinical laboratories, regulatory scientists, and laboratory inspectors. The focus of the group was restricted to fluorescence reporter reagents, although some common principles are shared by immunohistochemistry or immunocytochemistry techniques and noted where appropriate. The work product of this two year effort is the content of this special issue of this journal, which is published as 5 separate articles, this being Validation of Cell-based Fluorescence Assays: Practice Guidelines from the ICSH and ICCS - Part I - Rationale and aims. © 2013 International Clinical Cytometry Society. © 2013 International Clinical Cytometry Society.

DAFi: A directed recursive data filtering and clustering approach for improving and interpreting data clustering identification of cell populations from polychromatic flow cytometry data.

PubMed

Lee, Alexandra J; Chang, Ivan; Burel, Julie G; Lindestam Arlehamn, Cecilia S; Mandava, Aishwarya; Weiskopf, Daniela; Peters, Bjoern; Sette, Alessandro; Scheuermann, Richard H; Qian, Yu

2018-04-17

Computational methods for identification of cell populations from polychromatic flow cytometry data are changing the paradigm of cytometry bioinformatics. Data clustering is the most common computational approach to unsupervised identification of cell populations from multidimensional cytometry data. However, interpretation of the identified data clusters is labor-intensive. Certain types of user-defined cell populations are also difficult to identify by fully automated data clustering analysis. Both are roadblocks before a cytometry lab can adopt the data clustering approach for cell population identification in routine use. We found that combining recursive data filtering and clustering with constraints converted from the user manual gating strategy can effectively address these two issues. We named this new approach DAFi: Directed Automated Filtering and Identification of cell populations. Design of DAFi preserves the data-driven characteristics of unsupervised clustering for identifying novel cell subsets, but also makes the results interpretable to experimental scientists through mapping and merging the multidimensional data clusters into the user-defined two-dimensional gating hierarchy. The recursive data filtering process in DAFi helped identify small data clusters which are otherwise difficult to resolve by a single run of the data clustering method due to the statistical interference of the irrelevant major clusters. Our experiment results showed that the proportions of the cell populations identified by DAFi, while being consistent with those by expert centralized manual gating, have smaller technical variances across samples than those from individual manual gating analysis and the nonrecursive data clustering analysis. Compared with manual gating segregation, DAFi-identified cell populations avoided the abrupt cut-offs on the boundaries. DAFi has been implemented to be used with multiple data clustering methods including K-means, FLOCK, FlowSOM, and the ClusterR package. For cell population identification, DAFi supports multiple options including clustering, bisecting, slope-based gating, and reversed filtering to meet various autogating needs from different scientific use cases. © 2018 International Society for Advancement of Cytometry. © 2018 International Society for Advancement of Cytometry.

Tips and tricks for flow cytometry-based analysis and counting of microparticles.

PubMed

Poncelet, Philippe; Robert, Stéphane; Bailly, Nicolas; Garnache-Ottou, Francine; Bouriche, Tarik; Devalet, Bérangère; Segatchian, Jerard H; Saas, Philippe; Mullier, François

2015-10-01

Submicron-sized extra-cellular vesicles generated by budding from the external cell membranes, microparticles (MPs) are important actors in transfusion as well as in other medical specialties. After briefly positioning their role in the characterization of labile blood products, this technically oriented chapter aims to review practical points that need to be considered when trying to use flow cytometry for the analysis, characterization and absolute counting of MP subsets. Subjects of active discussions relative to instrumentation will include the choice of the trigger parameter, possible standardization approaches requiring instrument quality-control, origin and control of non-specific background and of coincidence artifacts, choice of the type of electronic signals, optimal sheath fluid and sample speed. Questions related to reagents will cover target antigens and receptors, multi-color reagents, negative controls, enumeration of MPs and limiting artifacts due to unexpected (micro-) coagulation of plasma samples. Newly detected problems are generating innovative solutions and flow cytometry will continue to remain the technology of choice for the analysis of MPs, in the domain of transfusion as well as in many diverse specialties. Copyright © 2015 Elsevier Ltd. All rights reserved.

Novel Quantitative Autophagy Analysis by Organelle Flow Cytometry after Cell Sonication

PubMed Central

Degtyarev, Michael; Reichelt, Mike; Lin, Kui

2014-01-01

Autophagy is a dynamic process of bulk degradation of cellular proteins and organelles in lysosomes. Current methods of autophagy measurement include microscopy-based counting of autophagic vacuoles (AVs) in cells. We have developed a novel method to quantitatively analyze individual AVs using flow cytometry. This method, OFACS (organelle flow after cell sonication), takes advantage of efficient cell disruption with a brief sonication, generating cell homogenates with fluorescently labeled AVs that retain their integrity as confirmed with light and electron microscopy analysis. These AVs could be detected directly in the sonicated cell homogenates on a flow cytometer as a distinct population of expected organelle size on a cytometry plot. Treatment of cells with inhibitors of autophagic flux, such as chloroquine or lysosomal protease inhibitors, increased the number of particles in this population under autophagy inducing conditions, while inhibition of autophagy induction with 3-methyladenine or knockdown of ATG proteins prevented this accumulation. This assay can be easily performed in a high-throughput format and opens up previously unexplored avenues for autophagy analysis. PMID:24489953

Semi-automatized segmentation method using image-based flow cytometry to study sperm physiology: the case of capacitation-induced tyrosine phosphorylation.

PubMed

Matamoros-Volante, Arturo; Moreno-Irusta, Ayelen; Torres-Rodriguez, Paulina; Giojalas, Laura; Gervasi, María G; Visconti, Pablo E; Treviño, Claudia L

2018-02-01

Is image-based flow cytometry a useful tool to study intracellular events in human sperm such as protein tyrosine phosphorylation or signaling processes? Image-based flow cytometry is a powerful tool to study intracellular events in a relevant number of sperm cells, which enables a robust statistical analysis providing spatial resolution in terms of the specific subcellular localization of the labeling. Sperm capacitation is required for fertilization. During this process, spermatozoa undergo numerous physiological changes, via activation of different signaling pathways, which are not completely understood. Classical approaches for studying sperm physiology include conventional microscopy, flow cytometry and Western blotting. These techniques present disadvantages for obtaining detailed subcellular information of signaling pathways in a relevant number of cells. This work describes a new semi-automatized analysis using image-based flow cytometry which enables the study, at the subcellular and population levels, of different sperm parameters associated with signaling. The increase in protein tyrosine phosphorylation during capacitation is presented as an example. Sperm cells were isolated from seminal plasma by the swim-up technique. We evaluated the intensity and distribution of protein tyrosine phosphorylation in sperm incubated in non-capacitation and capacitation-supporting media for 1 and 18 h under different experimental conditions. We used an antibody against FER kinase and pharmacological inhibitors in an attempt to identify the kinases involved in protein tyrosine phosphorylation during human sperm capacitation. Semen samples from normospermic donors were obtained by masturbation after 2-3 days of sexual abstinence. We used the innovative technique image-based flow cytometry and image analysis tools to segment individual images of spermatozoa. We evaluated and quantified the regions of sperm where protein tyrosine phosphorylation takes place at the subcellular level in a large number of cells. We also used immunocytochemistry and Western blot analysis. Independent experiments were performed with semen samples from seven different donors. Using image analysis tools, we developed a completely novel semi-automatic strategy useful for segmenting thousands of individual cell images obtained using image-based flow cytometry. Contrary to immunofluorescence which relies on the analysis of a limited sperm population and also on the observer, image-based flow cytometry allows for unbiased quantification and simultaneous localization of post-translational changes in an extended sperm population. Interestingly, important data can be independently analyzed by looking to the frame of interest. As an example, we evaluated the capacitation-associated increase in tyrosine phosphorylation in sperm incubated in non-capacitation and capacitation-supporting media for 1 and 18 h. As previously reported, protein tyrosine phosphorylation increases in a time-depending manner, but our method revealed that this increase occurs differentially among distinct sperm segments. FER kinase is reported to be the enzyme responsible for the increase in protein tyrosine phosphorylation in mouse sperm. Our Western blot analysis revealed for the first time the presence of this enzyme in human sperm. Using our segmentation strategy, we aimed to quantify the effect of pharmacological inhibition of FER kinase and found a marked reduction of protein tyrosine phosphorylation only in the flagellum, which corresponded to the physical localization of FER in human sperm. Our method provides an alternative strategy to study signaling markers associated with capacitation, such as protein tyrosine phosphorylation, in a fast and quantitative manner. None. This is an in vitro study performed under controlled conditions. Chemical inhibitors are not completely specific for the intended target; the possibility of side effects cannot be discarded. Our results demonstrate that the use of image-based flow cytometry is a very powerful tool to study sperm physiology. A large number of cells can be easily analyzed and information at the subcellular level can be obtained. As the segmentation process works with bright-field images, it can be extended to study expression of other proteins of interest using different antibodies or it can be used in living sperm to study intracellular parameters that can be followed using fluorescent dyes sensitive to the parameter of interest (e.g. pH, Ca2+). Therefore, this a versatile method that can be exploited to study several aspects of sperm physiology. This work was supported DGAPA (IN203116 to C. Treviño), Fronteras-CONACyT No. 71 and Eunice Kennedy Shriver National Institute of Child Health and Human Development NIH (RO1 HD38082) to P.E. Visconti and by a Lalor Foundation fellowship to M.G. Gervasi. A. Matamoros is a student of the Maestría en Ciencias Bioquímicas-UNAM program supported by CONACyT (416400) and DGAPA-UNAM. A. Moreno obtained a scholarship from Red MacroUniversidades and L. Giojalas obtained a schloarhip from CONICET and Universidad Nacional de Cordoba. The authors declare there are not conflicts of interest. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email:journals.permissions@oup.com

Simultaneous automatic scoring and co-registration of hormone receptors in tumor areas in whole slide images of breast cancer tissue slides.

PubMed

Trahearn, Nicholas; Tsang, Yee Wah; Cree, Ian A; Snead, David; Epstein, David; Rajpoot, Nasir

2017-06-01

Automation of downstream analysis may offer many potential benefits to routine histopathology. One area of interest for automation is in the scoring of multiple immunohistochemical markers to predict the patient's response to targeted therapies. Automated serial slide analysis of this kind requires robust registration to identify common tissue regions across sections. We present an automated method for co-localized scoring of Estrogen Receptor and Progesterone Receptor (ER/PR) in breast cancer core biopsies using whole slide images. Regions of tumor in a series of fifty consecutive breast core biopsies were identified by annotation on H&E whole slide images. Sequentially cut immunohistochemical stained sections were scored manually, before being digitally scanned and then exported into JPEG 2000 format. A two-stage registration process was performed to identify the annotated regions of interest in the immunohistochemistry sections, which were then scored using the Allred system. Overall correlation between manual and automated scoring for ER and PR was 0.944 and 0.883, respectively, with 90% of ER and 80% of PR scores within in one point or less of agreement. This proof of principle study indicates slide registration can be used as a basis for automation of the downstream analysis for clinically relevant biomarkers in the majority of cases. The approach is likely to be improved by implantation of safeguarding analysis steps post registration. © 2016 International Society for Advancement of Cytometry. © 2016 International Society for Advancement of Cytometry.

Imaging flow cytometry for phytoplankton analysis.

PubMed

Dashkova, Veronika; Malashenkov, Dmitry; Poulton, Nicole; Vorobjev, Ivan; Barteneva, Natasha S

2017-01-01

This review highlights the concepts and instrumentation of imaging flow cytometry technology and in particular its use for phytoplankton analysis. Imaging flow cytometry, a hybrid technology combining speed and statistical capabilities of flow cytometry with imaging features of microscopy, is rapidly advancing as a cell imaging platform that overcomes many of the limitations of current techniques and contributed significantly to the advancement of phytoplankton analysis in recent years. This review presents the various instrumentation relevant to the field and currently used for assessment of complex phytoplankton communities' composition and abundance, size structure determination, biovolume estimation, detection of harmful algal bloom species, evaluation of viability and metabolic activity and other applications. Also we present our data on viability and metabolic assessment of Aphanizomenon sp. cyanobacteria using Imagestream X Mark II imaging cytometer. Herein, we highlight the immense potential of imaging flow cytometry for microalgal research, but also discuss limitations and future developments. Copyright © 2016 Elsevier Inc. All rights reserved.

Extended Field Laser Confocal Microscopy (EFLCM): Combining automated Gigapixel image capture with in silico virtual microscopy

PubMed Central

Flaberg, Emilie; Sabelström, Per; Strandh, Christer; Szekely, Laszlo

2008-01-01

Background Confocal laser scanning microscopy has revolutionized cell biology. However, the technique has major limitations in speed and sensitivity due to the fact that a single laser beam scans the sample, allowing only a few microseconds signal collection for each pixel. This limitation has been overcome by the introduction of parallel beam illumination techniques in combination with cold CCD camera based image capture. Methods Using the combination of microlens enhanced Nipkow spinning disc confocal illumination together with fully automated image capture and large scale in silico image processing we have developed a system allowing the acquisition, presentation and analysis of maximum resolution confocal panorama images of several Gigapixel size. We call the method Extended Field Laser Confocal Microscopy (EFLCM). Results We show using the EFLCM technique that it is possible to create a continuous confocal multi-colour mosaic from thousands of individually captured images. EFLCM can digitize and analyze histological slides, sections of entire rodent organ and full size embryos. It can also record hundreds of thousands cultured cells at multiple wavelength in single event or time-lapse fashion on fixed slides, in live cell imaging chambers or microtiter plates. Conclusion The observer independent image capture of EFLCM allows quantitative measurements of fluorescence intensities and morphological parameters on a large number of cells. EFLCM therefore bridges the gap between the mainly illustrative fluorescence microscopy and purely quantitative flow cytometry. EFLCM can also be used as high content analysis (HCA) instrument for automated screening processes. PMID:18627634

Extended Field Laser Confocal Microscopy (EFLCM): combining automated Gigapixel image capture with in silico virtual microscopy.

PubMed

Flaberg, Emilie; Sabelström, Per; Strandh, Christer; Szekely, Laszlo

2008-07-16

Confocal laser scanning microscopy has revolutionized cell biology. However, the technique has major limitations in speed and sensitivity due to the fact that a single laser beam scans the sample, allowing only a few microseconds signal collection for each pixel. This limitation has been overcome by the introduction of parallel beam illumination techniques in combination with cold CCD camera based image capture. Using the combination of microlens enhanced Nipkow spinning disc confocal illumination together with fully automated image capture and large scale in silico image processing we have developed a system allowing the acquisition, presentation and analysis of maximum resolution confocal panorama images of several Gigapixel size. We call the method Extended Field Laser Confocal Microscopy (EFLCM). We show using the EFLCM technique that it is possible to create a continuous confocal multi-colour mosaic from thousands of individually captured images. EFLCM can digitize and analyze histological slides, sections of entire rodent organ and full size embryos. It can also record hundreds of thousands cultured cells at multiple wavelength in single event or time-lapse fashion on fixed slides, in live cell imaging chambers or microtiter plates. The observer independent image capture of EFLCM allows quantitative measurements of fluorescence intensities and morphological parameters on a large number of cells. EFLCM therefore bridges the gap between the mainly illustrative fluorescence microscopy and purely quantitative flow cytometry. EFLCM can also be used as high content analysis (HCA) instrument for automated screening processes.

CytometryML: a data standard which has been designed to interface with other standards

NASA Astrophysics Data System (ADS)

Leif, Robert C.

2007-02-01

Because of the differences in the requirements, needs, and past histories including existing standards of the creating organizations, a single encompassing cytology-pathology standard will not, in the near future, replace the multiple existing or under development standards. Except for DICOM and FCS, these standardization efforts are all based on XML. CytometryML is a collection of XML schemas, which are based on the Digital Imaging and Communications in Medicine (DICOM) and Flow Cytometry Standard (FCS) datatypes. The CytometryML schemas contain attributes that link them to the DICOM standard and FCS. Interoperability with DICOM has been facilitated by, wherever reasonable, limiting the difference between CytometryML and the previous standards to syntax. In order to permit the Resource Description Framework, RDF, to reference the CytometryML datatypes, id attributes have been added to many CytometryML elements. The Laboratory Digital Imaging Project (LDIP) Data Exchange Specification and the Flowcyt standards development effort employ RDF syntax. Documentation from DICOM has been reused in CytometryML. The unity of analytical cytology was demonstrated by deriving a microscope type and a flow cytometer type from a generic cytometry instrument type. The feasibility of incorporating the Flowcyt gating schemas into CytometryML has been demonstrated. CytometryML is being extended to include many of the new DICOM Working Group 26 datatypes, which describe patients, specimens, and analytes. In situations where multiple standards are being created, interoperability can be facilitated by employing datatypes based on a common set of semantics and building in links to standards that employ different syntax.

Flow cytometry: basic principles and applications.

PubMed

Adan, Aysun; Alizada, Günel; Kiraz, Yağmur; Baran, Yusuf; Nalbant, Ayten

2017-03-01

Flow cytometry is a sophisticated instrument measuring multiple physical characteristics of a single cell such as size and granularity simultaneously as the cell flows in suspension through a measuring device. Its working depends on the light scattering features of the cells under investigation, which may be derived from dyes or monoclonal antibodies targeting either extracellular molecules located on the surface or intracellular molecules inside the cell. This approach makes flow cytometry a powerful tool for detailed analysis of complex populations in a short period of time. This review covers the general principles and selected applications of flow cytometry such as immunophenotyping of peripheral blood cells, analysis of apoptosis and detection of cytokines. Additionally, this report provides a basic understanding of flow cytometry technology essential for all users as well as the methods used to analyze and interpret the data. Moreover, recent progresses in flow cytometry have been discussed in order to give an opinion about the future importance of this technology.

Flow cytometry shows added value in diagnosing lymphoma in brain biopsies.

PubMed

van der Meulen, Matthijs; Bromberg, Jacoline E C; Lam, King H; Dammers, Ruben; Langerak, Anton W; Doorduijn, Jeanette K; Kros, Johan M; van den Bent, Martin J; van der Velden, Vincent H J

2018-05-10

To assess the sensitivity, specificity and turnaround time of flow cytometric analysis on brain biopsies compared to histology plus immunohistochemistry analysis in tumors with clinical suspicion of lymphoma. All brain biopsies performed between 2010 and 2015 at our institution and analyzed by both immunohistochemistry and flow cytometry were included in this retrospective study. Immunohistochemistry was considered the gold standard. In a total of 77 biopsies from 71 patients, 49 lymphomas were diagnosed by immunohistochemistry, flow cytometry results were concordant in 71 biopsies (92,2%). We found a specificity and sensitivity of flow cytometry of 100% and 87,8%, respectively. The time between the biopsy and reporting the result (turnaround time) was significantly shorter for flow cytometry, compared to immunohistochemistry (median: 1 versus 5 days). Flow cytometry has a high specificity and can confirm the diagnosis of a lymphoma significantly faster than immunohistochemistry. This allows for rapid initiation of treatment in this highly aggressive tumor. However, since its sensitivity is less than 100%, we recommend to perform histology plus immunohistochemistry in parallel to flow cytometry. This article is protected by copyright. All rights reserved. © 2018 International Clinical Cytometry Society.

Elegant pH-Responsive Nanovehicle for Drug Delivery Based on Triazine Dendrimer Modified Magnetic Nanoparticles.

PubMed

Landarani-Isfahani, Amir; Moghadam, Majid; Mohammadi, Shima; Royvaran, Maryam; Moshtael-Arani, Naimeh; Rezaei, Saghar; Tangestaninejad, Shahram; Mirkhani, Valiollah; Mohammadpoor-Baltork, Iraj

2017-08-29

Owing to properties of magnetic nanoparticles and elegant three-dimensional macromolecule architectural features, dendrimeric structures have been investigated as nanoscale drug delivery systems. In this work, a novel magnetic nanocarrier, generation two (G2) triazine dendrimer modified Fe 3 O 4 @SiO 2 magnetic nanoparticles (MNP-G2), was designed, fabricated, and characterized by Fourier transform infrared (FT-IR), thermal gravimetric analysis (TGA), vibrating sample magnetometer (VSM), field emission scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM), and dynamic light scattering (DLS). The prepared MNP-G2 nanosystem offers a new formulation that combines the unique properties of MNPs and triazine dendrimer as a biocompatible material for biomedical applications. To demonstrate the potential of MNP-G2, the nanoparticles were loaded with methotrexate (MTX), a proven chemotherapy drug. The MTX-loaded MNP-G2 (MNP-G2/MTX) exhibited a high drug-loading capacity of MTX and the excellent ability for controlled drug release. The cytotoxicity of MNP-G2/MTX using an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide based assay and MCF-7, HeLa, and Caov-4 cell lines revealed that MNP-G2/MTX was more active against the tumor cells than the free drug in a mildly acidic environment. The results of hemolysis, hemagglutination, and coagulation assays confirmed the good blood safety of MNP-G2/MTX. Moreover, the cell uptake and intracellular distribution of MNP-G2/MTX were studied by flow cytometry analysis and confocal laser scanning microscopy (CLSM). This research suggests that MNP-G2/MTX with good biocompatibility and degradability can be selected as an ideal and effective drug carrier in targeted biomedicine studies especially anticancer applications.

Comprehensive analysis of mouse retinal mononuclear phagocytes.

PubMed

Lückoff, Anika; Scholz, Rebecca; Sennlaub, Florian; Xu, Heping; Langmann, Thomas

2017-06-01

The innate immune system is activated in a number of degenerative and inflammatory retinal disorders such as age-related macular degeneration (AMD). Retinal microglia, choroidal macrophages, and recruited monocytes, collectively termed 'retinal mononuclear phagocytes', are critical determinants of ocular disease outcome. Many publications have described the presence of these cells in mouse models for retinal disease; however, only limited aspects of their behavior have been uncovered, and these have only been uncovered using a single detection method. The workflow presented here describes a comprehensive analysis strategy that allows characterization of retinal mononuclear phagocytes in vivo and in situ. We present standardized working steps for scanning laser ophthalmoscopy of microglia from MacGreen reporter mice (mice expressing the macrophage colony-stimulating factor receptor GFP transgene throughout the mononuclear phagocyte system), quantitative analysis of Iba1-stained retinal sections and flat mounts, CD11b-based retinal flow cytometry, and qRT-PCR analysis of key microglia markers. The protocol can be completed within 3 d, and we present data from retinas treated with laser-induced choroidal neovascularization (CNV), bright white-light exposure, and Fam161a-associated inherited retinal degeneration. The assays can be applied to any of the existing mouse models for retinal disorders and may be valuable for documenting immune responses in studies for immunomodulatory therapies.

Early Detection of NSCLC Using Stromal Markers in Peripheral Blood

DTIC Science & Technology

2016-09-01

circulating myeloid cells, flow cytometry, RNA -sequencing, expression profiling. 3. ACCOMPLISHMENTS:  What were the major goals of the project...Subtask 2: Flow cytometry sorting of circulating myeloid cells. Subtask 3: RNA -Sequencing Subtask 4: RNA -seq data analysis Subtask 5: Feasible RT-PCR...accomplished the patient recruitment, flow cytometry sorting of circulating myeloid cells, RNA -sequencing of the samples. During the RNA - seq data analysis, we

High-speed bioimaging with frequency-division-multiplexed fluorescence confocal microscopy

NASA Astrophysics Data System (ADS)

Mikami, Hideharu; Harmon, Jeffrey; Ozeki, Yasuyuki; Goda, Keisuke

2017-04-01

We present methods of fluorescence confocal microscopy that enable unprecedentedly high frame rate of > 10,000 fps. The methods are based on a frequency-division multiplexing technique, which was originally developed in the field of communication engineering. Specifically, we achieved a broad bandwidth ( 400 MHz) of detection signals using a dual- AOD method and overcame limitations in frame rate, due to a scanning device, by using a multi-line focusing method, resulting in a significant increase in frame rate. The methods have potential biomedical applications such as observation of sub-millisecond dynamics in biological tissues, in-vivo three-dimensional imaging, and fluorescence imaging flow cytometry.

In vitro flow cytometry-based screening platform for cellulase engineering

PubMed Central

Körfer, Georgette; Pitzler, Christian; Vojcic, Ljubica; Martinez, Ronny; Schwaneberg, Ulrich

2016-01-01

Ultrahigh throughput screening (uHTS) plays an essential role in directed evolution for tailoring biocatalysts for industrial applications. Flow cytometry-based uHTS provides an efficient coverage of the generated protein sequence space by analysis of up to 107 events per hour. Cell-free enzyme production overcomes the challenge of diversity loss during the transformation of mutant libraries into expression hosts, enables directed evolution of toxic enzymes, and holds the promise to efficiently design enzymes of human or animal origin. The developed uHTS cell-free compartmentalization platform (InVitroFlow) is the first report in which a flow cytometry-based screened system has been combined with compartmentalized cell-free expression for directed cellulase enzyme evolution. InVitroFlow was validated by screening of a random cellulase mutant library employing a novel screening system (based on the substrate fluorescein-di-β-D-cellobioside), and yielded significantly improved cellulase variants (e.g. CelA2-H288F-M1 (N273D/H288F/N468S) with 13.3-fold increased specific activity (220.60 U/mg) compared to CelA2 wildtype: 16.57 U/mg). PMID:27184298

Quantifying CD4 receptor protein in two human CD4+ lymphocyte preparations for quantitative flow cytometry.

PubMed

Wang, Meiyao; Misakian, Martin; He, Hua-Jun; Bajcsy, Peter; Abbasi, Fatima; Davis, Jeffrey M; Cole, Kenneth D; Turko, Illarion V; Wang, Lili

2014-01-01

In our previous study that characterized different human CD4+ lymphocyte preparations, it was found that both commercially available cryopreserved peripheral blood mononuclear cells (PBMC) and a commercially available lyophilized PBMC (Cyto-Trol™) preparation fulfilled a set of criteria for serving as biological calibrators for quantitative flow cytometry. However, the biomarker CD4 protein expression level measured for T helper cells from Cyto-Trol was about 16% lower than those for cryopreserved PBMC and fresh whole blood using flow cytometry and mass cytometry. A primary reason was hypothesized to be due to steric interference in anti- CD4 antibody binding to the smaller sized lyophilized control cells. Targeted multiple reaction monitoring (MRM) mass spectrometry (MS) is used to quantify the copy number of CD4 receptor protein per CD4+ lymphocyte. Scanning electron microscopy (SEM) is utilized to assist searching the underlying reasons for the observed difference in CD4 receptor copy number per cell determined by MRM MS and CD4 expression measured previously by flow cytometry. The copy number of CD4 receptor proteins on the surface of the CD4+ lymphocyte in cryopreserved PBMCs and in lyophilized control cells is determined to be (1.45 ± 0.09) × 10(5) and (0.85 ± 0.11) × 10(5), respectively, averaged over four signature peptides using MRM MS. In comparison with cryopreserved PBMCs, there are more variations in the CD4 copy number in lyophilized control cells determined based on each signature peptide. SEM images of CD4+ lymphocytes from lyophilized control cells are very different when compared to the CD4+ T cells from whole blood and cryopreserved PBMC. Because of the lyophilization process applied to Cyto-Trol control cells, a lower CD4 density value, defined as the copy number of CD4 receptors per CD4+ lymphocyte, averaged over three different production lots is most likely explained by the loss of the CD4 receptors on damaged and/or broken microvilli where CD4 receptors reside. Steric hindrance of antibody binding and the association of CD4 receptors with other biomolecules likely contribute significantly to the nearly 50% lower CD4 receptor density value for cryopreserved PBMC determined from flow cytometry compared to the value obtained from MRM MS.

Supercontinuum white light lasers for flow cytometry

PubMed Central

Telford, William G.; Subach, Fedor V.; Verkhusha, Vladislav V.

2009-01-01

Excitation of fluorescent probes for flow cytometry has traditionally been limited to a few discrete laser lines, an inherent limitation in our ability to excite the vast array of fluorescent probes available for cellular analysis. 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 (~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 similar excitation levels to traditional lasers for a wide variety of fluorescent probes and expressible proteins. Sensitivity assessment using fluorescent bead arrays confirmed that the SC laser and traditional sources resulted in similar levels of detection sensitivity. Supercontinuum white light laser sources therefore have the potential to remove a significant barrier in flow cytometric analysis, namely the limitation of excitation wavelengths. Almost any visible wavelength range can be made available for excitation, allowing access to virtually any fluorescent probe, and permitting “fine-tuning” of excitation wavelength to particular probes. PMID:19072836

Biological activity of the azlactone derivative EPA-35 against Trypanosoma cruzi.

PubMed

de Azeredo, Camila Maria Oliveira; Ávila, Eloah Pereira; Pinheiro, Danielle Lobo Justo; Amarante, Giovanni Wilson; Soares, Maurilio José

2017-02-01

Chagas disease, caused by Trypanosoma cruzi, affects six to seven million people worldwide. Treatment is based on benznidazole, producing several side effects and debatable efficacy, highlighting the need for new alternative drugs. We investigated the activity of four C-4 functionalized azlactone derivatives (EPA-27, EPA-35, EPA-63 and EPA-91) as potential T. cruzi inhibitors. Screening with epimastigotes indicated EPA-35 as the best compound (IC50/24 h: 33 μM). This compound was 14.1 times more potent against intracellular amastigotes (IC50/24 h: 2.34 μM). Treatment of infected Vero cells for 72 h (up to 30 μM EPA-35) resulted in a dose-dependent decrease in number of trypomastigotes and amastigotes released in the supernatant, but the amastigote/trypomastigote ratio remained constant, indicating that amastigote growth was disturbed, but cell differentiation was unaffected. Analysis of treated epimastigotes by flow cytometry indicated that the plasma membrane remained intact, but there was a significant decrease in mitochondrial membrane potential. The pattern of cell distribution in the cell cycle stages (G1, G2, M) was unaltered in treated epimastigotes, indicating a trypanocidal rather than a trypanostatic activity. Scanning electron microscopy and flow cytometry showed epimastigotes with a round shape and decrease in cell size. Taken together, our data indicate that the EPA-35 is effective against T. cruzi. Synthetic transformation of EPA-35 into other derivatives may provide promising compounds for further evaluation against this parasite. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Dihydroartemisinin (DHA) induces caspase-3-dependent apoptosis in human lung adenocarcinoma ASTC-a-1 cells

PubMed Central

Lu, Ying-Ying; Chen, Tong-Sheng; Qu, Jun-Le; Pan, Wen-Liang; Sun, Lei; Wei, Xun-Bin

2009-01-01

Background Dihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin, isolated from the traditional Chinese herb Artemisia annua, is recommended as the first-line anti-malarial drug with low toxicity. DHA has been shown to possess promising anticancer activities and induce cancer cell death through apoptotic pathways, although the molecular mechanisms are not well understood. Methods In this study, cell counting kit (CCK-8) assay was employed to evaluate the survival of DHA-treated ASTC-a-1 cells. The induction of apoptosis was detected by Hoechst 33258 and PI staining as well as flow cytometry analysis. Collapse of mitochondrial transmembrane potential (ΔΨm) was measured by dynamic detection under a laser scanning confocal microscope and flow cytometry analysis using Rhodamine123. Caspase-3 activities measured with or without Z-VAD-fmk (a broad spectrum caspase inhibitor) pretreatment by FRET techniques, caspase-3 activity measurement, and western blotting analysis. Results Our results indicated that DHA induced apoptotic cell death in a dose- and time-dependent manner, which was accompanied by mitochondrial morphology changes, the loss of ΔΨm and the activation of caspase-3. Conclusion These results show for the first time that DHA can inhibit proliferation and induce apoptosis via caspase-3-dependent mitochondrial death pathway in ASTC-a-1 cells. Our work may provide evidence for further studies of DHA as a possible anticancer drug in the clinical treatment of lung adenocarcinoma. PMID:19272183

Normalization of mass cytometry data with bead standards

PubMed Central

Finck, Rachel; Simonds, Erin F.; Jager, Astraea; Krishnaswamy, Smita; Sachs, Karen; Fantl, Wendy; Pe’er, Dana; Nolan, Garry P.; Bendall, Sean C.

2013-01-01

Mass cytometry uses atomic mass spectrometry combined with isotopically pure reporter elements to currently measure as many as 40 parameters per single cell. As with any quantitative technology, there is a fundamental need for quality assurance and normalization protocols. In the case of mass cytometry, the signal variation over time due to changes in instrument performance combined with intervals between scheduled maintenance must be accounted for and then normalized. Here, samples were mixed with polystyrene beads embedded with metal lanthanides, allowing monitoring of mass cytometry instrument performance over multiple days of data acquisition. The protocol described here includes simultaneous measurements of beads and cells on the mass cytometer, subsequent extraction of the bead-based signature, and the application of an algorithm enabling correction of both short- and long-term signal fluctuations. The variation in the intensity of the beads that remains after normalization may also be used to determine data quality. Application of the algorithm to a one-month longitudinal analysis of a human peripheral blood sample reduced the range of median signal fluctuation from 4.9-fold to 1.3-fold. PMID:23512433

An introduction to mass cytometry: fundamentals and applications.

PubMed

Tanner, Scott D; Baranov, Vladimir I; Ornatsky, Olga I; Bandura, Dmitry R; George, Thaddeus C

2013-05-01

Mass cytometry addresses the analytical challenges of polychromatic flow cytometry by using metal atoms as tags rather than fluorophores and atomic mass spectrometry as the detector rather than photon optics. The many available enriched stable isotopes of the transition elements can provide up to 100 distinguishable reporting tags, which can be measured simultaneously because of the essential independence of detection provided by the mass spectrometer. We discuss the adaptation of traditional inductively coupled plasma mass spectrometry to cytometry applications. We focus on the generation of cytometry-compatible data and on approaches to unsupervised multivariate clustering analysis. Finally, we provide a high-level review of some recent benchmark reports that highlight the potential for massively multi-parameter mass cytometry.

Synergetic effects of ultrasound and slightly acidic electrolyzed water against Staphylococcus aureus evaluated by flow cytometry and electron microscopy.

PubMed

Li, Jiao; Ding, Tian; Liao, Xinyu; Chen, Shiguo; Ye, Xingqian; Liu, Donghong

2017-09-01

This study evaluated the synergetic effects of ultrasound and slightly acidic electrolyzed water (SAEW) on the inactivation of Staphylococcus aureus using flow cytometry and electron microscopy. The individual ultrasound treatment for 10min only resulted in 0.36logCFU/mL reductions of S. aureus, while the SAEW treatment alone for 10min resulted in 3.06logCFU/mL reductions. The log reductions caused by combined treatment were enhanced to 3.68logCFU/mL, which were greater than the sum of individual treatments. This phenomenon was referred to as synergistic effects. FCM analysis distinguished live and dead cells as well as revealed dynamic changes in the physiological states of S. aureus after different treatments. The combined treatment greatly reduced the number of viable but nonculturable (VBNC) bacteria to 0.07%; in contrast, a single ultrasound treatment for 10min induced the formation of VBNC cells to 45.75%. Scanning and transmission electron microscopy analysis revealed that greater damage to the appearance and ultrastructure of S. aureus were achieved after combined ultrasound-SAEW treatment compared to either treatment alone. These results indicated that combining ultrasound with SAEW is a promising sterilization technology with potential uses for environmental remediation and food preservation. Copyright © 2016 Elsevier B.V. All rights reserved.

Blockade of Tumor Cell TGF-Betas: A Strategy to Reverse Antiestrogen Resistance in Human Breast Cancer

DTIC Science & Technology

2002-01-01

the TM- FKHRL1 construct exhibited exclusive nuclear localization Cell Cycle Analysis by Flow Cytometry of the HA-tagged mutant under any experimental...distribution as measured by flow cytometry (Figure 8A). ALS AND METHODS. Consistent with its antiapoptotic effect, these results, addi- tion of TGFI3... flow cytometry . Under these conditions more than 95% of selected cells expressed GFP at the time of experiments. Immunoblot Analysis. Cells were

A Study of the Effects of High Power Pulsed 2450 MHz Microwaves, ELF modulated Microwaves, and ELF Fields on Human Lymphocytes and Selected Cell Lines

DTIC Science & Technology

1993-01-27

Considerable effect was expended in investigating shifts in intercellular calcium of one particular cell line, Jurket, using flow cytometry methods. No...culture. The following analysis were used to characterize the immortalized cell lines: flow cytometry , electron microscopy, two-dimensional protein gel...further characterized by flow cytometry , electron microscopy, two dimensional protein electrophoresis and nuclear run-off assay. Flow cytometric analysis of

Hypoxia and Prx1 in Malignant Progression of Prostate Cancer

DTIC Science & Technology

2006-09-01

Species (ROS) Formation The rate of ROS formation was determined by flow cytometry analysis using the probe 20,70-dichlorofluorescin diacetate (DCFH-DA...DA were subjected to 4-h hypoxia treatment. After the indicated time, fluorescent cells were analyzed by flow cytometry . Western Blot Analysis Equal...species (ROS) generation was measured by flow cytometry at 0.5, 1, 2, 3, 6, 12, or 24 h after hypoxia treatment. The rate of ROS generation increased

Hybrid Dispersion Laser Scanner

PubMed Central

Goda, K.; Mahjoubfar, A.; Wang, C.; Fard, A.; Adam, J.; Gossett, D. R.; Ayazi, A.; Sollier, E.; Malik, O.; Chen, E.; Liu, Y.; Brown, R.; Sarkhosh, N.; Di Carlo, D.; Jalali, B.

2012-01-01

Laser scanning technology is one of the most integral parts of today's scientific research, manufacturing, defense, and biomedicine. In many applications, high-speed scanning capability is essential for scanning a large area in a short time and multi-dimensional sensing of moving objects and dynamical processes with fine temporal resolution. Unfortunately, conventional laser scanners are often too slow, resulting in limited precision and utility. Here we present a new type of laser scanner that offers ∼1,000 times higher scan rates than conventional state-of-the-art scanners. This method employs spatial dispersion of temporally stretched broadband optical pulses onto the target, enabling inertia-free laser scans at unprecedented scan rates of nearly 100 MHz at 800 nm. To show our scanner's broad utility, we use it to demonstrate unique and previously difficult-to-achieve capabilities in imaging, surface vibrometry, and flow cytometry at a record 2D raster scan rate of more than 100 kHz with 27,000 resolvable points. PMID:22685627

[Fiat Lux. May be no more true in cytometry! Go to mass and spectrum but still stay classic].

PubMed

Idziorek, Thierry; Cazareth, Julie; Blanc, Catherine; Jouy, Nathalie; Bourdely, Pierre; Corneau, Aurélien

2018-05-01

The last decade has been an era of accelerated technological progress for flow cytometry. New technologies have been developed such as mass cytometry in which standard fluorochromes have been replaced by lanthanide-based non-radioactive metals and by spectral cytometry that measures the complete fluorescence spectrum. In this review, we schematically describe conventional, mass and spectral cytometry and present the plus and minus of each technology. © 2018 médecine/sciences – Inserm.

Validation of cell-based fluorescence assays: practice guidelines from the ICSH and ICCS - part IV - postanalytic considerations.

PubMed

Barnett, David; Louzao, Raaul; Gambell, Peter; De, Jitakshi; Oldaker, Teri; Hanson, Curtis A

2013-01-01

Flow cytometry and other technologies of cell-based fluorescence assays are as a matter of good laboratory practice required to validate all assays, which when in clinical practice may pass through regulatory review processes using criteria often defined with a soluble analyte in plasma or serum samples in mind. Recently the U.S. Food and Drug Administration (FDA) has entered into a public dialogue in the U.S. regarding their regulatory interest in laboratory developed tests (LDTs) or so-called home brew assays performed in clinical laboratories. The absence of well-defined guidelines for validation of cell-based assays using fluorescence detection has thus become a subject of concern for the International Council for Standardization of Haematology (ICSH) and International Clinical Cytometry Society (ICCS). Accordingly, a group of over 40 international experts in the areas of test development, test validation, and clinical practice of a variety of assay types using flow cytometry and/or morphologic image analysis were invited to develop a set of practical guidelines useful to in vitro diagnostic (IVD) innovators, clinical laboratories, regulatory scientists, and laboratory inspectors. The focus of the group was restricted to fluorescence reporter reagents, although some common principles are shared by immunohistochemistry or immunocytochemistry techniques and noted where appropriate. The work product of this two year effort is the content of this special issue of this journal, which is published as 5 separate articles, this being Validation of Cell-based Fluorescence Assays: Practice Guidelines from the ICSH and ICCS - Part IV - Postanalytic considerations. © 2013 International Clinical Cytometry Society.

Detection and isolation of rare cells by 2-step enrichment high-speed flow cytometry/cell sorting and single cell LEAP laser ablation

NASA Astrophysics Data System (ADS)

Zordan, M. D.; Leary, James F.

2011-02-01

The clonal isolation of rare cells, especially cancer and stem cells, in a population is important to the development of improved medical treatment. We have demonstrated that the Laser-Enabled Analysis and Processing (LEAP, Cyntellect Inc., San Diego, CA) instrument can be used to efficiently produce single cell clones by photoablative dilution. Additionally, we have also shown that cells present at low frequencies can be cloned by photoablative dilution after they are pre-enriched by flow cytometry based cell sorting. Circulating tumor cells were modeled by spiking isolated peripheral blood cells with cells from the lung carcinoma cell line A549. Flow cytometry based cell sorting was used to perform an enrichment sort of A549 cells directly into a 384 well plate. Photoablative dilution was performed with the LEAPTM instrument to remove any contaminating cells, and clonally isolate 1 side population cell per well. We were able to isolate and grow single clones of side population cells using this method at greater than 90% efficiency. We have developed a 2 step method that is able to perform the clonal isolation of rare cells based on a medically relevant functional phenotype.

An active, collaborative approach to learning skills in flow cytometry.

PubMed

Fuller, Kathryn; Linden, Matthew D; Lee-Pullen, Tracey; Fragall, Clayton; Erber, Wendy N; Röhrig, Kimberley J

2016-06-01

Advances in science education research have the potential to improve the way students learn to perform scientific interpretations and understand science concepts. We developed active, collaborative activities to teach skills in manipulating flow cytometry data using FlowJo software. Undergraduate students were given compensated clinical flow cytometry listmode output (FCS) files and asked to design a gating strategy to diagnose patients with different hematological malignancies on the basis of their immunophenotype. A separate cohort of research trainees was given uncompensated data files on which they performed their own compensation, calculated the antibody staining index, designed a sequential gating strategy, and quantified rare immune cell subsets. Student engagement, confidence, and perceptions of flow cytometry were assessed using a survey. Competency against the learning outcomes was assessed by asking students to undertake tasks that required understanding of flow cytometry dot plot data and gating sequences. The active, collaborative approach allowed students to achieve learning outcomes not previously possible with traditional teaching formats, for example, having students design their own gating strategy, without forgoing essential outcomes such as the interpretation of dot plots. In undergraduate students, favorable perceptions of flow cytometry as a field and as a potential career choice were correlated with student confidence but not the ability to perform flow cytometry data analysis. We demonstrate that this new pedagogical approach to teaching flow cytometry is beneficial for student understanding and interpretation of complex concepts. It should be considered as a useful new method for incorporating complex data analysis tasks such as flow cytometry into curricula. Copyright © 2016 The American Physiological Society.

MPQ-cytometry: a magnetism-based method for quantification of nanoparticle-cell interactions

NASA Astrophysics Data System (ADS)

Shipunova, V. O.; Nikitin, M. P.; Nikitin, P. I.; Deyev, S. M.

2016-06-01

Precise quantification of interactions between nanoparticles and living cells is among the imperative tasks for research in nanobiotechnology, nanotoxicology and biomedicine. To meet the challenge, a rapid method called MPQ-cytometry is developed, which measures the integral non-linear response produced by magnetically labeled nanoparticles in a cell sample with an original magnetic particle quantification (MPQ) technique. MPQ-cytometry provides a sensitivity limit 0.33 ng of nanoparticles and is devoid of a background signal present in many label-based assays. Each measurement takes only a few seconds, and no complicated sample preparation or data processing is required. The capabilities of the method have been demonstrated by quantification of interactions of iron oxide nanoparticles with eukaryotic cells. The total amount of targeted nanoparticles that specifically recognized the HER2/neu oncomarker on the human cancer cell surface was successfully measured, the specificity of interaction permitting the detection of HER2/neu positive cells in a cell mixture. Moreover, it has been shown that MPQ-cytometry analysis of a HER2/neu-specific iron oxide nanoparticle interaction with six cell lines of different tissue origins quantitatively reflects the HER2/neu status of the cells. High correlation of MPQ-cytometry data with those obtained by three other commonly used in molecular and cell biology methods supports consideration of this method as a prospective alternative for both quantifying cell-bound nanoparticles and estimating the expression level of cell surface antigens. The proposed method does not require expensive sophisticated equipment or highly skilled personnel and it can be easily applied for rapid diagnostics, especially under field conditions.Precise quantification of interactions between nanoparticles and living cells is among the imperative tasks for research in nanobiotechnology, nanotoxicology and biomedicine. To meet the challenge, a rapid method called MPQ-cytometry is developed, which measures the integral non-linear response produced by magnetically labeled nanoparticles in a cell sample with an original magnetic particle quantification (MPQ) technique. MPQ-cytometry provides a sensitivity limit 0.33 ng of nanoparticles and is devoid of a background signal present in many label-based assays. Each measurement takes only a few seconds, and no complicated sample preparation or data processing is required. The capabilities of the method have been demonstrated by quantification of interactions of iron oxide nanoparticles with eukaryotic cells. The total amount of targeted nanoparticles that specifically recognized the HER2/neu oncomarker on the human cancer cell surface was successfully measured, the specificity of interaction permitting the detection of HER2/neu positive cells in a cell mixture. Moreover, it has been shown that MPQ-cytometry analysis of a HER2/neu-specific iron oxide nanoparticle interaction with six cell lines of different tissue origins quantitatively reflects the HER2/neu status of the cells. High correlation of MPQ-cytometry data with those obtained by three other commonly used in molecular and cell biology methods supports consideration of this method as a prospective alternative for both quantifying cell-bound nanoparticles and estimating the expression level of cell surface antigens. The proposed method does not require expensive sophisticated equipment or highly skilled personnel and it can be easily applied for rapid diagnostics, especially under field conditions. Electronic supplementary information (ESI) available. See DOI: 10.1039/c6nr03507h

Photoacoustic and photothermal detection of circulating tumor cells, bacteria and nanoparticles in cerebrospinal fluid in vivo and ex vivo.

PubMed

Nedosekin, Dmitry A; Juratli, Mazen A; Sarimollaoglu, Mustafa; Moore, Christopher L; Rusch, Nancy J; Smeltzer, Mark S; Zharov, Vladimir P; Galanzha, Ekaterina I

2013-06-01

Circulating cells, bacteria, proteins, microparticles, and DNA in cerebrospinal fluid (CSF) are excellent biomarkers of many diseases, including cancer and infections. However, the sensitivity of existing methods is limited in their ability to detect rare CSF biomarkers at the treatable, early-stage of diseases. Here, we introduce novel CSF tests based on in vivo photoacoustic flow cytometry (PAFC) and ex vivo photothermal scanning cytometry. In the CSF of tumor-bearing mice, we molecularly detected in vivo circulating tumor cells (CTCs) before the development of breast cancer brain metastasis with 20-times higher sensitivity than with current assays. For the first time, we demonstrated assessing three pathways (i.e., blood, lymphatic, and CSF) of CTC dissemination, tracking nanoparticles in CSF in vivo and their imaging ex vivo. In label-free CSF samples, we counted leukocytes, erythrocytes, melanoma cells, and bacteria and imaged intracellular cytochromes, hemoglobin, melanin, and carotenoids, respectively. Taking into account the safety of PAFC, its translation for use in humans is expected to improve disease diagnosis beyond conventional detection limits. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Elucidation of Seventeen Human Peripheral Blood B cell Subsets and Quantification of the Tetanus Response Using a Density-Based Method for the Automated Identification of Cell Populations in Multidimensional Flow Cytometry Data

PubMed Central

Qian, Yu; Wei, Chungwen; Lee, F. Eun-Hyung; Campbell, John; Halliley, Jessica; Lee, Jamie A.; Cai, Jennifer; Kong, Megan; Sadat, Eva; Thomson, Elizabeth; Dunn, Patrick; Seegmiller, Adam C.; Karandikar, Nitin J.; Tipton, Chris; Mosmann, Tim; Sanz, Iñaki; Scheuermann, Richard H.

2011-01-01

Background Advances in multi-parameter flow cytometry (FCM) now allow for the independent detection of larger numbers of fluorochromes on individual cells, generating data with increasingly higher dimensionality. The increased complexity of these data has made it difficult to identify cell populations from high-dimensional FCM data using traditional manual gating strategies based on single-color or two-color displays. Methods To address this challenge, we developed a novel program, FLOCK (FLOw Clustering without K), that uses a density-based clustering approach to algorithmically identify biologically relevant cell populations from multiple samples in an unbiased fashion, thereby eliminating operator-dependent variability. Results FLOCK was used to objectively identify seventeen distinct B cell subsets in a human peripheral blood sample and to identify and quantify novel plasmablast subsets responding transiently to tetanus and other vaccinations in peripheral blood. FLOCK has been implemented in the publically available Immunology Database and Analysis Portal – ImmPort (http://www.immport.org) for open use by the immunology research community. Conclusions FLOCK is able to identify cell subsets in experiments that use multi-parameter flow cytometry through an objective, automated computational approach. The use of algorithms like FLOCK for FCM data analysis obviates the need for subjective and labor intensive manual gating to identify and quantify cell subsets. Novel populations identified by these computational approaches can serve as hypotheses for further experimental study. PMID:20839340

Initiation and termination of DNA replication during S phase in relation to cyclins D1, E and A, p21WAF1, Cdt1 and the p12 subunit of DNA polymerase δ revealed in individual cells by cytometry

PubMed Central

Darzynkiewicz, Zbigniew; Zhao, Hong; Zhang, Sufang; Marietta, Y.W.T. Lee; Ernest, Y.C. Lee; Zhang, Zhongtao

2015-01-01

During our recent studies on mechanism of the regulation of human DNA polymerase δ in preparation for DNA replication or repair, multiparameter imaging cytometry as exemplified by laser scanning cytometry (LSC) has been used to assess changes in expression of the following nuclear proteins associated with initiation of DNA replication: cyclin A, PCNA, Ki-67, p21WAF1, DNA replication factor Cdt1 and the smallest subunit of DNA polymerase δ, p12. In the present review, rather than focusing on Pol δ, we emphasize the application of LSC in these studies and outline possibilities offered by the concurrent differential analysis of DNA replication in conjunction with expression of the nuclear proteins. A more extensive analysis of the data on a correlation between rates of EdU incorporation, likely reporting DNA replication, and expression of these proteins, is presently provided. New data, specifically on the expression of cyclin D1 and cyclin E with respect to EdU incorporation as well as on a relationship between expression of cyclin A vs. p21WAF1 and Ki-67 vs. Cdt1, are also reported. Of particular interest is the observation that this approach makes it possible to assess the temporal sequence of degradation of cyclin D1, p21WAF1, Cdt1 and p12, each with respect to initiation of DNA replication and with respect to each other. Also the sequence or reappearance of these proteins in G2 after termination of DNA replication is assessed. The reviewed data provide a more comprehensive presentation of potential markers, whose presence or absence marks the DNA replicating cells. Discussed is also usefulness of these markers as indicators of proliferative activity in cancer tissues that may bear information on tumor progression and have a prognostic value. PMID:26059433

Initiation and termination of DNA replication during S phase in relation to cyclins D1, E and A, p21WAF1, Cdt1 and the p12 subunit of DNA polymerase δ revealed in individual cells by cytometry.

PubMed

Darzynkiewicz, Zbigniew; Zhao, Hong; Zhang, Sufang; Lee, Marietta Y W T; Lee, Ernest Y C; Zhang, Zhongtao

2015-05-20

During our recent studies on mechanism of the regulation of human DNA polymerase δ in preparation for DNA replication or repair, multiparameter imaging cytometry as exemplified by laser scanning cytometry (LSC) has been used to assess changes in expression of the following nuclear proteins associated with initiation of DNA replication: cyclin A, PCNA, Ki-67, p21(WAF1), DNA replication factor Cdt1 and the smallest subunit of DNA polymerase δ, p12. In the present review, rather than focusing on Pol δ, we emphasize the application of LSC in these studies and outline possibilities offered by the concurrent differential analysis of DNA replication in conjunction with expression of the nuclear proteins. A more extensive analysis of the data on a correlation between rates of EdU incorporation, likely reporting DNA replication, and expression of these proteins, is presently provided. New data, specifically on the expression of cyclin D1 and cyclin E with respect to EdU incorporation as well as on a relationship between expression of cyclin A vs. p21(WAF1) and Ki-67 vs. Cdt1, are also reported. Of particular interest is the observation that this approach makes it possible to assess the temporal sequence of degradation of cyclin D1, p21(WAF1), Cdt1 and p12, each with respect to initiation of DNA replication and with respect to each other. Also the sequence or reappearance of these proteins in G2 after termination of DNA replication is assessed. The reviewed data provide a more comprehensive presentation of potential markers, whose presence or absence marks the DNA replicating cells. Discussed is also usefulness of these markers as indicators of proliferative activity in cancer tissues that may bear information on tumor progression and have a prognostic value.

A flow-cytometry-based method to simplify the analysis and quantification of protein association to chromatin in mammalian cells

PubMed Central

Forment, Josep V.; Jackson, Stephen P.

2016-01-01

Protein accumulation on chromatin has traditionally been studied using immunofluorescence microscopy or biochemical cellular fractionation followed by western immunoblot analysis. As a way to improve the reproducibility of this kind of analysis, make it easier to quantify and allow a stream-lined application in high-throughput screens, we recently combined a classical immunofluorescence microscopy detection technique with flow cytometry1. In addition to the features described above, and by combining it with detection of both DNA content and DNA replication, this method allows unequivocal and direct assignment of cell-cycle distribution of protein association to chromatin without the need for cell culture synchronization. Furthermore, it is relatively quick (no more than a working day from sample collection to quantification), requires less starting material compared to standard biochemical fractionation methods and overcomes the need for flat, adherent cell types that are required for immunofluorescence microscopy. PMID:26226461

Polymer-Coated Hollow Mesoporous Silica Nanoparticles for Triple-Responsive Drug Delivery.

PubMed

Zhang, Yuanyuan; Ang, Chung Yen; Li, Menghuan; Tan, Si Yu; Qu, Qiuyu; Luo, Zhong; Zhao, Yanli

2015-08-19

In this study, pH, reduction and light triple-responsive nanocarriers based on hollow mesoporous silica nanoparticles (HMSNs) modified with poly(2-(diethylamino)ethyl methacrylate) (PDEAEMA) were developed via surface-initiated atom transfer radical polymerization. Both reduction-cleavable disulfide bond and light-cleavable o-nitrobenzyl ester were used as the linkages between HMSNs and pH-sensitive PDEAEMA polymer caps. A series of characterization techniques were applied to characterize and confirm the structures of the intermediates and final nanocarriers. Doxorubicin (DOX) was easily encapsulated into the nanocarriers with a high loading capacity, and quickly released in response to the stimuli of reducing agent, acid environment or UV light irradiation. In addition, flow cytometry analysis, confocal laser scanning microscopy observations and cytotoxicity studies indicated that the nanocarriers were efficiently internalized by HeLa cancer cells, exhibiting (i) enhanced release of DOX into the cytoplasm under external UV light irradiation, (ii) better cytotoxicity against HeLa cells, and (iii) superior control over drug delivery and release. Thus, the triple-responsive nanocarriers present highly promising potentials as a drug delivery platform for cancer therapy.

Polystyrene microspheres enable 10‐color compensation for immunophenotyping of primary human leukocytes

PubMed Central

Carr, Karen D.; Norman, John C.; Huye, Leslie; Hegde, Meenakshi

2015-01-01

Abstract Compensation is a critical process for the unbiased analysis of flow cytometry data. Numerous compensation strategies exist, including the use of bead‐based products. The purpose of this study was to determine whether beads, specifically polystyrene microspheres (PSMS) compare to the use of primary leukocytes for single color based compensation when conducting polychromatic flow cytometry. To do so, we stained individual tubes of both PSMS and leukocytes with panel specific antibodies conjugated to fluorochromes corresponding to fluorescent channels FL1‐FL10. We compared the matrix generated by PSMS to that generated using peripheral blood mononuclear cells (PBMC). Ideal for compensation is a sample with both a discrete negative population and a bright positive population. We demonstrate that PSMS display autofluorescence properties similar to PBMC. When comparing PSMS to PBMC for compensation PSMS yielded more evenly distributed and discrete negative and positive populations to use for compensation. We analyzed three donors' PBMC stained with our 10‐color T cell subpopulation panel using compensation generated by PSMS vs.PBMC and detected no significant differences in the population distribution. Panel specific antibodies bound to PSMS represent an invaluable valid tool to generate suitable compensation matrices especially when sample material is limited and/or the sample requires analysis of dynamically modulated or rare events. © 2015 The Authors. Cytometry Part A Published by Wiley Periodicals, Inc. PMID:26202733

Aptamer-facilitated mass cytometry.

PubMed

Mironov, Gleb G; Bouzekri, Alexandre; Watson, Jessica; Loboda, Olga; Ornatsky, Olga; Berezovski, Maxim V

2018-05-01

Mass cytometry is a novel cell-by-cell analysis technique, which uses elemental tags instead of fluorophores. Sample cells undergo rapid ionization in inductively coupled plasma and the ionized elemental tags are then analyzed by means of time-of-flight mass spectrometry. Benefits of the mass cytometry approach are in no need for compensation, the high number of detection channels (up to 100) and low background noise. In this work, we applied a biotinylated aptamer against human PTK7 receptor for characterization of positive (human acute lymphoblastic leukemia) and negative (human Burkitt's lymphoma) cells by a mass cytometry instrument. Our proof of principal experiments showed that biotinylated aptamers in conjunction with metal-labeled neutravidin can be successfully utilized for mass cytometry experiments at par with commercially available antibodies. Graphical abstract Biotinylated aptamers in conjunction with metal-labeled neutravidin bind to cell biomarkers, and then injected into the inductively coupled plasma (ICP) source, where cells are vaporized, atomized, and ionized in the plasma for subsequent mass spectrometry (MS) analysis of lanthanide metals.

A Method for the Interpretation of Flow Cytometry Data Using Genetic Algorithms.

PubMed

Angeletti, Cesar

2018-01-01

Flow cytometry analysis is the method of choice for the differential diagnosis of hematologic disorders. It is typically performed by a trained hematopathologist through visual examination of bidimensional plots, making the analysis time-consuming and sometimes too subjective. Here, a pilot study applying genetic algorithms to flow cytometry data from normal and acute myeloid leukemia subjects is described. Initially, Flow Cytometry Standard files from 316 normal and 43 acute myeloid leukemia subjects were transformed into multidimensional FITS image metafiles. Training was performed through introduction of FITS metafiles from 4 normal and 4 acute myeloid leukemia in the artificial intelligence system. Two mathematical algorithms termed 018330 and 025886 were generated. When tested against a cohort of 312 normal and 39 acute myeloid leukemia subjects, both algorithms combined showed high discriminatory power with a receiver operating characteristic (ROC) curve of 0.912. The present results suggest that machine learning systems hold a great promise in the interpretation of hematological flow cytometry data.

Microfluidic device for a rapid immobilization of zebrafish larvae in environmental scanning electron microscopy.

PubMed

Akagi, Jin; Zhu, Feng; Skommer, Joanna; Hall, Chris J; Crosier, Philip S; Cialkowski, Michal; Wlodkowic, Donald

2015-03-01

Small vertebrate model organisms have recently gained popularity as attractive experimental models that enhance our understanding of human tissue and organ development. Despite a large body of evidence using optical spectroscopy for the characterization of small model organism on chip-based devices, no attempts have been so far made to interface microfabricated technologies with environmental scanning electron microscopy (ESEM). Conventional scanning electron microscopy requires high vacuum environments and biological samples must be, therefore, submitted to many preparative procedures to dehydrate, fix, and subsequently stain the sample with gold-palladium deposition. This process is inherently low-throughput and can introduce many analytical artifacts. This work describes a proof-of-concept microfluidic chip-based system for immobilizing zebrafish larvae for ESEM imaging that is performed in a gaseous atmosphere, under low vacuum mode and without any need for sample staining protocols. The microfabricated technology provides a user-friendly and simple interface to perform ESEM imaging on zebrafish larvae. Presented lab-on-a-chip device was fabricated using a high-speed infrared laser micromachining in a biocompatible poly(methyl methacrylate) thermoplastic. It consisted of a reservoir with multiple semispherical microwells designed to hold the yolk of dechorionated zebrafish larvae. Immobilization of the larvae was achieved by a gentle suction generated during blotting of the medium. Trapping region allowed for multiple specimens to be conveniently positioned on the chip-based device within few minutes for ESEM imaging. © 2014 International Society for Advancement of Cytometry.

Hybrid Imaging Labels: Providing the Link Between Mass Spectrometry-Based Molecular Pathology and Theranostics

PubMed Central

Buckle, Tessa; van der Wal, Steffen; van Malderen, Stijn J.M.; Müller, Larissa; Kuil, Joeri; van Unen, Vincent; Peters, Ruud J.B.; van Bemmel, Margaretha E.M.; McDonnell, Liam A.; Velders, Aldrik H.; Koning, Frits; Vanhaeke, Frank; van Leeuwen, Fijs W. B.

2017-01-01

Background: Development of theranostic concepts that include inductively coupled plasma mass spectrometry (ICP-MS) and laser ablation ICP-MS (LA-ICP-MS) imaging can be hindered by the lack of a direct comparison to more standardly used methods for in vitro and in vivo evaluation; e.g. fluorescence or nuclear medicine. In this study a bimodal (or rather, hybrid) tracer that contains both a fluorescent dye and a chelate was used to evaluate the existence of a direct link between mass spectrometry (MS) and in vitro and in vivo molecular imaging findings using fluorescence and radioisotopes. At the same time, the hybrid label was used to determine whether the use of a single isotope label would allow for MS-based diagnostics. Methods: A hybrid label that contained both a DTPA chelate (that was coordinated with either 165Ho or 111In) and a Cy5 fluorescent dye was coupled to the chemokine receptor 4 (CXCR4) targeting peptide Ac-TZ14011 (hybrid-Cy5-Ac-TZ4011). This receptor targeting tracer was used to 1) validate the efficacy of (165Ho-based) mass-cytometry in determining the receptor affinity via comparison with fluorescence-based flow cytometry (Cy5), 2) evaluate the microscopic binding pattern of the tracer in tumor cells using both fluorescence confocal imaging (Cy5) and LA-ICP-MS-imaging (165Ho), 3) compare in vivo biodistribution patterns obtained with ICP-MS (165Ho) and radiodetection (111In) after intravenous administration of hybrid-Cy5-Ac-TZ4011 in tumor-bearing mice. Finally, LA-ICP-MS-imaging (165Ho) was linked to fluorescence-based analysis of excised tissue samples (Cy5). Results: Analysis with both mass-cytometry and flow cytometry revealed a similar receptor affinity, respectively 352 ± 141 nM and 245 ± 65 nM (p = 0.08), but with a much lower detection sensitivity for the first modality. In vitro LA-ICP-MS imaging (165Ho) enabled clear discrimination between CXCR4 positive and negative cells, but fluorescence microscopy was required to determine the intracellular distribution. In vivo biodistribution patterns obtained with ICP-MS (165Ho) and radiodetection (111In) of the hybrid peptide were shown to be similar. Assessment of tracer distribution in excised tissues revealed the location of tracer uptake with both LA-ICP-MS-imaging and fluorescence imaging. Conclusion: Lanthanide-isotope chelation expands the scope of fluorescent/radioactive hybrid tracers to include MS-based analytical tools such as mass-cytometry, ICP-MS and LA-ICP-MS imaging in molecular pathology. In contradiction to common expectations, MS detection using a single chelate imaging agent was shown to be feasible, enabling a direct link between nuclear medicine-based imaging and theranostic methods. PMID:28255355

Flow cytometry combined with viSNE for the analysis of microbial biofilms and detection of microplastics

PubMed Central

Sgier, Linn; Freimann, Remo; Zupanic, Anze; Kroll, Alexandra

2016-01-01

Biofilms serve essential ecosystem functions and are used in different technical applications. Studies from stream ecology and waste-water treatment have shown that biofilm functionality depends to a great extent on community structure. Here we present a fast and easy-to-use method for individual cell-based analysis of stream biofilms, based on stain-free flow cytometry and visualization of the high-dimensional data by viSNE. The method allows the combined assessment of community structure, decay of phototrophic organisms and presence of abiotic particles. In laboratory experiments, it allows quantification of cellular decay and detection of survival of larger cells after temperature stress, while in the field it enables detection of community structure changes that correlate with known environmental drivers (flow conditions, dissolved organic carbon, calcium) and detection of microplastic contamination. The method can potentially be applied to other biofilm types, for example, for inferring community structure for environmental and industrial research and monitoring. PMID:27188265

CMOS based image cytometry for detection of phytoplankton in ballast water.

PubMed

Pérez, J M; Jofre, M; Martínez, P; Yáñez, M A; Catalan, V; Parker, A; Veldhuis, M; Pruneri, V

2017-02-01

We introduce an image cytometer (I-CYT) for the analysis of phytoplankton in fresh and marine water environments. A linear quantification of cell numbers was observed covering several orders of magnitude using cultures of Tetraselmis and Nannochloropsis measured by autofluorescence in a laboratory environment. We assessed the functionality of the system outside the laboratory by phytoplankton quantification of samples taken from a marine water environment (Dutch Wadden Sea, The Netherlands) and a fresh water environment (Lake Ijssel, The Netherlands). The I-CYT was also employed to study the effects of two ballast water treatment systems (BWTS), based on chlorine electrolysis and UV sterilization, with the analysis including the vitality of the phytoplankton. For comparative study and benchmarking of the I-CYT, a standard flow cytometer was used. Our results prove a limit of detection (LOD) of 10 cells/ml with an accuracy between 0.7 and 0.5 log, and a correlation of 88.29% in quantification and 96.21% in vitality, with respect to the flow cytometry results.

Anti-methicillin Resistant Staphylococcus aureus Compound Isolation from Halophilic Bacillus amyloliquefaciens MHB1 and Determination of Its Mode of Action Using Electron Microscope and Flow Cytometry Analysis.

PubMed

Jeyanthi, Venkadapathi; Velusamy, Palaniyandi

2016-06-01

The aim of this study was to purify, characterize and evaluate the antibacterial activity of bioactive compound against methicillin-resistant Staphylococcus aureus (MRSA). The anti-MRSA compound was produced by a halophilic bacterial strain designated as MHB1. The MHB1 strain exhibited 99 % similarity to Bacillus amyloliquefaciens based on 16S rRNA gene analysis. The culture conditions of Bacillus amyloliquefaciens MHB1 were optimized using nutritional and environmental parameters for enhanced anti-MRSA compound production. The pure bioactive compound was isolated using silica gel column chromatography and Semi-preparative High-performance liquid chromatography (Semi-preparative HPLC). The Thin layer chromatography, Fourier transform infrared spectroscopy and proton NMR ((1)H NMR) analysis indicated the phenolic nature of the compound. The molecular mass of the purified compound was 507 Da as revealed by Liquid chromatography-mass spectrometry (LC-MS) analysis. The compound inhibited the growth of MRSA with minimum inhibitory concentration (MIC) of 62.5 µg mL(-1). MRSA bacteria exposed to 4× MIC of the compound and the cell viability was determined using flow cytometric analysis. Scanning electron microscope and Transmission electron microscope analysis was used to determine the ultrastructural changes in bacteria. This is the first report on isolation of anti-MRSA compound from halophilic B. amyloliquefaciens MHB1 and could act as a promising biocontrol agent.

Phenotypic and functional characterization of earthworm coelomocyte subsets: Linking light scatter-based cell typing and imaging of the sorted populations.

PubMed

Engelmann, Péter; Hayashi, Yuya; Bodó, Kornélia; Ernszt, Dávid; Somogyi, Ildikó; Steib, Anita; Orbán, József; Pollák, Edit; Nyitrai, Miklós; Németh, Péter; Molnár, László

2016-12-01

Flow cytometry is a common approach to study invertebrate immune cells including earthworm coelomocytes. However, the link between light-scatter- and microscopy-based phenotyping remains obscured. Here we show, by means of light scatter-based cell sorting, both subpopulations (amoebocytes and eleocytes) can be physically isolated with good sort efficiency and purity confirmed by downstream morphological and cytochemical applications. Immunocytochemical analysis using anti-EFCC monoclonal antibodies combined with phalloidin staining has revealed antigenically distinct, sorted subsets. Screening of lectin binding capacity indicated wheat germ agglutinin (WGA) as the strongest reactor to amoebocytes. This is further evidenced by WGA inhibition assays that suggest high abundance of N-acetyl-d-glucosamine in amoebocytes. Post-sort phagocytosis assays confirmed the functional differences between amoebocytes and eleocytes, with the former being in favor of bacterial engulfment. This study has proved successful in linking flow cytometry and microscopy analysis and provides further experimental evidence of phenotypic and functional heterogeneity in earthworm coelomocyte subsets. Copyright © 2016 Elsevier Ltd. All rights reserved.

Deconvoluting Post-Transplant Immunity: Cell Subset-Specific Mapping Reveals Pathways for Activation and Expansion of Memory T, Monocytes and B Cells

PubMed Central

Grigoryev, Yevgeniy A.; Kurian, Sunil M.; Avnur, Zafi; Borie, Dominic; Deng, Jun; Campbell, Daniel; Sung, Joanna; Nikolcheva, Tania; Quinn, Anthony; Schulman, Howard; Peng, Stanford L.; Schaffer, Randolph; Fisher, Jonathan; Mondala, Tony; Head, Steven; Flechner, Stuart M.; Kantor, Aaron B.; Marsh, Christopher; Salomon, Daniel R.

2010-01-01

A major challenge for the field of transplantation is the lack of understanding of genomic and molecular drivers of early post-transplant immunity. The early immune response creates a complex milieu that determines the course of ensuing immune events and the ultimate outcome of the transplant. The objective of the current study was to mechanistically deconvolute the early immune response by purifying and profiling the constituent cell subsets of the peripheral blood. We employed genome-wide profiling of whole blood and purified CD4, CD8, B cells and monocytes in tandem with high-throughput laser-scanning cytometry in 10 kidney transplants sampled serially pre-transplant, 1, 2, 4, 8 and 12 weeks. Cytometry confirmed early cell subset depletion by antibody induction and immunosuppression. Multiple markers revealed the activation and proliferative expansion of CD45RO+CD62L− effector memory CD4/CD8 T cells as well as progressive activation of monocytes and B cells. Next, we mechanistically deconvoluted early post-transplant immunity by serial monitoring of whole blood using DNA microarrays. Parallel analysis of cell subset-specific gene expression revealed a unique spectrum of time-dependent changes and functional pathways. Gene expression profiling results were validated with 157 different probesets matching all 65 antigens detected by cytometry. Thus, serial blood cell monitoring reflects the profound changes in blood cell composition and immune activation early post-transplant. Each cell subset reveals distinct pathways and functional programs. These changes illuminate a complex, early phase of immunity and inflammation that includes activation and proliferative expansion of the memory effector and regulatory cells that may determine the phenotype and outcome of the kidney transplant. PMID:20976225

Deconvoluting post-transplant immunity: cell subset-specific mapping reveals pathways for activation and expansion of memory T, monocytes and B cells.

PubMed

Grigoryev, Yevgeniy A; Kurian, Sunil M; Avnur, Zafi; Borie, Dominic; Deng, Jun; Campbell, Daniel; Sung, Joanna; Nikolcheva, Tania; Quinn, Anthony; Schulman, Howard; Peng, Stanford L; Schaffer, Randolph; Fisher, Jonathan; Mondala, Tony; Head, Steven; Flechner, Stuart M; Kantor, Aaron B; Marsh, Christopher; Salomon, Daniel R

2010-10-14

A major challenge for the field of transplantation is the lack of understanding of genomic and molecular drivers of early post-transplant immunity. The early immune response creates a complex milieu that determines the course of ensuing immune events and the ultimate outcome of the transplant. The objective of the current study was to mechanistically deconvolute the early immune response by purifying and profiling the constituent cell subsets of the peripheral blood. We employed genome-wide profiling of whole blood and purified CD4, CD8, B cells and monocytes in tandem with high-throughput laser-scanning cytometry in 10 kidney transplants sampled serially pre-transplant, 1, 2, 4, 8 and 12 weeks. Cytometry confirmed early cell subset depletion by antibody induction and immunosuppression. Multiple markers revealed the activation and proliferative expansion of CD45RO(+)CD62L(-) effector memory CD4/CD8 T cells as well as progressive activation of monocytes and B cells. Next, we mechanistically deconvoluted early post-transplant immunity by serial monitoring of whole blood using DNA microarrays. Parallel analysis of cell subset-specific gene expression revealed a unique spectrum of time-dependent changes and functional pathways. Gene expression profiling results were validated with 157 different probesets matching all 65 antigens detected by cytometry. Thus, serial blood cell monitoring reflects the profound changes in blood cell composition and immune activation early post-transplant. Each cell subset reveals distinct pathways and functional programs. These changes illuminate a complex, early phase of immunity and inflammation that includes activation and proliferative expansion of the memory effector and regulatory cells that may determine the phenotype and outcome of the kidney transplant.

Flow Cytometry with Gold Nanoparticles and their Clusters as scattering Contrast Agents: FDTD Simulation of Light-Cell Interaction

PubMed Central

Tanev, Stoyan; Sun, Wenbo; Pond, James; Tuchin, Valery V.; Zharov, Vladimir P.

2010-01-01

The formulation of the Finite-Difference Time-Domain (FDTD) approach is presented in the framework of its potential applications to in vivo flow cytometry based on light scattering. The consideration is focused on comparison of light scattering by a single biological cell alone in controlled refractive index matching conditions and by cells labeled by gold nanoparticles. The optical schematics including phase contrast (OPCM) microscopy as a prospective modality for in vivo flow cytometry is also analyzed. The validation of the FDTD approach for the simulation of flow cytometry may open a new avenue in the development of advanced cytometric techniques based on scattering effects from nanoscale targets. PMID:19670359

Multiplex, quantitative cellular analysis in large tissue volumes with clearing-enhanced 3D microscopy (Ce3D)

PubMed Central

Li, Weizhe; Germain, Ronald N.

2017-01-01

Organ homeostasis, cellular differentiation, signal relay, and in situ function all depend on the spatial organization of cells in complex tissues. For this reason, comprehensive, high-resolution mapping of cell positioning, phenotypic identity, and functional state in the context of macroscale tissue structure is critical to a deeper understanding of diverse biological processes. Here we report an easy to use method, clearing-enhanced 3D (Ce3D), which generates excellent tissue transparency for most organs, preserves cellular morphology and protein fluorescence, and is robustly compatible with antibody-based immunolabeling. This enhanced signal quality and capacity for extensive probe multiplexing permits quantitative analysis of distinct, highly intermixed cell populations in intact Ce3D-treated tissues via 3D histo-cytometry. We use this technology to demonstrate large-volume, high-resolution microscopy of diverse cell types in lymphoid and nonlymphoid organs, as well as to perform quantitative analysis of the composition and tissue distribution of multiple cell populations in lymphoid tissues. Combined with histo-cytometry, Ce3D provides a comprehensive strategy for volumetric quantitative imaging and analysis that bridges the gap between conventional section imaging and disassociation-based techniques. PMID:28808033

AirLab: a cloud-based platform to manage and share antibody-based single-cell research.

PubMed

Catena, Raúl; Özcan, Alaz; Jacobs, Andrea; Chevrier, Stephane; Bodenmiller, Bernd

2016-06-29

Single-cell analysis technologies are essential tools in research and clinical diagnostics. These methods include flow cytometry, mass cytometry, and other microfluidics-based technologies. Most laboratories that employ these methods maintain large repositories of antibodies. These ever-growing collections of antibodies, their multiple conjugates, and the large amounts of data generated in assays using specific antibodies and conditions makes a dedicated software solution necessary. We have developed AirLab, a cloud-based tool with web and mobile interfaces, for the organization of these data. AirLab streamlines the processes of antibody purchase, organization, and storage, antibody panel creation, results logging, and antibody validation data sharing and distribution. Furthermore, AirLab enables inventory of other laboratory stocks, such as primers or clinical samples, through user-controlled customization. Thus, AirLab is a mobile-powered and flexible tool that harnesses the capabilities of mobile tools and cloud-based technology to facilitate inventory and sharing of antibody and sample collections and associated validation data.

Identification of residual leukemic cells by flow cytometry in childhood B-cell precursor acute lymphoblastic leukemia: verification of leukemic state by flow-sorting and molecular/cytogenetic methods.

PubMed

Øbro, Nina F; Ryder, Lars P; Madsen, Hans O; Andersen, Mette K; Lausen, Birgitte; Hasle, Henrik; Schmiegelow, Kjeld; Marquart, Hanne V

2012-01-01

Reduction in minimal residual disease, measured by real-time quantitative PCR or flow cytometry, predicts prognosis in childhood B-cell precursor acute lymphoblastic leukemia. We explored whether cells reported as minimal residual disease by flow cytometry represent the malignant clone harboring clone-specific genomic markers (53 follow-up bone marrow samples from 28 children with B-cell precursor acute lymphoblastic leukemia). Cell populations (presumed leukemic and non-leukemic) were flow-sorted during standard flow cytometry-based minimal residual disease monitoring and explored by PCR and/or fluorescence in situ hybridization. We found good concordance between flow cytometry and genomic analyses in the individual flow-sorted leukemic (93% true positive) and normal (93% true negative) cell populations. Four cases with discrepant results had plausible explanations (e.g. partly informative immunophenotype and antigen modulation) that highlight important methodological pitfalls. These findings demonstrate that with sufficient experience, flow cytometry is reliable for minimal residual disease monitoring in B-cell precursor acute lymphoblastic leukemia, although rare cases require supplementary PCR-based monitoring.

Fluorescent genetic barcoding in mammalian cells for enhanced multiplexing capabilities in flow cytometry.

PubMed

Smurthwaite, Cameron A; Hilton, Brett J; O'Hanlon, Ryan; Stolp, Zachary D; Hancock, Bryan M; Abbadessa, Darin; Stotland, Aleksandr; Sklar, Larry A; Wolkowicz, Roland

2014-01-01

The discovery of the green fluorescent protein from Aequorea victoria has revolutionized the field of cell and molecular biology. Since its discovery a growing panel of fluorescent proteins, fluorophores and fluorescent-coupled staining methodologies, have expanded the analytical capabilities of flow cytometry. Here, we exploit the power of genetic engineering to barcode individual cells with genes encoding fluorescent proteins. For genetic engineering, we utilize retroviral technology, which allows for the expression of ectopic genetic information in a stable manner in mammalian cells. We have genetically barcoded both adherent and nonadherent cells with different fluorescent proteins. Multiplexing power was increased by combining both the number of distinct fluorescent proteins, and the fluorescence intensity in each channel. Moreover, retroviral expression has proven to be stable for at least a 6-month period, which is critical for applications such as biological screens. We have shown the applicability of fluorescent barcoded multiplexing to cell-based assays that rely themselves on genetic barcoding, or on classical staining protocols. Fluorescent genetic barcoding gives the cell an inherited characteristic that distinguishes it from its counterpart. Once cell lines are developed, no further manipulation or staining is required, decreasing time, nonspecific background associated with staining protocols, and cost. The increasing number of discovered and/or engineered fluorescent proteins with unique absorbance/emission spectra, combined with the growing number of detection devices and lasers, increases multiplexing versatility, making fluorescent genetic barcoding a powerful tool for flow cytometry-based analysis. © 2013 International Society for Advancement of Cytometry.

Aptamer-fluorescent silica nanoparticles bioconjugates based dual-color flow cytometry for specific detection of Staphylococcus aureus.

PubMed

He, Xiaoxiao; Li, Yuhong; He, Dinggen; Wang, Kemin; Shangguan, Jingfang; Shi, Hui

2014-07-01

This paper describes a sensitive and specific determination strategy for Staphylococcus aureus (S. aureus) detection using aptamer recognition and fluorescent silica nanoparticles (FSiNPs) label based dual-color flow cytometry assay (Aptamer/FSiNPs-DCFCM). In the protocol, an aptamer, having high affinity to S. aureus, was first covalently immobilized onto chloropropyl functionalized FSiNPs through a click chemistry approach to generate aptamer-nanoparticles bioconjugates (Aptamer/FSiNPs). Next, S. aureus was incubated with Aptamer/FSiNPs, and then stained with SYBR Green I (a special staining material for the duplex DNA). Upon target binding and nucleic acid staining with SYBR Green I, the S. aureus was determined using two-color flow cytometry. The method took advantage of the specificity of aptamer, signal amplification of FSiNPs label and decreased false positives of two-color flow cytometry assay. It was demonstrated that these Aptamer/FSiNPs could efficiently recognize and fluorescently label target S. aureus. Through multiparameter determination with flow cytometry, this assay allowed for detection of as low as 1.5 x 10(2) and 7.6 x 10(2) cells mL(-1) S. aureus in buffer and spiked milk, respectively, with higher sensitivity than the Aptamer/FITC based flow cytometry.

Multinode acoustic focusing for parallel flow cytometry

PubMed Central

Piyasena, Menake E.; Suthanthiraraj, Pearlson P. Austin; Applegate, Robert W.; Goumas, Andrew M.; Woods, Travis A.; López, Gabriel P.; Graves, Steven W.

2012-01-01

Flow cytometry can simultaneously measure and analyze multiple properties of single cells or particles with high sensitivity and precision. Yet, conventional flow cytometers have fundamental limitations with regards to analyzing particles larger than about 70 microns, analyzing at flow rates greater than a few hundred microliters per minute, and providing analysis rates greater than 50,000 per second. To overcome these limits, we have developed multi-node acoustic focusing flow cells that can position particles (as small as a red blood cell and as large as 107 microns in diameter) into as many as 37 parallel flow streams. We demonstrate the potential of such flow cells for the development of high throughput, parallel flow cytometers by precision focusing of flow cytometry alignment microspheres, red blood cells, and the analysis of CD4+ cellular immunophenotyping assay. This approach will have significant impact towards the creation of high throughput flow cytometers for rare cell detection applications (e.g. circulating tumor cells), applications requiring large particle analysis, and high volume flow cytometry. PMID:22239072

Final Report Nucleic Acid System - Hybrid PCR and Multiplex Assay Project Phase 2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Koopman, R P; Langlois, R G; Nasarabadi, S

2002-04-17

This report covers phase 2 (year 2) of the Nucleic Acid System--Hybrid PCR and Multiplex Assay project. The objective of the project is to reduce to practice the detection and identification of biological warfare pathogens by the nucleic acid recognition technique of PCR (polymerase chain reaction) in a multiplex mode using flow cytometry. The Hybrid instrument consists of a flow-through PCR module capable of handling a multiplexed PCR assay, a hybridizing module capable of hybridizing multiplexed PCR amplicons and beads, and a flow cytometer module for bead-based identification, all controlled by a single computer. Multiplex immunoassay using bead-based Luminex flowmore » cytometry is available, allowing rapid screening for many agents. PCR is highly specific and complements and verifies immunoassay. It can also be multiplexed and detection provided using the bead-based Luminex flow cytometer. This approach allows full access to the speed and 100-fold multiplex capability of flow cytometry for rapid screening as well as the accuracy and specificity of PCR. This project has two principal activities: (1) Design, build and test a prototype hybrid PCR/flow cytometer with the basic capabilities for rapid, broad spectrum detection and identification, and (2) Develop and evaluate multiplex flow analysis assay protocols and reagents for the simultaneous detection of PCR products. This project requires not only building operationally functional instrumentation but also developing the chemical assays for detection of priority pathogens. This involves development and evaluation of multiplex flow analysis assay protocols and reagents for the simultaneous detection of PCR products.« less

Purification, Characterization, and Mode of Action of Plantaricin GZ1-27, a Novel Bacteriocin against Bacillus cereus.

PubMed

Du, Hechao; Yang, Jie; Lu, Xiaohong; Lu, Zhaoxin; Bie, Xiaomei; Zhao, Haizhen; Zhang, Chong; Lu, Fengxia

2018-05-09

Bacillus cereus is an opportunistic pathogen that causes foodborne diseases. We isolated a novel bacteriocin, designated plantaricin GZ1-27, and elucidated its mode of action against B. cereus. Plantaricin GZ1-27 was purified using ammonium sulfate precipitation, gel-filtration chromatography, and RP-HPLC. MALDI-TOF/MS revealed that its molecular mass was 975 Da, and Q-TOF-MS/MS analysis predicted the amino acid sequence as VSGPAGPPGTH. Plantaricin GZ1-27 showed thermostability and pH stability. The antibacterial mechanism was investigated using flow cytometry, confocal laser-scanning microscopy, scanning and transmission electron microscopy, and RT-PCR, which revealed that GZ1-27 increased cell membrane permeability, triggered K + leakage and pore formation, damaged cell membrane integrity, altered cell morphology and intracellular organization, and reduced the expression of genes related to cytotoxin production, peptidoglycan synthesis, and cell division. These results suggest that plantaricin GZ1-27 effectively inhibits B. cereus at both the cellular and the molecular levels and is a potential natural food preservative targeting B. cereus.

Masks in Imaging Flow Cytometry

PubMed Central

Dominical, Venina; Samsel, Leigh; McCoy, J. Philip

2016-01-01

Data analysis in imaging flow cytometry incorporates elements of flow cytometry together with other aspects of morphological analysis of images. A crucial early step in this analysis is the creation of a mask to distinguish the portion of the image upon which further examination of specified features can be performed. Default masks are provided by the manufacturer of the imaging flow cytometer but additional custom masks can be created by the individual user for specific applications. Flawed or inaccurate masks can have a substantial negative impact on the overall analysis of a sample, thus great care must be taken to ensure the accuracy of masks. Here we discuss various types of masks and cite examples of their use. Furthermore we provide our insight for how to approach selecting and assessing the optimal mask for a specific analysis. PMID:27461256

Extracting Cell Stiffness from Real-Time Deformability Cytometry: Theory and Experiment.

PubMed

Mietke, Alexander; Otto, Oliver; Girardo, Salvatore; Rosendahl, Philipp; Taubenberger, Anna; Golfier, Stefan; Ulbricht, Elke; Aland, Sebastian; Guck, Jochen; Fischer-Friedrich, Elisabeth

2015-11-17

Cell stiffness is a sensitive indicator of physiological and pathological changes in cells, with many potential applications in biology and medicine. A new method, real-time deformability cytometry, probes cell stiffness at high throughput by exposing cells to a shear flow in a microfluidic channel, allowing for mechanical phenotyping based on single-cell deformability. However, observed deformations of cells in the channel not only are determined by cell stiffness, but also depend on cell size relative to channel size. Here, we disentangle mutual contributions of cell size and cell stiffness to cell deformation by a theoretical analysis in terms of hydrodynamics and linear elasticity theory. Performing real-time deformability cytometry experiments on both model spheres of known elasticity and biological cells, we demonstrate that our analytical model not only predicts deformed shapes inside the channel but also allows for quantification of cell mechanical parameters. Thereby, fast and quantitative mechanical sampling of large cell populations becomes feasible. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Extracting Cell Stiffness from Real-Time Deformability Cytometry: Theory and Experiment

PubMed Central

Mietke, Alexander; Otto, Oliver; Girardo, Salvatore; Rosendahl, Philipp; Taubenberger, Anna; Golfier, Stefan; Ulbricht, Elke; Aland, Sebastian; Guck, Jochen; Fischer-Friedrich, Elisabeth

2015-01-01

Cell stiffness is a sensitive indicator of physiological and pathological changes in cells, with many potential applications in biology and medicine. A new method, real-time deformability cytometry, probes cell stiffness at high throughput by exposing cells to a shear flow in a microfluidic channel, allowing for mechanical phenotyping based on single-cell deformability. However, observed deformations of cells in the channel not only are determined by cell stiffness, but also depend on cell size relative to channel size. Here, we disentangle mutual contributions of cell size and cell stiffness to cell deformation by a theoretical analysis in terms of hydrodynamics and linear elasticity theory. Performing real-time deformability cytometry experiments on both model spheres of known elasticity and biological cells, we demonstrate that our analytical model not only predicts deformed shapes inside the channel but also allows for quantification of cell mechanical parameters. Thereby, fast and quantitative mechanical sampling of large cell populations becomes feasible. PMID:26588562

Autologous Adipose-Derived Tissue Matrix Part I: Biologic Characteristics.

PubMed

Schendel, Stephen A

2017-10-01

Autologous collagen is an ideal soft tissue filler and may serve as a matrix for stem cell implantation and growth. Procurement of autologous collagen has been limited, though, secondary to a sufficient source. Liposuction is a widely performed and could be a source of autologous collagen. The amount of collagen and its composition in liposuctioned fat remains unknown. The purpose of this research was to characterize an adipose-derived tissue-based product created using ultrasonic cavitation and cryo-grinding. This study evaluated the cellular and protein composition of the final product. Fat was obtained from individuals undergoing routine liposuction and was processed by a 2 step process to obtain only the connective tissue. The tissue was then evaluated by scanning electronic microscope, Western blot analysis, and flow cytometry. Liposuctioned fat was obtained from 10 individuals with an average of 298 mL per subject. After processing an average of 1 mL of collagen matrix was obtained from each 100 mL of fat. Significant viable cell markers were present in descending order for adipocytes > CD90+ > CD105+ > CD45+ > CD19+ > CD144+ > CD34+. Western blot analysis showed collagen type II, III, IV, and other proteins. Scanning electronic microscope study showed a regular pattern of cross-linked, helical collagen. Additionally, vital staing demonstrated that the cells were still viable after processing. Collagen and cells can be easily obtained from liposuctioned fat by ultrasonic separation without alteration of the overall cellular composition of the tissue. Implantation results in new collagen and cellular growth. Collagen matrix with viable cells for autologous use can be obtained from liposuctioned fat and may provide long term results. 5. © 2017 The American Society for Aesthetic Plastic Surgery, Inc. Reprints and permission: journals.permissions@oup.com

Optofluidic time-stretch microscopy: recent advances

NASA Astrophysics Data System (ADS)

Lei, Cheng; Nitta, Nao; Ozeki, Yasuyuki; Goda, Keisuke

2018-06-01

Flow cytometry is an indispensable method for valuable applications in numerous fields such as immunology, pathology, pharmacology, molecular biology, and marine biology. Optofluidic time-stretch microscopy is superior to conventional flow cytometry methods for its capability to acquire high-quality images of single cells at a high-throughput exceeding 10,000 cells per second. This makes it possible to extract copious information from cellular images for accurate cell detection and analysis with the assistance of machine learning. Optofluidic time-stretch microscopy has proven its effectivity in various applications, including microalga-based biofuel production, evaluation of thrombotic disorders, as well as drug screening and discovery. In this review, we discuss the principles and recent advances of optofluidic time-stretch microscopy.

Optofluidic time-stretch microscopy: recent advances

NASA Astrophysics Data System (ADS)

Lei, Cheng; Nitta, Nao; Ozeki, Yasuyuki; Goda, Keisuke

2018-04-01

Flow cytometry is an indispensable method for valuable applications in numerous fields such as immunology, pathology, pharmacology, molecular biology, and marine biology. Optofluidic time-stretch microscopy is superior to conventional flow cytometry methods for its capability to acquire high-quality images of single cells at a high-throughput exceeding 10,000 cells per second. This makes it possible to extract copious information from cellular images for accurate cell detection and analysis with the assistance of machine learning. Optofluidic time-stretch microscopy has proven its effectivity in various applications, including microalga-based biofuel production, evaluation of thrombotic disorders, as well as drug screening and discovery. In this review, we discuss the principles and recent advances of optofluidic time-stretch microscopy.

One-dimensional acoustic standing waves in rectangular channels for flow cytometry.

PubMed

Austin Suthanthiraraj, Pearlson P; Piyasena, Menake E; Woods, Travis A; Naivar, Mark A; Lόpez, Gabriel P; Graves, Steven W

2012-07-01

Flow cytometry has become a powerful analytical tool for applications ranging from blood diagnostics to high throughput screening of molecular assemblies on microsphere arrays. However, instrument size, expense, throughput, and consumable use limit its use in resource poor areas of the world, as a component in environmental monitoring, and for detection of very rare cell populations. For these reasons, new technologies to improve the size and cost-to-performance ratio of flow cytometry are required. One such technology is the use of acoustic standing waves that efficiently concentrate cells and particles to the center of flow channels for analysis. The simplest form of this method uses one-dimensional acoustic standing waves to focus particles in rectangular channels. We have developed one-dimensional acoustic focusing flow channels that can be fabricated in simple capillary devices or easily microfabricated using photolithography and deep reactive ion etching. Image and video analysis demonstrates that these channels precisely focus single flowing streams of particles and cells for traditional flow cytometry analysis. Additionally, use of standing waves with increasing harmonics and in parallel microfabricated channels is shown to effectively create many parallel focused streams. Furthermore, we present the fabrication of an inexpensive optical platform for flow cytometry in rectangular channels and use of the system to provide precise analysis. The simplicity and low-cost of the acoustic focusing devices developed here promise to be effective for flow cytometers that have reduced size, cost, and consumable use. Finally, the straightforward path to parallel flow streams using one-dimensional multinode acoustic focusing, indicates that simple acoustic focusing in rectangular channels may also have a prominent role in high-throughput flow cytometry. Copyright © 2012 Elsevier Inc. All rights reserved.

Thymbra capitata essential oil as potential therapeutic agent against Gardnerella vaginalis biofilm-related infections.

PubMed

Machado, Daniela; Gaspar, Carlos; Palmeira-de-Oliveira, Ana; Cavaleiro, Carlos; Salgueiro, Lígia; Martinez-de-Oliveira, José; Cerca, Nuno

2017-04-01

To evaluate the antibacterial activity of Thymbra capitata essential oil and its main compound, carvacrol, against Gardnerella vaginalis grown planktonically and as biofilms, and its effect of vaginal lactobacilli. Minimal inhibitory concentration, minimal lethal concentration determination and flow cytometry analysis were used to assess the antibacterial effect against planktonic cells. Antibiofilm activity was measured through quantification of biomass and visualization of biofilm structure by confocal laser scanning microscopy. T. capitata essential oil and carvacrol exhibited a potent antibacterial activity against G. vaginalis cells. Antibiofilm activity was more evident with the essential oil than carvacrol. Furthermore, vaginal lactobacilli were significantly more tolerant to the essential oil. T. capitata essential oil stands up as a promising therapeutic agent against G. vaginalis biofilm-related infections.

Gating mass cytometry data by deep learning.

PubMed

Li, Huamin; Shaham, Uri; Stanton, Kelly P; Yao, Yi; Montgomery, Ruth R; Kluger, Yuval

2017-11-01

Mass cytometry or CyTOF is an emerging technology for high-dimensional multiparameter single cell analysis that overcomes many limitations of fluorescence-based flow cytometry. New methods for analyzing CyTOF data attempt to improve automation, scalability, performance and interpretation of data generated in large studies. Assigning individual cells into discrete groups of cell types (gating) involves time-consuming sequential manual steps, untenable for larger studies. We introduce DeepCyTOF, a standardization approach for gating, based on deep learning techniques. DeepCyTOF requires labeled cells from only a single sample. It is based on domain adaptation principles and is a generalization of previous work that allows us to calibrate between a target distribution and a source distribution in an unsupervised manner. We show that DeepCyTOF is highly concordant (98%) with cell classification obtained by individual manual gating of each sample when applied to a collection of 16 biological replicates of primary immune blood cells, even when measured across several instruments. Further, DeepCyTOF achieves very high accuracy on the semi-automated gating challenge of the FlowCAP-I competition as well as two CyTOF datasets generated from primary immune blood cells: (i) 14 subjects with a history of infection with West Nile virus (WNV), (ii) 34 healthy subjects of different ages. We conclude that deep learning in general, and DeepCyTOF specifically, offers a powerful computational approach for semi-automated gating of CyTOF and flow cytometry data. Our codes and data are publicly available at https://github.com/KlugerLab/deepcytof.git. yuval.kluger@yale.edu. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

Dynamic analysis of apoptosis using cyanine SYTO probes: From classical to microfluidic cytometry

PubMed Central

Wlodkowic, Donald; Skommer, Joanna; Faley, Shannon; Darzynkiewicz, Zbigniew; Cooper, Jonathan M.

2013-01-01

Cell death is a stochastic process, often initiated and/or executed in a multi-pathway/multi-organelle fashion. Therefore, high-throughput single-cell analysis platforms are required to provide detailed characterization of kinetics and mechanisms of cell death in heterogeneous cell populations. However, there is still a largely unmet need for inert fluorescent probes, suitable for prolonged kinetic studies. Here, we compare the use of innovative adaptation of unsymmetrical SYTO dyes for dynamic real-time analysis of apoptosis in conventional as well as microfluidic chip-based systems. We show that cyanine SYTO probes allow non-invasive tracking of intracellular events over extended time. Easy handling and “stain–no wash” protocols open up new opportunities for high-throughput analysis and live-cell sorting. Furthermore, SYTO probes are easily adaptable for detection of cell death using automated microfluidic chip-based cytometry. Overall, the combined use of SYTO probes and state-of-the-art Lab-on-a-Chip platform emerges as a cost effective solution for automated drug screening compared to conventional Annexin V or TUNEL assays. In particular, it should allow for dynamic analysis of samples where low cell number has so far been an obstacle, e.g. primary cancer stems cells or circulating minimal residual tumors. PMID:19298813

Development of CD3 cell quantitation algorithms for renal allograft biopsy rejection assessment utilizing open source image analysis software.

PubMed

Moon, Andres; Smith, Geoffrey H; Kong, Jun; Rogers, Thomas E; Ellis, Carla L; Farris, Alton B Brad

2018-02-01

Renal allograft rejection diagnosis depends on assessment of parameters such as interstitial inflammation; however, studies have shown interobserver variability regarding interstitial inflammation assessment. Since automated image analysis quantitation can be reproducible, we devised customized analysis methods for CD3+ T-cell staining density as a measure of rejection severity and compared them with established commercial methods along with visual assessment. Renal biopsy CD3 immunohistochemistry slides (n = 45), including renal allografts with various degrees of acute cellular rejection (ACR) were scanned for whole slide images (WSIs). Inflammation was quantitated in the WSIs using pathologist visual assessment, commercial algorithms (Aperio nuclear algorithm for CD3+ cells/mm 2 and Aperio positive pixel count algorithm), and customized open source algorithms developed in ImageJ with thresholding/positive pixel counting (custom CD3+%) and identification of pixels fulfilling "maxima" criteria for CD3 expression (custom CD3+ cells/mm 2 ). Based on visual inspections of "markup" images, CD3 quantitation algorithms produced adequate accuracy. Additionally, CD3 quantitation algorithms correlated between each other and also with visual assessment in a statistically significant manner (r = 0.44 to 0.94, p = 0.003 to < 0.0001). Methods for assessing inflammation suggested a progression through the tubulointerstitial ACR grades, with statistically different results in borderline versus other ACR types, in all but the custom methods. Assessment of CD3-stained slides using various open source image analysis algorithms presents salient correlations with established methods of CD3 quantitation. These analysis techniques are promising and highly customizable, providing a form of on-slide "flow cytometry" that can facilitate additional diagnostic accuracy in tissue-based assessments.

Mobile flow cytometer for mHealth.

PubMed

Balsam, Joshua; Bruck, Hugh Alan; Rasooly, Avraham

2015-01-01

Flow cytometry is used for cell counting and analysis in numerous clinical and environmental applications. However flow cytometry is not used in mHealth mainly because current flow cytometers are large, expensive, power-intensive devices designed to operate in a laboratory. Their design results in a lack of portability and makes them unsuitable for mHealth applications. Another limitation of current technology is the low volumetric throughput rates that are not suitable for rapid detection of rare cells.To address these limitations, we describe here a novel, low-cost, mobile flow cytometer based on wide-field imaging with a webcam for large volume and high throughput fluorescence detection of rare cells as a simulation for circulating tumor cells (CTCs) detection. The mobile flow cytometer uses a commercially available webcam capable of 187 frames per second video capture at a resolution of 320 × 240 pixels. For fluorescence detection, a 1 W 450 nm blue laser is used for excitation of Syto-9 fluorescently stained cells detected at 535 nm. A wide-field flow cell was developed for large volume analysis that allows for the linear velocity of target cells to be lower than in conventional hydrodynamic focusing flow cells typically used in cytometry. The mobile flow cytometer was found to be capable of detecting low concentrations at flow rates of 500 μL/min, suitable for rare cell detection in large volumes. The simplicity and low cost of this device suggests that it may have a potential clinical use for mHealth flow cytometry for resource-poor settings associated with global health.

Genomic Instability at Premalignant and Early Stages of Breast Cancer Development

DTIC Science & Technology

1999-08-01

by routine DNA flow cytometry vation. ERBB2 expression was detected with a to determine DNA index (DI). commercially available antibody (Oncogene Sci...supplements the information gained from ic microsatellite primers. We observed that the ploidy analysis by DNA flow cytometry alone. In DNA so obtained...preserved the proportionality of many cases where flow cytometry could not be per- the different alleles as found in the original sample. formed because the

Enzymatic signal amplification for sensitive detection of intracellular antigens by flow cytometry.

PubMed

Karkmann, U; Radbruch, A; Hölzel, V; Scheffold, A

1999-11-19

Flow cytometry is the method of choice for the analysis of single cells with respect to the expression of specific antigens. Antigens can be detected with specific antibodies either on the cell surface or within the cells, after fixation and permeabilization of the cell membrane. Using conventional fluorochrome-labeled antibodies several thousand antigens are required for clear-cut separation of positive and negative cells. More sensitive reagents, e.g., magnetofluorescent liposomes conjugated to specific antibodies permit the detection of less than 200 molecules per cell but cannot be used for the detection of intracellular antigens. Here, we describe an enzymatic amplification technique (intracellular tyramine-based signal amplification, ITSA) for the sensitive cytometric analysis of intracellular cytokines by immunofluorescence. This approach results in a 10 to 15-fold improvement of the signal-to-noise ratio compared to conventional fluorochrome labeled antibodies and permits the detection of as few as 300-400 intracellular antigens per cell.

Flow cytometry with gold nanoparticles and their clusters as scattering contrast agents: FDTD simulation of light-cell interaction.

PubMed

Tanev, Stoyan; Sun, Wenbo; Pond, James; Tuchin, Valery V; Zharov, Vladimir P

2009-09-01

The formulation of the finite-difference time-domain (FDTD) approach is presented in the framework of its potential applications to in-vivo flow cytometry based on light scattering. The consideration is focused on comparison of light scattering by a single biological cell alone in controlled refractive-index matching conditions and by cells labeled by gold nanoparticles. The optical schematics including phase contrast (OPCM) microscopy as a prospective modality for in-vivo flow cytometry is also analyzed. The validation of the FDTD approach for the simulation of flow cytometry may open up a new avenue in the development of advanced cytometric techniques based on scattering effects from nanoscale targets. 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Multivariate data analysis methods for the interpretation of microbial flow cytometric data.

PubMed

Davey, Hazel M; Davey, Christopher L

2011-01-01

Flow cytometry is an important technique in cell biology and immunology and has been applied by many groups to the analysis of microorganisms. This has been made possible by developments in hardware that is now sensitive enough to be used routinely for analysis of microbes. However, in contrast to advances in the technology that underpin flow cytometry, there has not been concomitant progress in the software tools required to analyse, display and disseminate the data and manual analysis, of individual samples remains a limiting aspect of the technology. We present two new data sets that illustrate common applications of flow cytometry in microbiology and demonstrate the application of manual data analysis, automated visualisation (including the first description of a new piece of software we are developing to facilitate this), genetic programming, principal components analysis and artificial neural nets to these data. The data analysis methods described here are equally applicable to flow cytometric applications with other cell types.

Experimental and theoretical study of light scattering by individual mature red blood cells by use of scanning flow cytometry and a discrete dipole approximation.

PubMed

Yurkin, Maxim A; Semyanov, Konstantin A; Tarasov, Peter A; Chernyshev, Andrei V; Hoekstra, Alfons G; Maltsev, Valeri P

2005-09-01

Elastic light scattering by mature red blood cells (RBCs) was theoretically and experimentally analyzed by use of the discrete dipole approximation (DDA) and scanning flow cytometry (SFC), respectively. SFC permits measurement of the angular dependence of the light-scattering intensity (indicatrix) of single particles. A mature RBC is modeled as a biconcave disk in DDA simulations of light scattering. We have studied the effect of RBC orientation related to the direction of the light incident upon the indicatrix. Numerical calculations of indicatrices for several axis ratios and volumes of RBC have been carried out. Comparison of the simulated indicatrices and indicatrices measured by SFC showed good agreement, validating the biconcave disk model for a mature RBC. We simulated the light-scattering output signals from the SFC with the DDA for RBCs modeled as a disk-sphere and as an oblate spheroid. The biconcave disk, the disk-sphere, and the oblate spheroid models have been compared for two orientations, i.e., face-on and rim-on incidence, relative to the direction of the incident beam. Only the oblate spheroid model for rim-on incidence gives results similar to those of the rigorous biconcave disk model.

Bioactivity Studies of β-Lactam Derived Polycyclic Fused Pyrroli-Dine/Pyrrolizidine Derivatives in Dentistry: In Vitro, In Vivo and In Silico Studies

PubMed Central

Winfred, Sofi Beaula; Mannivanan, Bhavani; Bhoopalan, Hemadev; Shankar, Venkatesh; Sekar, Sathiya; Venkatachalam, Deepa Parvathi; Pitani, Ravishankar; Nagendrababu, Venkateshbabu; Thaiman, Malini; Devivanayagam, Kandaswamy; Jayaraman, Jeyakanthan; Ragavachary, Raghunathan; Venkatraman, Ganesh

2015-01-01

The antibacterial activity of β-lactam derived polycyclic fused pyrrolidine/pyrrolizidine derivatives synthesized by 1, 3-dipolar cycloaddition reaction was evaluated against microbes involved in dental infection. Fifteen compounds were screened; among them compound 3 showed efficient antibacterial activity in an ex vivo dentinal tubule model and in vivo mice infectious model. In silico docking studies showed greater affinity to penicillin binding protein. Cell damage was observed under Scanning Electron Microscopy (SEM) which was further proved by Confocal Laser Scanning Microscope (CLSM) and quantified using Flow Cytometry by PI up-take. Compound 3 treated E. faecalis showed ROS generation and loss of membrane integrity was quantified by flow cytometry. Compound 3 was also found to be active against resistant E. faecalis strains isolated from failed root canal treatment cases. Further, compound 3 was found to be hemocompatible, not cytotoxic to normal mammalian NIH 3T3 cells and non mutagenic. It was concluded that β-lactam compound 3 exhibited promising antibacterial activity against E. faecalis involved in root canal infections and the mechanism of action was deciphered. The results of this research can be further implicated in the development of potent antibacterial medicaments with applications in dentistry. PMID:26185985

Streak Imaging Flow Cytometer for Rare Cell Analysis.

PubMed

Balsam, Joshua; Bruck, Hugh Alan; Ossandon, Miguel; Prickril, Ben; Rasooly, Avraham

2017-01-01

There is a need for simple and affordable techniques for cytology for clinical applications, especially for point-of-care (POC) medical diagnostics in resource-poor settings. However, this often requires adapting expensive and complex laboratory-based techniques that often require significant power and are too massive to transport easily. One such technique is flow cytometry, which has great potential for modification due to the simplicity of the principle of optical tracking of cells. However, it is limited in that regard due to the flow focusing technique used to isolate cells for optical detection. This technique inherently reduces the flow rate and is therefore unsuitable for rapid detection of rare cells which require large volume for analysis.To address these limitations, we developed a low-cost, mobile flow cytometer based on streak imaging. In our new configuration we utilize a simple webcam for optical detection over a large area associated with a wide-field flow cell. The new flow cell is capable of larger volume and higher throughput fluorescence detection of rare cells than the flow cells with hydrodynamic focusing used in conventional flow cytometry. The webcam is an inexpensive, commercially available system, and for fluorescence analysis we use a 1 W 450 nm blue laser to excite Syto-9 stained cells with emission at 535 nm. We were able to detect low concentrations of stained cells at high flow rates of 10 mL/min, which is suitable for rapidly analyzing larger specimen volumes to detect rare cells at appropriate concentration levels. The new rapid detection capabilities, combined with the simplicity and low cost of this device, suggest a potential for clinical POC flow cytometry in resource-poor settings associated with global health.

Fluorescent supramolecular micelles for imaging-guided cancer therapy

NASA Astrophysics Data System (ADS)

Sun, Mengmeng; Yin, Wenyan; Dong, Xinghua; Yang, Wantai; Zhao, Yuliang; Yin, Meizhen

2016-02-01

A novel smart fluorescent drug delivery system composed of a perylene diimide (PDI) core and block copolymer poly(d,l-lactide)-b-poly(ethyl ethylene phosphate) is developed and named as PDI-star-(PLA-b-PEEP)8. The biodegradable PDI-star-(PLA-b-PEEP)8 is a unimolecular micelle and can self-assemble into supramolecular micelles, called as fluorescent supramolecular micelles (FSMs), in aqueous media. An insoluble drug camptothecin (CPT) can be effectively loaded into the FSMs and exhibits pH-responsive release. Moreover, the FSMs with good biocompatibility can also be employed as a remarkable fluorescent probe for cell labelling because the maximum emission of PDI is beneficial for bio-imaging. The flow cytometry and confocal laser scanning microscopy analysis demonstrate that the micelles are easily endocytosed by cancer cells. In vitro and in vivo tumor growth-inhibitory studies reveal a better therapeutic effect of FSMs after CPT encapsulation when compared with the free CPT drug. The multifunctional FSM nanomedicine platform as a nanovehicle has great potential for fluorescence imaging-guided cancer therapy.A novel smart fluorescent drug delivery system composed of a perylene diimide (PDI) core and block copolymer poly(d,l-lactide)-b-poly(ethyl ethylene phosphate) is developed and named as PDI-star-(PLA-b-PEEP)8. The biodegradable PDI-star-(PLA-b-PEEP)8 is a unimolecular micelle and can self-assemble into supramolecular micelles, called as fluorescent supramolecular micelles (FSMs), in aqueous media. An insoluble drug camptothecin (CPT) can be effectively loaded into the FSMs and exhibits pH-responsive release. Moreover, the FSMs with good biocompatibility can also be employed as a remarkable fluorescent probe for cell labelling because the maximum emission of PDI is beneficial for bio-imaging. The flow cytometry and confocal laser scanning microscopy analysis demonstrate that the micelles are easily endocytosed by cancer cells. In vitro and in vivo tumor growth-inhibitory studies reveal a better therapeutic effect of FSMs after CPT encapsulation when compared with the free CPT drug. The multifunctional FSM nanomedicine platform as a nanovehicle has great potential for fluorescence imaging-guided cancer therapy. Electronic supplementary information (ESI) available. See DOI: 10.1039/c6nr00450d

Identification of microbes from the surfaces of food-processing lines based on the flow cytometric evaluation of cellular metabolic activity combined with cell sorting.

PubMed

Juzwa, W; Duber, A; Myszka, K; Białas, W; Czaczyk, K

2016-09-01

In this study the design of a flow cytometry-based procedure to facilitate the detection of adherent bacteria from food-processing surfaces was evaluated. The measurement of the cellular redox potential (CRP) of microbial cells was combined with cell sorting for the identification of microorganisms. The procedure enhanced live/dead cell discrimination owing to the measurement of the cell physiology. The microbial contamination of the surface of a stainless steel conveyor used to process button mushrooms was evaluated in three independent experiments. The flow cytometry procedure provided a step towards monitoring of contamination and enabled the assessment of microbial food safety hazards by the discrimination of active, mid-active and non-active bacterial sub-populations based on determination of their cellular vitality and subsequently single cell sorting to isolate microbial strains from discriminated sub-populations. There was a significant correlation (r = 0.97; p < 0.05) between the bacterial cell count estimated by the pour plate method and flow cytometry, despite there being differences in the absolute number of cells detected. The combined approach of flow cytometric CRP measurement and cell sorting allowed an in situ analysis of microbial cell vitality and the identification of species from defined sub-populations, although the identified microbes were limited to culturable cells.

Improving label-free detection of circulating melanoma cells by photoacoustic flow cytometry

NASA Astrophysics Data System (ADS)

Zhou, Huan; Wang, Qiyan; Pang, Kai; Zhou, Quanyu; Yang, Ping; He, Hao; Wei, Xunbin

2018-02-01

Melanoma is a kind of a malignant tumor of melanocytes with the properties of high mortality and high metastasis rate. The circulating melanoma cells with the high content of melanin can be detected by light absorption to diagnose and treat cancer at an early stage. Compared with conventional detection methods such as in vivo flow cytometry (IVFC) based on fluorescence, the in vivo photoacoustic flow cytometry (PAFC) utilizes melanin cells as biomarkers to collect the photoacoustic (PA) signals without toxic fluorescent dyes labeling in a non-invasive way. The information of target tumor cells is helpful for data analysis and cell counting. However, the raw signals in PAFC system contain numerous noises such as environmental noise, device noise and in vivo motion noise. Conventional denoising algorithms such as wavelet denoising (WD) method and means filter (MF) method are based on the local information to extract the data of clinical interest, which remove the subtle feature and leave many noises. To address the above questions, the nonlocal means (NLM) method based on nonlocal data has been proposed to suppress the noise in PA signals. Extensive experiments on in vivo PA signals from the mice with the injection of B16F10 cells in caudal vein have been conducted. All the results indicate that the NLM method has superior noise reduction performance and subtle information reservation.

Immunological tools: engaging students in the use and analysis of flow cytometry and enzyme-linked immunosorbent assay (ELISA).

PubMed

Ott, Laura E; Carson, Susan

2014-01-01

Flow cytometry and enzyme-linked immunosorbent assay (ELISA) are commonly used techniques associated with clinical and research applications within the immunology and medical fields. The use of these techniques is becoming increasingly valuable in many life science and engineering disciplines as well. Herein, we report the development and evaluation of a novel half-semester course that focused on introducing undergraduate and graduate students to advance conceptual and technical skills associated with flow cytometry and ELISA, with emphasis on applications, experimental design, and data analysis. This course was offered in the North Carolina State University Biotechnology Program over three semesters and consisted of weekly lectures and laboratories. Students performed and/or analyzed flow cytometry and ELISA in three separate laboratory exercises: (1) identification of transgenic zebrafish hematopoietic cells, (2) analysis of transfection efficiency, and (3) analysis of cytokine production upon lipopolysaccharide stimulation. Student learning outcomes were achieved as demonstrated by multiple means of assessment, including three laboratory reports, a data analysis laboratory practicum, and a cumulative final exam. Further, anonymous student self-assessment revealed increased student confidence in the knowledge and skill sets defined in the learning outcomes. Copyright © 2014 The International Union of Biochemistry and Molecular Biology.

Cell-cycle synchronisation of bloodstream forms of Trypanosoma brucei using Vybrant DyeCycle Violet-based sorting.

PubMed

Kabani, Sarah; Waterfall, Martin; Matthews, Keith R

2010-01-01

Studies on the cell-cycle of Trypanosoma brucei have revealed several unusual characteristics that differ from the model eukaryotic organisms. However, the inability to isolate homogenous populations of parasites in distinct cell-cycle stages has limited the analysis of trypanosome cell division and complicated the understanding of mutant phenotypes with possible impact on cell-cycle related events. Although hydroxyurea-induced cell-cycle arrest in procyclic and bloodstream forms has been applied recently with success, such block-release protocols can complicate the analysis of cell-cycle regulated events and have the potential to disrupt important cell-cycle checkpoints. An alternative approach based on flow cytometry of parasites stained with Vybrant DyeCycle Orange circumvents this problem, but is restricted to procyclic form parasites. Here, we apply Vybrant Dyecycle Violet staining coupled with flow cytometry to effectively select different cell-cycle stages of bloodstream form trypanosomes. Moreover, the sorted parasites remain viable, although synchrony is rapidly lost. This method enables cell-cycle enrichment of populations of trypanosomes in their mammal infective stage, particularly at the G1 phase.

Cell-cycle synchronisation of bloodstream forms of Trypanosoma brucei using Vybrant DyeCycle Violet-based sorting

PubMed Central

Kabani, Sarah; Waterfall, Martin; Matthews, Keith R.

2010-01-01

Studies on the cell-cycle of Trypanosoma brucei have revealed several unusual characteristics that differ from the model eukaryotic organisms. However, the inability to isolate homogenous populations of parasites in distinct cell-cycle stages has limited the analysis of trypanosome cell division and complicated the understanding of mutant phenotypes with possible impact on cell-cycle related events. Although hydroxyurea-induced cell-cycle arrest in procyclic and bloodstream forms has been applied recently with success, such block-release protocols can complicate the analysis of cell-cycle regulated events and have the potential to disrupt important cell-cycle checkpoints. An alternative approach based on flow cytometry of parasites stained with Vybrant DyeCycle Orange circumvents this problem, but is restricted to procyclic form parasites. Here, we apply Vybrant Dyecycle Violet staining coupled with flow cytometry to effectively select different cell-cycle stages of bloodstream form trypanosomes. Moreover, the sorted parasites remain viable, although synchrony is rapidly lost. This method enables cell-cycle enrichment of populations of trypanosomes in their mammal infective stage, particularly at the G1 phase. PMID:19729042

Genome survey sequencing of red swamp crayfish Procambarus clarkii.

PubMed

Shi, Linlin; Yi, Shaokui; Li, Yanhe

2018-06-21

Red swamp crayfish, Procambarus clarkii, presently is an important aquatic commercial species in China. The crayfish is a hot area of research focus, and its genetic improvement is quite urgent for the crayfish aquaculture in China. However, the knowledge of its genomic landscape is limited. In this study, a survey of P. clarkii genome was investigated based on Illumina's Solexa sequencing platform. Meanwhile, its genome size was estimated using flow cytometry. Interestingly, the genome size estimated is about 8.50 Gb by flow cytometry and 1.86 Gb with genome survey sequencing. Based on the assembled genome sequences, total of 136,962 genes and 152,268 exons were predicted, and the predicted genes ranged from 150 to 12,807 bp in length. The survey sequences could help accelerate the progress of gene discovery involved in genetic diversity and evolutionary analysis, even though it could not successfully applied for estimation of P. clarkii genome size.

Automated analysis of individual sperm cells using stain-free interferometric phase microscopy and machine learning.

PubMed

Mirsky, Simcha K; Barnea, Itay; Levi, Mattan; Greenspan, Hayit; Shaked, Natan T

2017-09-01

Currently, the delicate process of selecting sperm cells to be used for in vitro fertilization (IVF) is still based on the subjective, qualitative analysis of experienced clinicians using non-quantitative optical microscopy techniques. In this work, a method was developed for the automated analysis of sperm cells based on the quantitative phase maps acquired through use of interferometric phase microscopy (IPM). Over 1,400 human sperm cells from 8 donors were imaged using IPM, and an algorithm was designed to digitally isolate sperm cell heads from the quantitative phase maps while taking into consideration both the cell 3D morphology and contents, as well as acquire features describing sperm head morphology. A subset of these features was used to train a support vector machine (SVM) classifier to automatically classify sperm of good and bad morphology. The SVM achieves an area under the receiver operating characteristic curve of 88.59% and an area under the precision-recall curve of 88.67%, as well as precisions of 90% or higher. We believe that our automatic analysis can become the basis for objective and automatic sperm cell selection in IVF. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

The tumor affinity of chlorin e6 and its sonodynamic effects on non-small cell lung cancer.

PubMed

Chen, Bei; Zheng, Ruinian; Liu, Duan; Li, Baofeng; Lin, Jinrong; Zhang, Weimin

2013-03-01

Sonodynamic therapy (SDT) is a promising new approach for cancer therapy. The aim of this study was to investigate the tumor affinity of chlorin e6, a photosensitizer, and its sonodynamic effects on NSCLC. Human lung adenocarcinoma cells SPCA-1 and mice bearing SPCA-1 tumor xenograft were exposed to ultrasound in the presence or absence of chlorin e6. Chlorin e6 distribution was detected by laser scan confocal microscope. Cell apoptosis and necrosis were studied by flow cytometry analysis. Tumor size and weight were measured after different treatments. The concentration of chlorin e6 in tumor tissue was remarkably higher than that in normal muscle near tumor, and the difference was greatest at 18h (the fluorescence intensity was 5.38-fold higher in tumor than in muscle, P<0.05). In vivo, ultrasound (0.4-1.6W/cm(2)) or chlorin e6 (10-40mg/kg) alone had no remarkable anti-tumor effects, but the combination of ultrasound (1.6W/cm(2)) with chlorin e6 (SDT) hampered tumor growth significantly (P<0.05). Intraperitoneal injection of 40mg/kg chlorin e6 exerted no notable side effect on blood, liver and kidney function. Flow cytometry analysis showed that chlorin e6-mediated sonodynamic effect was mainly through the induction of cell necrosis. Chlorin e6 is a promising sonosensitizer and chlorin e6-mediated SDT may provide a new approach for NSCLC therapy. Copyright © 2012 Elsevier B.V. All rights reserved.

Establishing tools for early diagnosis of congenital toxoplasmosis: Flow cytometric IgG avidity assay as a confirmatory test for neonatal screening.

PubMed

de Castro Zacche-Tonini, Aline; Fonseca, Giuliana Schmidt França; de Jesus, Laura Néspoli Nassar Pansini; Barros, Geisa Baptista; Coelho-Dos-Reis, Jordana Grazziela Alves; Béla, Samantha Ribeiro; Machado, Anderson Silva; Carneiro, Ana Carolina Aguiar Vasconcelos; Andrade, Gláucia Manzan Queiroz; Vasconcelos-Santos, Daniel Vitor; Januário, José Nélio; Teixeira-Carvalho, Andréa; Vitor, Ricardo Wagner Almeida; Ferro, Eloísa Amália Vieira; Mineo, José Roberto; Martins-Filho, Olindo Assis; Lemos, Elenice Moreira

2017-12-01

The aim of this study was to evaluate the performance of conventional serology (Q-Preven™ and ELFAVIDAS™) and flow cytometry-based serologic tools for early serologic diagnosis of congenital toxoplasmosis. The study groups included prospectively confirmed cases of congenital toxoplasmosis (TOXO=88) and age-matching non-infected controls (NI=15).The results demonstrated that all samples tested positive/indeterminate for anti-T. gondii IgM screening at birth using air-dried whole blood samples. Serum samples collected at 30-45days after birth tested positive for ELFAVIDAS™ IgG in both groups. While all NI tested negative for ELFAVIDAS™ IgM and IgA, only 78% and 36% of TOXO tested positive for IgM and IgA, respectively. Flow cytometry-based anti-T. gondii IgM, IgA and IgG reactivity displayed moderate performance with low sensitivity (47.6%, 72.6% and 75.0%, respectively). Regardless the remarkable specificity of IgG1, IgG2 and IgG3 subclasses for early diagnosis, weak or moderate specificity was observed (Se=73.9%, 60.2% and 83.0%, respectively). The analysis of IgG avidity indices (AI) demonstrated the highest performance among the flow cytometry-based methods (Se=96.6%; Sp=93.3%), underscoring the low avidity index (AI<60%) within TOXO (97.0%) in contrast with the high avidity index (AI>60%) in NI (93%). Analysis of anti-T. gondii IgG and IgG3 reactivity for mother:infant paired samples may represent a relevant complementary tests for early diagnosis. In conclusion, a feasible high-standard algorithm (Accuracy=97.1%) was proposed consisting of Q-Preven™ IgM screening at birth, followed by ELFAVIDAS™ IgM and flow cytometric IgG avidity analysis at 30-45days after birth as a high performance tool for early serological diagnosis of congenital toxoplasmosis. Copyright © 2017. Published by Elsevier B.V.

Microengineering of artificial capillaries

NASA Astrophysics Data System (ADS)

Moldovan, Nicanor I.

2002-11-01

Biocompatibility and functionality of implanted inorganic medical devices is limited by the local reaction of the organism, with a recently recognized contribution of nearby microvasculature. We explored the possibility to microengineer pre-embedded microvascular networks in the surface of inorganic devices. The implants would thus function as carriers of pre-assembled microvessels, ready to expand, and contribute to local angiogenesis. Based on our own studies on the role played by local microtopography in angiogenesis (the tunneling concept), we have shown the feasibility of endothelial cells cultivation in grooves created on the surface of the materials to be implanted, either polymeric or silicon. In order to develop this new technology, we devised an in situ approach to the study of the cellular behavior on micropatterned surfaces, by use of Laser Scanning Cytometry (LSC). In this report I will present our results regarding the LSC analysis of endothelial cells cultivated in grooves made on the surface of silicon wafers, and the consequences of this treatment on endothelial physiology. When comparing the growth of endothelial cells on line patterned and non-patterned areas, in terms of several morphological parameters of cell nuclei, our data support the conclusion that lateral confinement of endothelial cells induces a quiescent state, possibly by inhibiting their ability to proliferate.

Enhanced Cellular Uptake and Pharmacokinetic Characteristics of Doxorubicin-Valine Amide Prodrug.

PubMed

Park, Yohan; Park, Ju-Hwan; Park, Suryeon; Lee, Song Yi; Cho, Kwan Hyung; Kim, Dae-Duk; Shim, Won-Sik; Yoon, In-Soo; Cho, Hyun-Jong; Maeng, Han-Joo

2016-09-22

In this study, we synthesized the valine (Val)-conjugated amide prodrug of doxorubicin (DOX) by the formation of amide bonds between DOX and Val. The synthesis of the DOX-Val prodrug was identified by a proton nuclear magnetic resonance (¹H-NMR) assay. In the MCF-7 cells (human breast adenocarcinoma cell; amino acid transporter-positive cell), the cellular accumulation efficiency of DOX-Val was higher than that of DOX according to the flow cytometry analysis data. Using confocal laser scanning microscopy (CLSM) imaging, it was confirmed that DOX-Val as well as DOX was mainly distributed in the nucleus of cancer cells. DOX-Val was intravenously administered to rats at a dose of 4 mg/kg, and the plasma concentrations of DOX-Val (prodrug) and DOX (formed metabolite) were quantitatively determined. Based on the systemic exposure (represented as area under the curve (AUC) values) of DOX-Val (prodrug) and DOX (formed metabolite), approximately half of DOX-Val seemed to be metabolized into DOX. However, it is expected that the remaining DOX-Val may exert improved cellular uptake efficiency in cancer cells after its delivery to the cancer region.

Synthetic aperture tomographic phase microscopy for 3D imaging of live cells in translational motion

PubMed Central

Lue, Niyom; Choi, Wonshik; Popescu, Gabriel; Badizadegan, Kamran; Dasari, Ramachandra R.; Feld, Michael S.

2009-01-01

We present a technique for 3D imaging of live cells in translational motion without need of axial scanning of objective lens. A set of transmitted electric field images of cells at successive points of transverse translation is taken with a focused beam illumination. Based on Hyugens’ principle, angular plane waves are synthesized from E-field images of a focused beam. For a set of synthesized angular plane waves, we apply a filtered back-projection algorithm and obtain 3D maps of refractive index of live cells. This technique, which we refer to as synthetic aperture tomographic phase microscopy, can potentially be combined with flow cytometry or microfluidic devices, and will enable high throughput acquisition of quantitative refractive index data from large numbers of cells. PMID:18825263

Diagnosis of Plasma Cell Dyscrasias and Monitoring of Minimal Residual Disease by Multiparametric Flow Cytometry

PubMed Central

Soh, Kah Teong; Tario, Joseph D.; Wallace, Paul K.

2018-01-01

Synopsis Plasma cell dyscrasia (PCD) is a heterogeneous disease which has seen a tremendous change in outcomes due to improved therapies. Over the last few decades, multiparametric flow cytometry has played an important role in the detection and monitoring of PCDs. Flow cytometry is a high sensitivity assay for early detection of minimal residual disease (MRD) that correlates well with progression-free survival and overall survival. Before flow cytometry can be effectively implemented in the clinical setting sample preparation, panel configuration, analysis, and gating strategies must be optimized to ensure accurate results. Current consensus methods and reporting guidelines for MRD testing are discussed. PMID:29128071

A cellular uptake and cytotoxicity properties study of gallic acid-loaded mesoporous silica nanoparticles on Caco-2 cells

NASA Astrophysics Data System (ADS)

Rashidi, Ladan; Vasheghani-Farahani, Ebrahim; Soleimani, Masoud; Atashi, Amir; Rostami, Khosrow; Gangi, Fariba; Fallahpour, Masoud; Tahouri, Mohammad Taher

2014-03-01

In this study, the effects of intracellular delivery of various concentrations of gallic acid (GA) as a semistable antioxidant, gallic acid-loaded mesoporous silica nanoparticles (MSNs-GA), and cellular uptake of nanoparticles into Caco-2 cells were investigated. MSNs were synthesized and loaded with GA, then characterized using transmission electron microscopy (TEM), scanning electron microscopy (SEM), Fourier transform infrared spectroscopy, N2 adsorption isotherms, X-ray diffraction, and thermal gravimetric analysis. The cytotoxicity of MSNs and MSNs-GA at low and high concentrations were studied by means of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) test and flow cytometry. MSNs did not show significant toxicity in various concentrations (0-500 μg/ml) on Caco-2 cells. For MSNs-GA, cell viability was reduced as a function of incubation time and different concentrations of nanoparticles. The in vitro GA release from MSNs-GA exhibited the same antitumor properties as free GA on Caco-2 cells. Flow cytometry results confirmed those obtained using MTT assay. TEM and fluorescent microscopy confirmed the internalization of MSNs by Caco-2 cells through nonspecific cellular uptake. MSNs can easily internalize into Caco-2 cells without deleterious effects on cell viability. The cell viability of Caco-2 cells was affected during MSNs-GA uptake. MSNs could be designed as suitable nanocarriers for antioxidants delivery.

Therapy of Prostate Cancer Using a Human Antibody Targeting the Type 1 Insulin-Like Growth Factor Receptor (IGF-IR)

DTIC Science & Technology

2009-09-01

euthanized, tumors harvested and portions processed for IHC, Western blot, flow cytometry , culture, and RNA analysis. If not enough tissue is available...temperature for 60 minutes. Samples were analyzed by flow cytometry using a BD FACScan. Data were analyzed with CellQuestPRO software. Evaluation of BrdUrd...were approved by the University of Washington Institutional Animal Care and Use Committee (IACUC). Flow cytometry . To measure tumor IGF-IR expression

Dose-Dependent Thresholds of 10-ns Electric Pulse Induced Plasma Membrane Disruption and Cytotoxicity in Multiple Cell Lines

DTIC Science & Technology

2011-01-01

normalized to parallel controls. Flow Cytometry and Confocal Microscopy Upon exposure to 10-ns EP, aliquots of the cellular suspension were added to a tube...Survival data was processed and plotted using GrapherH software (Golden Software, Golden, Colorado). Flow cytometry results were processed in C6 software...Accuri Cytometers, Inc., Ann Arbor, MI) and FCSExpress software (DeNovo Software, Los Angeles, CA). Final analysis and presentation of flow cytometry

Imaging Flow Cytometry Analysis to Identify Differences of Survival Motor Neuron Protein Expression in Patients With Spinal Muscular Atrophy.

PubMed

Arakawa, Reiko; Arakawa, Masayuki; Kaneko, Kaori; Otsuki, Noriko; Aoki, Ryoko; Saito, Kayoko

2016-08-01

Spinal muscular atrophy is a neurodegenerative disorder caused by the deficient expression of survival motor neuron protein in motor neurons. A major goal of disease-modifying therapy is to increase survival motor neuron expression. Changes in survival motor neuron protein expression can be monitored via peripheral blood cells in patients; therefore we tested the sensitivity and utility of imaging flow cytometry for this purpose. After the immortalization of peripheral blood lymphocytes from a human healthy control subject and two patients with spinal muscular atrophy type 1 with two and three copies of SMN2 gene, respectively, we used imaging flow cytometry analysis to identify significant differences in survival motor neuron expression. A bright detail intensity analysis was used to investigate differences in the cellular localization of survival motor neuron protein. Survival motor neuron expression was significantly decreased in cells derived from patients with spinal muscular atrophy relative to those derived from a healthy control subject. Moreover, survival motor neuron expression correlated with the clinical severity of spinal muscular atrophy according to SMN2 copy number. The cellular accumulation of survival motor neuron protein was also significantly decreased in cells derived from patients with spinal muscular atrophy relative to those derived from a healthy control subject. The benefits of imaging flow cytometry for peripheral blood analysis include its capacities for analyzing heterogeneous cell populations; visualizing cell morphology; and evaluating the accumulation, localization, and expression of a target protein. Imaging flow cytometry analysis should be implemented in future studies to optimize its application as a tool for spinal muscular atrophy clinical trials. Copyright © 2016 Elsevier Inc. All rights reserved.

Prognostic value of bone marrow involvement by clonal immunoglobulin gene rearrangements in follicular lymphoma

PubMed Central

Berget, Ellen; Helgeland, Lars; Liseth, Knut; Løkeland, Turid; Molven, Anders; Vintermyr, Olav Karsten

2014-01-01

Aims We aimed to evaluate the prognostic value of routine use of PCR amplification of immunoglobulin gene rearrangements in bone marrow (BM) staging in patients with follicular lymphoma (FL). Methods Clonal rearrangements were assessed by immunoglobulin heavy and light-chain gene rearrangement analysis in BM aspirates from 96 patients diagnosed with FL and related to morphological detection of BM involvement in biopsies. In 71 patients, results were also compared with concurrent flow cytometry analysis. Results BM involvement was detected by PCR in 34.4% (33/96) of patients. The presence of clonal rearrangements by PCR was associated with advanced clinical stage (I–III vs IV; p<0.001), high FL International Prognostic Index (FLIPI) score (0–1, 2 vs ≥3; p=0.003), and detection of BM involvement by morphology and flow cytometry analysis (p<0.001 for both). PCR-positive patients had a significantly poorer survival than PCR-negative patients (p=0.001, log-rank test). Thirteen patients positive by PCR but without morphologically detectable BM involvement, had significantly poorer survival than patients with negative morphology and negative PCR result (p=0.002). The poor survival associated with BM involvement by PCR was independent of the FLIPI score (p=0.007, Cox regression). BM involvement by morphology or flow cytometry did not show a significant impact on survival. Conclusions Our results showed that routine use of PCR-based clonality analysis significantly improved the prognostic impact of BM staging in patients with FL. BM involvement by PCR was also an independent adverse prognostic factor. PMID:25233852

Meta-analysis of two computer-assisted screening methods for diagnosing oral precancer and cancer.

PubMed

Ye, Xiaojing; Zhang, Jing; Tan, Yaqin; Chen, Guanying; Zhou, Gang

2015-11-01

The early diagnosis of oral precancer and cancer is crucial and could have the highest impact on improving survival rates. A meta-analysis was conducted to compare the accuracy between the OralCDx brush biopsy and DNA-image cytometry in diagnosing both conditions. Bibliographic databases were systematically searched for original relevant studies on the early diagnosis of oral precancer and oral cancer. Study characteristics were evaluated to determine the accuracy of the two screening strategies. Thirteen studies (eight of OralCDx brush biopsy and five of DNA-image cytometry) were identified as having reported on 1981 oral mucosa lesions. The meta-analysis found that the area under the summary receiver operating characteristic curves of the OralCDx brush biopsy and DNA-image cytometry were 0.8879 and 0.9885, respectively. The pooled sensitivity, specificity, and diagnostic odds ratio of the OralCDx brush biopsy were 86% (95% CI 81-90), 81% (95% CI 78-85), and 20.36 (95% CI 2.72-152.67), respectively, while these modalities of DNA-image cytometry were 89% (95% CI 83-94), 99% (95% CI 97-100), and 446.08 (95% CI 73.36-2712.43), respectively. Results of a pairwise comparison between each modality demonstrated that specificity, area under the curve (AUC), and Q(∗) index of DNA-image cytometry was significantly higher than that of the OralCDx brush biopsy (Z=2.821, p<0.05; Z=1.711, p<0.05; Z=1.727, p<0.05), but no significant difference in sensitivity was found (Z=1.520, p>0.05). In conclusion, the meta-analysis of the published studies indicated that DNA-image cytometry is more accurate than the OralCDx brush biopsy in diagnosing oral precancer and oral cancer. Copyright © 2015 Elsevier Ltd. All rights reserved.

Single-cell mass cytometry and transcriptome profiling reveal the impact of graphene on human immune cells.

PubMed

Orecchioni, Marco; Bedognetti, Davide; Newman, Leon; Fuoco, Claudia; Spada, Filomena; Hendrickx, Wouter; Marincola, Francesco M; Sgarrella, Francesco; Rodrigues, Artur Filipe; Ménard-Moyon, Cécilia; Cesareni, Gianni; Kostarelos, Kostas; Bianco, Alberto; Delogu, Lucia G

2017-10-24

Understanding the biomolecular interactions between graphene and human immune cells is a prerequisite for its utilization as a diagnostic or therapeutic tool. To characterize the complex interactions between graphene and immune cells, we propose an integrative analytical pipeline encompassing the evaluation of molecular and cellular parameters. Herein, we use single-cell mass cytometry to dissect the effects of graphene oxide (GO) and GO functionalized with amino groups (GONH 2 ) on 15 immune cell populations, interrogating 30 markers at the single-cell level. Next, the integration of single-cell mass cytometry with genome-wide transcriptome analysis shows that the amine groups reduce the perturbations caused by GO on cell metabolism and increase biocompatibility. Moreover, GONH 2 polarizes T-cell and monocyte activation toward a T helper-1/M1 immune response. This study describes an innovative approach for the analysis of the effects of nanomaterials on distinct immune cells, laying the foundation for the incorporation of single-cell mass cytometry on the experimental pipeline.

Magnetic fingerprints of rolling cells for quantitative flow cytometry in whole blood

NASA Astrophysics Data System (ADS)

Reisbeck, Mathias; Helou, Michael Johannes; Richter, Lukas; Kappes, Barbara; Friedrich, Oliver; Hayden, Oliver

2016-09-01

Over the past 50 years, flow cytometry has had a profound impact on preclinical and clinical applications requiring single cell function information for counting, sub-typing and quantification of epitope expression. At the same time, the workflow complexity and high costs of such optical systems still limit flow cytometry applications to specialized laboratories. Here, we present a quantitative magnetic flow cytometer that incorporates in situ magnetophoretic cell focusing for highly accurate and reproducible rolling of the cellular targets over giant magnetoresistance sensing elements. Time-of-flight analysis is used to unveil quantitative single cell information contained in its magnetic fingerprint. Furthermore, we used erythrocytes as a biological model to validate our methodology with respect to precise analysis of the hydrodynamic cell diameter, quantification of binding capacity of immunomagnetic labels, and discrimination of cell morphology. The extracted time-of-flight information should enable point-of-care quantitative flow cytometry in whole blood for clinical applications, such as immunology and primary hemostasis.

Identification of a small molecule yeast TORC1 inhibitor with a multiplex screen based on flow cytometry.

PubMed

Chen, Jun; Young, Susan M; Allen, Chris; Seeber, Andrew; Péli-Gulli, Marie-Pierre; Panchaud, Nicolas; Waller, Anna; Ursu, Oleg; Yao, Tuanli; Golden, Jennifer E; Strouse, J Jacob; Carter, Mark B; Kang, Huining; Bologa, Cristian G; Foutz, Terry D; Edwards, Bruce S; Peterson, Blake R; Aubé, Jeffrey; Werner-Washburne, Margaret; Loewith, Robbie J; De Virgilio, Claudio; Sklar, Larry A

2012-04-20

TOR (target of rapamycin) is a serine/threonine kinase, evolutionarily conserved from yeast to human, which functions as a fundamental controller of cell growth. The moderate clinical benefit of rapamycin in mTOR-based therapy of many cancers favors the development of new TOR inhibitors. Here we report a high-throughput flow cytometry multiplexed screen using five GFP-tagged yeast clones that represent the readouts of four branches of the TORC1 signaling pathway in budding yeast. Each GFP-tagged clone was differentially color-coded, and the GFP signal of each clone was measured simultaneously by flow cytometry, which allows rapid prioritization of compounds that likely act through direct modulation of TORC1 or proximal signaling components. A total of 255 compounds were confirmed in dose-response analysis to alter GFP expression in one or more clones. To validate the concept of the high-throughput screen, we have characterized CID 3528206, a small molecule most likely to act on TORC1 as it alters GFP expression in all five GFP clones in a manner analogous to that of rapamycin. We have shown that CID 3528206 inhibited yeast cell growth and that CID 3528206 inhibited TORC1 activity both in vitro and in vivo with EC(50)'s of 150 nM and 3.9 μM, respectively. The results of microarray analysis and yeast GFP collection screen further support the notion that CID 3528206 and rapamycin modulate similar cellular pathways. Together, these results indicate that the HTS has identified a potentially useful small molecule for further development of TOR inhibitors.

Time-gated flow cytometry: an ultra-high selectivity method to recover ultra-rare-event μ-targets in high-background biosamples

NASA Astrophysics Data System (ADS)

Jin, Dayong; Piper, James A.; Leif, Robert C.; Yang, Sean; Ferrari, Belinda C.; Yuan, Jingli; Wang, Guilan; Vallarino, Lidia M.; Williams, John W.

2009-03-01

A fundamental problem for rare-event cell analysis is auto-fluorescence from nontarget particles and cells. Time-gated flow cytometry is based on the temporal-domain discrimination of long-lifetime (>1 μs) luminescence-stained cells and can render invisible all nontarget cell and particles. We aim to further evaluate the technique, focusing on detection of ultra-rare-event 5-μm calibration beads in environmental water dirt samples. Europium-labeled 5-μm calibration beads with improved luminescence homogeneity and reduced aggregation were evaluated using the prototype UV LED excited time-gated luminescence (TGL) flow cytometer (FCM). A BD FACSAria flow cytometer was used to sort accurately a very low number of beads (<100 events), which were then spiked into concentrated samples of environmental water. The use of europium-labeled beads permitted the demonstration of specific detection rates of 100%+/-30% and 91%+/-3% with 10 and 100 target beads, respectively, that were mixed with over one million nontarget autofluorescent background particles. Under the same conditions, a conventional FCM was unable to recover rare-event fluorescein isothiocyanate (FITC) calibration beads. Preliminary results on Giardia detection are also reported. We have demonstrated the scientific value of lanthanide-complex biolabels in flow cytometry. This approach may augment the current method that uses multifluorescence-channel flow cytometry gating.

Monoclonal Antibody L1Mab-13 Detected Human PD-L1 in Lung Cancers.

PubMed

Yamada, Shinji; Itai, Shunsuke; Nakamura, Takuro; Yanaka, Miyuki; Chang, Yao-Wen; Suzuki, Hiroyoshi; Kaneko, Mika K; Kato, Yukinari

2018-04-01

Programmed cell death ligand-1 (PD-L1) is a type I transmembrane glycoprotein expressed on antigen-presenting cells. It is also expressed in several tumor cells such as melanoma and lung cancer cells. A strong correlation has been reported between human PD-L1 (hPD-L1) expression in tumor cells and negative prognosis in cancer patients. Here, a novel anti-hPD-L1 monoclonal antibody (mAb) L 1 Mab-13 (IgG 1 , kappa) was produced using a cell-based immunization and screening (CBIS) method. We investigated hPD-L1 expression in lung cancer using flow cytometry, Western blot, and immunohistochemical analyses. L 1 Mab-13 specifically reacted hPD-L1 of hPD-L1-overexpressed Chinese hamster ovary (CHO)-K1 cells and endogenous hPD-L1 of KMST-6 (human fibroblast) in flow cytometry and Western blot. Furthermore, L 1 Mab-13 reacted with lung cancer cell lines (EBC-1, Lu65, and Lu99) in flow cytometry and stained lung cancer tissues in a membrane-staining pattern in immunohistochemical analysis. These results indicate that a novel anti-hPD-L1 mAb, L 1 Mab-13, is very useful for detecting hPD-L1 of lung cancers in flow cytometry, Western blot, and immunohistochemical analyses.

Ultrasensitive automated RNA in situ hybridization for kappa and lambda light chain mRNA detects B-cell clonality in tissue biopsies with performance comparable or superior to flow cytometry.

PubMed

Guo, Ling; Wang, Zhen; Anderson, Courtney M; Doolittle, Emerald; Kernag, Siobhan; Cotta, Claudiu V; Ondrejka, Sarah L; Ma, Xiao-Jun; Cook, James R

2018-03-01

The assessment of B-cell clonality is a critical component of the evaluation of suspected lymphoproliferative disorders, but analysis from formalin-fixed, paraffin-embedded tissues can be challenging if fresh tissue is not available for flow cytometry. Immunohistochemical and conventional bright field in situ hybridization stains for kappa and lambda are effective for evaluation of plasma cells but are often insufficiently sensitive to detect the much lower abundance of light chains present in B-cells. We describe an ultrasensitive RNA in situ hybridization assay that has been adapted for use on an automated immunohistochemistry platform and compare results with flow cytometry in 203 consecutive tissues and 104 consecutive bone marrows. Overall, in 203 tissue biopsies, RNA in situ hybridization identified light chain-restricted B-cells in 85 (42%) vs 58 (29%) by flow cytometry. Within 83 B-cell non-Hodgkin lymphomas, RNA in situ hybridization identified restricted B-cells in 74 (89%) vs 56 (67%) by flow cytometry. B-cell clonality could be evaluated in only 23/104 (22%) bone marrow cases owing to poor RNA preservation, but evaluable cases showed 91% concordance with flow cytometry. RNA in situ hybridization allowed for recognition of biclonal/composite lymphomas not identified by flow cytometry and highlighted unexpected findings, such as coexpression of kappa and lambda RNA in 2 cases and the presence of lambda light chain RNA in a T lymphoblastic lymphoma. Automated RNA in situ hybridization showed excellent interobserver reproducibility for manual evaluation (average K=0.92), and an automated image analysis system showed high concordance (97%) with manual evaluation. Automated RNA in situ hybridization staining, which can be adopted on commonly utilized immunohistochemistry instruments, allows for the interpretation of clonality in the context of the morphological features in formalin-fixed, paraffin-embedded tissues with a clinical sensitivity similar or superior to flow cytometry.

Ultrasensitive Automated RNA in situ Hybridization for Kappa and Lambda Light Chain mRNA Detects B-cell Clonality in Tissue Biopsies with Performance Comparable or Superior to Flow Cytometry

PubMed Central

Guo, Ling; Wang, Zhen; Anderson, Courtney M.; Doolittle, Emerald; Kernag, Siobhan; Cotta, Claudiu V.; Ondrejka, Sarah L.; Ma, Xiao-Jun; Cook, James R.

2017-01-01

The assessment of B-cell clonality is a critical component of the evaluation of suspected lymphoproliferative disorders, but analysis from formalin fixed paraffin embedded tissues can be challenging if fresh tissue is not available for flow cytometry. Immunohistochemical and conventional bright field in situ hybridization stains for kappa and lambda are effective for evaluation of plasma cells, but are often insufficiently sensitive to detect the much lower abundance of light chains present in B cells. We describe an ultrasensitive RNA in situ hybridization assay which has been adapted for use on an automated immunohistochemistry platform and compare results with flow cytometry in 203 consecutive tissues and 104 consecutive bone marrows. Overall, in 203 tissue biopsies, RNA in situ hybridization identified light chain restricted B-cells in 85 (42%) vs. 58 (29%) by flow cytometry. Within 83 B-cell non-Hodgkin lymphomas, RNA in situ hybridization identified a restricted B-cells in 74 (89%) vs. 56 (67%) by flow cytometry. B-cell clonality could be evaluated in only 23/104 (22%) bone marrow cases due to poor RNA preservation, but evaluable cases showed 91% concordance with flow cytometry. RNA in situ hybridization allowed for recognition of biclonal/composite lymphomas not identified by flow cytometry, and highlighted unexpected findings, such as coexpression of kappa and lambda RNA in 2 cases and the presence of lambda light chain RNA in a T lymphoblastic lymphoma. Automated RNA in situ hybridization showed excellent interobserver reproducibility for manual evaluation (average K=0.92), and an automated image analysis system showed high concordance (97%) with manual evaluation. Automated RNA in situ hybridization staining, which can be adopted on commonly utilized immunohistochemistry instruments, allows for the interpretation of clonality in the context of the morphologic features in formalin fixed, paraffin embedded tissues with a clinical sensitivity similar or superior to flow cytometry. PMID:29052600

Cluster stability in the analysis of mass cytometry data.

PubMed

Melchiotti, Rossella; Gracio, Filipe; Kordasti, Shahram; Todd, Alan K; de Rinaldis, Emanuele

2017-01-01

Manual gating has been traditionally applied to cytometry data sets to identify cells based on protein expression. The advent of mass cytometry allows for a higher number of proteins to be simultaneously measured on cells, therefore providing a means to define cell clusters in a high dimensional expression space. This enhancement, whilst opening unprecedented opportunities for single cell-level analyses, makes the incremental replacement of manual gating with automated clustering a compelling need. To this aim many methods have been implemented and their successful applications demonstrated in different settings. However, the reproducibility of automatically generated clusters is proving challenging and an analytical framework to distinguish spurious clusters from more stable entities, and presumably more biologically relevant ones, is still missing. One way to estimate cell clusters' stability is the evaluation of their consistent re-occurrence within- and between-algorithms, a metric that is commonly used to evaluate results from gene expression. Herein we report the usage and importance of cluster stability evaluations, when applied to results generated from three popular clustering algorithms - SPADE, FLOCK and PhenoGraph - run on four different data sets. These algorithms were shown to generate clusters with various degrees of statistical stability, many of them being unstable. By comparing the results of automated clustering with manually gated populations, we illustrate how information on cluster stability can assist towards a more rigorous and informed interpretation of clustering results. We also explore the relationships between statistical stability and other properties such as clusters' compactness and isolation, demonstrating that whilst cluster stability is linked to other properties it cannot be reliably predicted by any of them. Our study proposes the introduction of cluster stability as a necessary checkpoint for cluster interpretation and contributes to the construction of a more systematic and standardized analytical framework for the assessment of cytometry clustering results. © 2016 International Society for Advancement of Cytometry. © 2016 International Society for Advancement of Cytometry.

Analysis of cellular autofluorescence in touch samples by flow cytometry: implications for front end separation of trace mixture evidence.

PubMed

Katherine Philpott, M; Stanciu, Cristina E; Kwon, Ye Jin; Bustamante, Eduardo E; Greenspoon, Susan A; Ehrhardt, Christopher J

2017-07-01

The goal of this study was to survey optical and biochemical variation in cell populations deposited onto a surface through touch or contact and identify specific features that may be used to distinguish and then sort cell populations from separate contributors in a trace biological mixture. Although we were not able to detect meaningful biochemical variation in touch samples deposited by different contributors through preliminary antibody surveys, we did observe distinct differences in red autofluorescence emissions (650-670 nm), with as much as a tenfold difference in mean fluorescence intensities observed between certain pairs of donors. Results indicate that the level of red autofluorescence in touch samples can be influenced by a donor's contact with specific material prior to handling the substrate from which cells were collected. In particular, we observed increased red autofluorescence in cells deposited subsequent to handling laboratory gloves, plant material, and certain types of marker ink, which could be easily visualized microscopically or using flow cytometry, and persisted after hand washing. To test whether these observed optical differences could potentially be used as the basis for a cell separation workflow, a controlled two-person touch mixture was separated into two fractions via fluorescence-activated cell sorting (FACS) using gating criteria based on intensity of 650-670 nm emissions and then subjected to DNA analysis. Genetic analysis of the sorted fractions provided partial DNA profiles that were consistent with separation of individual contributors from the mixture suggesting that variation in autofluorescence signatures, even if driven by extrinsic factors, may nonetheless be a useful means of isolating contributors to some touch mixtures. Graphical Abstract Conceptual workflow diagram. Trace biological mixtures containing cells from multiple individuals are analyzed by flow cytometry. Cells are then physically separated into two populations based on intensity of red autofluorescence using Fluorescence Activated Cell Sorting. Each isolated cell fraction is subjected to DNA analysis resulting in a DNA profile for each contributor.

Probing Tumor Microenvironment With In Vivo Phage Display

DTIC Science & Technology

2014-10-01

C). (C) Dot plots showing mCherry expression on the X axis and fibroblast activation protein ( FAP ) or rabbit isotype control staining in the Y...by flow cytometry-based cell sorting using an antibody against fibroblast activation protein ( FAP ). During the optimization steps, flow cytometry...expression of αvβ3 and αvβ5 integrins, neuropilin-1 (NRP-1), and fibroblast activation protein ( FAP ) in hb6011 CAFs was analyzed by flow cytometry

Flow Cytometry and Solid Organ Transplantation: A Perfect Match

PubMed Central

Maguire, Orla; Tario, Joseph D.; Shanahan, Thomas C.; Wallace, Paul K.; Minderman, Hans

2015-01-01

In the field of transplantation, flow cytometry serves a well-established role in pre-transplant crossmatching and monitoring immune reconstitution following hematopoietic stem cell transplantation. The capabilities of flow cytometers have continuously expanded and this combined with more detailed knowledge of the constituents of the immune system, their function and interaction and newly developed reagents to study these parameters have led to additional utility of flow cytometry-based analyses, particularly in the post-transplant setting. This review discusses the impact of flow cytometry on managing alloantigen reactions, monitoring opportunistic infections and graft rejection and gauging immunosuppression in the context of solid organ transplantation. PMID:25296232

Semi-quantitative estimation of cellular SiO2 nanoparticles using flow cytometry combined with X-ray fluorescence measurements.

PubMed

Choi, Seo Yeon; Yang, Nuri; Jeon, Soo Kyung; Yoon, Tae Hyun

2014-09-01

In this study, we have demonstrated feasibility of a semi-quantitative approach for the estimation of cellular SiO2 nanoparticles (NPs), which is based on the flow cytometry measurements of their normalized side scattering intensity. In order to improve our understanding on the quantitative aspects of cell-nanoparticle interactions, flow cytometry, transmission electron microscopy, and X-ray fluorescence experiments were carefully performed for the HeLa cells exposed to SiO2 NPs with different core diameters, hydrodynamic sizes, and surface charges. Based on the observed relationships among the experimental data, a semi-quantitative cellular SiO2 NPs estimation method from their normalized side scattering and core diameters was proposed, which can be applied for the determination of cellular SiO2 NPs within their size-dependent linear ranges. © 2014 International Society for Advancement of Cytometry.

Flow cytometry and conventional enumeration of microorganisms in ships' ballast water and marine samples.

PubMed

Joachimsthal, Eva L; Ivanov, Volodymyr; Tay, Joo-Hwa; Tay, Stephen T-L

2003-03-01

Conventional methods for bacteriological testing of water quality take long periods of time to complete. This makes them inappropriate for a shipping industry that is attempting to comply with the International Maritime Organization's anticipated regulations for ballast water discharge. Flow cytometry for the analysis of marine and ship's ballast water is a comparatively fast and accurate method. Compared to a 5% standard error for flow cytometry analysis the standard methods of culturing and epifluorescence analysis have errors of 2-58% and 10-30%, respectively. Also, unlike culturing methods, flow cytometry is capable of detecting both non-viable and viable but non-culturable microorganisms which can still pose health risks. The great variability in both cell concentrations and microbial content for the samples tested is an indication of the difficulties facing microbial monitoring programmes. The concentration of microorganisms in the ballast tank was generally lower than in local seawater. The proportion of aerobic, microaerophilic, and facultative anaerobic microorganisms present appeared to be influenced by conditions in the ballast tank. The gradual creation of anaerobic conditions in a ballast tank could lead to the accumulation of facultative anaerobic microorganisms, which might represent a potential source of pathogenic species.

The Influence of Shuttle-Shape Emodin Nanoparticles on the Streptococcus suis Biofilm.

PubMed

Ding, Wenya; Sun, Jin; Lian, He; Xu, Changgeng; Liu, Xin; Zheng, Sidi; Zhang, Dong; Han, Xiaopeng; Liu, Yanyan; Chen, Xueying; God Spower, Bello O; Li, Yanhua

2018-01-01

Biofilm is one of the most important physiological protective barriers of the Streptococcus suis ( S. suis ), and it is also one of the primary causes of hindrance to drug infiltration, reduction of bactericidal effects, and the development of antibiotic resistance. In order to intervene or eliminate S. suis biofilm, shuttle-shape emodin-loaded nanoparticles were developed in our study. The emodin nanoparticles were prepared by emodin and gelatin-cyclodextrin which was synthesized as drug carrier, and the nanoparticles were 174 nm in size, -4.64 mv in zeta potential, and exhibited a sustained emodin release. Moreover, the delivery kinetics of nanoparticles were also explored in our study. The confocal laser scanning microscopy and colony forming unit enumeration experiment indicated that nanoparticles could increase drug infiltration and uptake by biofilm. The flow cytometry system analysis showed that nanoparticles could be up taken by 99% of the bacteria cells. TCP assay and scanning electron microscopy showed that the nanoparticles had better effect on biofilm inhibition and elimination when compared with emodin solution. These results revealed that the emodin nanoparticles had a better therapeutic effect on the S. suis biofilm in vitro .

Optofluidic Fluorescent Imaging Cytometry on a Cell Phone

PubMed Central

Zhu, Hongying; Mavandadi, Sam; Coskun, Ahmet F.; Yaglidere, Oguzhan; Ozcan, Aydogan

2012-01-01

Fluorescent microscopy and flow cytometry are widely used tools in biomedical sciences. Cost-effective translation of these technologies to remote and resource-limited environments could create new opportunities especially for telemedicine applications. Toward this direction, here we demonstrate the integration of imaging cytometry and fluorescent microscopy on a cell phone using a compact, lightweight, and cost-effective optofluidic attachment. In this cell-phone-based optofluidic imaging cytometry platform, fluorescently labeled particles or cells of interest are continuously delivered to our imaging volume through a disposable microfluidic channel that is positioned above the existing camera unit of the cell phone. The same microfluidic device also acts as a multilayered optofluidic waveguide and efficiently guides our excitation light, which is butt-coupled from the side facets of our microfluidic channel using inexpensive light-emitting diodes. Since the excitation of the sample volume occurs through guided waves that propagate perpendicular to the detection path, our cell-phone camera can record fluorescent movies of the specimens as they are flowing through the microchannel. The digital frames of these fluorescent movies are then rapidly processed to quantify the count and the density of the labeled particles/cells within the target solution of interest. We tested the performance of our cell-phone-based imaging cytometer by measuring the density of white blood cells in human blood samples, which provided a decent match to a commercially available hematology analyzer. We further characterized the imaging quality of the same platform to demonstrate a spatial resolution of ~2 μm. This cell-phone-enabled optofluidic imaging flow cytometer could especially be useful for rapid and sensitive imaging of bodily fluids for conducting various cell counts (e.g., toward monitoring of HIV+ patients) or rare cell analysis as well as for screening of water quality in remote and resource-poor settings. PMID:21774454

Optofluidic fluorescent imaging cytometry on a cell phone.

PubMed

Zhu, Hongying; Mavandadi, Sam; Coskun, Ahmet F; Yaglidere, Oguzhan; Ozcan, Aydogan

2011-09-01

Fluorescent microscopy and flow cytometry are widely used tools in biomedical sciences. Cost-effective translation of these technologies to remote and resource-limited environments could create new opportunities especially for telemedicine applications. Toward this direction, here we demonstrate the integration of imaging cytometry and fluorescent microscopy on a cell phone using a compact, lightweight, and cost-effective optofluidic attachment. In this cell-phone-based optofluidic imaging cytometry platform, fluorescently labeled particles or cells of interest are continuously delivered to our imaging volume through a disposable microfluidic channel that is positioned above the existing camera unit of the cell phone. The same microfluidic device also acts as a multilayered optofluidic waveguide and efficiently guides our excitation light, which is butt-coupled from the side facets of our microfluidic channel using inexpensive light-emitting diodes. Since the excitation of the sample volume occurs through guided waves that propagate perpendicular to the detection path, our cell-phone camera can record fluorescent movies of the specimens as they are flowing through the microchannel. The digital frames of these fluorescent movies are then rapidly processed to quantify the count and the density of the labeled particles/cells within the target solution of interest. We tested the performance of our cell-phone-based imaging cytometer by measuring the density of white blood cells in human blood samples, which provided a decent match to a commercially available hematology analyzer. We further characterized the imaging quality of the same platform to demonstrate a spatial resolution of ~2 μm. This cell-phone-enabled optofluidic imaging flow cytometer could especially be useful for rapid and sensitive imaging of bodily fluids for conducting various cell counts (e.g., toward monitoring of HIV+ patients) or rare cell analysis as well as for screening of water quality in remote and resource-poor settings.

Three-dimensional DNA image cytometry by optical projection tomographic microscopy for early cancer diagnosis.

PubMed

Agarwal, Nitin; Biancardi, Alberto M; Patten, Florence W; Reeves, Anthony P; Seibel, Eric J

2014-04-01

Aneuploidy is typically assessed by flow cytometry (FCM) and image cytometry (ICM). We used optical projection tomographic microscopy (OPTM) for assessing cellular DNA content using absorption and fluorescence stains. OPTM combines some of the attributes of both FCM and ICM and generates isometric high-resolution three-dimensional (3-D) images of single cells. Although the depth of field of the microscope objective was in the submicron range, it was extended by scanning the objective's focal plane. The extended depth of field image is similar to a projection in a conventional x-ray computed tomography. These projections were later reconstructed using computed tomography methods to form a 3-D image. We also present an automated method for 3-D nuclear segmentation. Nuclei of chicken, trout, and triploid trout erythrocyte were used to calibrate OPTM. Ratios of integrated optical densities extracted from 50 images of each standard were compared to ratios of DNA indices from FCM. A comparison of mean square errors with thionin, hematoxylin, Feulgen, and SYTOX green was done. Feulgen technique was preferred as it showed highest stoichiometry, least variance, and preserved nuclear morphology in 3-D. The addition of this quantitative biomarker could further strengthen existing classifiers and improve early diagnosis of cancer using 3-D microscopy.

High-throughput microfluidic line scan imaging for cytological characterization

NASA Astrophysics Data System (ADS)

Hutcheson, Joshua A.; Powless, Amy J.; Majid, Aneeka A.; Claycomb, Adair; Fritsch, Ingrid; Balachandran, Kartik; Muldoon, Timothy J.

2015-03-01

Imaging cells in a microfluidic chamber with an area scan camera is difficult due to motion blur and data loss during frame readout causing discontinuity of data acquisition as cells move at relatively high speeds through the chamber. We have developed a method to continuously acquire high-resolution images of cells in motion through a microfluidics chamber using a high-speed line scan camera. The sensor acquires images in a line-by-line fashion in order to continuously image moving objects without motion blur. The optical setup comprises an epi-illuminated microscope with a 40X oil immersion, 1.4 NA objective and a 150 mm tube lens focused on a microfluidic channel. Samples containing suspended cells fluorescently stained with 0.01% (w/v) proflavine in saline are introduced into the microfluidics chamber via a syringe pump; illumination is provided by a blue LED (455 nm). Images were taken of samples at the focal plane using an ELiiXA+ 8k/4k monochrome line-scan camera at a line rate of up to 40 kHz. The system's line rate and fluid velocity are tightly controlled to reduce image distortion and are validated using fluorescent microspheres. Image acquisition was controlled via MATLAB's Image Acquisition toolbox. Data sets comprise discrete images of every detectable cell which may be subsequently mined for morphological statistics and definable features by a custom texture analysis algorithm. This high-throughput screening method, comparable to cell counting by flow cytometry, provided efficient examination including counting, classification, and differentiation of saliva, blood, and cultured human cancer cells.

Non-invasive determination of conjugative transfer of plasmids bearing antibiotic-resistance genes in biofilm-bound bacteria: effects of substrate loading and antibiotic selection

PubMed Central

Ma, Hongyan; Bryers, James D.

2012-01-01

Biofilms cause much of all human microbial infections. Attempts to eradicate biofilm-based infections rely on disinfectants and antibiotics. Unfortunately, biofilm bacteria are significantly less responsive to antibiotic stressors than their planktonic counterparts. Sublethal doses of antibiotics can actually enhance biofilm formation. Here, we have developed a non-invasive microscopic image analyses to quantify plasmid conjugation within a developing biofilm. Corroborating destructive samples were analyzed by a cultivation-independent flow cytometry analysis and a selective plate count method to cultivate transconjugants. Increases in substrate loading altered biofilm 3-D architecture and subsequently affected the frequency of plasmid conjugation (decreases at least two times) in the absence of any antibiotic selective pressure. More importantly, donor populations in biofilms exposed to a sublethal dose of kanamycin exhibited enhanced transfer efficiency of plasmids containing the kanamycin resistance gene, up to tenfold. However, when stressed with a different antibiotic, imipenem, transfer of plasmids containing the kanR+ gene was not enhanced. These preliminary results suggest biofilm bacteria “sense” antibiotics to which they are resistant, which enhances the spread of that resistance. Confocal scanning microscopy coupled with our non-invasive image analysis was able to estimate plasmid conjugative transfer efficiency either averaged over the entire biofilm landscape or locally with individual biofilm clusters. PMID:22669634

Subnuclear foci quantification using high-throughput 3D image cytometry

NASA Astrophysics Data System (ADS)

Wadduwage, Dushan N.; Parrish, Marcus; Choi, Heejin; Engelward, Bevin P.; Matsudaira, Paul; So, Peter T. C.

2015-07-01

Ionising radiation causes various types of DNA damages including double strand breaks (DSBs). DSBs are often recognized by DNA repair protein ATM which forms gamma-H2AX foci at the site of the DSBs that can be visualized using immunohistochemistry. However most of such experiments are of low throughput in terms of imaging and image analysis techniques. Most of the studies still use manual counting or classification. Hence they are limited to counting a low number of foci per cell (5 foci per nucleus) as the quantification process is extremely labour intensive. Therefore we have developed a high throughput instrumentation and computational pipeline specialized for gamma-H2AX foci quantification. A population of cells with highly clustered foci inside nuclei were imaged, in 3D with submicron resolution, using an in-house developed high throughput image cytometer. Imaging speeds as high as 800 cells/second in 3D were achieved by using HiLo wide-field depth resolved imaging and a remote z-scanning technique. Then the number of foci per cell nucleus were quantified using a 3D extended maxima transform based algorithm. Our results suggests that while most of the other 2D imaging and manual quantification studies can count only up to about 5 foci per nucleus our method is capable of counting more than 100. Moreover we show that 3D analysis is significantly superior compared to the 2D techniques.

Immunophenotype Discovery, Hierarchical Organization, and Template-Based Classification of Flow Cytometry Samples

DOE PAGES

Azad, Ariful; Rajwa, Bartek; Pothen, Alex

2016-08-31

We describe algorithms for discovering immunophenotypes from large collections of flow cytometry samples and using them to organize the samples into a hierarchy based on phenotypic similarity. The hierarchical organization is helpful for effective and robust cytometry data mining, including the creation of collections of cell populations’ characteristic of different classes of samples, robust classification, and anomaly detection. We summarize a set of samples belonging to a biological class or category with a statistically derived template for the class. Whereas individual samples are represented in terms of their cell populations (clusters), a template consists of generic meta-populations (a group ofmore » homogeneous cell populations obtained from the samples in a class) that describe key phenotypes shared among all those samples. We organize an FC data collection in a hierarchical data structure that supports the identification of immunophenotypes relevant to clinical diagnosis. A robust template-based classification scheme is also developed, but our primary focus is in the discovery of phenotypic signatures and inter-sample relationships in an FC data collection. This collective analysis approach is more efficient and robust since templates describe phenotypic signatures common to cell populations in several samples while ignoring noise and small sample-specific variations. We have applied the template-based scheme to analyze several datasets, including one representing a healthy immune system and one of acute myeloid leukemia (AML) samples. The last task is challenging due to the phenotypic heterogeneity of the several subtypes of AML. However, we identified thirteen immunophenotypes corresponding to subtypes of AML and were able to distinguish acute promyelocytic leukemia (APL) samples with the markers provided. Clinically, this is helpful since APL has a different treatment regimen from other subtypes of AML. Core algorithms used in our data analysis are available in the flowMatch package at www.bioconductor.org. It has been downloaded nearly 6,000 times since 2014.« less

Immunophenotype Discovery, Hierarchical Organization, and Template-Based Classification of Flow Cytometry Samples

DOE Office of Scientific and Technical Information (OSTI.GOV)

Azad, Ariful; Rajwa, Bartek; Pothen, Alex

We describe algorithms for discovering immunophenotypes from large collections of flow cytometry samples and using them to organize the samples into a hierarchy based on phenotypic similarity. The hierarchical organization is helpful for effective and robust cytometry data mining, including the creation of collections of cell populations’ characteristic of different classes of samples, robust classification, and anomaly detection. We summarize a set of samples belonging to a biological class or category with a statistically derived template for the class. Whereas individual samples are represented in terms of their cell populations (clusters), a template consists of generic meta-populations (a group ofmore » homogeneous cell populations obtained from the samples in a class) that describe key phenotypes shared among all those samples. We organize an FC data collection in a hierarchical data structure that supports the identification of immunophenotypes relevant to clinical diagnosis. A robust template-based classification scheme is also developed, but our primary focus is in the discovery of phenotypic signatures and inter-sample relationships in an FC data collection. This collective analysis approach is more efficient and robust since templates describe phenotypic signatures common to cell populations in several samples while ignoring noise and small sample-specific variations. We have applied the template-based scheme to analyze several datasets, including one representing a healthy immune system and one of acute myeloid leukemia (AML) samples. The last task is challenging due to the phenotypic heterogeneity of the several subtypes of AML. However, we identified thirteen immunophenotypes corresponding to subtypes of AML and were able to distinguish acute promyelocytic leukemia (APL) samples with the markers provided. Clinically, this is helpful since APL has a different treatment regimen from other subtypes of AML. Core algorithms used in our data analysis are available in the flowMatch package at www.bioconductor.org. It has been downloaded nearly 6,000 times since 2014.« less

Multi-parametric analysis of phagocyte antimicrobial responses using imaging flow cytometry.

PubMed

Havixbeck, Jeffrey J; Wong, Michael E; More Bayona, Juan A; Barreda, Daniel R

2015-08-01

We feature a multi-parametric approach based on an imaging flow cytometry platform for examining phagocyte antimicrobial responses against the gram-negative bacterium Aeromonas veronii. This pathogen is known to induce strong inflammatory responses across a broad range of animal species, including humans. We examined the contribution of A. veronii to the induction of early phagocyte inflammatory processes in RAW 264.7 murine macrophages in vitro. We found that A. veronii, both in live or heat-killed forms, induced similar levels of macrophage activation based on NF-κB translocation. Although these macrophages maintained high levels of viability following heat-killed or live challenges with A. veronii, we identified inhibition of macrophage proliferation as early as 1h post in vitro challenge. The characterization of phagocytic responses showed a time-dependent increase in phagocytosis upon A. veronii challenge, which was paired with a robust induction of intracellular respiratory burst responses. Interestingly, despite the overall increase in the production of reactive oxygen species (ROS) among RAW 264.7 macrophages, we found a significant reduction in the production of ROS among the macrophage subset that had bound A. veronii. Phagocytic uptake of the pathogen further decreased ROS production levels, even beyond those of unstimulated controls. Overall, this multi-parametric imaging flow cytometry-based approach allowed for segregation of unique phagocyte sub-populations and examination of their downstream antimicrobial responses, and should contribute to improved understanding of phagocyte responses against Aeromonas and other pathogens. Copyright © 2015 Elsevier B.V. All rights reserved.

Stochastic Individual-Based Modeling of Bacterial Growth and Division Using Flow Cytometry.

PubMed

García, Míriam R; Vázquez, José A; Teixeira, Isabel G; Alonso, Antonio A

2017-01-01

A realistic description of the variability in bacterial growth and division is critical to produce reliable predictions of safety risks along the food chain. Individual-based modeling of bacteria provides the theoretical framework to deal with this variability, but it requires information about the individual behavior of bacteria inside populations. In this work, we overcome this problem by estimating the individual behavior of bacteria from population statistics obtained with flow cytometry. For this objective, a stochastic individual-based modeling framework is defined based on standard assumptions during division and exponential growth. The unknown single-cell parameters required for running the individual-based modeling simulations, such as cell size growth rate, are estimated from the flow cytometry data. Instead of using directly the individual-based model, we make use of a modified Fokker-Plank equation. This only equation simulates the population statistics in function of the unknown single-cell parameters. We test the validity of the approach by modeling the growth and division of Pediococcus acidilactici within the exponential phase. Estimations reveal the statistics of cell growth and division using only data from flow cytometry at a given time. From the relationship between the mother and daughter volumes, we also predict that P. acidilactici divide into two successive parallel planes.

Flow cytometry in the post fluorescence era.

PubMed

Nolan, Garry P

2011-12-01

While flow cytometry once enabled researchers to examine 10--15 cell surface parameters, new mass flow cytometry technology enables interrogation of up to 45 parameters on a single cell. This new technology has increased understanding of cell expression and how cells differentiate during hematopoiesis. Using this information, knowledge of leukemia cell biology has also increased. Other new technologies, such as SPADE analysis and single cell network profiling (SCNP), are enabling researchers to put different cancers into more biologically similar categories and have the potential to enable more personalized medicine. Copyright © 2011. Published by Elsevier Ltd.

Small lasers in flow cytometry.

PubMed

Telford, William G

2004-01-01

Laser technology has made tremendous advances in recent years, particularly in the area of diode and diode-pumped solid state sources. Flow cytometry has been a direct beneficiary of these advances, as these small, low-maintenance, inexpensive lasers with reasonable power outputs are integrated into flow cytometers. In this chapter we review the contribution and potential of solid-state lasers to flow cytometry, and show several examples of these novel sources integrated into production flow cytometers. Technical details and critical parameters for successful application of these lasers for biomedical analysis are reviewed.

Examination pf Potential Anti-Tumor Activity of N-Thiolated B-Lactam Antibiotics in Nude Mice Bearing Human Breast Tumors

DTIC Science & Technology

2005-08-01

temperature in the dark, and then analyzed by flow cytometry within 3 hr of staining. 2.7. Caspase-3/-7 activity assay To measure cell-free caspase-3/-7...were treated with 50 mM of lactam 12 for the indicated hours. (A) Measurement of sub-G1 DNA content by flow cytometry analysis. The percentage of sub...Daniel for critical reading of the manuscript. We also appreciate the assistance of the Flow Cytometry Core at H. Lee Moffitt Cancer Center

Augmenting Trastuzumab Therapy against Breast Cancer through Selective Activation of NK Cells

DTIC Science & Technology

2014-12-01

purity as defined by CD3-CD56+ flow cytometry ) and activation (>50% expression of CD137). Breast cancer cell lines including MCF7 (A and E...purity as defined by CD3-CD56+ flow cytometry ) and activation (>50% expression of CD137). Chromium-labeled breast cancer cell lines including MCF7 (A...and Whiteside, T.L. 2007. A novel multiparametric flow cytometry -based cytotoxicity assay simultaneously immunophenotypes effector cells: comparisons

Managing Multi-center Flow Cytometry Data for Immune Monitoring

PubMed Central

White, Scott; Laske, Karoline; Welters, Marij JP; Bidmon, Nicole; van der Burg, Sjoerd H; Britten, Cedrik M; Enzor, Jennifer; Staats, Janet; Weinhold, Kent J; Gouttefangeas, Cécile; Chan, Cliburn

2014-01-01

With the recent results of promising cancer vaccines and immunotherapy1–5, immune monitoring has become increasingly relevant for measuring treatment-induced effects on T cells, and an essential tool for shedding light on the mechanisms responsible for a successful treatment. Flow cytometry is the canonical multi-parameter assay for the fine characterization of single cells in solution, and is ubiquitously used in pre-clinical tumor immunology and in cancer immunotherapy trials. Current state-of-the-art polychromatic flow cytometry involves multi-step, multi-reagent assays followed by sample acquisition on sophisticated instruments capable of capturing up to 20 parameters per cell at a rate of tens of thousands of cells per second. Given the complexity of flow cytometry assays, reproducibility is a major concern, especially for multi-center studies. A promising approach for improving reproducibility is the use of automated analysis borrowing from statistics, machine learning and information visualization21–23, as these methods directly address the subjectivity, operator-dependence, labor-intensive and low fidelity of manual analysis. However, it is quite time-consuming to investigate and test new automated analysis techniques on large data sets without some centralized information management system. For large-scale automated analysis to be practical, the presence of consistent and high-quality data linked to the raw FCS files is indispensable. In particular, the use of machine-readable standard vocabularies to characterize channel metadata is essential when constructing analytic pipelines to avoid errors in processing, analysis and interpretation of results. For automation, this high-quality metadata needs to be programmatically accessible, implying the need for a consistent Application Programming Interface (API). In this manuscript, we propose that upfront time spent normalizing flow cytometry data to conform to carefully designed data models enables automated analysis, potentially saving time in the long run. The ReFlow informatics framework was developed to address these data management challenges. PMID:26085786

Data Standards for Flow Cytometry

PubMed Central

SPIDLEN, JOSEF; GENTLEMAN, ROBERT C.; HAALAND, PERRY D.; LANGILLE, MORGAN; MEUR, NOLWENN LE; OCHS, MICHAEL F.; SCHMITT, CHARLES; SMITH, CLAYTON A.; TREISTER, ADAM S.; BRINKMAN, RYAN R.

2009-01-01

Flow cytometry (FCM) is an analytical tool widely used for cancer and HIV/AIDS research, and treatment, stem cell manipulation and detecting microorganisms in environmental samples. Current data standards do not capture the full scope of FCM experiments and there is a demand for software tools that can assist in the exploration and analysis of large FCM datasets. We are implementing a standardized approach to capturing, analyzing, and disseminating FCM data that will facilitate both more complex analyses and analysis of datasets that could not previously be efficiently studied. Initial work has focused on developing a community-based guideline for recording and reporting the details of FCM experiments. Open source software tools that implement this standard are being created, with an emphasis on facilitating reproducible and extensible data analyses. As well, tools for electronic collaboration will assist the integrated access and comprehension of experiments to empower users to collaborate on FCM analyses. This coordinated, joint development of bioinformatics standards and software tools for FCM data analysis has the potential to greatly facilitate both basic and clinical research—impacting a notably diverse range of medical and environmental research areas. PMID:16901228

Assessing FRET using spectral techniques.

PubMed

Leavesley, Silas J; Britain, Andrea L; Cichon, Lauren K; Nikolaev, Viacheslav O; Rich, Thomas C

2013-10-01

Förster resonance energy transfer (FRET) techniques have proven invaluable for probing the complex nature of protein-protein interactions, protein folding, and intracellular signaling events. These techniques have traditionally been implemented with the use of one or more fluorescence band-pass filters, either as fluorescence microscopy filter cubes, or as dichroic mirrors and band-pass filters in flow cytometry. In addition, new approaches for measuring FRET, such as fluorescence lifetime and acceptor photobleaching, have been developed. Hyperspectral techniques for imaging and flow cytometry have also shown to be promising for performing FRET measurements. In this study, we have compared traditional (filter-based) FRET approaches to three spectral-based approaches: the ratio of acceptor-to-donor peak emission, linear spectral unmixing, and linear spectral unmixing with a correction for direct acceptor excitation. All methods are estimates of FRET efficiency, except for one-filter set and three-filter set FRET indices, which are included for consistency with prior literature. In the first part of this study, spectrofluorimetric data were collected from a CFP-Epac-YFP FRET probe that has been used for intracellular cAMP measurements. All comparisons were performed using the same spectrofluorimetric datasets as input data, to provide a relevant comparison. Linear spectral unmixing resulted in measurements with the lowest coefficient of variation (0.10) as well as accurate fits using the Hill equation. FRET efficiency methods produced coefficients of variation of less than 0.20, while FRET indices produced coefficients of variation greater than 8.00. These results demonstrate that spectral FRET measurements provide improved response over standard, filter-based measurements. Using spectral approaches, single-cell measurements were conducted through hyperspectral confocal microscopy, linear unmixing, and cell segmentation with quantitative image analysis. Results from these studies confirmed that spectral imaging is effective for measuring subcellular, time-dependent FRET dynamics and that additional fluorescent signals can be readily separated from FRET signals, enabling multilabel studies of molecular interactions. © 2013 International Society for Advancement of Cytometry. Copyright © 2013 International Society for Advancement of Cytometry.

Quantification of telomere length by FISH and laser scanning cytometry

NASA Astrophysics Data System (ADS)

Mahoney, John E.; Sahin, Ergun; Jaskelioff, Mariela; Chin, Lynda; DePinho, Ronald A.; Protopopov, Alexei I.

2008-02-01

Telomeres play a critical role in the maintenance of chromosomal stability. Telomere erosion, coupled with loss of DNA damage checkpoint function, results in genomic instability that promotes the development of cancer. The critical role of telomere dynamics in cancer has motivated the development of technologies designed to monitor telomere reserves in a highly quantitative and high-throughput manner in humans and model organisms. To this end, we have adapted and modified two established technologies, telomere-FISH and laser scanning cytometry. Specifically, we have produced a number of enhancements to the iCys LSC (CompuCyte) package including software updates, use of 60X dry objectives, and increased spatial resolution by 0.2 um size of stage steps. In addition, the 633 nm HeNe laser was replaced with a 532 nm green diode laser to better match the viewing options. Utilization of telomere-deficient mouse cells with short dysfunctional telomeres and matched telomerase reconstituted cultures demonstrated significantly higher mean integral specific fluorescence values for mTR transfectants relative to empty vector controls: 4.485M vs. 1.362M (p<0.0001). Histograms of average telomere intensities for individual cells were obtained and demonstrated intercellular heterogeneity in telomere lengths. The validation of the approach derives from a strong correlation between iCys LSC values and Southern blotting. This validated method greatly increases our experimental throughput and objectivity.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vasdekis, Andreas E.; Stephanopoulos, Gregory

The sampling and manipulation of cells down to the individual has been of substantial interest since the very beginning of Life Sciences. Herein, our objective is to highlight the most recent developments in single cell manipulation, as well as pioneering ones. First, flow-through methods will be discussed, namely methods in which the single cells flow continuously in an ordered manner during their analysis. This section will be followed by confinement techniques that enable cell isolation and confinement in one, two- or three-dimensions. Flow cytometry and droplet microfluidics are the two most common methods of flow-through analysis. While both are high-throughputmore » techniques, their difference lays in the fact that the droplet encapsulated cells experience a restricted and personal microenvironment, while in flow cytometry cells experience similar nutrient and stimuli initial concentrations. These methods are rather well established; however, they recently enabled immense strides in single cell phenotypic analysis, namely the identification and analysis of metabolically distinct individuals from an isogenic population using both droplet microfluidics and flow cytometry.« less

Fever of unknown origin (FUO): CMV infectious mononucleosis or lymphoma?

PubMed

Cunha, Burke A; Chawla, Karishma

2018-07-01

Fever of unknown origin (FUO) refers to fevers of > 101 °F that persist for > 3 weeks and remain undiagnosed after a focused inpatient or outpatient workup. FUO may be due to infectious, malignant/neoplastic, rheumatic/inflammatory, or miscellaneous disorders. The FUO category determines the focus of the diagnostic workup. In the case presented of an FUO in a young woman, there were clinical findings of both CMV infectious mononucleosis or a lymphoma, e.g., highly elevated ESR, elevated ferritin levels, and elevated ACE level, β-2 microglobulins. The indium scan showed intense splenic uptake. Lymph node biopsy, PET scan, and flow cytometry were negative for lymphoma. CMV infectious mononucleosis was the diagnosis, and she made a slow recovery.

CyTOF workflow: differential discovery in high-throughput high-dimensional cytometry datasets

PubMed Central

Nowicka, Malgorzata; Krieg, Carsten; Weber, Lukas M.; Hartmann, Felix J.; Guglietta, Silvia; Becher, Burkhard; Levesque, Mitchell P.; Robinson, Mark D.

2017-01-01

High dimensional mass and flow cytometry (HDCyto) experiments have become a method of choice for high throughput interrogation and characterization of cell populations.Here, we present an R-based pipeline for differential analyses of HDCyto data, largely based on Bioconductor packages. We computationally define cell populations using FlowSOM clustering, and facilitate an optional but reproducible strategy for manual merging of algorithm-generated clusters. Our workflow offers different analysis paths, including association of cell type abundance with a phenotype or changes in signaling markers within specific subpopulations, or differential analyses of aggregated signals. Importantly, the differential analyses we show are based on regression frameworks where the HDCyto data is the response; thus, we are able to model arbitrary experimental designs, such as those with batch effects, paired designs and so on. In particular, we apply generalized linear mixed models to analyses of cell population abundance or cell-population-specific analyses of signaling markers, allowing overdispersion in cell count or aggregated signals across samples to be appropriately modeled. To support the formal statistical analyses, we encourage exploratory data analysis at every step, including quality control (e.g. multi-dimensional scaling plots), reporting of clustering results (dimensionality reduction, heatmaps with dendrograms) and differential analyses (e.g. plots of aggregated signals). PMID:28663787

Novel nuclei isolation buffer for flow cytometric genome size estimation of Zingiberaceae: a comparison with common isolation buffers

PubMed Central

Sadhu, Abhishek; Bhadra, Sreetama; Bandyopadhyay, Maumita

2016-01-01

Background and Aims Cytological parameters such as chromosome numbers and genome sizes of plants are used routinely for studying evolutionary aspects of polyploid plants. Members of Zingiberaceae show a wide range of inter- and intrageneric variation in their reproductive habits and ploidy levels. Conventional cytological study in this group of plants is severely hampered by the presence of diverse secondary metabolites, which also affect their genome size estimation using flow cytometry. None of the several nuclei isolation buffers used in flow cytometry could be used very successfully for members of Zingiberaceae to isolate good quality nuclei from both shoot and root tissues. Methods The competency of eight nuclei isolation buffers was compared with a newly formulated buffer, MB01, in six different genera of Zingiberaceae based on the fluorescence intensity of propidium iodide-stained nuclei using flow cytometric parameters, namely coefficient of variation of the G0/G1 peak, debris factor and nuclei yield factor. Isolated nuclei were studied using fluorescence microscopy and bio-scanning electron microscopy to analyse stain–nuclei interaction and nuclei topology, respectively. Genome contents of 21 species belonging to these six genera were determined using MB01. Key Results Flow cytometric parameters showed significant differences among the analysed buffers. MB01 exhibited the best combination of analysed parameters; photomicrographs obtained from fluorescence and electron microscopy supported the superiority of MB01 buffer over other buffers. Among the 21 species studied, nuclear DNA contents of 14 species are reported for the first time. Conclusions Results of the present study substantiate the enhanced efficacy of MB01, compared to other buffers tested, in the generation of acceptable cytograms from all species of Zingiberaceae studied. Our study facilitates new ways of sample preparation for further flow cytometric analysis of genome size of other members belonging to this highly complex polyploid family. PMID:27594649

Novel nuclei isolation buffer for flow cytometric genome size estimation of Zingiberaceae: a comparison with common isolation buffers.

PubMed

Sadhu, Abhishek; Bhadra, Sreetama; Bandyopadhyay, Maumita

2016-11-01

Cytological parameters such as chromosome numbers and genome sizes of plants are used routinely for studying evolutionary aspects of polyploid plants. Members of Zingiberaceae show a wide range of inter- and intrageneric variation in their reproductive habits and ploidy levels. Conventional cytological study in this group of plants is severely hampered by the presence of diverse secondary metabolites, which also affect their genome size estimation using flow cytometry. None of the several nuclei isolation buffers used in flow cytometry could be used very successfully for members of Zingiberaceae to isolate good quality nuclei from both shoot and root tissues. The competency of eight nuclei isolation buffers was compared with a newly formulated buffer, MB01, in six different genera of Zingiberaceae based on the fluorescence intensity of propidium iodide-stained nuclei using flow cytometric parameters, namely coefficient of variation of the G 0 /G 1 peak, debris factor and nuclei yield factor. Isolated nuclei were studied using fluorescence microscopy and bio-scanning electron microscopy to analyse stain-nuclei interaction and nuclei topology, respectively. Genome contents of 21 species belonging to these six genera were determined using MB01. Flow cytometric parameters showed significant differences among the analysed buffers. MB01 exhibited the best combination of analysed parameters; photomicrographs obtained from fluorescence and electron microscopy supported the superiority of MB01 buffer over other buffers. Among the 21 species studied, nuclear DNA contents of 14 species are reported for the first time. Results of the present study substantiate the enhanced efficacy of MB01, compared to other buffers tested, in the generation of acceptable cytograms from all species of Zingiberaceae studied. Our study facilitates new ways of sample preparation for further flow cytometric analysis of genome size of other members belonging to this highly complex polyploid family. © The Author 2016. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

50 years LASERS: in vitro diagnostics, clinical applications and perspectives.

PubMed

Spyropoulos, Basile

2011-01-01

1960 Theodore Maiman built the first Ruby-LASER, starting-point for half a century of R&D on Biomedical LASER continuous improvement. The purpose of this paper is to contribute a review of the often disregarded, however, extremely important Industrial Property documents of LASER-based in vitro Diagnostics devices. It is an attempt to sketch-out the patent-trail leading towards the modern Biomedical Laboratory and to offer an introduction to the employment of "exotic" systems, such as the Free Electron LASER (FEL), that are expected to focus on the fundamental processes of life, following chemical reactions and biological processes as they happen, on unprecedented time and size scales. There are various in vitro LASER applications, however, the most important ones include: Hybrid Coulter Principle-LASER Hematology Analyzers. Flow Cytometry systems. Fluorescent in situ Hybridization (FISH Techniques). Confocal LASER Scanning Microscopy and Cytometry. From the first fluorescence-based flow Cytometry device developed in 1968 by Wolfgang Göhde until nowadays, numerous improvements and new features related to these devices appeared. The relevant industrial property milestone-documents and their overall numeral trends are presented. In 1971, J. Madey invented and developed the Free Electron LASER (FEL), a vacuum-tube that uses a beam of relativistic electrons passing through a periodic, transverse magnetic field (wiggler) to produce coherent radiation, contained in an optical cavity defined by mirrors. A resonance condition that involves the energy of the electron beam, the strength of the magnetic field, and the periodicity of the magnet determines the wavelength of the radiation. The FEL Coherent Light Sources like the Linac Coherent Light Source (LCLS) at Stanford, CA, USA or the Xray Free Electron LASER (XFEL) at Hamburg, Germany, will work much like a high-speed (< 100 femtoseconds) camera, enabling scientists to take stop-motion pictures, on the nanoscale, of atoms and molecules in motion. The curve of FEL-related patents of the last 20 years is much smoother than the corresponding one for in vitro Diagnostics conventional LASERS. If the diodes brought a LASER into almost everyone's pocket, the above-mentioned super-imaging systems are huge facilities of enormous cost--the price to steal a look at the fundamental processes of life.

Quantitative analysis of gold and carbon nanoparticles in mammalian cells by flow cytometry light scattering

NASA Astrophysics Data System (ADS)

Zhou, Gang; Liu, Naicheng; Wang, Zhenheng; Shi, Tongguo; Gan, Jingjing; Wang, Zhenzhen; Zhang, Junfeng

2017-02-01

Nanoparticle-based applications for diagnostics and therapeutics have been extensively studied. These applications require a profound understanding of the fate of nanoparticles (NPs) in cellular environments. However, until now, few analytical methods are available and most of them rely on fluorescent properties or special elements of NPs; therefore, for NPs without observable optical properties or special elements, the existing methods are hardly applicable. In this study, we introduce a flow cytometry light scattering (FCLS)-based approach that quantifies in situ NPs accurately in mammalian cells. Continuous cells of heterogeneous human epithelial colorectal adenocarcinoma (Caco-2 cells), mouse peritoneal macrophages (MPM), and human adenocarcinomic alveolar basal epithelia (A549 cells) were cultured with NPs with certain concentrations and size. The intensity of the flow cytometric side scattered light, which indicates the quantity of NPs in the cells, was analyzed. The result shows an accurate size- and dose-dependent uptake of Au NPs (5, 30, 250 nm) in Caco-2 cells. The size- and dose- dependence of Au NPs (5, 30, 250 nm) and carbon NPs (50, 500 nm) in cells was validated by transmission electron microscope (TEM). This paper demonstrates the great potential of flow cytometry light scattering in the quantitative study of the size and dose effect on in situ metallic or non-metallic NPs in mammalian cells.

Interaction study between synthetic glycoconjugate ligands and endocytic receptors using flow cytometry.

PubMed

Yura, Hirofumi; Ishihara, Masayuki; Kanatani, Yasuhiro; Takase, Bonpei; Hattori, Hidemi; Suzuki, Shinya; Kawakami, Mitsuyuki; Matsui, Takemi

2006-04-01

Flow cytometric analysis of synthetic galactosyl polymers, asialofetuin and LDL derivatives labeled with FITC (Fluorescein Isothiocyanate) was carried out to determine the phenotypes of endocytic receptors, such as asialoglycoprotein (ASPG) and the LDL receptor, on various types of cells. When FITC-labeled galactosyl polystyrene (GalCPS), being a synthetic ligand of ASPG, was applied to rat hepatocytes and human cancer cells (Hep G2 and Chang Liver), surface fluorescence intensities varied according to receptor expression on the cells. The fluorescence intensity originates from the calcium-dependent binding of the FITC-labeled GalCPS. Although unaltered by pre-treatment with glucosyl polystyrene (GluCPS), fetuin and LDL, the fluorescence intensity was suppressed by pre-treatment with (non-labeled) GalCPS and asialofetuin. Flow cytometry allowed us to demonstrate that the calcium-dependent binding of FITC-labeled LDL (prepared from rabbits) upon the addition of 17alpha-ethinyl estradiol enhances LDL receptor expression, and the expression is suppressed upon the addition of a monoclonal antibody to the LDL receptor. The binding efficiency based on the combination of FITC-labeled ligands suggests a possible application for the classification of cell types and conditions corresponding to endocytic receptor expression without the need for immuno-active antibodies or radiolabeled substances. Furthermore, the synthetic glycoconjugate (GalCPS) is shown to be a sensitive and useful marker for classification based on cell phenotype using flow cytometry.

Label-free high-throughput imaging flow cytometry

NASA Astrophysics Data System (ADS)

Mahjoubfar, A.; Chen, C.; Niazi, K. R.; Rabizadeh, S.; Jalali, B.

2014-03-01

Flow cytometry is an optical method for studying cells based on their individual physical and chemical characteristics. It is widely used in clinical diagnosis, medical research, and biotechnology for analysis of blood cells and other cells in suspension. Conventional flow cytometers aim a laser beam at a stream of cells and measure the elastic scattering of light at forward and side angles. They also perform single-point measurements of fluorescent emissions from labeled cells. However, many reagents used in cell labeling reduce cellular viability or change the behavior of the target cells through the activation of undesired cellular processes or inhibition of normal cellular activity. Therefore, labeled cells are not completely representative of their unaltered form nor are they fully reliable for downstream studies. To remove the requirement of cell labeling in flow cytometry, while still meeting the classification sensitivity and specificity goals, measurement of additional biophysical parameters is essential. Here, we introduce an interferometric imaging flow cytometer based on the world's fastest continuous-time camera. Our system simultaneously measures cellular size, scattering, and protein concentration as supplementary biophysical parameters for label-free cell classification. It exploits the wide bandwidth of ultrafast laser pulses to perform blur-free quantitative phase and intensity imaging at flow speeds as high as 10 meters per second and achieves nanometer-scale optical path length resolution for precise measurements of cellular protein concentration.

Articular Cartilage Repair Through Muscle Cell-Based Tissue Engineering

DTIC Science & Technology

2010-03-01

results suggest that sFlt-1 has more of an enhancing effect in vivo. With cell markers and flow cytometry , investiga- tors at our laboratory have...were analyzed by flow cytometry . They were immunostained by desmin, vimentin and MyoD and their chondrogenic potential was evaluated under the...M1, M2, and M3) and 3 F-MDSC populations (F1, F2, and F3) were characterized by flow cytometry for CD34 and Sca1 expression. MDSCs were labeled with

Annual Progress Report FY-92. Volume 1

DTIC Science & Technology

1993-01-21

Billups, L Flow Cytom Resh Psychologist 12 0180 CS Hamm, C DCI 7 DESCRIPTION GRADE MOS BRANCH NAME ACTIVITY Kyle Metabolic Unit Nursing Service Supv...3349 Salata, Kalman PhD. Mitogen-Inducible T Suppressor Cell 13 Assay by Flow Cytometry (12/89) 3350 Salata, Kalman PhD. Flow Cytometric Analysis of...17 Immunotherapy (3/90) 3354 Salata, Kalman PhD. Two Way Mixed Lymphocyte Culture: 18 Analysis by Two Color Flow Cytometry (4/90) 3355 Salata, Kalman

78 FR 5186 - Clinical Flow Cytometry in Hematologic Malignancies; Public Workshop; Request for Comments

Federal Register 2010, 2011, 2012, 2013, 2014

2013-01-24

... in the diagnosis of leukemia and lymphoma and more recently in the detection of minimal residual... chronic lymphocytic leukemia (CLL); (3) Third-party flow cytometry data analysis software; and (4... held February 27, 2013 (77 FR 76051, December 26, 2012). An FDA workshop for acute lymphocytic leukemia...

Applications of flow cytometry to toxicological mycotoxin effects in cultured mammalian cells: a review.

PubMed

Juan-García, Ana; Manyes, Lara; Ruiz, María-José; Font, Guillermina

2013-06-01

This review gives an overview of flow cytometry applications to toxicological studies of several physiological target sites of mycotoxins on different mammalian cell lines. Mycotoxins are secondary metabolites of fungi that may be present in food, feed, air and water. The increasing presence of mycotoxins in crops, their wide distribution in the food chain, and their potential for toxicity demonstrate the need for further knowledge. Flow cytometry has become a valuable tool in mycotoxin studies in recent years for the rapid analysis of single cells in a mixture. In toxicology, the power of these methods lies in the possibility of determining a wide range of cell parameters, providing valuable information to elucidate cell growth and viability, metabolic activity, mitochondrial membrane potential and membrane integrity mechanisms. There are studies using flow cytometry technique on Alternaria, Aspergillus, Fusarium and Penicillium mycotoxins including information about cell type, assay conditions and functional parameters. Most of the studies collected in the literature are on deoxynivalenol and zearalenone mycotoxins. Cell cycle analysis and apoptosis are the processes more widely investigated. Copyright © 2013 Elsevier Ltd. All rights reserved.

Hyaluronic acid-coated liposomes for targeted delivery of paclitaxel, in-vitro characterization and in-vivo evaluation.

PubMed

Ravar, Fatemeh; Saadat, Ebrahim; Gholami, Mehdi; Dehghankelishadi, Pouya; Mahdavi, Mehdi; Azami, Samira; Dorkoosh, Farid A

2016-05-10

Breast cancer is the leading cause of cancer death in women. Chemotherapy is regarded as the most essential strategy in inhibiting the proliferation of tumor cells. Paclitaxel is a widely used taxane; however, the side effects of available Cremophor-based formulations and also the limitations of passive targeting uncovered an essential need to develop tumor-specific targeted nanocarriers. A hyaluronic acid targeted liposomal formulation of paclitaxel was prepared in which, hyaluronic acid was electrostatistically attracted to the surface of liposomes. Liposomes, had a particle size of 106.4±3.2nm, a weakly negative zeta potential of -9.7±0.8mV and an acceptable encapsulation efficiency of 92.1±1.7%. The release profile of liposomes in buffer showed that 95% of PTX was released during 40h. Confocal laser scanning microscopy and flow cytometry analysis showed the greater cellular internalization of coumarin-loaded liposomes compared to free coumarin. MTT assay on 4T1 and T47D cells demonstrated the stronger cytotoxic activity of liposomes in comparison to free paclitaxel. Cell cycle analysis showed that cells were mainly blocked at G2/M phases after 48h treatment with liposomes. In vivo real time imaging on 4T1 tumor-bearing mice revealed that the liposomal formulation mainly accumulated in the tumor area. Liposomes also had better antitumor efficacy against Cremophor-based formulation. In conclusion, hyaluronic acid targeted paclitaxel liposome can serve as a promising targeted formulation of paclitaxel for future cancer chemotherapy. Copyright © 2016 Elsevier B.V. All rights reserved.

[Effects of ezrin silencing on pancreatic cancer cell line Panc-1].

PubMed

Meng, Yun-xiao; Yu, Shuang-ni; Lu, Zhao-hui; Chen, Jie

2012-12-01

To explore the effects of ezrin silencing on pancreatic cancer cell line Panc-1. Pancreatic cancer cell line Panc-1 was transfected with ezrin silencing plasmid. The proliferation and the cell cycle status were determined by CCK-8 assay and flow cytometry analysis, respectively. Cellular membrane protrusions/microvilli formation were visualized by scanning election microscopy. Colony formation assay was used to determine the cell anchor-independent growth ability in vitro. Trans-filter migration and invasion assays were performed with 8 µm pore inserts in a 24-well BioCoat chamber with/without Matrigel. Ezrin silencing decreased cellular protrusions/microvilli formation, anchorage-independent growth, cell migration and invasion, but had no effects on cell proliferation in vitro and cell cycle, in pancreatic cancer cell line Panc-1. Ezrin expression affects the cellular protrusions/microvilli formation, anchorage-independent growth, cell migration and invasion in pancreatic cancer cell line Panc-1.

The detailed analysis of the changes of murine dendritic cells (DCs) induced by thymic peptide

PubMed Central

Hu, Xiaofang; Zheng, Wei; Wang, Lu; Wan, Nan; Wang, Bing; Li, Weiwei; Hua, Hui; Hu, Xu; Shan, Fengping

2012-01-01

The aim of present research is to analyze the detailed changes of dendritic cells (DCs) induced by pidotimod(PTD). These impacts on DCs of both bone marrow derived DCs and established DC2.4 cell line were assessed with use of conventional scanning electron microscopy (SEM), flow cytometry (FCM), transmission electron microscopy (TEM), cytochemistry assay FITC-dextran, bio-assay and enzyme linked immunosorbent assay (ELISA). We demonstrated the ability of PTD to induce DC phynotypic and functional maturation as evidenced by higher expression of key surface molecules such as MHC II, CD80 and CD86. The functional tests proved the downregulation of ACP inside the DCs, occurred when phagocytosis of DCs decreased, with simultaneously antigen presentation increased toward maturation. Finally, PTD also stimulated production of more cytokine IL-12 and less TNF-α. Therefore it is concluded that PTD can markedly exert positive induction to murine DCs. PMID:22863756

Metal induced changes in trivalent chromium resistant Alcaligenes faecalis VITSIM2.

PubMed

Matilda, Shiny C; Shanthi, Chittibabu

2017-05-01

The changes induced in bacterial strains under stress conditions provide an insight into metal resistance strategies. Trivalent chromium resistant bacterium were isolated and identified by 16S rRNA gene sequencing and designated as Alcaligenes faecalis VITSIM2. The growth pattern was monitored. The organism also showed resistance to copper, cadmium, and certain antibiotics. The differentially expressed proteins in SDS PAGE were identified by mass spectrometry as flagellin and 50S ribosomal L36 protein. The morphological changes were identified by scanning electron microscopy. The changes in the cell wall content were estimated by peptidoglycan analysis and transformation of phosphates was detected by 31 P NMR. Flow cytometry was employed to measure the membrane integrity, esterase activity and intracellular pH. In conclusion spectrum of proteomic, physiological, and morphological alterations was observed that aid the organism to overcome chromium stress. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Development of an unbiased, semi-automated approach for classifying plasma cell immunophenotype following multicolor flow cytometry of bone marrow aspirates.

PubMed

Post, Steven R; Post, Ginell R; Nikolic, Dejan; Owens, Rebecca; Insuasti-Beltran, Giovanni

2018-03-24

Despite increased usage of multiparameter flow cytometry (MFC) to assess diagnosis, prognosis, and therapeutic efficacy (minimal residual disease, MRD) in plasma cell neoplasms (PCNs), standardization of methodology and data analysis is suboptimal. We investigated the utility of using the mean and median fluorescence intensities (FI) obtained from MFC to objectively describe parameters that distinguish plasma cell (PC) phenotypes. In this retrospective study, flow cytometry results from bone marrow aspirate specimens from 570 patients referred to the Myeloma Institute at UAMS were evaluated. Mean and median FI data were obtained from 8-color MFC of non-neoplastic, malignant, and mixed PC populations using antibodies to CD38, CD138, CD19, CD20, CD27, CD45, CD56, and CD81. Of 570 cases, 252 cases showed only non-neoplastic PCs, 168 showed only malignant PCs, and 150 showed mixed PC populations. Statistical analysis of median FI data for each CD marker showed no difference in expression intensity on non-neoplastic and malignant PCs, between pure and mixed PC populations. ROC analysis of the median FI of CD expression in non-neoplastic and malignant PCs was used to develop an algorithm to convert quantitative FI values to qualitative assessments including "negative," "positive," "dim," and "heterogeneous" expression. FI data derived from 8-color MFC can be used to define marker expression on PCs. Translation of FI data from Infinicyt software to an Excel worksheet streamlines workflow and eliminates transcriptional errors when generating flow reports. © 2018 International Clinical Cytometry Society. © 2018 International Clinical Cytometry Society.

SCANS (Shipping Cask ANalysis System) a microcomputer-based analysis system for shipping cask design review: User`s manual to Version 3a. Volume 1, Revision 2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mok, G.C.; Thomas, G.R.; Gerhard, M.A.

SCANS (Shipping Cask ANalysis System) is a microcomputer-based system of computer programs and databases developed at the Lawrence Livermore National Laboratory (LLNL) for evaluating safety analysis reports on spent fuel shipping casks. SCANS is an easy-to-use system that calculates the global response to impact loads, pressure loads and thermal conditions, providing reviewers with an independent check on analyses submitted by licensees. SCANS is based on microcomputers compatible with the IBM-PC family of computers. The system is composed of a series of menus, input programs, cask analysis programs, and output display programs. All data is entered through fill-in-the-blank input screens thatmore » contain descriptive data requests. Analysis options are based on regulatory cases described in the Code of Federal Regulations 10 CFR 71 and Regulatory Guides published by the US Nuclear Regulatory Commission in 1977 and 1978.« less

Flow cytometry immunophenotyping in integrated diagnostics of patients with newly diagnosed cytopenia: one tube 10-color 14-antibody screening panel and 3-tube extensive panel for detection of MDS-related features.

PubMed

Porwit, A; Rajab, A

2015-05-01

Acute leukemia, myelodysplastic syndromes (MDS), myeloproliferative neoplasms and lymphomas are the most prevalent diagnoses in adults presenting with new onset cytopenia. Here, we describe two 10-color panels of surface markers (screening and comprehensive panel) applied at the Flow Cytometry Laboratory, University Health Network, Toronto, ON, Canada. A 10-color flow cytometry is applied using the stain-lyse-wash sample preparation method. In patients with <10% blasts and no clear involvement by hematological malignancy based on cytomorphological evaluation of bone marrow (BM) smear, the recently published one-tube 10-color 14-antibody screening panel is applied. This panel allows detection of major B- and T-cell abnormalities, enumeration of cells in blast region (CD45 dim), and gives insight into myeloid BM compartment, including calculation of four-parameter score for MDS-related abnormalities. In patients who present with ≥10 - <20% blasts in blood or BM smears, a comprehensive three-tube panel of surface markers is used up front. The analysis is focused on the detection of abnormal antigen expression patterns not seen in normal/reactive BM, according to the guidelines developed by International/European LeukemiaNet Working Group for Flow Cytometry in MDS. In patients with ≥20% blasts, an additional tube is added to allow the detection of cytoplasmic markers necessary to diagnose mixed phenotype acute leukemia. © 2015 John Wiley & Sons Ltd.

Drug testing and flow cytometry analysis on a large number of uniform sized tumor spheroids using a microfluidic device

NASA Astrophysics Data System (ADS)

Patra, Bishnubrata; Peng, Chien-Chung; Liao, Wei-Hao; Lee, Chau-Hwang; Tung, Yi-Chung

2016-02-01

Three-dimensional (3D) tumor spheroid possesses great potential as an in vitro model to improve predictive capacity for pre-clinical drug testing. In this paper, we combine advantages of flow cytometry and microfluidics to perform drug testing and analysis on a large number (5000) of uniform sized tumor spheroids. The spheroids are formed, cultured, and treated with drugs inside a microfluidic device. The spheroids can then be harvested from the device without tedious operation. Due to the ample cell numbers, the spheroids can be dissociated into single cells for flow cytometry analysis. Flow cytometry provides statistical information in single cell resolution that makes it feasible to better investigate drug functions on the cells in more in vivo-like 3D formation. In the experiments, human hepatocellular carcinoma cells (HepG2) are exploited to form tumor spheroids within the microfluidic device, and three anti-cancer drugs: Cisplatin, Resveratrol, and Tirapazamine (TPZ), and their combinations are tested on the tumor spheroids with two different sizes. The experimental results suggest the cell culture format (2D monolayer vs. 3D spheroid) and spheroid size play critical roles in drug responses, and also demonstrate the advantages of bridging the two techniques in pharmaceutical drug screening applications.

Application of a Short Intracellular pH Method to Flow Cytometry for Determining Saccharomyces cerevisiae Vitality ▿

PubMed Central

Weigert, Claudia; Steffler, Fabian; Kurz, Tomas; Shellhammer, Thomas H.; Methner, Frank-Jürgen

2009-01-01

The measurement of yeast's intracellular pH (ICP) is a proven method for determining yeast vitality. Vitality describes the condition or health of viable cells as opposed to viability, which defines living versus dead cells. In contrast to fluorescence photometric measurements, which show only average ICP values of a population, flow cytometry allows the presentation of an ICP distribution. By examining six repeated propagations with three separate growth phases (lag, exponential, and stationary), the ICP method previously established for photometry was transferred successfully to flow cytometry by using the pH-dependent fluorescent probe 5,6-carboxyfluorescein. The correlation between the two methods was good (r2 = 0.898, n = 18). With both methods it is possible to track the course of growth phases. Although photometry did not yield significant differences between exponentially and stationary phases (P = 0.433), ICP via flow cytometry did (P = 0.012). Yeast in an exponential phase has a unimodal ICP distribution, reflective of a homogeneous population; however, yeast in a stationary phase displays a broader ICP distribution, and subpopulations could be defined by using the flow cytometry method. In conclusion, flow cytometry yielded specific evidence of the heterogeneity in vitality of a yeast population as measured via ICP. In contrast to photometry, flow cytometry increases information about the yeast population's vitality via a short measurement, which is suitable for routine analysis. PMID:19581482

Cytometry metadata in XML

NASA Astrophysics Data System (ADS)

Leif, Robert C.; Leif, Stephanie H.

2016-04-01

Introduction: The International Society for Advancement of Cytometry (ISAC) has created a standard for the Minimum Information about a Flow Cytometry Experiment (MIFlowCyt 1.0). CytometryML will serve as a common metadata standard for flow and image cytometry (digital microscopy). Methods: The MIFlowCyt data-types were created, as is the rest of CytometryML, in the XML Schema Definition Language (XSD1.1). The datatypes are primarily based on the Flow Cytometry and the Digital Imaging and Communication (DICOM) standards. A small section of the code was formatted with standard HTML formatting elements (p, h1, h2, etc.). Results:1) The part of MIFlowCyt that describes the Experimental Overview including the specimen and substantial parts of several other major elements has been implemented as CytometryML XML schemas (www.cytometryml.org). 2) The feasibility of using MIFlowCyt to provide the combination of an overview, table of contents, and/or an index of a scientific paper or a report has been demonstrated. Previously, a sample electronic publication, EPUB, was created that could contain both MIFlowCyt metadata as well as the binary data. Conclusions: The use of CytometryML technology together with XHTML5 and CSS permits the metadata to be directly formatted and together with the binary data to be stored in an EPUB container. This will facilitate: formatting, data- mining, presentation, data verification, and inclusion in structured research, clinical, and regulatory documents, as well as demonstrate a publication's adherence to the MIFlowCyt standard, promote interoperability and should also result in the textual and numeric data being published using web technology without any change in composition.

Spaceflight Flow Cytometry: Design Challenges and Applications

NASA Technical Reports Server (NTRS)

Pappas, Dimitri; Kao, Shih-Hsin; Jeevarajan, Antony S.

2004-01-01

Future space exploration missions will require analytical technology capable of providing both autonomous medical care to the crew and investigative capabilities to researchers. While several promising candidate technologies exist for further development, flow cytometry is an attractive technology as it offers both crew health and a wide array of biochemistry and immunology assays. While flow cytometry has been widely used for cellular analysis in both clinical and research settings, the requirements for proper operation in spaceflight impose constraints on any instrument designs. The challenges of designing a spaceflight-ready flow cytometer are discussed, as well as some preliminary results using a prototype system.

An open-source solution for advanced imaging flow cytometry data analysis using machine learning.

PubMed

Hennig, Holger; Rees, Paul; Blasi, Thomas; Kamentsky, Lee; Hung, Jane; Dao, David; Carpenter, Anne E; Filby, Andrew

2017-01-01

Imaging flow cytometry (IFC) enables the high throughput collection of morphological and spatial information from hundreds of thousands of single cells. This high content, information rich image data can in theory resolve important biological differences among complex, often heterogeneous biological samples. However, data analysis is often performed in a highly manual and subjective manner using very limited image analysis techniques in combination with conventional flow cytometry gating strategies. This approach is not scalable to the hundreds of available image-based features per cell and thus makes use of only a fraction of the spatial and morphometric information. As a result, the quality, reproducibility and rigour of results are limited by the skill, experience and ingenuity of the data analyst. Here, we describe a pipeline using open-source software that leverages the rich information in digital imagery using machine learning algorithms. Compensated and corrected raw image files (.rif) data files from an imaging flow cytometer (the proprietary .cif file format) are imported into the open-source software CellProfiler, where an image processing pipeline identifies cells and subcellular compartments allowing hundreds of morphological features to be measured. This high-dimensional data can then be analysed using cutting-edge machine learning and clustering approaches using "user-friendly" platforms such as CellProfiler Analyst. Researchers can train an automated cell classifier to recognize different cell types, cell cycle phases, drug treatment/control conditions, etc., using supervised machine learning. This workflow should enable the scientific community to leverage the full analytical power of IFC-derived data sets. It will help to reveal otherwise unappreciated populations of cells based on features that may be hidden to the human eye that include subtle measured differences in label free detection channels such as bright-field and dark-field imagery. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Quantitative evaluation of Candia antarctica lipase B displayed on the cell surface of a Pichia pastoris based on an FS anchor system.

PubMed

Liang, Xing-xiang; Wang, Bei-bei; Sun, Yu-fei; Lin, Ying; Han, Shuang-yan; Zheng, Sui-ping; Cui, Tang-bing

2013-03-01

A new approach is described to quantify the number of enzyme molecules, such as Candia antarctica lipase B, that are displayed on the cell surface of Pichia pastoris. Enhanced green fluorescent protein (EGFP) and Candida antarctica lipase B (CALB) were fused and displayed on the surface of P. pastoris by linking to the anchor flocculation functional domain of FLO1p from Saccharomyces cerevisiae. Confocal laser scanning microscopy, flow cytometry, and fluorescence spectrophotometry were used to monitor the fluorescence intensity of fused EGFP. Combined with the corresponding protein concentration detected in the medium, a standard curve describing the relationship between the fusion protein concentration and fluorescence intensity were obtained and could be used to number CALB displayed on the cell surface. The results showed that approx. 10(4) molecules of CALB molecules were immobilized on the single P. pastoris cell wall based on FS anchor system.

Immunological Tools: Engaging Students in the Use and Analysis of Flow Cytometry and Enzyme-linked Immunosorbent Assay (ELISA)

ERIC Educational Resources Information Center

Ott, Laura E.; Carson, Susan

2014-01-01

Flow cytometry and enzyme-linked immunosorbent assay (ELISA) are commonly used techniques associated with clinical and research applications within the immunology and medical fields. The use of these techniques is becoming increasingly valuable in many life science and engineering disciplines as well. Herein, we report the development and…

A benchmark for evaluation of algorithms for identification of cellular correlates of clinical outcomes.

PubMed

Aghaeepour, Nima; Chattopadhyay, Pratip; Chikina, Maria; Dhaene, Tom; Van Gassen, Sofie; Kursa, Miron; Lambrecht, Bart N; Malek, Mehrnoush; McLachlan, G J; Qian, Yu; Qiu, Peng; Saeys, Yvan; Stanton, Rick; Tong, Dong; Vens, Celine; Walkowiak, Sławomir; Wang, Kui; Finak, Greg; Gottardo, Raphael; Mosmann, Tim; Nolan, Garry P; Scheuermann, Richard H; Brinkman, Ryan R

2016-01-01

The Flow Cytometry: Critical Assessment of Population Identification Methods (FlowCAP) challenges were established to compare the performance of computational methods for identifying cell populations in multidimensional flow cytometry data. Here we report the results of FlowCAP-IV where algorithms from seven different research groups predicted the time to progression to AIDS among a cohort of 384 HIV+ subjects, using antigen-stimulated peripheral blood mononuclear cell (PBMC) samples analyzed with a 14-color staining panel. Two approaches (FlowReMi.1 and flowDensity-flowType-RchyOptimyx) provided statistically significant predictive value in the blinded test set. Manual validation of submitted results indicated that unbiased analysis of single cell phenotypes could reveal unexpected cell types that correlated with outcomes of interest in high dimensional flow cytometry datasets. © 2015 International Society for Advancement of Cytometry.

Line-Focused Optical Excitation of Parallel Acoustic Focused Sample Streams for High Volumetric and Analytical Rate Flow Cytometry.

PubMed

Kalb, Daniel M; Fencl, Frank A; Woods, Travis A; Swanson, August; Maestas, Gian C; Juárez, Jaime J; Edwards, Bruce S; Shreve, Andrew P; Graves, Steven W

2017-09-19

Flow cytometry provides highly sensitive multiparameter analysis of cells and particles but has been largely limited to the use of a single focused sample stream. This limits the analytical rate to ∼50K particles/s and the volumetric rate to ∼250 μL/min. Despite the analytical prowess of flow cytometry, there are applications where these rates are insufficient, such as rare cell analysis in high cellular backgrounds (e.g., circulating tumor cells and fetal cells in maternal blood), detection of cells/particles in large dilute samples (e.g., water quality, urine analysis), or high-throughput screening applications. Here we report a highly parallel acoustic flow cytometer that uses an acoustic standing wave to focus particles into 16 parallel analysis points across a 2.3 mm wide optical flow cell. A line-focused laser and wide-field collection optics are used to excite and collect the fluorescence emission of these parallel streams onto a high-speed camera for analysis. With this instrument format and fluorescent microsphere standards, we obtain analysis rates of 100K/s and flow rates of 10 mL/min, while maintaining optical performance comparable to that of a commercial flow cytometer. The results with our initial prototype instrument demonstrate that the integration of key parallelizable components, including the line-focused laser, particle focusing using multinode acoustic standing waves, and a spatially arrayed detector, can increase analytical and volumetric throughputs by orders of magnitude in a compact, simple, and cost-effective platform. Such instruments will be of great value to applications in need of high-throughput yet sensitive flow cytometry analysis.

Elucidation of the critical epitope of an anti-EGFR monoclonal antibody EMab-134.

PubMed

Kaneko, Mika K; Yamada, Shinji; Itai, Shunsuke; Chang, Yao-Wen; Nakamura, Takuro; Yanaka, Miyuki; Kato, Yukinari

2018-07-01

The epidermal growth factor receptor (EGFR) is a type-1 transmembrane receptor tyrosine kinase, which activates the downstream signaling cascades in many tumors, such as oral and lung cancers. We previously developed EMab-134, a novel anti-EGFR monoclonal antibody (mAb), which reacts with endogenous EGFR-expressing cancer cell lines and normal cells independent of glycosylation in Western blotting, flow cytometry, and immunohistochemical analysis. EMab-134 showed very high sensitivity (94.7%) to oral squamous cell carcinomas in immunohistochemical analysis. In this study, we performed enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunohistochemical analysis to determine the epitope of EMab-134. A blocking peptide (375-394 amino acids of EGFR) neutralized the EMab-134 reaction against oral cancer cells in flow cytometry and immunohistochemistry. The minimum epitope of EMab-134 was found to be the 377- RGDSFTHTPP -386 sequence. Our findings can be applied for the production of more functional anti-EGFR mAbs that in turn can be used for antitumor treatments.

Determination of critical epitope of PcMab-47 against human podocalyxin.

PubMed

Itai, Shunsuke; Yamada, Shinji; Kaneko, Mika K; Kato, Yukinari

2018-07-01

Podocalyxin (PODXL) is a type I transmembrane protein, which is highly glycosylated. PODXL is expressed in some types of human cancer tissues including oral, breast, and lung cancer tissues and may promote tumor growth, invasion, and metastasis. We previously produced PcMab-47, a novel anti-PODXL monoclonal antibody (mAb) which reacts with endogenous PODXL-expressing cancer cell lines and normal cells independently of glycosylation in Western blot, flow cytometry, and immunohistochemical analysis. In this study, we used enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunohistochemical analysis to determine the epitope of PcMab-47. The minimum epitope of PcMab-47 was found to be Asp207, His208, Leu209, and Met210. A blocking peptide containing this minimum epitope completely neutralized PcMab-47 reaction against oral cancer cells by flow cytometry and immunohistochemical analysis. These findings could lead to the production of more functional anti-PODXL mAbs, which are advantageous for antitumor activities.

Liquid Crystal Droplet-Based Amplification of Microvesicles that are Shed by Mammalian Cells

PubMed Central

Tan, Lie Na; Wiepz, Gregory J.; Miller, Daniel S.; Shusta, Eric V.; Abbott, Nicholas L.

2014-01-01

Membrane-derived microvesicles (MVs) shed by cells are being investigated for their role in intercellular communication and as potential biomarkers of disease, but facile and sensitive methods for their analysis do not exist. Here we demonstrate new principles for analysis of MVs that use micrometer-sized droplets of liquid crystals (LCs) to amplify MVs that are selectively captured via antibody-mediated interactions. The influence of the MVs on the micrometer-sized LC droplets is shown to be readily quantified via use of flow cytometry. The methodology was developed using MVs shed by epidermoid carcinoma A431 cells that contain epidermal growth factor receptor (EGFR) as an important and representative example of MVs containing signaling proteins that play a central role in cancer. The LC droplets were found to be sensitive to 106 MVs containing EGFR (relative to controls using isotype control antibody) and to possess a dynamic range of response across several orders of magnitude. Because the 100 nm-sized MVs captured via EGFR generate an optical response in the micrometer-sized LC droplets that can be readily detected by flow cytometry in light scattering mode, the approach possesses significant advantages over direct detection of MVs by flow cytometry. The LC droplets are also substantially more sensitive than techniques such as immunoblotting because the lipid-component of the MVs serves to amplify the antibody-mediated capture of the target proteins in the MVs. Other merits of the approach are defined and discussed in the paper. PMID:24667742

Microfluidic sorting and multimodal typing of cancer cells in self-assembled magnetic arrays.

PubMed

Saliba, Antoine-Emmanuel; Saias, Laure; Psychari, Eleni; Minc, Nicolas; Simon, Damien; Bidard, François-Clément; Mathiot, Claire; Pierga, Jean-Yves; Fraisier, Vincent; Salamero, Jean; Saada, Véronique; Farace, Françoise; Vielh, Philippe; Malaquin, Laurent; Viovy, Jean-Louis

2010-08-17

We propose a unique method for cell sorting, "Ephesia," using columns of biofunctionalized superparamagnetic beads self-assembled in a microfluidic channel onto an array of magnetic traps prepared by microcontact printing. It combines the advantages of microfluidic cell sorting, notably the application of a well controlled, flow-activated interaction between cells and beads, and those of immunomagnetic sorting, notably the use of batch-prepared, well characterized antibody-bearing beads. On cell lines mixtures, we demonstrated a capture yield better than 94%, and the possibility to cultivate in situ the captured cells. A second series of experiments involved clinical samples--blood, pleural effusion, and fine needle aspirates--issued from healthy donors and patients with B-cell hematological malignant tumors (leukemia and lymphoma). The immunophenotype and morphology of B-lymphocytes were analyzed directly in the microfluidic chamber, and compared with conventional flow cytometry and visual cytology data, in a blind test. Immunophenotyping results using Ephesia were fully consistent with those obtained by flow cytometry. We obtained in situ high resolution confocal three-dimensional images of the cell nuclei, showing intranuclear details consistent with conventional cytological staining. Ephesia thus provides a powerful approach to cell capture and typing allowing fully automated high resolution and quantitative immunophenotyping and morphological analysis. It requires at least 10 times smaller sample volume and cell numbers than cytometry, potentially increasing the range of indications and the success rate of microbiopsy-based diagnosis, and reducing analysis time and cost.

Novel full-spectral flow cytometry with multiple spectrally-adjacent fluorescent proteins and fluorochromes and visualization of in vivo cellular movement.

PubMed

Futamura, Koji; Sekino, Masashi; Hata, Akihiro; Ikebuchi, Ryoyo; Nakanishi, Yasutaka; Egawa, Gyohei; Kabashima, Kenji; Watanabe, Takeshi; Furuki, Motohiro; Tomura, Michio

2015-09-01

Flow cytometric analysis with multicolor fluoroprobes is an essential method for detecting biological signatures of cells. Here, we present a new full-spectral flow cytometer (spectral-FCM). Unlike conventional flow cytometer, this spectral-FCM acquires the emitted fluorescence for all probes across the full-spectrum from each cell with 32 channels sequential PMT unit after dispersion with prism, and extracts the signals of each fluoroprobe based on the spectral shape of each fluoroprobe using unique algorithm in high speed, high sensitive, accurate, automatic and real-time. The spectral-FCM detects the continuous changes in emission spectra from green to red of the photoconvertible protein, KikGR with high-spectral resolution and separates spectrally-adjacent fluoroprobes, such as FITC (Emission peak (Em) 519 nm) and EGFP (Em 507 nm). Moreover, the spectral-FCM can measure and subtract autofluorescence of each cell providing increased signal-to-noise ratios and improved resolution of dim samples, which leads to a transformative technology for investigation of single cell state and function. These advances make it possible to perform 11-color fluorescence analysis to visualize movement of multilinage immune cells by using KikGR-expressing mice. Thus, the novel spectral flow cytometry improves the combinational use of spectrally-adjacent various FPs and multicolor fluorochromes in metabolically active cell for the investigation of not only the immune system but also other research and clinical fields of use. © 2015 International Society for Advancement of Cytometry.

Microfluidic sorting and multimodal typing of cancer cells in self-assembled magnetic arrays

PubMed Central

Saliba, Antoine-Emmanuel; Saias, Laure; Psychari, Eleni; Minc, Nicolas; Simon, Damien; Bidard, François-Clément; Mathiot, Claire; Pierga, Jean-Yves; Fraisier, Vincent; Salamero, Jean; Saada, Véronique; Farace, Françoise; Vielh, Philippe; Malaquin, Laurent; Viovy, Jean-Louis

2010-01-01

We propose a unique method for cell sorting, “Ephesia,” using columns of biofunctionalized superparamagnetic beads self-assembled in a microfluidic channel onto an array of magnetic traps prepared by microcontact printing. It combines the advantages of microfluidic cell sorting, notably the application of a well controlled, flow-activated interaction between cells and beads, and those of immunomagnetic sorting, notably the use of batch-prepared, well characterized antibody-bearing beads. On cell lines mixtures, we demonstrated a capture yield better than 94%, and the possibility to cultivate in situ the captured cells. A second series of experiments involved clinical samples—blood, pleural effusion, and fine needle aspirates— issued from healthy donors and patients with B-cell hematological malignant tumors (leukemia and lymphoma). The immunophenotype and morphology of B-lymphocytes were analyzed directly in the microfluidic chamber, and compared with conventional flow cytometry and visual cytology data, in a blind test. Immunophenotyping results using Ephesia were fully consistent with those obtained by flow cytometry. We obtained in situ high resolution confocal three-dimensional images of the cell nuclei, showing intranuclear details consistent with conventional cytological staining. Ephesia thus provides a powerful approach to cell capture and typing allowing fully automated high resolution and quantitative immunophenotyping and morphological analysis. It requires at least 10 times smaller sample volume and cell numbers than cytometry, potentially increasing the range of indications and the success rate of microbiopsy-based diagnosis, and reducing analysis time and cost. PMID:20679245

Apigenin induced apoptosis in esophageal carcinoma cells by destruction membrane structures.

PubMed

Zhu, Haiyan; Jin, Hua; Pi, Jiang; Bai, Haihua; Yang, Fen; Wu, Chaomin; Jiang, Jinhuan; Cai, Jiye

2016-07-01

Apigenin has shown to have killing effects on some kinds of solid tumor cells. However, the changes in cell membrane induced by apigenin on subcellular- or nanometer-level were still unclear. In this work, human esophageal cancer cells (EC9706 and KYSE150 cells) were employed as cell model to detect the cytotoxicity of apigenin, including cell growth inhibition, apoptosis induction, membrane toxicity, etc. MTT assay showed that apigenin could remarkably inhibit the growth and proliferation in both types of cells. Annexin V/PI-based flow cytometry analysis showed that the cytotoxic effects of apigenin in KYSE150 cells were mainly through early apoptosis induction, while in EC9706 cells, necrosis, and apoptosis were both involved in cell death. The morphological and ultrastructural properties induced by apigenin were investigated at single cellular- or nanometer-level using atomic force microscopy (AFM). Additionally, lactate dehydrogenase (LDH) leakage was measured to assess the changes in membrane permeability. The results indicated that apigenin increased the membrane permeability and caused leakage of LDH, which was consistent with damages on membrane ultrastructure detected by AFM. Therefore, membrane toxicity, including membrane ultrastructure damages and enhanced membrane permeability, played vital roles in apigenin induced human esophageal cancer cell apoptosis. SCANNING 38:322-328, 2016. © 2015 Wiley Periodicals, Inc. © Wiley Periodicals, Inc.

Cholesterol-modified poly(lactide-co-glycolide) nanoparticles for tumor-targeted drug delivery.

PubMed

Lee, Jeong-Jun; Lee, Song Yi; Park, Ju-Hwan; Kim, Dae-Duk; Cho, Hyun-Jong

2016-07-25

Poly(lactide-co-glycolide)-cholesterol (PLGA-C)-based nanoparticles (NPs) were developed for the tumor-targeted delivery of curcumin (CUR). PLGA-C/CUR NPs with ∼200nm mean diameter, narrow size distribution, and neutral zeta potential were fabricated by a modified emulsification-solvent evaporation method. The existence of cholesterol moiety in PLGA-C copolymer was confirmed by proton nuclear magnetic resonance ((1)H NMR) analysis. In vitro stability of developed NPs after 24h incubation was confirmed in phosphate buffered saline (PBS) and serum media. Sustained (∼6days) and pH-responsive drug release profiles from PLGA-C NPs were presented. Blank PLGA and PLGA-C NPs exhibited a negligible cytotoxicity in Hep-2 (human laryngeal carcinoma) cells in the tested concentration range. According to the results of flow cytometry and confocal laser scanning microscopy (CLSM) studies, PLGA-C NPs presented an improved cellular accumulation efficiency, compared to PLGA NPs, in Hep-2 cells. Enhanced in vivo tumor targetability of PLGA-C NPs, compared to PLGA NPs, in Hep-2 tumor-xenografted mouse model was also verified by a real-time near-infrared fluorescence (NIRF) imaging study. Developed PLGA-C NPs may be a candidate of efficient and biocompatible nanosystems for tumor-targeted drug delivery and cancer imaging. Copyright © 2016 Elsevier B.V. All rights reserved.

High spatial variability of phytoplankton assessed by flow cytometry, in a dynamic productive coastal area, in spring: The eastern English Channel

NASA Astrophysics Data System (ADS)

Bonato, Simon; Christaki, Urania; Lefebvre, Alain; Lizon, Fabrice; Thyssen, Melilotus; Artigas, Luis Felipe

2015-03-01

The distribution of phytoplankton (from pico-to microphytoplankton) was investigated, at single-cell level and at high spatial resolution, during an oceanographic cruise across the eastern English Channel (EEC) between April 27 and 29, 2012. Seawater was continuously collected from surface waters and analysed on board at high frequency (one sample every 10 min), by using a new generation of pulse-shape recording scanning flow cytometer (CytoSense, Cytobuoy©). A Bray-Curtis matrix analysis based on phytoplankton composition allowed the discrimination of 4 communities. Within these communities, abundance, cell size as well as single cell and total red fluorescence of 8 phytoplankton groups were measured. Picoeukaryotes and Synechococcus spp cells dominated the mid Channel and most of the English waters monitored, whereas waters off Eastbourne as well as French coastal waters (under remote and direct estuarine influence) were characterized by the dominance of Phaeocystis globosa haploid and diploid cells. Most of the total red fluorescence signal, which correlated with chlorophyll a concentrations, was attributable to P. globosa and, to a lesser extent, to diatoms. In addition to sub-mesoscale variation within phytoplankton communities, the single-cell features within each phytoplankton group gave information about the physiological status of individual phytoplankton cells.

Functional analysis and classification of phytoplankton based on data from an automated flow cytometer.

PubMed

Malkassian, Anthony; Nerini, David; van Dijk, Mark A; Thyssen, Melilotus; Mante, Claude; Gregori, Gerald

2011-04-01

Analytical flow cytometry (FCM) is well suited for the analysis of phytoplankton communities in fresh and sea waters. The measurement of light scatter and autofluorescence properties of particles by FCM provides optical fingerprints, which enables different phytoplankton groups to be separated. A submersible version of the CytoSense flow cytometer (the CytoSub) has been designed for in situ autonomous sampling and analysis, making it possible to monitor phytoplankton at a short temporal scale and obtain accurate information about its dynamics. For data analysis, a manual clustering is usually performed a posteriori: data are displayed on histograms and scatterplots, and group discrimination is made by drawing and combining regions (gating). The purpose of this study is to provide greater objectivity in the data analysis by applying a nonmanual and consistent method to automatically discriminate clusters of particles. In other words, we seek for partitioning methods based on the optical fingerprints of each particle. As the CytoSense is able to record the full pulse shape for each variable, it quickly generates a large and complex dataset to analyze. The shape, length, and area of each curve were chosen as descriptors for the analysis. To test the developed method, numerical experiments were performed on simulated curves. Then, the method was applied and validated on phytoplankton cultures data. Promising results have been obtained with a mixture of various species whose optical fingerprints overlapped considerably and could not be accurately separated using manual gating. Copyright © 2011 International Society for Advancement of Cytometry.

Review of methods to probe single cell metabolism and bioenergetics

DOE PAGES

Vasdekis, Andreas E.; Stephanopoulos, Gregory

2014-10-31

The sampling and manipulation of cells down to the individual has been of substantial interest since the very beginning of Life Sciences. Herein, our objective is to highlight the most recent developments in single cell manipulation, as well as pioneering ones. First, flow-through methods will be discussed, namely methods in which the single cells flow continuously in an ordered manner during their analysis. This section will be followed by confinement techniques that enable cell isolation and confinement in one, two- or three-dimensions. Flow cytometry and droplet microfluidics are the two most common methods of flow-through analysis. While both are high-throughputmore » techniques, their difference lays in the fact that the droplet encapsulated cells experience a restricted and personal microenvironment, while in flow cytometry cells experience similar nutrient and stimuli initial concentrations. These methods are rather well established; however, they recently enabled immense strides in single cell phenotypic analysis, namely the identification and analysis of metabolically distinct individuals from an isogenic population using both droplet microfluidics and flow cytometry.« less

Combination of suberoylanilide hydroxamic acid with heavy ion therapy shows promising effects in infantile sarcoma cell lines

PubMed Central

2011-01-01

Introduction The pan-HDAC inhibitor (HDACI) suberoylanilide hydroxamic acid (SAHA) has previously shown to be a radio-sensitizer to conventional photon radiotherapy (XRT) in pediatric sarcoma cell lines. Here, we investigate its effect on the response of two sarcoma cell lines and a normal tissue cell line to heavy ion irradiation (HIT). Materials and methods Clonogenic assays after different doses of heavy ions were performed. DNA damage and repair were evaluated by measuring γH2AX via flow-cytometry. Apoptosis and cell cycle analysis were also measured via flow cytometry. Protein expression of repair proteins, p53 and p21 were measured using immunoblot analysis. Changes of nuclear architecture after treatment with SAHA and HIT were observed in one of the sarcoma cell lines via light microscopy after staining towards chromatin and γH2AX. Results Corresponding with previously reported photon data, SAHA lead to an increase of sensitivity to heavy ions along with an increase of DSB and apoptosis in the two sarcoma cell lines. In contrast, in the osteoblast cell line (hFOB 1.19), the combination of SAHA and HIT showed a significant radio-protective effect. Laser scanning microscopy revealed no significant morphologic changes after HIT compared to the combined treatment with SAHA. Immunoblot analysis revealed no significant up or down regulation of p53. However, p21 was significantly increased by SAHA and combination treatment as compared to HIT only in the two sarcoma cell lines - again in contrast to the osteoblast cell line. Changes in the repair kinetics of DSB p53-independent apoptosis with p21 involvement may be part of the underlying mechanisms for radio-sensitization by SAHA. Conclusion Our in vitro data suggest an increase of the therapeutic ratio by the combination of SAHA with HIT in infantile sarcoma cell lines. PMID:21933400

Consensus guidelines on plasma cell myeloma minimal residual disease analysis and reporting.

PubMed

Arroz, Maria; Came, Neil; Lin, Pei; Chen, Weina; Yuan, Constance; Lagoo, Anand; Monreal, Mariela; de Tute, Ruth; Vergilio, Jo-Anne; Rawstron, Andy C; Paiva, Bruno

2016-01-01

Major heterogeneity between laboratories in flow cytometry (FC) minimal residual disease (MRD) testing in multiple myeloma (MM) must be overcome. Cytometry societies such as the International Clinical Cytometry Society and the European Society for Clinical Cell Analysis recognize a strong need to establish minimally acceptable requirements and recommendations to perform such complex testing. A group of 11 flow cytometrists currently performing FC testing in MM using different instrumentation, panel designs (≥ 6-color) and analysis software compared the procedures between their respective laboratories and reviewed the literature to propose a consensus guideline on flow-MRD analysis and reporting in MM. Consensus guidelines support i) the use of minimum of five initial gating parameters (CD38, CD138, CD45, forward, and sideward light scatter) within the same aliquot for accurate identification of the total plasma cell compartment; ii) the analysis of potentially aberrant phenotypic markers and to report the antigen expression pattern on neoplastic plasma cells as being reduced, normal or increased, when compared to a normal reference plasma cell immunophenotype (obtained using the same instrument and parameters); and iii) the percentage of total bone marrow plasma cells plus the percentages of both normal and neoplastic plasma cells within the total bone marrow plasma cell compartment, and over total bone marrow cells. Consensus guidelines on minimal current and future MRD analyses should target a lower limit of detection of 0.001%, and ideally a limit of quantification of 0.001%, which requires at least 3 × 10(6) and 5 × 10(6) bone marrow cells to be measured, respectively. © 2015 International Clinical Cytometry Society.

Scalable clustering algorithms for continuous environmental flow cytometry.

PubMed

Hyrkas, Jeremy; Clayton, Sophie; Ribalet, Francois; Halperin, Daniel; Armbrust, E Virginia; Howe, Bill

2016-02-01

Recent technological innovations in flow cytometry now allow oceanographers to collect high-frequency flow cytometry data from particles in aquatic environments on a scale far surpassing conventional flow cytometers. The SeaFlow cytometer continuously profiles microbial phytoplankton populations across thousands of kilometers of the surface ocean. The data streams produced by instruments such as SeaFlow challenge the traditional sample-by-sample approach in cytometric analysis and highlight the need for scalable clustering algorithms to extract population information from these large-scale, high-frequency flow cytometers. We explore how available algorithms commonly used for medical applications perform at classification of such a large-scale, environmental flow cytometry data. We apply large-scale Gaussian mixture models to massive datasets using Hadoop. This approach outperforms current state-of-the-art cytometry classification algorithms in accuracy and can be coupled with manual or automatic partitioning of data into homogeneous sections for further classification gains. We propose the Gaussian mixture model with partitioning approach for classification of large-scale, high-frequency flow cytometry data. Source code available for download at https://github.com/jhyrkas/seaflow_cluster, implemented in Java for use with Hadoop. hyrkas@cs.washington.edu Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Modeling of cytometry data in logarithmic space: When is a bimodal distribution not bimodal?

PubMed

Erez, Amir; Vogel, Robert; Mugler, Andrew; Belmonte, Andrew; Altan-Bonnet, Grégoire

2018-02-16

Recent efforts in systems immunology lead researchers to build quantitative models of cell activation and differentiation. One goal is to account for the distributions of proteins from single-cell measurements by flow cytometry or mass cytometry as readout of biological regulation. In that context, large cell-to-cell variability is often observed in biological quantities. We show here that these readouts, viewed in logarithmic scale may result in two easily-distinguishable modes, while the underlying distribution (in linear scale) is unimodal. We introduce a simple mathematical test to highlight this mismatch. We then dissect the flow of influence of cell-to-cell variability proposing a graphical model which motivates higher-dimensional analysis of the data. Finally we show how acquiring additional biological information can be used to reduce uncertainty introduced by cell-to-cell variability, helping to clarify whether the data is uni- or bimodal. This communication has cautionary implications for manual and automatic gating strategies, as well as clustering and modeling of single-cell measurements. © 2018 International Society for Advancement of Cytometry. © 2018 International Society for Advancement of Cytometry.

Evaluation of Leishmania species reactivity in human serologic diagnosis of leishmaniasis.

PubMed

Silvestre, Ricardo; Santarém, Nuno; Teixeira, Lúcia; Cunha, Joana; Schallig, Henk; Cordeiro-da-Silva, Anabela

2009-08-01

The sensitivities and specificities of IgG-ELISA and IgG flow cytometry based techniques using different Leishmania species were determined using sera collected from 40 cutaneous or visceral leishmaniasis patients. The flow cytometry technique, using promastigote parasite forms, performed better than total soluble extract IgG-ELISA. At the species level, the use of Leishmania amazonensis and Leishmania major as antigens in enzyme linked immunosorbent assay (ELISA) decreased the overall sensitivity. To assess the specificity of these tests, sera from malaria, toxoplasmosis, amoebiasis, schistosomiasis, and leprosy patients were used. We also included sera from Leishmania non-infected endemic individuals. The cutaneous species displayed a decreased specificity in both assays. Although more sensitive, flow cytometry using promastigote parasite forms generally presented lower levels of specificity when compared with total extract of IgG-ELISA. Overall, the results of the study show the potential of IgG flow cytometry for the diagnosis of leishmaniasis. Although highly sensitive, a refinement of the flow cytometry method should be performed to improve the overall specificity.

Doxorubicin-mediated radiosensitivity in multicellular spheroids from a lung cancer cell line is enhanced by composite micelle encapsulation

PubMed Central

Xu, Wen-Hong; Han, Min; Dong, Qi; Fu, Zhi-Xuan; Diao, Yuan-Yuan; Liu, Hai; Xu, Jing; Jiang, Hong-Liang; Zhang, Su-Zhan; Zheng, Shu; Gao, Jian-Qing; Wei, Qi-Chun

2012-01-01

Background The purpose of this study is to evaluate the efficacy of composite doxorubicinloaded micelles for enhancing doxorubicin radiosensitivity in multicellular spheroids from a non-small cell lung cancer cell line. Methods A novel composite doxorubicin-loaded micelle consisting of polyethylene glycolpolycaprolactone/Pluronic P105 was developed, and carrier-mediated doxorubicin accumulation and release from multicellular spheroids was evaluated. We used confocal laser scanning microscopy and flow cytometry to study the accumulation and efflux of doxorubicin from A549 multicellular spheroids. Doxorubicin radiosensitization and the combined effects of irradiation and doxorubicin on cell migration and proliferation were compared for the different doxorubicin delivery systems. Results Confocal laser scanning microscopy and quantitative flow cytometry studies both verified that, for equivalent doxorubicin concentrations, composite doxorubicin-loaded micelles significantly enhanced cellular doxorubicin accumulation and inhibited doxorubicin release. Colony-forming assays demonstrated that composite doxorubicin-loaded micelles are radiosensitive, as shown by significantly reduced survival of cells treated by radiation + composite micelles compared with those treated with radiation + free doxorubicin or radiation alone. The multicellular spheroid migration area and growth ability verified higher radiosensitivity for the composite micelles loaded with doxorubicin than for free doxorubicin. Conclusion Our composite doxorubicin-loaded micelle was demonstrated to have radiosensitization. Doxorubicin loading in the composite micelles significantly increased its cellular uptake, improved drug retention, and enhanced its antitumor effect relative to free doxorubicin, thereby providing a novel approach for treatment of cancer. PMID:22679376

The role of RhD agglutination for the detection of weak D red cells by anti-D flow cytometry.

PubMed

Grey, D E; Davies, J I; Connolly, M; Fong, E A; Erber, W N

2005-04-01

Anti-D flow cytometry is an accurate method for quantifying feto-maternal haemorrhage (FMH). However, weak D red cells with <1000 RhD sites are not detectable using this methodology but are immunogenic. As quantitation of RhD sites is not practical, an alternative approach is required to identify those weak D fetal red cells where anti-D flow cytometry is inappropriate. We describe a simple algorithm based on RhD agglutination and flow cytometry peak separation. All weak D (n = 34) gave weak agglutination with RUM-1 on immediate spin (grading

Isolation of purified oocyst walls and sporocysts from Toxoplasma gondii.

PubMed

Everson, William V; Ware, Michael W; Dubey, J P; Lindquist, H D Alan

2002-01-01

Toxoplasma gondii oocysts are environmentally resistant and can infect virtually all warm-blooded hosts, including humans and livestock. Little is known about the biochemical basis for this resistance of oocysts, and mechanism for excystation of T. gondii sporozoites. The objective of the present study was to evaluate different methods (mechanical fragmentation, gradients, flow cytometry) to separate and purify T. gondii oocyst walls and sporocysts. Oocyst walls were successfully separated and purified using iodixanol gradients. Sporocysts were successfully separated and purified using iodixanol and Percoll gradients. Purification was also achieved by flow cytometry. Flow cytometry with fluorescence-activated cell sorting (FACS) yielded analytical quantities of oocyst walls and intact sporocysts. Flow cytometry with FACS also proved useful for quantitation of purity obtained following iodixanol gradient fractionation. Methods reported in this paper will be useful for analytical purposes, such as proteomic analysis of components unique to this life cycle stage, development of detection methods, or excystation studies.

The role of flow cytometry in companion animal diagnostic medicine.

PubMed

Tarrant, Jacqueline M

2005-11-01

Flow cytometry is a powerful tool for characterising the composition of complex cell populations. The accuracy and precision of this technology for describing and enumerating cells exceeds traditional methods. The number of diagnostic veterinary laboratories with access to a dedicated machine is increasing, and there is the potential to offer a clinical flow cytometry service. The improved availability of monoclonal antibodies (mAb) to cell markers expressed by the leukocytes of companion animals, permits the implementation of comprehensive mAb panels suitable for diagnosis of lympho- and myeloproliferative disease. Reticulated erythrocyte and platelet quantification, antiglobulin assays for immune-mediated cytopenias, lymphocyte subset analysis, and immunophenotyping of lymphoma and leukemia, have been validated for companion animal samples on the flow cytometer. It is now timely to consider the role of flow cytometry in diagnostic practice, and the requirement for quality assurance and standardization of testing procedures.

Flow cytometry as a tool for analyzing changes in Plasmodium falciparum cell cycle following treatment with indol compounds.

PubMed

Schuck, Desirée Cigaran; Ribeiro, Ramira Yuri; Nery, Arthur A; Ulrich, Henning; Garcia, Célia R S

2011-11-01

Melatonin and its derivatives modulate the Plasmodium falciparum and Plasmodium chabaudi cell cycle. Flow cytometry was employed together with the nucleic acid dye YOYO-1 allowing precise discrimination between mono- and multinucleated forms of P. falciparum-infected red blood cell. The use of YOYO-1 permitted excellent discrimination between uninfected and infected red blood cells as well as between early and late parasite stages. Fluorescence intensities of schizont-stage parasites were about 10-fold greater than those of ring-trophozoite form parasites. Melatonin and related indolic compounds including serotonin, N-acetyl-serotonin and tryptamine induced an increase in the percentage of multinucleated forms compared to non-treated control cultures. YOYO-1 staining of infected erythrocyte and subsequent flow cytometry analysis provides a powerful tool in malaria research for screening of bioactive compounds. Copyright © 2011 International Society for Advancement of Cytometry.

RchyOptimyx: Cellular Hierarchy Optimization for Flow Cytometry

PubMed Central

Aghaeepour, Nima; Jalali, Adrin; O’Neill, Kieran; Chattopadhyay, Pratip K.; Roederer, Mario; Hoos, Holger H.; Brinkman, Ryan R.

2013-01-01

Analysis of high-dimensional flow cytometry datasets can reveal novel cell populations with poorly understood biology. Following discovery, characterization of these populations in terms of the critical markers involved is an important step, as this can help to both better understand the biology of these populations and aid in designing simpler marker panels to identify them on simpler instruments and with fewer reagents (i.e., in resource poor or highly regulated clinical settings). However, current tools to design panels based on the biological characteristics of the target cell populations work exclusively based on technical parameters (e.g., instrument configurations, spectral overlap, and reagent availability). To address this shortcoming, we developed RchyOptimyx (cellular hieraRCHY OPTIMization), a computational tool that constructs cellular hierarchies by combining automated gating with dynamic programming and graph theory to provide the best gating strategies to identify a target population to a desired level of purity or correlation with a clinical outcome, using the simplest possible marker panels. RchyOptimyx can assess and graphically present the trade-offs between marker choice and population specificity in high-dimensional flow or mass cytometry datasets. We present three proof-of-concept use cases for RchyOptimyx that involve 1) designing a panel of surface markers for identification of rare populations that are primarily characterized using their intracellular signature; 2) simplifying the gating strategy for identification of a target cell population; 3) identification of a non-redundant marker set to identify a target cell population. PMID:23044634

Comparison of methods for in-house screening of HLA-B*57:01 to prevent abacavir hypersensitivity in HIV-1 care.

PubMed

De Spiegelaere, Ward; Philippé, Jan; Vervisch, Karen; Verhofstede, Chris; Malatinkova, Eva; Kiselinova, Maja; Trypsteen, Wim; Bonczkowski, Pawel; Vogelaers, Dirk; Callens, Steven; Ruelle, Jean; Kabeya, Kabamba; De Wit, Stephane; Van Acker, Petra; Van Sandt, Vicky; Emonds, Marie-Paule; Coucke, Paul; Sermijn, Erica; Vandekerckhove, Linos

2015-01-01

Abacavir is a nucleoside reverse transcriptase inhibitor used as part of combination antiretroviral therapy in HIV-1-infected patients. Because this drug can cause a hypersensitivity reaction that is correlated with the presence of the HLA-B*57:01 allotype, screening for the presence of HLA-B*57:01 is recommended before abacavir initiation. Different genetic assays have been developed for HLA-B*57:01 screening, each with specific sensitivity, turnaround time and assay costs. Here, a new real-time PCR (qPCR) based analysis is described and compared to sequence specific primer PCR with capillary electrophoresis (SSP PCR CE) on 149 patient-derived samples, using sequence specific oligonucleotide hybridization combined with high resolution SSP PCR as gold standard. In addition to these PCR based methods, a complementary approach was developed using flow cytometry with an HLA-B17 specific monoclonal antibody as a pre-screening assay to diminish the number of samples for genetic testing. All three assays had a maximum sensitivity of >99. However, differences in specificity were recorded, i.e. 84.3%, 97.2% and >99% for flow cytometry, qPCR and SSP PCR CE respectively. Our data indicate that the most specific and sensitive of the compared methods is the SSP PCR CE. Flow cytometry pre-screening can substantially decrease the number of genetic tests for HLA-B*57:01 typing in a clinical setting.

Flow cytometry for receptor analysis from ex-vivo brain tissue in adult rat.

PubMed

Benoit, A; Guillamin, M; Aitken, P; Smith, P F; Philoxene, B; Sola, B; Poulain, L; Coquerel, A; Besnard, S

2018-07-01

Flow cytometry allows single-cell analysis of peripheral biological samples and is useful in many fields of research and clinical applications, mainly in hematology, immunology, and oncology. In the neurosciences, the flow cytometry separation method was first applied to stem cell extraction from healthy or cerebral tumour tissue and was more recently tested in order to phenotype brain cells, hippocampal neurogenesis, and to detect prion proteins. However, it remains sparsely applied in quantifying membrane receptors in relation to synaptic plasticity. We aimed to optimize a flow cytometric procedure for receptor quantification in neurons and non-neurons. A neural dissociation process, myelin separation, fixation, and membrane permeability procedures were optimized to maximize cell survival and analysis in hippocampal tissue obtained from adult rodents. We then aimed to quantify membrane muscarinic acetylcholine receptors (mAChRs) in rats with and without bilateral vestibular loss (BVL). mAChR's were quantified for neuronal and non-neuronal cells in the hippocampus and striatum following BVL. At day 30 but not at day 7 following BVL, there was a significant increase (P ≤ 0.05) in the percentage of neurons expressing M 2/4 mAChRs in both the hippocampus and the striatum. Here, we showed that flow cytometry appears to be a reliable method of membrane receptor quantification in ex-vivo brain tissue. Copyright © 2018 Elsevier B.V. All rights reserved.

Implementation of Mass Cytometry as a Tool for Mechanism of Action Studies in Inflammatory Bowel Disease.

PubMed

Tyler, Christopher J; Pérez-Jeldres, Tamara; Ehinger, Erik; Capaldo, Brian; Karuppuchamy, Thangaraj; Boyer, Joshua D; Patel, Derek; Dulai, Parambir; Boland, Brigid S; Lannigan, Joanne; Eckmann, Lars; Ernst, Peter B; Sandborn, William J; Ho, Samuel B; Rivera-Nieves, Jesús

2018-06-08

Novel therapeutics for inflammatory bowel disease (IBD) are under development, yet mechanistic readouts at the tissue level are lacking. Techniques to assess intestinal immune composition could represent a valuable tool for mechanism of action (MOA) studies of novel drugs. Mass cytometry enables analysis of intestinal inflammatory cell infiltrate and corresponding molecular fingerprints with unprecedented resolution. Here, we aimed to optimize the methodology for isolation and cryopreservation of cells from intestinal tissue to allow for the potential implementation of mass cytometry in MOA studies. We investigated key technical issues, including minimal tissue requirements, cell isolation protocols, and cell storage, using intestinal biopsies and peripheral blood from healthy individuals. High-dimensional mass cytometry was employed for the analyses of biopsy-derived intestinal cellular subsets. Dithiothreitol and mechanical dissociation decreased epithelial cell contamination and allowed for isolation of adequate cell numbers from 2 to 4 colonic or ileal biopsies (6 × 104±2 × 104) after a 20-minute collagenase digestion, allowing for reliable detection of most major immune cell subsets. Biopsies and antibody-labeled mononuclear cells could be cryopreserved for later processing and acquisition (viability > 70%; P < 0.05). Mass cytometry represents a unique tool for deep immunophenotyping intestinal cell composition. This technique has the potential to facilitate analysis of drug actions at the target tissue by identifying specific cellular subsets and their molecular signatures. Its widespread implementation may impact not only IBD research but also other gastrointestinal conditions where inflammatory cells play a role in pathogenesis.

Digital Analysis and Sorting of Fluorescence Lifetime by Flow Cytometry

PubMed Central

Houston, Jessica P.; Naivar, Mark A.; Freyer, James P.

2010-01-01

Frequency-domain flow cytometry techniques are combined with modifications to the digital signal processing capabilities of the Open Reconfigurable Cytometric Acquisition System (ORCAS) to analyze fluorescence decay lifetimes and control sorting. Real-time fluorescence lifetime analysis is accomplished by rapidly digitizing correlated, radiofrequency modulated detector signals, implementing Fourier analysis programming with ORCAS’ digital signal processor (DSP) and converting the processed data into standard cytometric list mode data. To systematically test the capabilities of the ORCAS 50 MS/sec analog-to-digital converter (ADC) and our DSP programming, an error analysis was performed using simulated light scatter and fluorescence waveforms (0.5–25 ns simulated lifetime), pulse widths ranging from 2 to 15 µs, and modulation frequencies from 2.5 to 16.667 MHz. The standard deviations of digitally acquired lifetime values ranged from 0.112 to >2 ns, corresponding to errors in actual phase shifts from 0.0142° to 1.6°. The lowest coefficients of variation (<1%) were found for 10-MHz modulated waveforms having pulse widths of 6 µs and simulated lifetimes of 4 ns. Direct comparison of the digital analysis system to a previous analog phase-sensitive flow cytometer demonstrated similar precision and accuracy on measurements of a range of fluorescent microspheres, unstained cells and cells stained with three common fluorophores. Sorting based on fluorescence lifetime was accomplished by adding analog outputs to ORCAS and interfacing with a commercial cell sorter with a radiofrequency modulated solid-state laser. Two populations of fluorescent microspheres with overlapping fluorescence intensities but different lifetimes (2 and 7 ns) were separated to ~98% purity. Overall, the digital signal acquisition and processing methods we introduce present a simple yet robust approach to phase-sensitive measurements in flow cytometry. The ability to simply and inexpensively implement this system on a commercial flow sorter will both allow better dissemination of this technology and better exploit the traditionally underutilized parameter of fluorescence lifetime. PMID:20662090

Chromatin texture, DNA index, and S-phase fraction in primary breast carcinoma cells analysed by laserscanning cytometry.

PubMed

Kuliffay, P; Sanislo, L; Galbavy, S

2010-01-01

Laser scanning cytometry (LSC) is a slide-based technique capable of measuring a number of biological parameters both in immobilised cell suspensions and in formalin-fixed paraffin-embedded tissue sections. High proliferation rate in surgically removed breast tumours is an unfavourable prognostic factor. In node negative cases it can help distinguish patients with higher risk for distant metastases from those with a lower risk. In a prospective study we investigated 140 breast tumours, of which 113 were invasive ductal carcinomas, 11 were invasive lobular carcinomas, and 16 tumours were of other histological types. Cells for LSC investigations were prepared from fresh, surgically removed tumours by mechanical disintegration. After fixation the cells were stained with FITC-conjugated anti-cytokeratin (CK-FITC) to distinguish CK+ tumour cells from CK- stroma, and with propidium iodide to stain DNA. We identified three S-phase fraction (SPF) groups, with low (30 patients), moderate (54 patients), and high SPF (51 patients). Thirty-seven tumours were diploid, 83 were aneuploid, while 5 tumours had a bimodal distribution of DNA content. Chromatin texture values were increasing in the respective subclasses from the hypodiploid group to the tetraploid/hypertetraploid group. The measurement of DNA content and SPF of tumours by LSC completed by and correlated with other biological properties of the tumour cells may be a useful tool in assessing prognosis and clinical outcome of patients with breast cancer. (Tab. 5, Fig. 4, Ref. 18). Full Text (Free, PDF) www.bmj.sk.

Mode of action of plectasin-derived peptides against gas gangrene-associated Clostridium perfringens type A.

PubMed

Zheng, Xueling; Wang, Xiumin; Teng, Da; Mao, Ruoyu; Hao, Ya; Yang, Na; Zong, Lifen; Wang, Jianhua

2017-01-01

NZ2114 and MP1102 are novel plectasin-derived peptides with potent activity against Gram-positive bacteria. The antibacterial characteristics and mechanism of NZ2114 and MP1102 against gas gangrene-associated Clostridium perfringens were studied for the first time. The minimal inhibitory concentration and minimal bactericidal concentration of NZ2114 and MP1102 against resistant C. perfringens type A strain CVCC 46 were 0.91 μM. Based on the fractional inhibitory concentration index (FICI) result, an additive or synergic effect was observed between NZ2114 (FICI = 0.5~0.75) or MP1102 (FICI = 0.375~1.0) and antibiotics. The flow cytometry, scanning and transmission electron microscopy analysis showed that both NZ2114 and MP1102 induced obviously membrane damage, such as the leakage of cellular materials, partial disappearance of the cell membrane and membrane peeling, as well as retracting cytoplasm and ghost cell. The gel retardation and circular dichroism (CD) detection showed that NZ2114 and MP1102 could bind to C. perfringens genomic DNA and change the DNA conformation. Moreover, NZ2114 also interfered with the double helix and unwind the genomic DNA. The cell cycle analysis showed that C. perfringens CVCC 46 cells exposed to NZ2114 and MP1102 were arrested at the phase I. These data indicated that both NZ2114 and MP1102 have potential as new antimicrobial agents for gas gangrene infection resulting from resistant C. perfringens.

Mode of action of plectasin-derived peptides against gas gangrene-associated Clostridium perfringens type A

PubMed Central

Zheng, Xueling; Wang, Xiumin; Teng, Da; Mao, Ruoyu; Hao, Ya; Yang, Na; Zong, Lifen

2017-01-01

NZ2114 and MP1102 are novel plectasin-derived peptides with potent activity against Gram-positive bacteria. The antibacterial characteristics and mechanism of NZ2114 and MP1102 against gas gangrene-associated Clostridium perfringens were studied for the first time. The minimal inhibitory concentration and minimal bactericidal concentration of NZ2114 and MP1102 against resistant C. perfringens type A strain CVCC 46 were 0.91 μM. Based on the fractional inhibitory concentration index (FICI) result, an additive or synergic effect was observed between NZ2114 (FICI = 0.5~0.75) or MP1102 (FICI = 0.375~1.0) and antibiotics. The flow cytometry, scanning and transmission electron microscopy analysis showed that both NZ2114 and MP1102 induced obviously membrane damage, such as the leakage of cellular materials, partial disappearance of the cell membrane and membrane peeling, as well as retracting cytoplasm and ghost cell. The gel retardation and circular dichroism (CD) detection showed that NZ2114 and MP1102 could bind to C. perfringens genomic DNA and change the DNA conformation. Moreover, NZ2114 also interfered with the double helix and unwind the genomic DNA. The cell cycle analysis showed that C. perfringens CVCC 46 cells exposed to NZ2114 and MP1102 were arrested at the phase I. These data indicated that both NZ2114 and MP1102 have potential as new antimicrobial agents for gas gangrene infection resulting from resistant C. perfringens. PMID:28934314

RNA Flow Cytometry Using the Branched DNA Technique.

PubMed

Soh, Kah Teong; Wallace, Paul K

2018-01-01

The systematic modulation of mRNA and proteins governs the complicated and intermingled biological functions of our cells. Traditionally, transcriptomic technologies such as DNA microarray and RNA-Seq have been used to identify, characterize, and profile gene expression data. These are, however, considered bulk methods as they are unable to measure gene expression at the single-cell level, unless the cells are pre-sorted. Branched DNA is a flow cytometry-based detection platform that has been developed recently to measure mRNA at the single-cell level. Originally adapted from microscopy, the current system has been modified to achieve compatibility with the detection of surface and intracellular antigens using monoclonal antibodies conjugated to fluorochromes, thus permitting simultaneous detection of mRNAs and proteins. The Branched DNA method offers a variety of advantages when compared to traditional or standard methods used for the quantification of mRNA, such as (a) the detection of specific mRNA on a per cell basis, (b) an alternate detection tool when the measurement of a protein is technically infeasible (i.e., no quality antibody exists) or the epitope is not assessable, and (c) correlate the analysis of mRNA with protein. Compared to earlier attempts at measuring nucleic acid by flow cytometry, the hybridization temperature applied in the Branched DNA assay is much lower, thus preserving the integrity of cellular structures for further characterization. It also has greatly increased specificity and sensitivity. Here, we provide detailed instruction for performing the Branched DNA method using it in a model system to correlate the expression of CD8 mRNA and CD8 protein by flow cytometry.

Evaluation of flow cytometric HIT assays in relation to an IgG-Specific immunoassay and clinical outcome.

PubMed

Kerényi, Adrienne; Beke Debreceni, Ildikó; Oláh, Zsolt; Ilonczai, Péter; Bereczky, Zsuzsanna; Nagy, Béla; Muszbek, László; Kappelmayer, János

2017-09-01

Heparin-induced thrombocytopenia (HIT) is a severe side effect of heparin treatment caused by platelet activating IgG antibodies generated against the platelet factor 4 (PF4)-heparin complex. Thrombocytopenia and thrombosis are the leading clinical symptoms of HIT. The clinical pretest probability of HIT was evaluated by the 4T score system. Laboratory testing of HIT was performed by immunological detection of antibodies against PF4-heparin complex (EIA) and two functional assays. Heparin-dependent activation of donor platelets by patient plasma was detected by flow cytometry. Increased binding of Annexin-V to platelets and elevated number of platelet-derived microparticles (PMP) were the indicators of platelet activation. EIA for IgG isotype HIT antibodies was performed in 405 suspected HIT patients. Based on negative EIA results, HIT was excluded in 365 (90%) of cases. In 40 patients with positive EIA test result functional tests were performed. Platelet activating antibodies were detected in 17 cases by Annexin V binding. PMP count analysis provided nearly identical results. The probability of a positive flow cytometric assay result was higher in patients with elevated antibody titer. 71% of patients with positive EIA and functional assay had thrombosis. EIA is an important first line laboratory test in the diagnosis of HIT; however, HIT must be confirmed by a functional test. Annexin V binding and PMP assays using flow cytometry are functional HIT tests convenient in a clinical diagnostic laboratory. The positive results of functional assays may predict the onset of thrombosis. © 2016 International Clinical Cytometry Society. © 2016 International Clinical Cytometry Society.

Biomass measurement by flow cytometry during solid-state fermentation of basidiomycetes.

PubMed

Steudler, Susanne; Böhmer, Ulrike; Weber, Jost; Bley, Thomas

2015-02-01

Solid-state fermentation (SSF) is a robust process that is well suited to the on-site cultivation of basidiomycetes that produce enzymes for the treatment of lignocellulosics. Reliable methods for biomass quantification are essential for the analysis of fungal growth kinetics. However, direct biomass determination is not possible during SSF because the fungi grow into the substrate and use it as a nutrient source. This necessitates the use of indirect methods that are either very laborious and time consuming or can only provide biomass measurements during certain growth periods. Here, we describe the development and optimization of a new rapid method for fungal biomass determination during SSF that is based on counting fungal nuclei by flow cytometry. Fungal biomass was grown on an organic substrate and its concentration was measured by isolating the nuclei from the fungal hyphae after cell disruption, staining them with SYTOX(®) Green, and then counting them using a flow cytometer. A calibration curve relating the dry biomass of the samples to their concentrations of nuclei was established. Multiple buffers and disruption methods were tested. The results obtained were compared with values determined using the method of ergosterol determination, a classical technique for fungal biomass measurement during SSF. Our new approach can be used to measure fungal biomass on a range of different scales, from 15 mL cultures to a laboratory reactor with a working volume of 10 L (developed by the Research Center for Medical Technology and Biotechnology (fzmb GmbH)). © 2014 International Society for Advancement of Cytometry. © 2014 International Society for Advancement of Cytometry.

Polychromatic flow cytometry is more sensitive than microscopy in detecting small monoclonal plasma cell populations.

PubMed

Tran, Daniel N; Smith, Sandy A B C; Brown, David A; Parker, Andrew J C; Joseph, Joanne E; Armstrong, Nicola; Sewell, William A

2017-03-01

There is an emerging role for flow cytometry (FC) in the assessment of small populations of plasma cells (PC). However, FC's utility has been questioned due to consistent underestimation of the percentage of PC compared to microscopy. A retrospective study was performed on bone marrow samples analysed by 8-colour FC. Plasma cell populations were classified as polyclonal or monoclonal based on FC analysis. FC findings were compared with microscopy of aspirates, histology and immunohistochemistry of trephine biopsies, and immunofixation (IFX) of serum and/or urine. FC underestimated PC compared to aspirate and trephine microscopy. The 10% diagnostic cutoff for MM on aspirate microscopy corresponded to a 3.5% cutoff on FC. Abnormal plasma cell morphology by aspirate microscopy and clonality by FC correlated in 229 of 294 cases (78%). However, in 50 cases, FC demonstrated a monoclonal population but microscopy reported no abnormality. In 15 cases, abnormalities were reported by microscopy but not by FC. Clonality assessment by trephine microscopy and FC agreed in 251/280 cases (90%), but all 29 discordant cases were monoclonal by FC and not monoclonal by microscopy. These cases had fewer PC and proportionally more polyclonal PC, and when IFX detected a paraprotein, it had the same light chain as in the PC determined by FC. FC was more sensitive in detecting monoclonal populations that were small or accompanied by polyclonal PC. This study supports the inclusion of FC in the evaluation of PC, especially in the assessment of small populations. © 2016 International Clinical Cytometry Society. © 2016 International Clinical Cytometry Society.

Genome size and chromosome number in velvet worms (Onychophora).

PubMed

Jeffery, Nicholas W; Oliveira, Ivo S; Gregory, T Ryan; Rowell, David M; Mayer, Georg

2012-12-01

The Onychophora (velvet worms) represents a small group of invertebrates (~180 valid species), which is commonly united with Tardigrada and Arthropoda in a clade called Panarthropoda. As with the majority of invertebrate taxa, genome size data are very limited for the Onychophora, with only one previously published estimate. Here we use both flow cytometry and Feulgen image analysis densitometry to provide genome size estimates for seven species of velvet worms from both major subgroups, Peripatidae and Peripatopsidae, along with karyotype data for each species. Genome sizes in these species range from roughly 5-19 pg, with densitometric estimates being slightly larger than those obtained by flow cytometry for all species. Chromosome numbers range from 2n = 8 to 2n = 54. No relationship is evident between genome size, chromosome number, or reproductive mode. Various avenues for future genomic research are presented based on these results.

Accuracy of Automated Flow Cytometry-Based Leukocyte Counts To Rule Out Urinary Tract Infection in Febrile Children: a Prospective Cross-Sectional Study

PubMed Central

Duong, Hong Phuoc; Wissing, Karl Martin; Tram, Nathalie; Mascart, Georges; Lepage, Philippe

2016-01-01

Automated flow cytometry of urine remains an incompletely validated method to rule out urinary tract infection (UTI) in children. This cross-sectional analytical study was performed to compare the predictive values of flow cytometry and a dipstick test as initial diagnostic tests for UTI in febrile children and prospectively included 1,106 children (1,247 episodes). Urine culture was used as the gold standard test for diagnosing UTI. The performance of screening tests to diagnose UTI were established using receiver operating characteristic (ROC) analysis. Among these 1,247 febrile episodes, 221 UTIs were diagnosed (17.7% [95% confidence interval {CI}, 15.6 to 19.8%]). The area under the ROC curve for flow cytometry white blood cell (WBC) counts (0.99 [95% CI, 0.98 to 0.99]) was significantly superior to that for red blood cell (0.74 [95% CI, 0.70 to 0.78]) and bacterial counts (0.89 [95% CI, 0.87 to 0.92]) (P < 0.001). Urinary WBC counts also had a significantly higher area under the ROC curve than that of the leukocyte esterase (LE) dipstick (0.92 [95% CI, 0.90 to 0.94]), nitrite dipstick (0.83 [95% CI, 0.80 to 0.87]), or the combination of positive LE and/or nitrite dipstick (0.91 [95% CI, 0.89 to 0.93]) test (P < 0.001). The presence of ≥35 WBC/μl of urine was the best cutoff point, yielding both a high sensitivity (99.5% [95% CI, 99 to 100%]) and an acceptable specificity (80.6% [95% CI, 78 to 83%]). Using this cutoff point would have reduced the number of samples sent to the laboratory for culture by 67%. In conclusion, the determination of urinary WBC counts by flow cytometry provides optimal performance as an initial diagnostic test for UTI in febrile children. PMID:27682127

Distinction between asymptomatic monoclonal B-cell lymphocytosis with cyclin D1 overexpression and mantle cell lymphoma: from molecular profiling to flow cytometry.

PubMed

Espinet, Blanca; Ferrer, Ana; Bellosillo, Beatriz; Nonell, Lara; Salar, Antonio; Fernández-Rodríguez, Concepción; Puigdecanet, Eulàlia; Gimeno, Javier; Garcia-Garcia, Mar; Vela, Maria Carmen; Luño, Elisa; Collado, Rosa; Navarro, José Tomás; de la Banda, Esmeralda; Abrisqueta, Pau; Arenillas, Leonor; Serrano, Cristina; Lloreta, Josep; Miñana, Belén; Cerutti, Andrea; Florensa, Lourdes; Orfao, Alberto; Sanz, Ferran; Solé, Francesc; Dominguez-Sola, David; Serrano, Sergio

2014-02-15

According to current diagnostic criteria, mantle cell lymphoma (MCL) encompasses the usual, aggressive variants and rare, nonnodal cases with monoclonal asymptomatic lymphocytosis, cyclin D1-positive (MALD1). We aimed to understand the biology behind this clinical heterogeneity and to identify markers for adequate identification of MALD1 cases. We compared 17 typical MCL cases with a homogeneous group of 13 untreated MALD1 cases (median follow-up, 71 months). We conducted gene expression profiling with functional analysis in five MCL and five MALD1. Results were validated in 12 MCL and 8 MALD1 additional cases by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and in 24 MCL and 13 MALD1 cases by flow cytometry. Classification and regression trees strategy was used to generate an algorithm based on CD38 and CD200 expression by flow cytometry. We found 171 differentially expressed genes with enrichment of neoplastic behavior and cell proliferation signatures in MCL. Conversely, MALD1 was enriched in gene sets related to immune activation and inflammatory responses. CD38 and CD200 were differentially expressed between MCL and MALD1 and confirmed by flow cytometry (median CD38, 89% vs. 14%; median CD200, 0% vs. 24%, respectively). Assessment of both proteins allowed classifying 85% (11 of 13) of MALD1 cases whereas 15% remained unclassified. SOX11 expression by qRT-PCR was significantly different between MCL and MALD1 groups but did not improve the classification. We show for the first time that MALD1, in contrast to MCL, is characterized by immune activation and driven by inflammatory cues. Assessment of CD38/CD200 by flow cytometry is useful to distinguish most cases of MALD1 from MCL in the clinical setting. MALD1 should be identified and segregated from the current MCL category to avoid overdiagnosis and unnecessary treatment. ©2013 AACR

Distinction between Asymptomatic Monoclonal B-cell Lymphocytosis with Cyclin D1 Overexpression and Mantle Cell Lymphoma: From Molecular Profiling to Flow Cytometry

PubMed Central

Espinet, Blanca; Ferrer, Ana; Bellosillo, Beatriz; Nonell, Lara; Salar, Antonio; Fernández-Rodríguez, Concepción; Puigdecanet, Eulàlia; Gimeno, Javier; Garcia-Garcia, Mar; Carmen Vela, Maria; Luño, Elisa; Collado, Rosa; Navarro, José Tomás; de la Banda, Esmeralda; Abrisqueta, Pau; Arenillas, Leonor; Serrano, Cristina; Lloreta, Josep; Miñana, Belén; Cerutti, Andrea; Florensa, Lourdes; Orfao, Alberto; Sanz, Ferran; Solé, Francesc; Dominguez-Sola, David; Serrano, Sergio

2015-01-01

Purpose According to current diagnostic criteria, mantle cell lymphoma (MCL) encompasses the usual, aggressive variants and rare, nonnodal cases with monoclonal asymptomatic lymphocytosis, cyclin D1–positive (MALD1). We aimed to understand the biology behind this clinical heterogeneity and to identify markers for adequate identification of MALD1 cases. Experimental Design We compared 17 typical MCL cases with a homogeneous group of 13 untreated MALD1 cases (median follow-up, 71 months). We conducted gene expression profiling with functional analysis in five MCL and five MALD1. Results were validated in 12 MCL and 8 MALD1 additional cases by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and in 24 MCL and 13 MALD1 cases by flow cytometry. Classification and regression trees strategy was used to generate an algorithm based on CD38 and CD200 expression by flow cytometry. Results We found 171 differentially expressed genes with enrichment of neoplastic behavior and cell proliferation signatures in MCL. Conversely, MALD1 was enriched in gene sets related to immune activation and inflammatory responses. CD38 and CD200 were differentially expressed between MCL and MALD1 and confirmed by flow cytometry (median CD38, 89% vs. 14%; median CD200, 0% vs. 24%, respectively). Assessment of both proteins allowed classifying 85% (11 of 13) of MALD1 cases whereas 15% remained unclassified. SOX11 expression by qRT-PCR was significantly different between MCL and MALD1 groups but did not improve the classification. Conclusion We show for the first time that MALD1, in contrast to MCL, is characterized by immune activation and driven by inflammatory cues. Assessment of CD38/CD200 by flow cytometry is useful to distinguish most cases of MALD1 from MCL in the clinical setting. MALD1 should be identified and segregated from the current MCL category to avoid overdiagnosis and unnecessary treatment. PMID:24352646

Improved signal recovery for flow cytometry based on ‘spatially modulated emission’

NASA Astrophysics Data System (ADS)

Quint, S.; Wittek, J.; Spang, P.; Levanon, N.; Walther, T.; Baßler, M.

2017-09-01

Recently, the technique of ‘spatially modulated emission’ has been introduced (Baßler et al 2008 US Patent 0080181827A1; Kiesel et al 2009 Appl. Phys. Lett. 94 041107; Kiesel et al 2011 Cytometry A 79A 317-24) improving the signal-to-noise ratio (SNR) for detecting bio-particles in the field of flow cytometry. Based on this concept, we developed two advanced signal processing methods which further enhance the SNR and selectivity for cell detection. The improvements are achieved by adapting digital filtering methods from RADAR technology and mainly address inherent offset elimination, increased signal dynamics and moreover reduction of erroneous detections due to processing artifacts. We present a comprehensive theory on SNR gain and provide experimental results of our concepts.

Phenotypic assays for Mycobacterium tuberculosis infection.

PubMed

Song, Ok-Ryul; Deboosere, Nathalie; Delorme, Vincent; Queval, Christophe J; Deloison, Gaspard; Werkmeister, Elisabeth; Lafont, Frank; Baulard, Alain; Iantomasi, Raffaella; Brodin, Priscille

2017-10-01

Tuberculosis (TB) is still a major global threat, killing more than one million persons each year. With the constant increase of Mycobacterium tuberculosis strains resistant to first- and second-line drugs, there is an urgent need for the development of new drugs to control the propagation of TB. Although screenings of small molecules on axenic M. tuberculosis cultures were successful for the identification of novel putative anti-TB drugs, new drugs in the development pipeline remains scarce. Host-directed therapy may represent an alternative for drug development against TB. Indeed, M. tuberculosis has multiple specific interactions within host phagocytes, which may be targeted by small molecules. In order to enable drug discovery strategies against microbes residing within host macrophages, we developed multiple fluorescence-based HT/CS phenotypic assays monitoring the intracellular replication of M. tuberculosis as well as its intracellular trafficking. What we propose here is a population-based, multi-parametric analysis pipeline that can be used to monitor the intracellular fate of M. tuberculosis and the dynamics of cellular events such as phagosomal maturation (acidification and permeabilization), zinc poisoning system or lipid body accumulation. Such analysis allows the quantification of biological events considering the host-pathogen interplay and may thus be derived to other intracellular pathogens. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brousseau, P.; Fugere, N.; Coderre, D.

Immunotoxic effects of environmental exposure to chemical contaminants can be evaluated by monitoring cellular and functional parameters of the immune system of sentinel species. In this scope, the earthworm may represent a relevant sentinel species to determine the level of toxicity linked to soil contaminants or to test the efficacy of remediation protocols. In this work, coelomocytes were incubated in vitro for 18 hours with mercury, cadmium, zinc or lead at concentrations ranging from 10{sup {minus}9} to 10{sup {minus}4}M. The analysis of phagocytosis by flow cytometry revealed that this natural response was impaired at non cytotoxic concentrations of mercury, cadmiummore » and zinc. Moreover, the analysis of cells obtained from coelomic fluid, based on the combination of low angle forward scatter (FSC) and side scatter (SSC) allowed to discriminate between two distinct populations of coelomocytes. With the use of fluorescent probes, such as carboxyfluorescin diacetate (CFDA), dichlorofluorescin diacetate (DCFDA) and chloromethyl fluorescin diacetate (CFDA), to study the esterase activity and Fluo-3 to measure free cytoplasmic calcium, the results showed that the first discrimination between the two populations of cells based on size and complexity could be further accentuated on the basis of their metabolic activities. In summary, the data make very attractive the use of flow cytometry to study cellular and functional parameters of the earthworm.« less

Fishing on chips: up-and-coming technological advances in analysis of zebrafish and Xenopus embryos.

PubMed

Zhu, Feng; Skommer, Joanna; Huang, Yushi; Akagi, Jin; Adams, Dany; Levin, Michael; Hall, Chris J; Crosier, Philip S; Wlodkowic, Donald

2014-11-01

Biotests performed on small vertebrate model organisms provide significant investigative advantages as compared with bioassays that employ cell lines, isolated primary cells, or tissue samples. The main advantage offered by whole-organism approaches is that the effects under study occur in the context of intact physiological milieu, with all its intercellular and multisystem interactions. The gap between the high-throughput cell-based in vitro assays and low-throughput, disproportionally expensive and ethically controversial mammal in vivo tests can be closed by small model organisms such as zebrafish or Xenopus. The optical transparency of their tissues, the ease of genetic manipulation and straightforward husbandry, explain the growing popularity of these model organisms. Nevertheless, despite the potential for miniaturization, automation and subsequent increase in throughput of experimental setups, the manipulation, dispensing and analysis of living fish and frog embryos remain labor-intensive. Recently, a new generation of miniaturized chip-based devices have been developed for zebrafish and Xenopus embryo on-chip culture and experimentation. In this work, we review the critical developments in the field of Lab-on-a-Chip devices designed to alleviate the limits of traditional platforms for studies on zebrafish and clawed frog embryo and larvae. © 2014 International Society for Advancement of Cytometry. © 2014 International Society for Advancement of Cytometry.

Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids

PubMed Central

Beaumont, Kimberley A.; Anfosso, Andrea; Ahmed, Farzana

2015-01-01

Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to traditional two-dimensional (2D) cell culture. The spheroid model is able to mimic the effects of cell-cell interaction, hypoxia and nutrient deprivation, and drug penetration. One characteristic of this model is the development of a necrotic core, surrounded by a ring of G1 arrested cells, with proliferating cells on the outer layers of the spheroid. Of interest in the cancer field is how different regions of the spheroid respond to drug therapies as well as genetic or environmental manipulation. We describe here the use of the fluorescence ubiquitination cell cycle indicator (FUCCI) system along with cytometry and image analysis using commercial software to characterize the cell cycle status of cells with respect to their position inside melanoma spheroids. These methods may be used to track changes in cell cycle status, gene/protein expression or cell viability in different sub-regions of tumor spheroids over time and under different conditions. PMID:26779761

Quantitative Magnetic Separation of Particles and Cells using Gradient Magnetic Ratcheting

PubMed Central

Murray, Coleman; Pao, Edward; Tseng, Peter; Aftab, Shayan; Kulkarni, Rajan; Rettig, Matthew; Di Carlo, Dino

2016-01-01

Extraction of rare target cells from biosamples is enabling for life science research. Traditional rare cell separation techniques, such as magnetic activated cell sorting (MACS), are robust but perform coarse, qualitative separations based on surface antigen expression. We report a quantitative magnetic separation technology using high-force magnetic ratcheting over arrays of magnetically soft micro-pillars with gradient spacing, and use the system to separate and concentrate magnetic beads based on iron oxide content (IOC) and cells based on surface expression. The system consists of a microchip of permalloy micro-pillar arrays with increasing lateral pitch and a mechatronic device to generate a cycling magnetic-field. Particles with higher IOC separate and equilibrate along the miro-pillar array at larger pitches. We develop a semi-analytical model that predicts behavior for particles and cells. Using the system, LNCaP cells were separated based on the bound quantity of 1μm anti-EpCAM particles as a metric for expression. The ratcheting cytometry system was able to resolve a ±13 bound particle differential, successfully distinguishing LNCaP from PC3 populations based on EpCAM expression, correlating with flow cytometry analysis. As a proof of concept, EpCAM-labeled cells from patient blood were isolated with 74% purity, demonstrating potential towards a quantitative magnetic separation instrument. PMID:26890496

Label-free counting of circulating cells by in vivo photoacoustic flow cytometry

NASA Astrophysics Data System (ADS)

Zhou, Quanyu; Yang, Ping; Wang, Qiyan; Pang, Kai; Zhou, Hui; He, Hao; Wei, Xunbin

2018-02-01

Melanoma, developing from melanocytes, is the most serious type of skin cancer. Circulating melanoma cells, the prognosis marker for metastasis, are present in the circulation at the early stage. Thus, quantitative detection of rare circulating melanoma cells is essential for monitoring tumor metastasis and prognosis evaluation. Compared with in vitro assays, in vivo flow cytometry is able to identify circulating tumor cells without drawing blood. Here, we built in vivo photoacoustic flow cytometry based on the high absorption coefficient of melanoma cells, which is applied to labelfree counting of circulating melanoma cells in tumor-bearing mice.

Optimizing transformations for automated, high throughput analysis of flow cytometry data

PubMed Central

2010-01-01

Background In a high throughput setting, effective flow cytometry data analysis depends heavily on proper data preprocessing. While usual preprocessing steps of quality assessment, outlier removal, normalization, and gating have received considerable scrutiny from the community, the influence of data transformation on the output of high throughput analysis has been largely overlooked. Flow cytometry measurements can vary over several orders of magnitude, cell populations can have variances that depend on their mean fluorescence intensities, and may exhibit heavily-skewed distributions. Consequently, the choice of data transformation can influence the output of automated gating. An appropriate data transformation aids in data visualization and gating of cell populations across the range of data. Experience shows that the choice of transformation is data specific. Our goal here is to compare the performance of different transformations applied to flow cytometry data in the context of automated gating in a high throughput, fully automated setting. We examine the most common transformations used in flow cytometry, including the generalized hyperbolic arcsine, biexponential, linlog, and generalized Box-Cox, all within the BioConductor flowCore framework that is widely used in high throughput, automated flow cytometry data analysis. All of these transformations have adjustable parameters whose effects upon the data are non-intuitive for most users. By making some modelling assumptions about the transformed data, we develop maximum likelihood criteria to optimize parameter choice for these different transformations. Results We compare the performance of parameter-optimized and default-parameter (in flowCore) data transformations on real and simulated data by measuring the variation in the locations of cell populations across samples, discovered via automated gating in both the scatter and fluorescence channels. We find that parameter-optimized transformations improve visualization, reduce variability in the location of discovered cell populations across samples, and decrease the misclassification (mis-gating) of individual events when compared to default-parameter counterparts. Conclusions Our results indicate that the preferred transformation for fluorescence channels is a parameter- optimized biexponential or generalized Box-Cox, in accordance with current best practices. Interestingly, for populations in the scatter channels, we find that the optimized hyperbolic arcsine may be a better choice in a high-throughput setting than current standard practice of no transformation. However, generally speaking, the choice of transformation remains data-dependent. We have implemented our algorithm in the BioConductor package, flowTrans, which is publicly available. PMID:21050468

Optimizing transformations for automated, high throughput analysis of flow cytometry data.

PubMed

Finak, Greg; Perez, Juan-Manuel; Weng, Andrew; Gottardo, Raphael

2010-11-04

In a high throughput setting, effective flow cytometry data analysis depends heavily on proper data preprocessing. While usual preprocessing steps of quality assessment, outlier removal, normalization, and gating have received considerable scrutiny from the community, the influence of data transformation on the output of high throughput analysis has been largely overlooked. Flow cytometry measurements can vary over several orders of magnitude, cell populations can have variances that depend on their mean fluorescence intensities, and may exhibit heavily-skewed distributions. Consequently, the choice of data transformation can influence the output of automated gating. An appropriate data transformation aids in data visualization and gating of cell populations across the range of data. Experience shows that the choice of transformation is data specific. Our goal here is to compare the performance of different transformations applied to flow cytometry data in the context of automated gating in a high throughput, fully automated setting. We examine the most common transformations used in flow cytometry, including the generalized hyperbolic arcsine, biexponential, linlog, and generalized Box-Cox, all within the BioConductor flowCore framework that is widely used in high throughput, automated flow cytometry data analysis. All of these transformations have adjustable parameters whose effects upon the data are non-intuitive for most users. By making some modelling assumptions about the transformed data, we develop maximum likelihood criteria to optimize parameter choice for these different transformations. We compare the performance of parameter-optimized and default-parameter (in flowCore) data transformations on real and simulated data by measuring the variation in the locations of cell populations across samples, discovered via automated gating in both the scatter and fluorescence channels. We find that parameter-optimized transformations improve visualization, reduce variability in the location of discovered cell populations across samples, and decrease the misclassification (mis-gating) of individual events when compared to default-parameter counterparts. Our results indicate that the preferred transformation for fluorescence channels is a parameter- optimized biexponential or generalized Box-Cox, in accordance with current best practices. Interestingly, for populations in the scatter channels, we find that the optimized hyperbolic arcsine may be a better choice in a high-throughput setting than current standard practice of no transformation. However, generally speaking, the choice of transformation remains data-dependent. We have implemented our algorithm in the BioConductor package, flowTrans, which is publicly available.

flowVS: channel-specific variance stabilization in flow cytometry

DOE Office of Scientific and Technical Information (OSTI.GOV)

Azad, Ariful; Rajwa, Bartek; Pothen, Alex

Comparing phenotypes of heterogeneous cell populations from multiple biological conditions is at the heart of scientific discovery based on flow cytometry (FC). When the biological signal is measured by the average expression of a biomarker, standard statistical methods require that variance be approximately stabilized in populations to be compared. Since the mean and variance of a cell population are often correlated in fluorescence-based FC measurements, a preprocessing step is needed to stabilize the within-population variances.

flowVS: channel-specific variance stabilization in flow cytometry

DOE PAGES

Azad, Ariful; Rajwa, Bartek; Pothen, Alex

2016-07-28

Comparing phenotypes of heterogeneous cell populations from multiple biological conditions is at the heart of scientific discovery based on flow cytometry (FC). When the biological signal is measured by the average expression of a biomarker, standard statistical methods require that variance be approximately stabilized in populations to be compared. Since the mean and variance of a cell population are often correlated in fluorescence-based FC measurements, a preprocessing step is needed to stabilize the within-population variances.

Photon spectroscopy by picoseconds differential Geiger-mode Si photomultiplier

NASA Astrophysics Data System (ADS)

Yamamoto, Masanobu; Hernandez, Keegan; Robinson, J. Paul

2018-02-01

The pixel array silicon photomultiplier (SiPM) is known as an excellent photon sensor with picoseconds avalanche process with the capacity for millions amplification of photoelectrons. In addition, a higher quantum efficiency(QE), small size, low bias voltage, light durability are attractive features for biological applications. The primary disadvantage is the limited dynamic range due to the 50ns recharge process and a high dark count which is an additional hurdle. We have developed a wide dynamic Si photon detection system applying ultra-fast differentiation signal processing, temperature control by thermoelectric device and Giga photon counter with 9 decimal digits dynamic range. The tested performance is six orders of magnitude with 600ps pulse width and sub-fW sensitivity. Combined with 405nm laser illumination and motored monochromator, Laser Induced Fluorescence Photon Spectrometry (LIPS) has been developed with a scan range from 200 900nm at maximum of 500nm/sec and 1nm FWHM. Based on the Planck equation E=hν, this photon counting spectrum provides a fundamental advance in spectral analysis by digital processing. Advantages include its ultimate sensitivity, theoretical linearity, as well as quantitative and logarithmic analysis without use of arbitrary units. Laser excitation is also useful for evaluation of photobleaching or oxidation in materials by higher energy illumination. Traditional typical photocurrent detection limit is about 1pW which includes millions of photons, however using our system it is possible to evaluate the photon spectrum and determine background noise and auto fluorescence(AFL) in optics in any cytometry or imaging system component. In addition, the photon-stream digital signal opens up a new approach for picosecond time-domain analysis. Photon spectroscopy is a powerful method for analysis of fluorescence and optical properties in biology.

ImmunoPET imaging of tissue factor expression in pancreatic cancer with 89Zr-Df-ALT-836.

PubMed

Hernandez, Reinier; England, Christopher G; Yang, Yunan; Valdovinos, Hector F; Liu, Bai; Wong, Hing C; Barnhart, Todd E; Cai, Weibo

2017-10-28

Overexpression of tissue factor (TF) has been associated with increased tumor growth, tumor angiogenesis, and metastatic potential in many malignancies, including pancreatic cancer. Additionally, high TF expression was shown to strongly correlate with poor prognoses and decreased survival in pancreatic cancer patients. Herein, we exploited the potential targeting of TF for positron emission tomography (PET) imaging of pancreatic cancer. The TF-targeted tracer was developed through radiolabeling of the anti-human TF monoclonal antibody (ALT-836) with 89 Zr. The tracer was characterized by fluorescence microscopy and flow cytometry assays in BXPC-3 and PANC-1 cells, two pancreatic cancer cell lines with high and low TF expression levels, respectively. Non-invasive PET scans were acquired in tumor-bearing mice injected with 89 Zr-Df-ALT-836. Additionally, ex vivo biodistribution, blocking, and histological studies were performed to establish the affinity and specificity of 89 Zr-Df-ALT-836 for TF in vivo. 89 Zr-labeling of Df-ALT-836 was achieved in high yield and good specific activity. Flow cytometry and microscopy studies revealed no detectable difference in TF-binding affinity between ALT-836 and Df-ALT-836 in vitro. Longitudinal PET scans unveiled a lasting and prominent 89 Zr-Df-ALT-836 uptake in BXPC-3 tumors (peak at 31.5±6.0%ID/g at 48h post-injection; n=3), which was significantly abrogated (2.3±0.5%ID/g at 48h post-injection; n=3) when mice were pre-injected with a blocking dose (50mg/kg) of unlabeled ALT-836. Ex vivo biodistribution data confirmed the accuracy of the PET results, and histological analysis correlated high tumor uptake with in situ TF expression. Taken together, these results attest to the excellent affinity and TF-specificity of 89 Zr-Df-ALT-836. With elevated, persistent, and specific accumulation in TF-positive BXPC-3 tumors, PET imaging using 89 Zr-Df-ALT-836 promises to open new avenues for improving future diagnosis, stratification, and treatment response assessment in pancreatic cancer patients. Copyright © 2017 Elsevier B.V. All rights reserved.

Sensitive detection of Escherichia coli O157:H7 in food and water by immunomagnetic separation and solid-phase laser cytometry

NASA Technical Reports Server (NTRS)

Pyle, B. H.; Broadaway, S. C.; McFeters, G. A.

1999-01-01

Rapid, direct methods are needed to assess active bacterial populations in water and foods. Our objective was to determine the efficiency of bacterial detection by immunomagnetic separation (IMS) and the compatibility of IMS with cyanoditolyl tetrazolium chloride (CTC) incubation to determine respiratory activity, using the pathogen Escherichia coli O157:H7. Counterstaining with a specific fluorescein-conjugated anti-O157 antibody (FAb) following CTC incubation was used to allow confirmation and visualization of bacteria by epifluorescence microscopy. Broth-grown E. coli O157:H7 was used to inoculate fresh ground beef (<17% fat), sterile 0.1% peptone, or water. Inoculated meat was diluted and homogenized in a stomacher and then incubated with paramagnetic beads coated with anti-O157 specific antibody. After IMS, cells with magnetic beads attached were stained with CTC and then an anti-O157 antibody-fluorescein isothiocyanate conjugate and filtered for microscopic enumeration or solid-phase laser cytometry. Enumeration by laser scanning permitted detection of ca. 10 CFU/g of ground beef or <10 CFU/ml of liquid sample. With inoculated meat, the regression results for log-transformed respiring FAb-positive counts of cells recovered on beads versus sorbitol-negative plate counts in the inoculum were as follows: intercept = 1.06, slope = 0.89, and r2 = 0. 95 (n = 13). The corresponding results for inoculated peptone were as follows: intercept = 0.67, slope = 0.88, and r2 = 0.98 (n = 24). Recovery of target bacteria on beads by the IMS-CTC-FAb method, compared with recovery by sorbitol MacConkey agar plating, yielded greater numbers (beef, 6.0 times; peptone, 3.0 times; water, 2.4 times). Thus, within 5 to 7 h, the IMS-CTC-FAb method detected greater numbers of E. coli O157 cells than were detected by plating. The results show that the IMS-CTC-FAb technique with enumeration by either fluorescence microscopy or solid-phase laser scanning cytometry gave results that compared favorably with plating following IMS.

Large-scale detection of antigen-specific T cells using peptide-MHC-I multimers labeled with DNA barcodes.

PubMed

Bentzen, Amalie Kai; Marquard, Andrea Marion; Lyngaa, Rikke; Saini, Sunil Kumar; Ramskov, Sofie; Donia, Marco; Such, Lina; Furness, Andrew J S; McGranahan, Nicholas; Rosenthal, Rachel; Straten, Per Thor; Szallasi, Zoltan; Svane, Inge Marie; Swanton, Charles; Quezada, Sergio A; Jakobsen, Søren Nyboe; Eklund, Aron Charles; Hadrup, Sine Reker

2016-10-01

Identification of the peptides recognized by individual T cells is important for understanding and treating immune-related diseases. Current cytometry-based approaches are limited to the simultaneous screening of 10-100 distinct T-cell specificities in one sample. Here we use peptide-major histocompatibility complex (MHC) multimers labeled with individual DNA barcodes to screen >1,000 peptide specificities in a single sample, and detect low-frequency CD8 T cells specific for virus- or cancer-restricted antigens. When analyzing T-cell recognition of shared melanoma antigens before and after adoptive cell therapy in melanoma patients, we observe a greater number of melanoma-specific T-cell populations compared with cytometry-based approaches. Furthermore, we detect neoepitope-specific T cells in tumor-infiltrating lymphocytes and peripheral blood from patients with non-small cell lung cancer. Barcode-labeled pMHC multimers enable the combination of functional T-cell analysis with large-scale epitope recognition profiling for the characterization of T-cell recognition in various diseases, including in small clinical samples.

rFRET: A comprehensive, Matlab-based program for analyzing intensity-based ratiometric microscopic FRET experiments.

PubMed

Nagy, Peter; Szabó, Ágnes; Váradi, Tímea; Kovács, Tamás; Batta, Gyula; Szöllősi, János

2016-04-01

Fluorescence or Förster resonance energy transfer (FRET) remains one of the most widely used methods for assessing protein clustering and conformation. Although it is a method with solid physical foundations, many applications of FRET fall short of providing quantitative results due to inappropriate calibration and controls. This shortcoming is especially valid for microscopy where currently available tools have limited or no capability at all to display parameter distributions or to perform gating. Since users of multiparameter flow cytometry usually apply these tools, the absence of these features in applications developed for microscopic FRET analysis is a significant limitation. Therefore, we developed a graphical user interface-controlled Matlab application for the evaluation of ratiometric, intensity-based microscopic FRET measurements. The program can calculate all the necessary overspill and spectroscopic correction factors and the FRET efficiency and it displays the results on histograms and dot plots. Gating on plots and mask images can be used to limit the calculation to certain parts of the image. It is an important feature of the program that the calculated parameters can be determined by regression methods, maximum likelihood estimation (MLE) and from summed intensities in addition to pixel-by-pixel evaluation. The confidence interval of calculated parameters can be estimated using parameter simulations if the approximate average number of detected photons is known. The program is not only user-friendly, but it provides rich output, it gives the user freedom to choose from different calculation modes and it gives insight into the reliability and distribution of the calculated parameters. © 2016 International Society for Advancement of Cytometry. © 2016 International Society for Advancement of Cytometry.

Flow cytometry analysis of hormone receptors on human peripheral blood mononuclear cells to identify stress-induced neuroendocrine effects

NASA Technical Reports Server (NTRS)

Meehan, R. T.

1986-01-01

Understanding the role of circulating peptide hormones in the pathogenesis of space-flight induced disorders would be greatly facilitated by a method which monitors chronic levels of hormones and their effects upon in vivo cell physiology. Single and simultaneous multiparameter flow cytometry analysis was employed to identify subpopulations of mononuclear cells bearing receptors for ACTH, Endorphin, and Somatomedin-C using monoclonal antibodies and monospecific antisera with indirect immunofluorescence. Blood samples were obtained from normal donors and subjects participating in decompression chamber studies (acute stress), medical student academic examination (chronic stress), and a drug study (Dexamethasone). Preliminary results indicate most ACTH and Endorphin receptor positive cells are monocytes and B-cells, exhibit little diurnal variation but the relative percentages of receptor positive cells are influenced by exposure to various stressors and ACTH inhibition. This study demonstrates the capability of flow cytometry analysis to study cell surface hormone receptor regulation which should allow insight into neuroendocrine modulation of the immune and other cellular systems during exposure to stress or microgravity.

Cellular vacuolation and mitochondrial-associated factors induced by Clostridium perfringens epsilon toxin detected using acoustic flow cytometry.

PubMed

Ferrarezi, Marina C; Curci, Vera C L M; Cardoso, Tereza C

2013-12-01

Epsilon toxin (ETX) produced by Clostridium perfringens types B and D is a potent toxin that is responsible for fatal enterotoxaemia. In vitro, ETX, which is considered as a pore-forming toxin, forms a heptamer in Madin-Darby canine kidney (MDCK) cell membranes, which is considered to be a pre-pore stage. After binding of the ETX, vacuoles inside cell cytoplasm are produced. ETX causes decreased levels of essential coenzymes required for host cell energy. Here, we optimized and applied acoustic flow cytometry analysis in order to gain further insight into ETX-pathogenesis. Using acoustic flow cytometer analysis, which considered highly sensitive, ETX-exposed MDCK cells revealed mitochondrial membrane decreases followed by 25.48% and 45.45% of the exposed cells expressing the Bax and BCL-2 proteins at a pre-pore stage, respectively. These results together with high cytotoxicity and visualization of cell vacuoles, demonstrates that acoustic flow cytometry analysis potentially represents an effective tool to study ETX pathogenesis. Copyright © 2013. Published by Elsevier Ltd.

Multiplex polymerase chain reaction on FTA cards vs. flow cytometry for B-lymphocyte clonality.

PubMed

Dictor, Michael; Skogvall, Ingela; Warenholt, Janina; Rambech, Eva

2007-01-01

Two-colour flow cytometry was compared with multiplex PCR with capillary electrophoresis for clonality determination in specific categories of B-cell lymphoma. FTA cards were evaluated for preserving DNA from node imprints and expediting molecular analysis. A single-tube multiplex PCR targeted IGH and lymphoma-specific translocations in DNA extracted from 180 frozen lymphoid tissues and DNA bound to FTA cards from 192 fresh tissues and 137 aspirates. PCR results were compared with flow cytometry in the extracted and aspirated samples. Overall, single-tube multiplex PCR sensitivity was equivalent in the sample groups (intergroup range 79%-91%). False negatives were associated with tumour origin in the follicle centre. Multiplex PCR and flow cytometry were equally sensitive and together detected 98% of B-cell lymphomas. Additional two-tube targeting of IGK suggested an overall molecular sensitivity >90%. False positive (pseudoclonal) single-tube multiplex PCR was associated with necrosis and sparse lymphocytes. Multiplex PCR using template DNA bound to an FTA card effectively detects B-lymphocyte clonality, obviates DNA extraction and refrigeration, and can be used without diminished sensitivity in fine needle aspirates or node imprints as a replacement for or complement to flow cytometry at any point in the diagnostic work-up.

DNA Detection by Flow Cytometry using PNA-Modified Metal-Organic Framework Particles.

PubMed

Mejia-Ariza, Raquel; Rosselli, Jessica; Breukers, Christian; Manicardi, Alex; Terstappen, Leon W M M; Corradini, Roberto; Huskens, Jurriaan

2017-03-23

A DNA-sensing platform is developed by exploiting the easy surface functionalization of metal-organic framework (MOF) particles and their highly parallelized fluorescence detection by flow cytometry. Two strategies were employed to functionalize the surface of MIL-88A, using either covalent or non-covalent interactions, resulting in alkyne-modified and biotin-modified MIL-88A, respectively. Covalent surface coupling of an azide-dye and the alkyne-MIL-88A was achieved by means of a click reaction. Non-covalent streptavidin-biotin interactions were employed to link biotin-PNA to biotin-MIL-88A particles mediated by streptavidin. Characterization by confocal imaging and flow cytometry demonstrated that DNA can be bound selectively to the MOF surface. Flow cytometry provided quantitative data of the interaction with DNA. Making use of the large numbers of particles that can be simultaneously processed by flow cytometry, this MOF platform was able to discriminate between fully complementary, single-base mismatched, and randomized DNA targets. © 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

The development and validation of different decision-making tools to predict urine culture growth out of urine flow cytometry parameter.

PubMed

Müller, Martin; Seidenberg, Ruth; Schuh, Sabine K; Exadaktylos, Aristomenis K; Schechter, Clyde B; Leichtle, Alexander B; Hautz, Wolf E

2018-01-01

Patients presenting with suspected urinary tract infection are common in every day emergency practice. Urine flow cytometry has replaced microscopic urine evaluation in many emergency departments, but interpretation of the results remains challenging. The aim of this study was to develop and validate tools that predict urine culture growth out of urine flow cytometry parameter. This retrospective study included all adult patients that presented in a large emergency department between January and July 2017 with a suspected urinary tract infection and had a urine flow cytometry as well as a urine culture obtained. The objective was to identify urine flow cytometry parameters that reliably predict urine culture growth and mixed flora growth. The data set was split into a training (70%) and a validation set (30%) and different decision-making approaches were developed and validated. Relevant urine culture growth (respectively mixed flora growth) was found in 40.2% (7.2% respectively) of the 613 patients included. The number of leukocytes and bacteria in flow cytometry were highly associated with urine culture growth, but mixed flora growth could not be sufficiently predicted from the urine flow cytometry parameters. A decision tree, predictive value figures, a nomogram, and a cut-off table to predict urine culture growth from bacteria and leukocyte count were developed, validated and compared. Urine flow cytometry parameters are insufficient to predict mixed flora growth. However, the prediction of urine culture growth based on bacteria and leukocyte count is highly accurate and the developed tools should be used as part of the decision-making process of ordering a urine culture or starting an antibiotic therapy if a urogenital infection is suspected.

The development and validation of different decision-making tools to predict urine culture growth out of urine flow cytometry parameter

PubMed Central

Seidenberg, Ruth; Schuh, Sabine K.; Exadaktylos, Aristomenis K.; Schechter, Clyde B.; Leichtle, Alexander B.; Hautz, Wolf E.

2018-01-01

Objective Patients presenting with suspected urinary tract infection are common in every day emergency practice. Urine flow cytometry has replaced microscopic urine evaluation in many emergency departments, but interpretation of the results remains challenging. The aim of this study was to develop and validate tools that predict urine culture growth out of urine flow cytometry parameter. Methods This retrospective study included all adult patients that presented in a large emergency department between January and July 2017 with a suspected urinary tract infection and had a urine flow cytometry as well as a urine culture obtained. The objective was to identify urine flow cytometry parameters that reliably predict urine culture growth and mixed flora growth. The data set was split into a training (70%) and a validation set (30%) and different decision-making approaches were developed and validated. Results Relevant urine culture growth (respectively mixed flora growth) was found in 40.2% (7.2% respectively) of the 613 patients included. The number of leukocytes and bacteria in flow cytometry were highly associated with urine culture growth, but mixed flora growth could not be sufficiently predicted from the urine flow cytometry parameters. A decision tree, predictive value figures, a nomogram, and a cut-off table to predict urine culture growth from bacteria and leukocyte count were developed, validated and compared. Conclusions Urine flow cytometry parameters are insufficient to predict mixed flora growth. However, the prediction of urine culture growth based on bacteria and leukocyte count is highly accurate and the developed tools should be used as part of the decision-making process of ordering a urine culture or starting an antibiotic therapy if a urogenital infection is suspected. PMID:29474463

Development of fluorescent glucose bioprobes and their application on real-time and quantitative monitoring of glucose uptake in living cells.

PubMed

Lee, Hyang Yeon; Lee, Jae Jeong; Park, Jongmin; Park, Seung Bum

2011-01-03

We developed a novel fluorescent glucose bioprobe, GB2-Cy3, for the real-time and quantitative monitoring of glucose uptake in living cells. We synthesized a series of fluorescent glucose analogues by adding Cy3 fluorophores to the α-anomeric position of D-glucose through various linkers. Systematic and quantitative analysis of these Cy3-labeled glucose analogues revealed that GB2-Cy3 was the ideal fluorescent glucose bioprobe. The cellular uptake of this probe competed with the cellular uptake of D-glucose in the media and was mediated by a glucose-specific transport system, and not by passive diffusion. Flow cytometry and fluorescence microscopy analyses revealed that GB2-Cy3 is ten times more sensitive than 2-NBDG, a leading fluorescent glucose bioprobe. GB2-Cy3 can also be utilized for the quantitative flow cytometry monitoring of glucose uptake in metabolically active C2C12 myocytes under various treatment conditions. As opposed to a glucose uptake assay performed by using radioisotope-labeled deoxy-D-glucose and a scintillation counter, GB2-Cy3 allows the real-time monitoring of glucose uptake in living cells under various experimental conditions by using fluorescence microscopy or confocal laser scanning microscopy (CLSM). Therefore, we believe that GB2-Cy3 can be utilized in high-content screening (HCS) for the discovery of novel therapeutic agents and for making significant advances in biomedical studies and diagnosis of various diseases, especially metabolic diseases. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Novel full‐spectral flow cytometry with multiple spectrally‐adjacent fluorescent proteins and fluorochromes and visualization of in vivo cellular movement

PubMed Central

Futamura, Koji; Sekino, Masashi; Hata, Akihiro; Ikebuchi, Ryoyo; Nakanishi, Yasutaka; Egawa, Gyohei; Kabashima, Kenji; Watanabe, Takeshi; Furuki, Motohiro

2015-01-01

Abstract Flow cytometric analysis with multicolor fluoroprobes is an essential method for detecting biological signatures of cells. Here, we present a new full‐spectral flow cytometer (spectral‐FCM). Unlike conventional flow cytometer, this spectral‐FCM acquires the emitted fluorescence for all probes across the full‐spectrum from each cell with 32 channels sequential PMT unit after dispersion with prism, and extracts the signals of each fluoroprobe based on the spectral shape of each fluoroprobe using unique algorithm in high speed, high sensitive, accurate, automatic and real‐time. The spectral‐FCM detects the continuous changes in emission spectra from green to red of the photoconvertible protein, KikGR with high‐spectral resolution and separates spectrally‐adjacent fluoroprobes, such as FITC (Emission peak (Em) 519 nm) and EGFP (Em 507 nm). Moreover, the spectral‐FCM can measure and subtract autofluorescence of each cell providing increased signal‐to‐noise ratios and improved resolution of dim samples, which leads to a transformative technology for investigation of single cell state and function. These advances make it possible to perform 11‐color fluorescence analysis to visualize movement of multilinage immune cells by using KikGR‐expressing mice. Thus, the novel spectral flow cytometry improves the combinational use of spectrally‐adjacent various FPs and multicolor fluorochromes in metabolically active cell for the investigation of not only the immune system but also other research and clinical fields of use. © 2015 The Authors. Cytometry Part A Published by Wiley Periodicals, Inc. on behalf of ISAC PMID:26217952

Progress on an implementation of MIFlowCyt in XML

NASA Astrophysics Data System (ADS)

Leif, Robert C.; Leif, Stephanie H.

2015-03-01

Introduction: The International Society for Advancement of Cytometry (ISAC) Data Standards Task Force (DSTF) has created a standard for the Minimum Information about a Flow Cytometry Experiment (MIFlowCyt 1.0). The CytometryML schemas, are based in part upon the Flow Cytometry Standard and Digital Imaging and Communication (DICOM) standards. CytometryML has and will be extended and adapted to include MIFlowCyt, as well as to serve as a common standard for flow and image cytometry (digital microscopy). Methods: The MIFlowCyt data-types were created, as is the rest of CytometryML, in the XML Schema Definition Language (XSD1.1). Individual major elements of the MIFlowCyt schema were translated into XML and filled with reasonable data. A small section of the code was formatted with HTML formatting elements. Results: The differences in the amount of detail to be recorded for 1) users of standard techniques including data analysts and 2) others, such as method and device creators, laboratory and other managers, engineers, and regulatory specialists required that separate data-types be created to describe the instrument configuration and components. A very substantial part of the MIFlowCyt element that describes the Experimental Overview part of the MIFlowCyt and substantial parts of several other major elements have been developed. Conclusions: The future use of structured XML tags and web technology should facilitate searching of experimental information, its presentation, and inclusion in structured research, clinical, and regulatory documents, as well as demonstrate in publications adherence to the MIFlowCyt standard. The use of CytometryML together with XML technology should also result in the textual and numeric data being published using web technology without any change in composition. Preliminary testing indicates that CytometryML XML pages can be directly formatted with the combination of HTML and CSS.

Discovering cell types in flow cytometry data with random matrix theory

NASA Astrophysics Data System (ADS)

Shen, Yang; Nussenblatt, Robert; Losert, Wolfgang

Flow cytometry is a widely used experimental technique in immunology research. During the experiments, peripheral blood mononuclear cells (PBMC) from a single patient, labeled with multiple fluorescent stains that bind to different proteins, are illuminated by a laser. The intensity of each stain on a single cell is recorded and reflects the amount of protein expressed by that cell. The data analysis focuses on identifying specific cell types related to a disease. Different cell types can be identified by the type and amount of protein they express. To date, this has most often been done manually by labelling a protein as expressed or not while ignoring the amount of expression. Using a cross correlation matrix of stain intensities, which contains both information on the proteins expressed and their amount, has been largely ignored by researchers as it suffers from measurement noise. Here we present an algorithm to identify cell types in flow cytometry data which uses random matrix theory (RMT) to reduce noise in a cross correlation matrix. We demonstrate our method using a published flow cytometry data set. Compared with previous analysis techniques, we were able to rediscover relevant cell types in an automatic way. Department of Physics, University of Maryland, College Park, MD 20742.

Flow cytometry analysis of inflammatory cells isolated from the sciatic nerve and DRG after chronic constriction injury in mice.

PubMed

Liu, Liping; Yin, Yan; Li, Fei; Malhotra, Charvi; Cheng, Jianguo

2017-06-01

Cellular responses to nerve injury play a central role in the pathogenesis of neuropathic pain. However, the analysis of site specific cellular responses to nerve injury and neuropathic pain is limited to immunohistochemistry staining with numerous limitations. We proposed to apply flow cytometry to overcome some of the limitations and developed two protocols for isolation of cells from small specimens of the sciatic nerve and dorsal root ganglion (DRG) in mice. RESULTS AND COMPARASION WITH EXISTING: methods We found that both the non-enzymatic and enzymatic approaches were highly effective in harvesting a sufficient number of cells for flow cytometry analysis in normal and pathological conditions. The total number of cells in the injury site of the sciatic and its DRGs increased significantly 14days after chronic constriction injury (CCI) of the sciatic nerve, compared to sham surgery control or the contralateral control. The enzymatic approach yielded a significantly higher total number of cells and CD45 negative cells, suggesting that this approach allows for harvest of more resident cells, compared to the non-enzymatic method. The percentage of CD45 + /CD11b + cells was significantly increased in the sciatic nerve but not in the DRG. These results were consistent with both protocols. We thus offer two simple and effective protocols that allow for application of flow cytometry to the investigation of cellular and molecular mechanisms of neuropathic pain. Copyright © 2017 Elsevier B.V. All rights reserved.

Development of scan analysis techniques employing a small computer. Final report, February 1, 1963--July 31, 1976

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kuhl, D.E.

1976-08-05

During the thirteen year duration of this contract the goal has been to develop and apply computer based analysis of radionuclide scan data so as to make available improved diagnostic information based on a knowledge of localized quantitative estimates of radionuclide concentration. Results are summarized. (CH)

Use of flow cytometry, fluorescence microscopy, and PCR-based techniques to assess intraspecific and interspecific matings of Armillaria species

Treesearch

Mee-Sook Kim; Ned B. Klopfenstein; Geral I. McDonald; Kathiravetpillai Arumuganathan

2001-01-01

For assessments of intraspecific mating using flow cytometry and fluorescence microscopy, two compatible basidiospore-derived isolates were selected from each of four parental basidiomata of North American Biological Species (NABS) X. The nuclear status in NABS X varied with basidiospore-derived isolates. Nuclei within basidiospore-derived isolates existed as haploids...

Development of small and inexpensive digital data acquisition systems using a microcontroller-based approach†

PubMed Central

Naivar, Mark A.; Wilder, Mark E.; Habbersett, Robert C.; Woods, Travis A.; Sebba, David S.; Nolan, John P.; Graves, Steven W.

2014-01-01

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 and resolution required for most flow cytometry applications. It is also capable of millisecond time resolution, full waveform collection, and selective triggering of data collection from a CCD camera. The capability of our demonstration system suggests that the use of microcontrollers for flow cytometry digital data-acquisition will be increasingly valuable for extending the life of older cytometers and provides a compelling data-system design approach for low-cost, portable flow cytometers. PMID:19852060

Development of small and inexpensive digital data acquisition systems using a microcontroller-based approach.

PubMed

Naivar, Mark A; Wilder, Mark E; Habbersett, Robert C; Woods, Travis A; Sebba, David S; Nolan, John P; Graves, Steven W

2009-12-01

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 and resolution required for most flow cytometry applications. It is also capable of millisecond time resolution, full waveform collection, and selective triggering of data collection from a CCD camera. The capability of our demonstration system suggests that the use of microcontrollers for flow cytometry digital data-acquisition will be increasingly valuable for extending the life of older cytometers and provides a compelling data-system design approach for low-cost, portable flow cytometers.

Data File Standard for Flow Cytometry, version FCS 3.1.

PubMed

Spidlen, Josef; Moore, Wayne; Parks, David; Goldberg, Michael; Bray, Chris; Bierre, Pierre; Gorombey, Peter; Hyun, Bill; Hubbard, Mark; Lange, Simon; Lefebvre, Ray; Leif, Robert; Novo, David; Ostruszka, Leo; Treister, Adam; Wood, James; Murphy, Robert F; Roederer, Mario; Sudar, Damir; Zigon, Robert; Brinkman, Ryan R

2010-01-01

The flow cytometry data file standard provides the specifications needed to completely describe flow cytometry data sets within the confines of the file containing the experimental data. In 1984, the first Flow Cytometry Standard format for data files was adopted as FCS 1.0. This standard was modified in 1990 as FCS 2.0 and again in 1997 as FCS 3.0. 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 volume, information about originality of the data file, and support for plate and well identification in high throughput, plate based experiments. Please see the normative version of the FCS 3.1 specification in Supporting Information for this manuscript (or at http://www.isac-net.org/ in the Current standards section) for a complete list of changes.

Data File Standard for Flow Cytometry, Version FCS 3.1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Spidlen, Josef; Moore, Wayne; Parks, David

2009-11-10

The flow cytometry data file standard provides the specifications needed to completely describe flow cytometry data sets within the confines of the file containing the experimental data. In 1984, the first Flow Cytometry Standard format for data files was adopted as FCS 1.0. This standard was modified in 1990 as FCS 2.0 and again in 1997 as FCS 3.0. 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 allowsmore » 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 volume, information about originality of the data file, and support for plate and well identification in high throughput, plate based experiments. Please see the normative version of the FCS 3.1 specification in Supporting Information for this manuscript (or at http://www.isac-net.org/ in the Current standards section) for a complete list of changes.« less

Advanced detection and measurement of cells on membrane from peripheral blood by laser scanning cytometry (LSC) in early stage breast cancer patients.

PubMed

Sanislo, L; Kuliffay, P; Sedlak, J; Kausitz, J; Galbavy, S

2010-01-01

The aim of our study was the potential detection of circulating tumour cells (CTCs) in early stage breast cancer patients. Our approach was cell microfiltration through polycarbonate membrane as a concentration method suitable for CTC selection in peripheral blood. The isolated cells on membrane were further analysed by laser scanning cytometry. Sixteen patients were enrolled in the study, of which 13 had early stage breast carcinoma and 3 patients had metastatic breast carcinoma. The analyses were performed from 9 ml of peripheral blood, in one patient blood was drawn twice. Blood samples were taken after adjuvant chemotherapy but prior to adjuvant radiotherapy. The control group consisted of 12 clinically healthy subjects. In the control group 3 subjects out of 12 had 1 CTC, the mean CTC numbers being 0.25 +/- 0.45. In the early stage breast cancer patients 0-36 CTCs were detected (mean 13.9 +/- 12.9 CTCs. 10 patients out of 13 had more than 2 CTCs (62%). The detection and measurement of cells on membrane is a simple and reproducible method of detection of CTCs in peripheral blood. Sensitivity of the method is 88.5%. Detection of CTCs seems to be a promising method for the monitoring of adjuvant therapy in early stage breast cancer patients and for the identification of high risk patients in whom elevated numbers of CTCs are persisting following the termination of adjuvant therapy (Tab. 1, Fig. 4, Ref. 35). Full Text (Free, PDF) www.bmj.sk.

Comprehensive Mass Cytometry Analysis of Cell Cycle, Activation, and Coinhibitory Receptors Expression in CD4 T Cells from Healthy and HIV-Infected Individuals.

PubMed

Corneau, Aurélien; Cosma, Antonio; Even, Sophie; Katlama, Christine; Le Grand, Roger; Frachet, Véronique; Blanc, Catherine; Autran, Brigitte

2017-01-01

Mass cytometry allows large multiplex analysis of cell cycle stages together with differentiation, activation, and exhaustion markers, allowing further assessment of the quiescence status of resting CD4 T cells. Peripheral blood CD4 T lymphocytes from 8 individuals, 4 healthy donors, and 4 HIV-infected on antiretroviral treatment (T) were stained with the same 26 monoclonal antibodies and dyes targeting surface and intracellular markers of differentiation, activation, exhaustion, and cell cycle stages. Samples were run on a CYTOF-2. Patterns of naïve [TN] CD4 T cells strongly differed from all other memory subsets central-memory (CM), transitional-memory (TM), effector-memory (EM), and terminally differentiated RA-expressing (TEMRA) subsets, while stem-cell memory (SCM) and T follicular-helper cells (TfH) were close to CM and TM cells with the highest percentages in cell cycle. EM and TEMRA were the most altered by HIV infection, with an increased frequency of activated and cycling cells. Activation markers and coinhibitory receptor expression differed among cell cycle stages, with HLA-DR fitting better than CD25 or CD38 with cycle, and opposite PD-1 gradients along differentiation and cell cycle. "Resting" DR-CD25- CD4+ T cells contained similar amounts of cells in G1 than the activated DR ± CD25± ones but three fold lower cells in S-G2-M. This broad multiplex mass cytometry analysis demonstrates some subsets of the so-called "resting" CD25-DR- CD4+ T cells contain noticeable amounts of cells into cycle or expressing coinhibitory receptors, opening new avenues for a redefinition of resting peripheral blood CD4 T cells harboring the HIV reservoirs. © 2016 International Clinical Cytometry Society. © 2016 International Clinical Cytometry Society.

FuGEFlow: data model and markup language for flow cytometry.

PubMed

Qian, Yu; Tchuvatkina, Olga; Spidlen, Josef; Wilkinson, Peter; Gasparetto, Maura; Jones, Andrew R; Manion, Frank J; Scheuermann, Richard H; Sekaly, Rafick-Pierre; Brinkman, Ryan R

2009-06-16

Flow cytometry technology is widely used in both health care and research. The rapid expansion of flow cytometry applications has outpaced the development of data storage and analysis tools. Collaborative efforts being taken to eliminate this gap include building common vocabularies and ontologies, designing generic data models, and defining data exchange formats. The Minimum Information about a Flow Cytometry Experiment (MIFlowCyt) standard was recently adopted by the International Society for Advancement of Cytometry. This standard guides researchers on the information that should be included in peer reviewed publications, but it is insufficient for data exchange and integration between computational systems. The Functional Genomics Experiment (FuGE) formalizes common aspects of comprehensive and high throughput experiments across different biological technologies. We have extended FuGE object model to accommodate flow cytometry data and metadata. We used the MagicDraw modelling tool to design a UML model (Flow-OM) according to the FuGE extension guidelines and the AndroMDA toolkit to transform the model to a markup language (Flow-ML). We mapped each MIFlowCyt term to either an existing FuGE class or to a new FuGEFlow class. The development environment was validated by comparing the official FuGE XSD to the schema we generated from the FuGE object model using our configuration. After the Flow-OM model was completed, the final version of the Flow-ML was generated and validated against an example MIFlowCyt compliant experiment description. The extension of FuGE for flow cytometry has resulted in a generic FuGE-compliant data model (FuGEFlow), which accommodates and links together all information required by MIFlowCyt. The FuGEFlow model can be used to build software and databases using FuGE software toolkits to facilitate automated exchange and manipulation of potentially large flow cytometry experimental data sets. Additional project documentation, including reusable design patterns and a guide for setting up a development environment, was contributed back to the FuGE project. We have shown that an extension of FuGE can be used to transform minimum information requirements in natural language to markup language in XML. Extending FuGE required significant effort, but in our experiences the benefits outweighed the costs. The FuGEFlow is expected to play a central role in describing flow cytometry experiments and ultimately facilitating data exchange including public flow cytometry repositories currently under development.

Evaluation of the platelet counting by Abbott CELL-DYN SAPPHIRE haematology analyser compared with flow cytometry.

PubMed

Grimaldi, E; Del Vecchio, L; Scopacasa, F; Lo Pardo, C; Capone, F; Pariante, S; Scalia, G; De Caterina, M

2009-04-01

The Abbot Cell-Dyn Sapphire is a new generation haematology analyser. The system uses optical/fluorescence flow cytometry in combination with electronic impedance to produce a full blood count. Optical and impedance are the default methods for platelet counting while automated CD61-immunoplatelet analysis can be run as selectable test. The aim of this study was to determine the platelet count performance of the three counting methods available on the instrument and to compare the results with those provided by Becton Dickinson FACSCalibur flow cytometer used as reference method. A lipid interference experiment was also performed. Linearity, carryover and precision were good, and satisfactory agreement with reference method was found for the impedance, optical and CD61-immunoplatelet analysis, although this latter provided the closest results in comparison with flow cytometry. In the lipid interference experiment, a moderate inaccuracy of optical and immunoplatelet counts was observed starting from a very high lipid value.

Multiplexed mass cytometry profiling of cellular states perturbed by small-molecule regulators

PubMed Central

Bodenmiller, Bernd; Zunder, Eli R.; Finck, Rachel; Chen, Tiffany J.; Savig, Erica S.; Bruggner, Robert V.; Simonds, Erin F.; Bendall, Sean C.; Sachs, Karen; Krutzik, Peter O.; Nolan, Garry P.

2013-01-01

The ability to comprehensively explore the impact of bio-active molecules on human samples at the single-cell level can provide great insight for biomedical research. Mass cytometry enables quantitative single-cell analysis with deep dimensionality, but currently lacks high-throughput capability. Here we report a method termed mass-tag cellular barcoding (MCB) that increases mass cytometry throughput by sample multiplexing. 96-well format MCB was used to characterize human peripheral blood mononuclear cell (PBMC) signaling dynamics, cell-to-cell communication, the signaling variability between 8 donors, and to define the impact of 27 inhibitors on this system. For each compound, 14 phosphorylation sites were measured in 14 PBMC types, resulting in 18,816 quantified phosphorylation levels from each multiplexed sample. This high-dimensional systems-level inquiry allowed analysis across cell-type and signaling space, reclassified inhibitors, and revealed off-target effects. MCB enables high-content, high-throughput screening, with potential applications for drug discovery, pre-clinical testing, and mechanistic investigation of human disease. PMID:22902532

Benchmarking of a treatment planning system for spot scanning proton therapy: Comparison and analysis of robustness to setup errors of photon IMRT and proton SFUD treatment plans of base of skull meningioma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Harding, R., E-mail: ruth.harding2@wales.nhs.uk; Trnková, P.; Lomax, A. J.

Purpose: Base of skull meningioma can be treated with both intensity modulated radiation therapy (IMRT) and spot scanned proton therapy (PT). One of the main benefits of PT is better sparing of organs at risk, but due to the physical and dosimetric characteristics of protons, spot scanned PT can be more sensitive to the uncertainties encountered in the treatment process compared with photon treatment. Therefore, robustness analysis should be part of a comprehensive comparison between these two treatment methods in order to quantify and understand the sensitivity of the treatment techniques to uncertainties. The aim of this work was tomore » benchmark a spot scanning treatment planning system for planning of base of skull meningioma and to compare the created plans and analyze their robustness to setup errors against the IMRT technique. Methods: Plans were produced for three base of skull meningioma cases: IMRT planned with a commercial TPS [Monaco (Elekta AB, Sweden)]; single field uniform dose (SFUD) spot scanning PT produced with an in-house TPS (PSI-plan); and SFUD spot scanning PT plan created with a commercial TPS [XiO (Elekta AB, Sweden)]. A tool for evaluating robustness to random setup errors was created and, for each plan, both a dosimetric evaluation and a robustness analysis to setup errors were performed. Results: It was possible to create clinically acceptable treatment plans for spot scanning proton therapy of meningioma with a commercially available TPS. However, since each treatment planning system uses different methods, this comparison showed different dosimetric results as well as different sensitivities to setup uncertainties. The results confirmed the necessity of an analysis tool for assessing plan robustness to provide a fair comparison of photon and proton plans. Conclusions: Robustness analysis is a critical part of plan evaluation when comparing IMRT plans with spot scanned proton therapy plans.« less

Determination of allergenic load and pollen count of Cupressus arizonica pollen by flow cytometry using Cup a1 polyclonal antibody.

PubMed

Benítez, Francisco Moreno; Camacho, Antonio Letrán; Del Cuvillo Bernal, Alfonso; de Medina, Pedro Lobatón Sánchez; Cózar, Francisco J García; Romeu, Ma Luisa Espinazo

2013-07-10

Background: There is an increase in the incidence of pollen related allergy, thus information on pollen schedules would be a great asset for physicians to improve the clinical care of patients. Like cypress pollen sensitization shows a high prevalence among the causes of allergic rhinitis, and therefore it is of interest to use it like a model of study, distinguishing cypress pollen, pollen count and allergenic load level. In this work, we use a flow cytometry based technique to obtain both Cupressus arizonica pollen count and allergenic load, using specific rabbit polyclonal antibody Cup a1 and its comparison with optical microscopy technique measurement. Methods: Airborne samples were collected from Burkard Spore-Trap and Burkard Cyclone Cupressus arizonica pollen was studied using specific rabbit polyclonal antibody Cup a1, labelled with AlexaFluor ® 488 or 750 and analysed by Flow Cytometry in both an EPICS XL and Cyan ADP cytometers (Beckman Coulter ® ). Optical microscopy study was realized with a Leica optical microscope. Bland & Altman was used to determine agreement between both techniques measured. Results: We can identify three different populations based on rabbit polyclonal antibody Cup a1 staining. The main region (44.5%) had 97.3% recognition, a second region (25%) with 28% and a third region (30.5%) with 68% respectively. Immunofluorescence and confocal microscopy showed that main region corresponds to whole pollen grains, the second region are pollen without exine and the third region is constituted by smaller particles with allergenic properties. Pollen schedule shows a higher correlation measured by optical microscopy and flow cytometry in the pollen count with a p-value: 0.0008E -2 and 0.0002 with regard to smaller particles, so the Bland & Altman measurement showed a good correlation between them, p-value: 0,0003. Conclusion: Determination of pollen count and allergenic load by flow cytometry represents an important tool in the determination of airborne respiratory allergens. We showed that not only whole pollen but also smaller particles could induce allergic sensitization. This is the first study where flow cytometry is used for calculating pollen counts and allergenic load. © 2013 Clinical Cytometry Society. Copyright © 2013 Clinical Cytometry Society.

Erythrocyte membrane based cationic polymer-mcDNA complexes as an efficient gene delivery system.

PubMed

Huang, Ping; Zhao, Jing; Wei, Chiju; Hou, Xiaohu; Chen, Pingzhang; Tan, Yan; He, Cheng-Yi; Wang, Zhiyong; Chen, Zhi-Ying

2016-12-20

Gene therapy has great promise for the treatment of obtained and inherited serious diseases. However, the lack of safe and efficient gene delivery systems remains a barrier for their clinical application. Here, we reported a potential gene delivery vehicle composed of the erythrocyte membrane and cationic polymers, for example the XtremeGENE from Roche and the ε-caprolactone modified polyethylenimine. In addition to high efficiency, this system showed negligible cytotoxicity compared to the two cationic polymers alone in various cell lines, including human embryonic kidney cells (293T), human liver cancer cells (Huh7 and HepG2), murine dendritic cells (DC2.4) and human umbilical cord mesenchymal stem cells (Hu-MSCs). Moreover, the results of confocal laser scanning microscopy and flow cytometry suggested that the cell uptake of this gene vector was improved and might be introduced by the fusion interaction between the erythrocyte membrane and targeted cells.Thus, all the results revealed that the erythrocyte membrane based gene delivery system might be able to serve as an excellent gene delivery system.

Genetic diversity and population structure of Musa accessions in ex situ conservation

PubMed Central

2013-01-01

Background Banana cultivars are mostly derived from hybridization between wild diploid subspecies of Musa acuminata (A genome) and M. balbisiana (B genome), and they exhibit various levels of ploidy and genomic constitution. The Embrapa ex situ Musa collection contains over 220 accessions, of which only a few have been genetically characterized. Knowledge regarding the genetic relationships and diversity between modern cultivars and wild relatives would assist in conservation and breeding strategies. Our objectives were to determine the genomic constitution based on Internal Transcribed Spacer (ITS) regions polymorphism and the ploidy of all accessions by flow cytometry and to investigate the population structure of the collection using Simple Sequence Repeat (SSR) loci as co-dominant markers based on Structure software, not previously performed in Musa. Results From the 221 accessions analyzed by flow cytometry, the correct ploidy was confirmed or established for 212 (95.9%), whereas digestion of the ITS region confirmed the genomic constitution of 209 (94.6%). Neighbor-joining clustering analysis derived from SSR binary data allowed the detection of two major groups, essentially distinguished by the presence or absence of the B genome, while subgroups were formed according to the genomic composition and commercial classification. The co-dominant nature of SSR was explored to analyze the structure of the population based on a Bayesian approach, detecting 21 subpopulations. Most of the subpopulations were in agreement with the clustering analysis. Conclusions The data generated by flow cytometry, ITS and SSR supported the hypothesis about the occurrence of homeologue recombination between A and B genomes, leading to discrepancies in the number of sets or portions from each parental genome. These phenomenons have been largely disregarded in the evolution of banana, as the “single-step domestication” hypothesis had long predominated. These findings will have an impact in future breeding approaches. Structure analysis enabled the efficient detection of ancestry of recently developed tetraploid hybrids by breeding programs, and for some triploids. However, for the main commercial subgroups, Structure appeared to be less efficient to detect the ancestry in diploid groups, possibly due to sampling restrictions. The possibility of inferring the membership among accessions to correct the effects of genetic structure opens possibilities for its use in marker-assisted selection by association mapping. PMID:23497122

Two-Photon Flow Cytometry

NASA Technical Reports Server (NTRS)

Zhog, Cheng Frank; Ye, Jing Yong; Norris, Theodore B.; Myc, Andrzej; Cao, Zhengyl; Bielinska, Anna; Thomas, Thommey; Baker, James R., Jr.

2004-01-01

Flow cytometry is a powerful technique for obtaining quantitative information from fluorescence in cells. Quantitation is achieved by assuring a high degree of uniformity in the optical excitation and detection, generally by using a highly controlled flow such as is obtained via hydrodynamic focusing. In this work, we demonstrate a two-beam, two- channel detection and two-photon excitation flow cytometry (T(sup 3)FC) system that enables multi-dye analysis to be performed very simply, with greatly relaxed requirements on the fluid flow. Two-photon excitation using a femtosecond near-infrared (NIR) laser has the advantages that it enables simultaneous excitation of multiple dyes and achieves very high signal-to-noise ratio through simplified filtering and fluorescence background reduction. By matching the excitation volume to the size of a cell, single-cell detection is ensured. Labeling of cells by targeted nanoparticles with multiple fluorophores enables normalization of the fluorescence signal and thus ratiometric measurements under nonuniform excitation. Quantitative size measurements can also be done even under conditions of nonuniform flow via a two-beam layout. This innovative detection scheme not only considerably simplifies the fluid flow system and the excitation and collection optics, it opens the way to quantitative cytometry in simple and compact microfluidics systems, or in vivo. Real-time detection of fluorescent microbeads in the vasculature of mouse ear demonstrates the ability to do flow cytometry in vivo. The conditions required to perform quantitative in vivo cytometry on labeled cells will be presented.

Vitrectomy for the diagnosis and management of uveitis of unknown cause.

PubMed

Margolis, Ron; Brasil, Oswaldo F M; Lowder, Careen Y; Singh, Rishi P; Kaiser, Peter K; Smith, Scott D; Perez, Victor L; Sonnie, Christine; Sears, Jonathan E

2007-10-01

To determine the diagnostic yield of tests commonly used for vitreous fluid analysis in eyes with suspected intraocular infection or malignancy. Noncomparative interventional case series. Forty-four consecutive patients (45 eyes) treated from 1998 through 2006 with posterior segment inflammation who underwent pars plana vitrectomy for diagnostic purposes. Vitreous specimens obtained via pars plana vitrectomy were analyzed by microbiologic culture, cytologic analysis, and flow cytometry. Diagnostic yield and sensitivity of each test performed on vitreous specimens and visual outcomes of eyes that underwent diagnostic vitrectomy (DVx). Preoperative diagnoses were infection in 15 eyes and malignancy in 30 eyes. Overall, vitreous analysis identified a specific cause in 9 (20%) of 45 eyes. The overall sensitivity of DVx was 63.6%. The sensitivities of individual tests were: culture, 50%; cytologic analysis, 66.7%; and flow cytometry, 83.3%. The yields of diagnostic tests were: culture, 5.7%; cytologic analysis, 14.3%; and flow cytometry, 20.6%. Final diagnoses were infection in 6 eyes, malignancy in 9 eyes, and idiopathic in 30 eyes. Mean visual acuity improved significantly in the first 6 months after DVx. Visual acuity improved in 60% of eyes, with 37.8% of eyes improving by 3 lines or more. Analysis of vitreous fluid by widely available tests is useful in identifying intraocular infection or malignancy. Most patients experienced a substantial improvement in vision.

Role of flow-cytometric immunophenotyping in prediction of BCR/ABL1 gene rearrangement in adult B-cell acute lymphoblastic leukemia.

PubMed

Corrente, Francesco; Bellesi, Silvia; Metafuni, Elisabetta; Puggioni, Pier Luigi; Marietti, Sara; Ciminello, Angela Maria; Za, Tommaso; Sorà, Federica; Fianchi, Luana; Sica, Simona; De Stefano, Valerio; Chiusolo, Patrizia

2018-05-01

We performed a retrospective analysis of 88 adult patients with B-ALL diagnosed in our center by a flow-cytometric assessment. Immunophenotypic expression of leukemic cells was explored by simultaneous evaluation of positivity, percentage of expressing cells and median fluorescence intensity (MFI). BCR/ABL1 fusion transcripts were assessed by RT-PCR analysis and were identified in 36 patients (40.9%). CD10 and CD34 were positive in the totality of BCR/ABL1-positive cases. Patients with gene rearrangement had a greater frequency of CD66c, CD13 and CD33 positivity compared with BCR/ABL1-negative cases. Moreover, BCR/ABL1-positive cases exhibited a greater median percentage and MFI values of CD13, CD33, CD66c, CD10, CD34 and CD25 expressions, but a lower median percentage and MFI values of CD38 and CD22 expressions than patients without gene rearrangement. Multivariate logistic regression analysis showed that CD10, CD38 and CD13 expressions were independent predictors for the presence of BCR/ABL1 rearrangement. Predictive probabilities of molecular occurrence based on these markers are proposed. © 2017 International Clinical Cytometry Society. © 2017 International Clinical Cytometry Society.

CytometryML with DICOM and FCS

NASA Astrophysics Data System (ADS)

Leif, Robert C.

2018-02-01

Abstract: Flow Cytometry Standard, FCS, and Digital Imaging and Communications in Medicine standard, DICOM, are based on extensive, superb domain knowledge, However, they are isolated systems, do not take advantage of data structures, require special programs to read and write the data, lack the capability to interoperate or work with other standards and FCS lacks many of the datatypes necessary for clinical laboratory data. The large overlap between imaging and flow cytometry provides strong evidence that both modalities should be covered by the same standard. Method: The XML Schema Definition Language, XSD 1.1 was used to translate FCS and/or DICOM objects. A MIFlowCyt file was tested with published values. Results: Previously, a significant part of an XML standard based upon a combination of FCS and DICOM has been implemented and validated with MIFlowCyt data. Strongly typed translations of FCS keywords have been constructed in XML. These keywords contain links to their DICOM and FCS equivalents.

Metal-chelating polymers by anionic ring-opening polymerization and their use in quantitative mass cytometry.

PubMed

Illy, Nicolas; Majonis, Daniel; Herrera, Isaac; Ornatsky, Olga; Winnik, Mitchell A

2012-08-13

Metal-chelating polymers (MCPs) are important reagents for multiplexed immunoassays based on mass cytometry. The role of the polymer is to carry multiple copies of individual metal isotopes, typically as lanthanide ions, and to provide a reactive functionality for convenient attachment to a monoclonal antibody (mAb). For this application, the optimum combination of chain length, backbone structure, end group, pendant groups, and synthesis strategy has yet to be determined. Here we describe the synthesis of a new type of MCP based on anionic ring-opening polymerization of an activated cyclopropane (the diallyl ester of 1,1-cyclopropane dicarboxylic acid) using a combination of 2-furanmethanethiol and a phosphazene base as the initiator. This reaction takes place with rigorous control over molecular weight, yielding a polymer with a narrow molecular weight distribution, reactive pendant groups for introducing a metal chelator, and a functional end group with orthogonal reactivity for attaching the polymer to the mAbs. Following the ring-opening polymerization, a two-step transformation introduced diethylenetriaminepentaacetic acid (DTPA) chelating groups on each pendant group. The polymers were characterized by NMR, size exclusion chromatography (SEC), and thermogravimetric analysis (TGA). The binding properties toward Gd(3+) as a prototypical lanthanide (Ln) ion were also studied by isothermal titration calorimetry (ITC). Attachment to a mAb involves a Diels-Alder reaction of the terminal furan with a bismaleimide, followed by a Michael addition of a thiol on the mAb, generated by mild reduction of a disulfide bond in the hinge region. Polymer samples with a number average degree of polymerization of 35, with a binding capacity of 49.5 ± 6 Ln(3+) ions per chain, were loaded with 10 different types of Ln ions and conjugated to 10 different mAbs. A suite of metal-tagged Abs was tested by mass cytometry in a 10-plex single cell analysis of human adult peripheral blood, allowing us to quantify the antibody binding capacity of 10 different cell surface antigens associated with specific cell types.

Easy performance of 6-color confocal immunofluorescence with 4-laser line microscopes.

PubMed

Eissing, Nathalie; Heger, Lukas; Baranska, Anna; Cesnjevar, Robert; Büttner-Herold, Maike; Söder, Stephan; Hartmann, Arndt; Heidkamp, Gordon F; Dudziak, Diana

2014-09-01

Confocal laser scanning microscopy is an advanced technique for imaging tissue samples in vitro and in vivo at high optical resolution. The development of new fluorochrome variants do not only make it possible to perform multicolor flow cytometry of single cells, but in combination with high resolution laser scanning systems also to investigate the distribution of cells in lymphoid tissues by confocal immunofluorescence analyses, thus allowing the distinction of various cell populations directly in the tissue. Here, we provide a protocol for the visualization of at least six differently fluorochrome-labeled antibodies at the same time using a conventional confocal laser scanning microscope with four laser lines (405 nm, 488 nm, 555 nm, and 639 nm laser wavelength) in both murine and human tissue samples. We further demonstrate that compensation correction algorithms are not necessary to reduce spillover of fluorochromes into other channels when the used fluorochromes are combined according to their specific emission bands and the varying Stokes shift for co-excited fluorochromes with the same laser line. Copyright © 2014 Elsevier B.V. All rights reserved.

Laser fluorescence fluctuation excesses in molecular immunology experiments

NASA Astrophysics Data System (ADS)

Galich, N. E.; Filatov, M. V.

2007-04-01

A novel approach to statistical analysis of flow cytometry fluorescence data have been developed and applied for population analysis of blood neutrophils stained with hydroethidine during respiratory burst reaction. The staining based on intracellular oxidation hydroethidine to ethidium bromide, which intercalate into cell DNA. Fluorescence of the resultant product serves as a measure of the neutrophil ability to generate superoxide radicals after induction respiratory burst reaction by phorbol myristate acetate (PMA). It was demonstrated that polymorphonuclear leukocytes of persons with inflammatory diseases showed a considerably changed response. Cytofluorometric histograms obtained have unique information about condition of neutrophil population what might to allow a determination of the pathology processes type connecting with such inflammation. A novel approach to histogram analysis is based on analysis of high-momentum dynamic of distribution. The features of fluctuation excesses of distribution have unique information about disease under consideration.

Quantitative Magnetic Separation of Particles and Cells Using Gradient Magnetic Ratcheting.

PubMed

Murray, Coleman; Pao, Edward; Tseng, Peter; Aftab, Shayan; Kulkarni, Rajan; Rettig, Matthew; Di Carlo, Dino

2016-04-13

Extraction of rare target cells from biosamples is enabling for life science research. Traditional rare cell separation techniques, such as magnetic activated cell sorting, are robust but perform coarse, qualitative separations based on surface antigen expression. A quantitative magnetic separation technology is reported using high-force magnetic ratcheting over arrays of magnetically soft micropillars with gradient spacing, and the system is used to separate and concentrate magnetic beads based on iron oxide content (IOC) and cells based on surface expression. The system consists of a microchip of permalloy micropillar arrays with increasing lateral pitch and a mechatronic device to generate a cycling magnetic field. Particles with higher IOC separate and equilibrate along the miropillar array at larger pitches. A semi-analytical model is developed that predicts behavior for particles and cells. Using the system, LNCaP cells are separated based on the bound quantity of 1 μm anti-epithelial cell adhesion molecule (EpCAM) particles as a metric for expression. The ratcheting cytometry system is able to resolve a ±13 bound particle differential, successfully distinguishing LNCaP from PC3 populations based on EpCAM expression, correlating with flow cytometry analysis. As a proof-of-concept, EpCAM-labeled cells from patient blood are isolated with 74% purity, demonstrating potential toward a quantitative magnetic separation instrument. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Quantitative image cytometry measurements of lipids, DNA, CD45 and cytokeratin for circulating tumor cell identification in a model system

NASA Astrophysics Data System (ADS)

Futia, Gregory L.; Qamar, Lubna; Behbakht, Kian; Gibson, Emily A.

2016-04-01

Circulating tumor cell (CTC) identification has applications in both early detection and monitoring of solid cancers. The rarity of CTCs, expected at ~1-50 CTCs per million nucleated blood cells (WBCs), requires identifying methods based on biomarkers with high sensitivity and specificity for accurate identification. Discovery of biomarkers with ever higher sensitivity and specificity to CTCs is always desirable to potentially find more CTCs in cancer patients thus increasing their clinical utility. Here, we investigate quantitative image cytometry measurements of lipids with the biomarker panel of DNA, Cytokeratin (CK), and CD45 commonly used to identify CTCs. We engineered a device for labeling suspended cell samples with fluorescent antibodies and dyes. We used it to prepare samples for 4 channel confocal laser scanning microscopy. The total data acquired at high resolution from one sample is ~ 1.3 GB. We developed software to perform the automated segmentation of these images into regions of interest (ROIs) containing individual cells. We quantified image features of total signal, spatial second moment, spatial frequency second moment, and their product for each ROI. We performed measurements on pure WBCs, cancer cell line MCF7 and mixed samples. Multivariable regressions and feature selection were used to determine combination features that are more sensitive and specific than any individual feature separately. We also demonstrate that computation of spatial characteristics provides higher sensitivity and specificity than intensity alone. Statistical models allowed quantification of the required sensitivity and specificity for detecting small levels of CTCs in a human blood sample.

Multispectral Imaging Broadens Cellular Analysis

NASA Technical Reports Server (NTRS)

2007-01-01

Amnis Corporation, a Seattle-based biotechnology company, developed ImageStream to produce sensitive fluorescence images of cells in flow. The company responded to an SBIR solicitation from Ames Research Center, and proposed to evaluate several methods of extending the depth of field for its ImageStream system and implement the best as an upgrade to its commercial products. This would allow users to view whole cells at the same time, rather than just one section of each cell. Through Phase I and II SBIR contracts, Ames provided Amnis the funding the company needed to develop this extended functionality. For NASA, the resulting high-speed image flow cytometry process made its way into Medusa, a life-detection instrument built to collect, store, and analyze sample organisms from erupting hydrothermal vents, and has the potential to benefit space flight health monitoring. On the commercial end, Amnis has implemented the process in ImageStream, combining high-resolution microscopy and flow cytometry in a single instrument, giving researchers the power to conduct quantitative analyses of individual cells and cell populations at the same time, in the same experiment. ImageStream is also built for many other applications, including cell signaling and pathway analysis; classification and characterization of peripheral blood mononuclear cell populations; quantitative morphology; apoptosis (cell death) assays; gene expression analysis; analysis of cell conjugates; molecular distribution; and receptor mapping and distribution.

Determination of allergenic load and pollen count of Cupressus arizonica pollen by flow cytometry using Cup a1 polyclonal antibody.

PubMed

Benítez, Francisco Moreno; Camacho, Antonio Letrán; del Cuvillo Bernal, Alfonso; de Medina, Pedro Lobatón Sánchez; García Cózar, Francisco J; Romeu, Marisa Espinazo

2014-01-01

There is an increase in the incidence of pollen related allergy, thus information on pollen schedules would be a great asset for physicians to improve the clinical care of patients. Like cypress pollen sensitization shows a high prevalence among the causes of allergic rhinitis, and therefore it is of interest to use it like a model of study, distinguishing cypress pollen, pollen count, and allergenic load level. In this work, we use a flow cytometry based technique to obtain both Cupressus arizonica pollen count and allergenic load, using specific rabbit polyclonal antibody Cup a1 and its comparison with optical microscopy technique measurement. Airborne samples were collected from Burkard Spore-Trap and Burkard Cyclone Cupressus arizonica pollen was studied using specific rabbit polyclonal antibody Cup a1, labeled with AlexaFluor(®) 488 or 750 and analysed by Flow Cytometry in both an EPICS XL and Cyan ADP cytometers (Beckman Coulter(®) ). Optical microscopy study was realized with a Leica optical microscope. Bland and Altman was used to determine agreement between both techniques measured. We can identify three different populations based on rabbit polyclonal antibody Cup a1 staining. The main region (44.5%) had 97.3% recognition, a second region (25%) with 28% and a third region (30.5%) with 68% respectively. Immunofluorescence and confocal microscopy showed that main region corresponds to whole pollen grains, the second region are pollen without exine and the third region is constituted by smaller particles with allergenic properties. Pollen schedule shows a higher correlation measured by optical microscopy and flow cytometry in the pollen count with a P-value: 0.0008 E(-2) and 0.0002 with regard to smaller particles, so the Bland and Altman measurement showed a good correlation between them, P-value: 0.0003. Determination of pollen count and allergenic load by flow cytometry represents an important tool in the determination of airborne respiratory allergens. We showed that not only whole pollen but also smaller particles could induce allergic sensitization. This is the first study where flow cytometry is used for calculating pollen counts and allergenic load. Copyright © 2013 Clinical Cytometry Society.

Intracellular drug release from curcumin-loaded PLGA nanoparticles induces G2/M block in breast cancer cells.

PubMed

Verderio, Paolo; Bonetti, Paolo; Colombo, Miriam; Pandolfi, Laura; Prosperi, Davide

2013-03-11

PLGA nanoparticles are among the most studied polymer nanoformulations for several drugs against different kinds of malignant diseases, thanks to their in vivo stability and tumor localization exploiting the well-documented "enhanced permeation and retention" (EPR) effect. In this paper, we have developed uniform curcumin-bearing PLGA nanoparticles by a single-emulsion process, which exhibited a curcumin release following a Fickian-law diffusion over 10 days in vitro. PLGA nanoparticles were about 120 nm in size, as determined by dynamic light scattering, with a surface negative charge of -30 mV. The loading ratio of encapsulated drug in our PLGA nanoformulation was 8 wt%. PLGA encapsulation provided efficient protection of curcumin from environment, as determined by fluorescence emission experiments. Next, we have investigated the possibility to study the intracellular degradation of nanoparticles associated with a specific G2/M blocking effect on MCF7 breast cancer cells caused by curcumin release in the cytoplasm, which provided direct evidence on the mechanism of action of our nanocomplex. This study was carried out using Annexin V-based cell death analysis, MTT assessment of proliferation, flow cytometry, and confocal laser scanning microscopy. PLGA nanoparticles proved to be completely safe, suggesting a potential utilization of this nanocomplex to improve the intrinsically poor bioavailability of curcumin for the treatment of severe malignant breast cancer.

Mechanism of action of a novel recombinant peptide, MP1102, against Clostridium perfringens type C.

PubMed

Zong, Lifen; Teng, Da; Wang, Xiumin; Mao, Ruoyu; Yang, Na; Hao, Ya; Wang, Jianhua

2016-06-01

This work is the first to report the antibacterial characteristics and antibacterial mechanisms of MP1102, which is a variant of NZ2114, against pathogenic Clostridium perfringens. MP1102 exhibited strong antimicrobial activity against C. perfringens strains CVCC 61, CVCC 1163, and CVCC 2032 at a low minimal inhibitory concentration (MIC) of 0.91 μM. MP1102 showed anti-C. perfringens activity over a wide pH range of 2.0 and 10.0, high thermal stability from 20 to 80 °C, and remarkable resistance to pepsin. The fractional inhibitory concentration index (FICI) indicated an additive or synergic effect between MP1102 and bacitracin zinc, nisin, vancomycin, virginiamycin, aureomycin, and ampicillin against C. perfringens (FICI = 0.3125-1.0). To further elucidate the antibacterial mechanism of MP1102, its effect on the C. perfringens CVCC 61 cell membrane and intracellular DNA was studied. Flow cytometry and scanning electron microscopy (SEM) indicated that MP1102 treatment resulted in the release of cellular contents by damaging the membrane. A DNA gel retardation and circular dichroism analysis demonstrated that MP1102 interacted with DNA and intercalated into the DNA base pairs. A cell cycle assay demonstrated that MP1102 affected cellular functions, such as DNA synthesis. These results suggested that MP1102 exhibited potential as a new antimicrobial agent against C. perfringens infections.

Enhancement of Anti-Inflammatory Activity of Curcumin Using Phosphatidylserine-Containing Nanoparticles in Cultured Macrophages.

PubMed

Wang, Ji; Kang, Yu-Xia; Pan, Wen; Lei, Wan; Feng, Bin; Wang, Xiao-Juan

2016-06-20

Macrophages are one kind of innate immune cells, and produce a variety of inflammatory cytokines in response to various stimuli, such as oxidized low density lipoprotein found in the pathogenesis of atherosclerosis. In this study, the effect of phosphatidylserine on anti-inflammatory activity of curcumin-loaded nanostructured lipid carriers was investigated using macrophage cultures. Different amounts of phosphatidylserine were used in the preparation of curcumin nanoparticles, their physicochemical properties and biocompatibilities were then compared. Cellular uptake of the nanoparticles was investigated using a confocal laser scanning microscope and flow cytometry analysis in order to determine the optimal phosphatidylserine concentration. In vitro anti-inflammatory activities were evaluated in macrophages to test whether curcumin and phosphatidylserine have interactive effects on macrophage lipid uptake behavior and anti-inflammatory responses. Here, we showed that macrophage uptake of phosphatidylserine-containing nanostructured lipid carriers increased with increasing amount of phosphatidylserine in the range of 0%-8%, and decreased when the phosphatidylserine molar ratio reached over 12%. curcumin-loaded nanostructured lipid carriers significantly inhibited lipid accumulation and pro-inflammatory factor production in cultured macrophages, and evidently promoted release of anti-inflammatory cytokines, when compared with curcumin or phosphatidylserine alone. These results suggest that the delivery system using PS-based nanoparticles has great potential for efficient delivery of drugs such as curcumin, specifically targeting macrophages and modulation of their anti-inflammatory functions.

Different rates of DNA replication at early versus late S-phase sections: multiscale modeling of stochastic events related to DNA content/EdU (5-ethynyl-2'deoxyuridine) incorporation distributions.

PubMed

Li, Biao; Zhao, Hong; Rybak, Paulina; Dobrucki, Jurek W; Darzynkiewicz, Zbigniew; Kimmel, Marek

2014-09-01

Mathematical modeling allows relating molecular events to single-cell characteristics assessed by multiparameter cytometry. In the present study we labeled newly synthesized DNA in A549 human lung carcinoma cells with 15-120 min pulses of EdU. All DNA was stained with DAPI and cellular fluorescence was measured by laser scanning cytometry. The frequency of cells in the ascending (left) side of the "horseshoe"-shaped EdU/DAPI bivariate distributions reports the rate of DNA replication at the time of entrance to S phase while their frequency in the descending (right) side is a marker of DNA replication rate at the time of transition from S to G2 phase. To understand the connection between molecular-scale events and scatterplot asymmetry, we developed a multiscale stochastic model, which simulates DNA replication and cell cycle progression of individual cells and produces in silico EdU/DAPI scatterplots. For each S-phase cell the time points at which replication origins are fired are modeled by a non-homogeneous Poisson Process (NHPP). Shifted gamma distributions are assumed for durations of cell cycle phases (G1, S and G2 M), Depending on the rate of DNA synthesis being an increasing or decreasing function, simulated EdU/DAPI bivariate graphs show predominance of cells in left (early-S) or right (late-S) side of the horseshoe distribution. Assuming NHPP rate estimated from independent experiments, simulated EdU/DAPI graphs are nearly indistinguishable from those experimentally observed. This finding proves consistency between the S-phase DNA-replication rate based on molecular-scale analyses, and cell population kinetics ascertained from EdU/DAPI scatterplots and demonstrates that DNA replication rate at entrance to S is relatively slow compared with its rather abrupt termination during S to G2 transition. Our approach opens a possibility of similar modeling to study the effect of anticancer drugs on DNA replication/cell cycle progression and also to quantify other kinetic events that can be measured during S-phase. © 2014 International Society for Advancement of Cytometry.

Barcoding of live human peripheral blood mononuclear cells for multiplexed mass cytometry.

PubMed

Mei, Henrik E; Leipold, Michael D; Schulz, Axel Ronald; Chester, Cariad; Maecker, Holden T

2015-02-15

Mass cytometry is developing as a means of multiparametric single-cell analysis. In this study, we present an approach to barcoding separate live human PBMC samples for combined preparation and acquisition on a cytometry by time of flight instrument. Using six different anti-CD45 Ab conjugates labeled with Pd104, Pd106, Pd108, Pd110, In113, and In115, respectively, we barcoded up to 20 samples with unique combinations of exactly three different CD45 Ab tags. Cell events carrying more than or less than three different tags were excluded from analyses during Boolean data deconvolution, allowing for precise sample assignment and the electronic removal of cell aggregates. Data from barcoded samples matched data from corresponding individually stained and acquired samples, at cell event recoveries similar to individual sample analyses. The approach greatly reduced technical noise and minimizes unwanted cell doublet events in mass cytometry data, and it reduces wet work and Ab consumption. It also eliminates sample-to-sample carryover and the requirement of instrument cleaning between samples, thereby effectively reducing overall instrument runtime. Hence, CD45 barcoding facilitates accuracy of mass cytometric immunophenotyping studies, thus supporting biomarker discovery efforts, and it should be applicable to fluorescence flow cytometry as well. Copyright © 2015 by The American Association of Immunologists, Inc.

Statistical performance of image cytometry for DNA, lipids, cytokeratin, & CD45 in a model system for circulation tumor cell detection.

PubMed

Futia, Gregory L; Schlaepfer, Isabel R; Qamar, Lubna; Behbakht, Kian; Gibson, Emily A

2017-07-01

Detection of circulating tumor cells (CTCs) in a blood sample is limited by the sensitivity and specificity of the biomarker panel used to identify CTCs over other blood cells. In this work, we present Bayesian theory that shows how test sensitivity and specificity set the rarity of cell that a test can detect. We perform our calculation of sensitivity and specificity on our image cytometry biomarker panel by testing on pure disease positive (D + ) populations (MCF7 cells) and pure disease negative populations (D - ) (leukocytes). In this system, we performed multi-channel confocal fluorescence microscopy to image biomarkers of DNA, lipids, CD45, and Cytokeratin. Using custom software, we segmented our confocal images into regions of interest consisting of individual cells and computed the image metrics of total signal, second spatial moment, spatial frequency second moment, and the product of the spatial-spatial frequency moments. We present our analysis of these 16 features. The best performing of the 16 features produced an average separation of three standard deviations between D + and D - and an average detectable rarity of ∼1 in 200. We performed multivariable regression and feature selection to combine multiple features for increased performance and showed an average separation of seven standard deviations between the D + and D - populations making our average detectable rarity of ∼1 in 480. Histograms and receiver operating characteristics (ROC) curves for these features and regressions are presented. We conclude that simple regression analysis holds promise to further improve the separation of rare cells in cytometry applications. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

A Survey of Flow Cytometry Data Analysis Methods

PubMed Central

Bashashati, Ali; Brinkman, Ryan R.

2009-01-01

Flow cytometry (FCM) is widely used in health research and in treatment for a variety of tasks, such as in the diagnosis and monitoring of leukemia and lymphoma patients, providing the counts of helper-T lymphocytes needed to monitor the course and treatment of HIV infection, the evaluation of peripheral blood hematopoietic stem cell grafts, and many other diseases. In practice, FCM data analysis is performed manually, a process that requires an inordinate amount of time and is error-prone, nonreproducible, nonstandardized, and not open for re-evaluation, making it the most limiting aspect of this technology. This paper reviews state-of-the-art FCM data analysis approaches using a framework introduced to report each of the components in a data analysis pipeline. Current challenges and possible future directions in developing fully automated FCM data analysis tools are also outlined. PMID:20049163

Ultrafast Microfluidic Cellular Imaging by Optical Time-Stretch.

PubMed

Lau, Andy K S; Wong, Terence T W; Shum, Ho Cheung; Wong, Kenneth K Y; Tsia, Kevin K

2016-01-01

There is an unmet need in biomedicine for measuring a multitude of parameters of individual cells (i.e., high content) in a large population efficiently (i.e., high throughput). This is particularly driven by the emerging interest in bringing Big-Data analysis into this arena, encompassing pathology, drug discovery, rare cancer cell detection, emulsion microdroplet assays, to name a few. This momentum is particularly evident in recent advancements in flow cytometry. They include scaling of the number of measurable colors from the labeled cells and incorporation of imaging capability to access the morphological information of the cells. However, an unspoken predicament appears in the current technologies: higher content comes at the expense of lower throughput, and vice versa. For example, accessing additional spatial information of individual cells, imaging flow cytometers only achieve an imaging throughput ~1000 cells/s, orders of magnitude slower than the non-imaging flow cytometers. In this chapter, we introduce an entirely new imaging platform, namely optical time-stretch microscopy, for ultrahigh speed and high contrast label-free single-cell (in a ultrafast microfluidic flow up to 10 m/s) imaging and analysis with an ultra-fast imaging line-scan rate as high as tens of MHz. Based on this technique, not only morphological information of the individual cells can be obtained in an ultrafast manner, quantitative evaluation of cellular information (e.g., cell volume, mass, refractive index, stiffness, membrane tension) at nanometer scale based on the optical phase is also possible. The technology can also be integrated with conventional fluorescence measurements widely adopted in the non-imaging flow cytometers. Therefore, these two combinatorial and complementary measurement capabilities in long run is an attractive platform for addressing the pressing need for expanding the "parameter space" in high-throughput single-cell analysis. This chapter provides the general guidelines of constructing the optical system for time stretch imaging, fabrication and design of the microfluidic chip for ultrafast fluidic flow, as well as the image acquisition and processing.

DNA-based probes for flow cytometry analysis of endocytosis and recycling.

PubMed

Dumont, Claire; Czuba, Ewa; Chen, Moore; Villadangos, Jose A; Johnston, Angus P R; Mintern, Justine D

2017-04-01

The internalization of proteins plays a key role in cell development, cell signaling and immunity. We have previously developed a specific hybridization internalization probe (SHIP) to quantitate the internalization of proteins and particles into cells. Herein, we extend the utility of SHIP to examine both the endocytosis and recycling of surface receptors using flow cytometry. SHIP was used to monitor endocytosis of membrane-bound transferrin receptor (TFR) and its soluble ligand transferrin (TF). SHIP enabled measurements of the proportion of surface molecules internalized, the internalization kinetics and the proportion and rate of internalized molecules that recycle to the cell surface with time. Using this method, we have demonstrated the internalization and recycling of holo-TF and an antibody against the TFR behave differently. This assay therefore highlights the implications of receptor internalization and recycling, where the internalization of the receptor-antibody complex behaves differently to the receptor-ligand complex. In addition, we observe distinct internalization patterns for these molecules expressed by different subpopulations of primary cells. SHIP provides a convenient and high throughput technique for analysis of trafficking parameters for both cell surface receptors and their ligands. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Optimization of flow cytometric detection and cell sorting of transgenic Plasmodium parasites using interchangeable optical filters

PubMed Central

2012-01-01

Background Malaria remains a major cause of morbidity and mortality worldwide. Flow cytometry-based assays that take advantage of fluorescent protein (FP)-expressing malaria parasites have proven to be valuable tools for quantification and sorting of specific subpopulations of parasite-infected red blood cells. However, identification of rare subpopulations of parasites using green fluorescent protein (GFP) labelling is complicated by autofluorescence (AF) of red blood cells and low signal from transgenic parasites. It has been suggested that cell sorting yield could be improved by using filters that precisely match the emission spectrum of GFP. Methods Detection of transgenic Plasmodium falciparum parasites expressing either tdTomato or GFP was performed using a flow cytometer with interchangeable optical filters. Parasitaemia was evaluated using different optical filters and, after optimization of optics, the GFP-expressing parasites were sorted and analysed by microscopy after cytospin preparation and by imaging cytometry. Results A new approach to evaluate filter performance in flow cytometry using two-dimensional dot blot was developed. By selecting optical filters with narrow bandpass (BP) and maximum position of filter emission close to GFP maximum emission in the FL1 channel (510/20, 512/20 and 517/20; dichroics 502LP and 466LP), AF was markedly decreased and signal-background improve dramatically. Sorting of GFP-expressing parasite populations in infected red blood cells at 90 or 95% purity with these filters resulted in 50-150% increased yield when compared to the standard filter set-up. The purity of the sorted population was confirmed using imaging cytometry and microscopy of cytospin preparations of sorted red blood cells infected with transgenic malaria parasites. Discussion Filter optimization is particularly important for applications where the FP signal and percentage of positive events are relatively low, such as analysis of parasite-infected samples with in the intention of gene-expression profiling and analysis. The approach outlined here results in substantially improved yield of GFP-expressing parasites, and requires decreased sorting time in comparison to standard methods. It is anticipated that this protocol will be useful for a wide range of applications involving rare events. PMID:22950515

Toxicant inhibition in activated sludge: fractionation of the physiological status of bacteria.

PubMed

Foladori, P; Bruni, L; Tamburini, S

2014-09-15

In wastewater treatment plants the sensitivity of activated sludge to a toxicant depends on the toxicity test chosen, and thus the use of more than one test is suggested. The physiological status of bacteria in response to toxicants was analysed by flow cytometry to distinguish intact, permeabilised, active cells and cells disrupted. Results were compared with respirometry and bioluminescence bioassay (Vibrio fischeri). 3,5-Dichlorophenol (DCP) was used as reference xenobiotic. DCP has a strong effect on cellular integrity, causing an increase in permeabilised and disrupted cells. A reduction of 44-80% of intact cells with 6-30 mgDCP/L for 5h was found. Inhibition of active cells was 25-49%, at 6-30 mgDCP/L for 5h. The bioluminescence bioassay resulted oversensitive to DCP compared to tests based on activated sludge, while oxygen uptake rate was affected similarly to intact cells measured by flow cytometry. Landfill leachate was tested: a detrimental impact on both cellular integrity and enzymatic activity was observed. Reduction of intact cells and active cells was by 32% and 61% respectively after addition of 50% (v/v) of leachate for 5h. The flow cytometry analysis proposed here might be widely applicable in the monitoring of various toxicants and in other aquatic biosystems. Copyright © 2014 Elsevier B.V. All rights reserved.

Flow cytometry-assisted rapid isolation of recombinant Plasmodium berghei parasites exemplified by functional analysis of aquaglyceroporin

PubMed Central

Kenthirapalan, Sanketha; Waters, Andrew P.; Matuschewski, Kai; Kooij, Taco W.A.

2012-01-01

The most critical bottleneck in the generation of recombinant Plasmodium berghei parasites is the mandatory in vivo cloning step following successful genetic manipulation. This study describes a new technique for rapid selection of recombinant P. berghei parasites. The method is based on flow cytometry to isolate isogenic parasite lines and represents a major advance for the field, in that it will speed the generation of recombinant parasites as well as cut down on animal use significantly. High expression of GFP during blood infection, a prerequisite for robust separation of transgenic lines by flow cytometry, was achieved. Isogenic recombinant parasite populations were isolated even in the presence of a 100-fold excess of wild-type (WT) parasites. Aquaglyceroporin (AQP) loss-of-function mutants and parasites expressing a tagged AQP were generated to validate this approach. aqp− parasites grow normally within the WT phenotypic range during blood infection of NMRI mice. Similarly, colonization of the insect vector and establishment of an infection after mosquito transmission were unaffected, indicating that AQP is dispensable for life cycle progression in vivo under physiological conditions, refuting its use as a suitable drug target. Tagged AQP localized to perinuclear structures and not the parasite plasma membrane. We suggest that flow-cytometric isolation of isogenic parasites overcomes the major roadblock towards a genome-scale repository of mutant and transgenic malaria parasite lines. PMID:23137753

Isomorphic red blood cells using automated urine flow cytometry is a reliable method in diagnosis of bladder cancer.

PubMed

Muto, Satoru; Sugiura, Syo-Ichiro; Nakajima, Akiko; Horiuchi, Akira; Inoue, Masahiro; Saito, Keisuke; Isotani, Shuji; Yamaguchi, Raizo; Ide, Hisamitsu; Horie, Shigeo

2014-10-01

We aimed to identify patients with a chief complaint of hematuria who could safely avoid unnecessary radiation and instrumentation in the diagnosis of bladder cancer (BC), using automated urine flow cytometry to detect isomorphic red blood cells (RBCs) in urine. We acquired urine samples from 134 patients over the age of 35 years with a chief complaint of hematuria and a positive urine occult blood test or microhematuria. The data were analyzed using the UF-1000i (®) (Sysmex Co., Ltd., Kobe, Japan) automated urine flow cytometer to determine RBC morphology, which was classified as isomorphic or dysmorphic. The patients were divided into two groups (BC versus non-BC) for statistical analysis. Multivariate logistic regression analysis was used to determine the predictive value of flow cytometry versus urine cytology, the bladder tumor antigen test, occult blood in urine test, and microhematuria test. BC was confirmed in 26 of 134 patients (19.4 %). The area under the curve for RBC count using the automated urine flow cytometer was 0.94, representing the highest reference value obtained in this study. Isomorphic RBCs were detected in all patients in the BC group. On multivariate logistic regression analysis, only isomorphic RBC morphology was significantly predictive for BC (p < 0.001). Analytical parameters such as sensitivity, specificity, positive predictive value, and negative predictive value of isomorphic RBCs in urine were 100.0, 91.7, 74.3, and 100.0 %, respectively. Detection of urinary isomorphic RBCs using automated urine flow cytometry is a reliable method in the diagnosis of BC with hematuria.

A Stem Cell-Seeded Nanofibrous Scaffold for Auditory Nerve Replacement

DTIC Science & Technology

2013-10-01

the brightest GFP+ cells by flow cytometry and compared these with GFP- cells (Figure 1A-C). The transfected cells showed robust GFP expression even...al., 2011), but no normative data were provided on SGN loss by cochlear turn and, in contrast to our results, those authors reported no impact on...A) Flow cytometry analysis to identify GFP+ and GFP- cells. The large cluster of cells on the left represent the GFP- cells and exhibited similar

Comparative analysis of minimal residual disease detection using four-color flow cytometry, consensus IgH-PCR, and quantitative IgH PCR in CLL after allogeneic and autologous stem cell transplantation.

PubMed

Böttcher, S; Ritgen, M; Pott, C; Brüggemann, M; Raff, T; Stilgenbauer, S; Döhner, H; Dreger, P; Kneba, M

2004-10-01

The clinically most suitable method for minimal residual disease (MRD) detection in chronic lymphocytic leukemia is still controversial. We prospectively compared MRD assessment in 158 blood samples of 74 patients with CLL after stem cell transplantation (SCT) using four-color flow cytometry (MRD flow) in parallel with consensus IgH-PCR and ASO IgH real-time PCR (ASO IgH RQ-PCR). In 25 out of 106 samples (23.6%) with a polyclonal consensus IgH-PCR pattern, MRD flow still detected CLL cells, proving higher sensitivity of flow cytometry over PCR-genescanning with consensus IgH-primers. Of 92 samples, 14 (15.2%) analyzed in parallel by MRD flow and by ASO IgH RQ-PCR were negative by our flow cytometric assay but positive by PCR, thus demonstrating superior sensitivity of RQ-PCR with ASO primers. Quantitative MRD levels measured by both methods correlated well (r=0.93). MRD detection by flow and ASO IgH RQ-PCR were equally suitable to monitor MRD kinetics after allogeneic SCT, but the PCR method detected impending relapses after autologous SCT earlier. An analysis of factors that influence sensitivity and specificity of flow cytometry for MRD detection allowed to devise further improvements of this technique.

Flow cytometric analysis of extracellular vesicle subsets in plasma: impact of swarm by particles of non-interest.

PubMed

Libregts, S F W M; Arkesteijn, G J A; Németh, A; Nolte-'t Hoen, E N M; Wauben, M H M

2018-05-20

Essentials Extracellular vesicles (EVs) in biological fluids are promising biomarkers for disease. Fluorescence-based flow cytometric analysis is suitable to detect low abundant EV subsets. Particles of non-interest can induce false-positive light scatter and fluorescent signals. Interference of particles of non-interest can be monitored by analyzing serial dilutions. Background Extracellular vesicles (EVs) in plasma are increasingly being recognized as potential biomarkers. EV analysis for diagnostic purposes should be robust and should allow analysis of EV subsets with a wide range of abundance and in a large number of patient samples. Flow cytometry offers possibilities to meet these criteria, as it allows multiparameter analysis of individual EVs. However, analysis of plasma EVs is challenging, because of their size and heterogeneity, and the presence of other submicrometer-sized particles in plasma that could interfere with EV analysis. Objectives To explore whether fluorescence-based flow cytometric analysis of EV subsets is suitable when the EVs of interest are present in low abundance in a background of non-labeled or differently labeled EVs and particles. Methods Fluorescently labeled EVs of interest were spiked at different ratios in full plasma, purified plasma components, or (non-)fluorescent polystyrene beads, and subsequently analyzed by flow cytometry with fluorescence threshold triggering. Results We found that light scatter detection of low-abundance or rare EV subsets during fluorescence threshold triggering was severely affected by particles of non-interest, owing to coincidence and swarming. Importantly, we show that interfering particles labeled with different fluorophores induced false-positive fluorescent signals on the particles of interest. These unwanted effects could only be discerned and controlled by performing serial dilutions and analyzing light scatter and fluorescence parameters. Conclusions We demonstrate how particles of non-interest in plasma can impact on the light scatter and fluorescence detection of low-abundance EVs of interest during fluorescence-based flow cytometric analysis, and provide a means to prevent erroneous data interpretation. © 2018 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.

Human treated dentin matrices combined with Zn-doped, Mg-based bioceramic scaffolds and human dental pulp stem cells towards targeted dentin regeneration.

PubMed

Bakopoulou, Athina; Papachristou, Eleni; Bousnaki, Maria; Hadjichristou, Christina; Kontonasaki, Eleana; Theocharidou, Anna; Papadopoulou, Lambrini; Kantiranis, Nikolaos; Zachariadis, George; Leyhausen, Gabriele; Geurtsen, Werner; Koidis, Petros

2016-08-01

This study aimed to investigate the potential of Mg-based bioceramic scaffolds combined with human treated-dentin matrices (hTDMs) and dentinogenesis-related morphogens to promote odontogenic differentiation and dentin-like tissue formation by Dental Pulp Stem Cells-DPSCs. DPSC cultures were established and characterized by flow cytometry. Experimental cavities were prepared inside crowns of extracted teeth and demineralized by EDTA (hTDMs). Zn-doped, Mg-based bioceramic scaffolds, synthesized by the sol-gel technique, were hosted inside the hTDMs. DPSCs were spotted inside the hTDMs/scaffold constructs with/without additional exposure to DMP-1 or BMP-2 (100ng/ml, 24h). Scanning Electron Microscopy-SEM, live/dead fluorescence staining and MTT assay were used to evaluate cell attachment and viability; Real time PCR for expression of osteo/odontogenic markers; Inductively Coupled Plasma-Atomic Emission Spectrometry-ICP/AES for scaffold elemental release analysis; ELISA for hTDM growth factor release analysis; SEM and X-ray Diffraction-XRD for structural/chemical characterization of the regenerated tissues. Scaffolds constantly released low concentrations of Mg(2+), Ca(2+), Zn(2+) and Si(4+), while hTDMs growth factors, like DMP-1, BMP-2 and TGFβ-1. hTDMs/scaffold constructs supported DPSC viability, inducing their rapid odontogenic shift, indicated by upregulation of DSPP, BMP-2, osteocalcin and osterix expression. Newly-formed Ca-P tissue overspread the scaffolds partially transforming into bioapatite. Exposure to DMP-1 or BMP-2 pronouncedly enhanced odontogenic differentiation phenomena. This is the first study to validate that combining the bioactivity and ion releasing properties of bioceramic materials with growth factor release by treated natural dentin further supported by exogenous addition of key dentinogenesis-related morphogens (DMP-1, BMP-2) can be a promising strategy for targeted dentin regeneration. Copyright © 2016 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Sizing up arthropod genomes: an evaluation of the impact of environmental variation on genome size estimates by flow cytometry and the use of qPCR as a method of estimation.

PubMed

Gregory, T Ryan; Nathwani, Paula; Bonnett, Tiffany R; Huber, Dezene P W

2013-09-01

A study was undertaken to evaluate both a pre-existing method and a newly proposed approach for the estimation of nuclear genome sizes in arthropods. First, concerns regarding the reliability of the well-established method of flow cytometry relating to impacts of rearing conditions on genome size estimates were examined. Contrary to previous reports, a more carefully controlled test found negligible environmental effects on genome size estimates in the fly Drosophila melanogaster. Second, a more recently touted method based on quantitative real-time PCR (qPCR) was examined in terms of ease of use, efficiency, and (most importantly) accuracy using four test species: the flies Drosophila melanogaster and Musca domestica and the beetles Tribolium castaneum and Dendroctonus ponderosa. The results of this analysis demonstrated that qPCR has the tendency to produce substantially different genome size estimates from other established techniques while also being far less efficient than existing methods.

Optimized signal detection and analysis methods for in vivo photoacoustic flow cytometry

NASA Astrophysics Data System (ADS)

Wang, Qiyan; Zhou, Quanyu; Yang, Ping; Wang, Xiaoling; Niu, Zhenyu; Suo, Yuanzhen; He, Hao; Gao, Wenyuan; Tang, Shuo; Wei, Xunbin

2017-02-01

Melanoma is known as a malignant tumor of melanocytes, which usually appear in the blood circulation at the metastasis stage of cancer. Thus the detection of circulating melanoma cells is useful for early diagnosis and therapy of cancer. Here we have developed an in vivo photoacoustic flow cytometry (PAFC) based on the photoacoustic effect to detect melanoma cells. However, the raw signals we obtain from the target cells contain noises such as environmental sonic noises and electronic noises. Therefore we apply correlation comparison and feature separation methods to the detection and verification of the in vivo signals. Due to similar shape and structure of cells, the photoacoustic signals usually have similar vibration mode. By analyzing the correlations and the signal features in time domain and frequency domain, we are able to provide a method for separating photoacoustic signals generated by target cells from background noises. The method introduced here has proved to optimize the signal acquisition and signal processing, which can improve the detection accuracy in PAFC.

Analysis of Cellular DNA Content by Flow Cytometry.

PubMed

Darzynkiewicz, Zbigniew; Huang, Xuan; Zhao, Hong

2017-10-02

Cellular DNA content can be measured by flow cytometry with the aim of : (1) revealing cell distribution within the major phases of the cell cycle, (2) estimating frequency of apoptotic cells with fractional DNA content, and/or (3) disclosing DNA ploidy of the measured cell population. In this unit, simple and universally applicable methods for staining fixed cells are presented, as are methods that utilize detergents and/or proteolytic treatment to permeabilize cells and make DNA accessible to fluorochrome. Additionally, supravital cell staining with Hoechst 33342, which is primarily used for sorting live cells based on DNA-content differences for their subsequent culturing, is described. Also presented are methods for staining cell nuclei isolated from paraffin-embedded tissues. Available algorithms are listed for deconvolution of DNA-content-frequency histograms to estimate percentage of cells in major phases of the cell cycle and frequency of apoptotic cells with fractional DNA content. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley and Sons, Inc.

Analysis of Cellular DNA Content by Flow Cytometry.

PubMed

Darzynkiewicz, Zbigniew; Huang, Xuan; Zhao, Hong

2017-11-01

Cellular DNA content can be measured by flow cytometry with the aim of : (1) revealing cell distribution within the major phases of the cell cycle, (2) estimating frequency of apoptotic cells with fractional DNA content, and/or (3) disclosing DNA ploidy of the measured cell population. In this unit, simple and universally applicable methods for staining fixed cells are presented, as are methods that utilize detergents and/or proteolytic treatment to permeabilize cells and make DNA accessible to fluorochrome. Additionally, supravital cell staining with Hoechst 33342, which is primarily used for sorting live cells based on DNA-content differences for their subsequent culturing, is described. Also presented are methods for staining cell nuclei isolated from paraffin-embedded tissues. Available algorithms are listed for deconvolution of DNA-content-frequency histograms to estimate percentage of cells in major phases of the cell cycle and frequency of apoptotic cells with fractional DNA content. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley and Sons, Inc.

Clustergrammer, a web-based heatmap visualization and analysis tool for high-dimensional biological data

PubMed Central

Fernandez, Nicolas F.; Gundersen, Gregory W.; Rahman, Adeeb; Grimes, Mark L.; Rikova, Klarisa; Hornbeck, Peter; Ma’ayan, Avi

2017-01-01

Most tools developed to visualize hierarchically clustered heatmaps generate static images. Clustergrammer is a web-based visualization tool with interactive features such as: zooming, panning, filtering, reordering, sharing, performing enrichment analysis, and providing dynamic gene annotations. Clustergrammer can be used to generate shareable interactive visualizations by uploading a data table to a web-site, or by embedding Clustergrammer in Jupyter Notebooks. The Clustergrammer core libraries can also be used as a toolkit by developers to generate visualizations within their own applications. Clustergrammer is demonstrated using gene expression data from the cancer cell line encyclopedia (CCLE), original post-translational modification data collected from lung cancer cells lines by a mass spectrometry approach, and original cytometry by time of flight (CyTOF) single-cell proteomics data from blood. Clustergrammer enables producing interactive web based visualizations for the analysis of diverse biological data. PMID:28994825

Accurate measurement of peripheral blood mononuclear cell concentration using image cytometry to eliminate RBC-induced counting error.

PubMed

Chan, Leo Li-Ying; Laverty, Daniel J; Smith, Tim; Nejad, Parham; Hei, Hillary; Gandhi, Roopali; Kuksin, Dmitry; Qiu, Jean

2013-02-28

Peripheral blood mononuclear cells (PBMCs) have been widely researched in the fields of immunology, infectious disease, oncology, transplantation, hematological malignancy, and vaccine development. Specifically, in immunology research, PBMCs have been utilized to monitor concentration, viability, proliferation, and cytokine production from immune cells, which are critical for both clinical trials and biomedical research. The viability and concentration of isolated PBMCs are traditionally measured by manual counting with trypan blue (TB) using a hemacytometer. One of the common issues of PBMC isolation is red blood cell (RBC) contamination. The RBC contamination can be dependent on the donor sample and/or technical skill level of the operator. RBC contamination in a PBMC sample can introduce error to the measured concentration, which can pass down to future experimental assays performed on these cells. To resolve this issue, RBC lysing protocol can be used to eliminate potential error caused by RBC contamination. In the recent years, a rapid fluorescence-based image cytometry system has been utilized for bright-field and fluorescence imaging analysis of cellular characteristics (Nexcelom Bioscience LLC, Lawrence, MA). The Cellometer image cytometry system has demonstrated the capability of automated concentration and viability detection in disposable counting chambers of unpurified mouse splenocytes and PBMCs stained with acridine orange (AO) and propidium iodide (PI) under fluorescence detection. In this work, we demonstrate the ability of Cellometer image cytometry system to accurately measure PBMC concentration, despite RBC contamination, by comparison of five different total PBMC counting methods: (1) manual counting of trypan blue-stained PBMCs in hemacytometer, (2) manual counting of PBMCs in bright-field images, (3) manual counting of acetic acid lysing of RBCs with TB-stained PBMCs, (4) automated counting of acetic acid lysing of RBCs with PI-stained PBMCs, and (5) AO/PI dual staining method. The results show comparable total PBMC counting among all five methods, which validate the AO/PI staining method for PBMC measurement in the image cytometry method. Copyright © 2012 Elsevier B.V. All rights reserved.

A flow-cytometry-based method for detecting simultaneously five allergens in a complex food matrix.

PubMed

Otto, Gaetan; Lamote, Amandine; Deckers, Elise; Dumont, Valery; Delahaut, Philippe; Scippo, Marie-Louise; Pleck, Jessica; Hillairet, Caroline; Gillard, Nathalie

2016-12-01

To avoid carry-over contamination with allergens, food manufacturers implement quality control strategies relying primarily on detection of allergenic proteins by ELISA. Although sensitive and specific, this method allowed detection of only one allergen per analysis and effective control policies were thus based on multiplying the number of tests done in order to cover the whole range of allergens. We present in this work an immunoassay for the simultaneous detection of milk, egg, peanut, mustard and crustaceans in cookies samples. The method was based on a combination of flow cytometry with competitive ELISA where microbeads were used as sorbent surface. The test was able to detect the presence of the five allergens with median inhibitory concentrations (IC50) ranging from 2.5 to 15 mg/kg according to the allergen to be detected. The lowest concentrations of contaminants inducing a significant difference of signal between non-contaminated controls and test samples were 2 mg/kg of peanut, 5 mg/kg of crustaceans, 5 mg/kg of milk, 5 mg/kg of mustard and 10 mg/kg of egg. Assay sensitivity was influenced by the concentration of primary antibodies added to the sample extract for the competition and by the concentration of allergenic proteins bound to the surface of the microbeads.

Automated Assessment of Disease Progression in Acute Myeloid Leukemia by Probabilistic Analysis of Flow Cytometry Data

PubMed Central

Rajwa, Bartek; Wallace, Paul K.; Griffiths, Elizabeth A.; Dundar, Murat

2017-01-01

Objective Flow cytometry (FC) is a widely acknowledged technology in diagnosis of acute myeloid leukemia (AML) and has been indispensable in determining progression of the disease. Although FC plays a key role as a post-therapy prognosticator and evaluator of therapeutic efficacy, the manual analysis of cytometry data is a barrier to optimization of reproducibility and objectivity. This study investigates the utility of our recently introduced non-parametric Bayesian framework in accurately predicting the direction of change in disease progression in AML patients using FC data. Methods The highly flexible non-parametric Bayesian model based on the infinite mixture of infinite Gaussian mixtures is used for jointly modeling data from multiple FC samples to automatically identify functionally distinct cell populations and their local realizations. Phenotype vectors are obtained by characterizing each sample by the proportions of recovered cell populations, which are in turn used to predict the direction of change in disease progression for each patient. Results We used 200 diseased and non-diseased immunophenotypic panels for training and tested the system with 36 additional AML cases collected at multiple time points. The proposed framework identified the change in direction of disease progression with accuracies of 90% (9 out of 10) for relapsing cases and 100% (26 out of 26) for the remaining cases. Conclusions We believe that these promising results are an important first step towards the development of automated predictive systems for disease monitoring and continuous response evaluation. Significance Automated measurement and monitoring of therapeutic response is critical not only for objective evaluation of disease status prognosis but also for timely assessment of treatment strategies. PMID:27416585

Consistency of the initial cell acquisition procedure is critical to the standardization of CD34+ cell enumeration by flow cytometry: results of a pairwise analysis of umbilical cord blood units and cryopreserved aliquots.

PubMed

Flores, Ana I; McKenna, David H; Montalbán, M Angeles; De la Cruz, Javier; Wagner, John E; Bornstein, Rafael

2009-04-01

The CD34+ cell content is a predictive factor for engraftment and survival after umbilical cord blood (UCB) transplantation. The high variability in the CD34 assay results in different recommended cell doses for infusion across transplant centers and also limits the clinical utility of the CD34+ cell counts provided by cord blood banks (CBBs). This bi-institutional study was intended to understand the sources of this variability. The level of CD34 agreement between the University of Minnesota (UM) and the Madrid CBB (MCBB) was evaluated on 50 UCB units before and after cryopreservation. Two cryopreserved vials per unit were thawed and processed at both laboratories. Dual-platform ISHAGE-based flow cytometry was used for CD34 enumeration. Postthaw nucleated cell recoveries were similar. However, whereas CD34+ cell enumeration before freezing was 0.35 +/- 0.22 percent, the results after thawing were 0.98 +/- 0.65 and 0.57 +/- 0.39 percent at UM and MCBB, respectively. Bland-Altman plots analysis ruled out the interchangeability of MCBB and UM CD34 values. Differences in the initial cell acquisition settings accounted for most of the CD34 discrepancy, which was no longer present after normalization of the forward scatter threshold for cell acquisition. The standardization of CD34+ cell enumeration by flow cytometry is strongly reliant on a consistent initial cell acquisition procedure. The interlaboratory variation can be minimized by using frozen cell aliquots as reference samples. Both requisites should be considered for CD34 testing and UCB unit selection by regulatory institutions involved with cord blood banking and transplantation.

Stem and Progenitor Cell Subsets Are Affected by JAK2 Signaling and Can Be Monitored by Flow Cytometry

PubMed Central

Iida, Ryuji; Welner, Robert S.; Zhao, Wanke; Alberola-lla, José; Medina, Kay L.; Zhao, Zhizhuang Joe; Kincade, Paul W.

2014-01-01

Although extremely rare, hematopoietic stem cells (HSCs) are divisible into subsets that differ with respect to differentiation potential and cell surface marker expression. For example, we recently found that CD86− CD150+ CD48− HSCs have limited potential for lymphocyte production. This could be an important new tool for studying hematological abnormalities. Here, we analyzed HSC subsets with a series of stem cell markers in JAK2V617F transgenic (Tg) mice, where the mutation is sufficient to cause myeloproliferative neoplasia with lymphocyte deficiency. Total numbers of HSC were elevated 3 to 20 fold in bone marrow of JAK2V617F mice. Careful analysis suggested the accumulation involved multiple HSC subsets, but particularly those characterized as CD150HI CD86− CD18L°CD41+ and excluding Hoechst dye. Real-Time PCR analysis of their HSC revealed that the erythropoiesis associated gene transcripts Gata1, Klf1 and Epor were particularly high. Flow cytometry analyses based on two differentiation schemes for multipotent progenitors (MPP) also suggested alteration by JAK2 signals. The low CD86 on HSC and multipotent progenitors paralleled the large reductions we found in lymphoid progenitors, but the few that were produced functioned normally when sorted and placed in culture. Either of two HSC subsets conferred disease when transplanted. Thus, flow cytometry can be used to observe the influence of abnormal JAK2 signaling on stem and progenitor subsets. Markers that similarly distinguish categories of human HSCs might be very valuable for monitoring such conditions. They could also serve as indicators of HSC fitness and suitability for transplantation. PMID:24699465

PSIDD3: Post-Scan Ultrasonic Data Display System for the Windows-Based PC Including Fuzzy Logic Analysis

NASA Technical Reports Server (NTRS)

Lovelace, Jeffrey J.; Cios, Krzysztof J.; Roth, Don J.; Cao, Wei

2000-01-01

Post-Scan Interactive Data Display (PSIDD) III is a user-oriented Windows-based system that facilitates the display and comparison of ultrasonic contact data. The system is optimized to compare ultrasonic measurements made at different locations within a material or at different stages of material degradation. PSIDD III provides complete analysis of the primary wave forms in the time and frequency domains along with the calculation of several frequency dependent properties including Phase Velocity and Attenuation Coefficient and several frequency independent properties, like the Cross Correlation Velocity. The system allows image generation on all of the frequency dependent properties at any available frequency (limited by the bandwidth used in the scans) and on any of the frequency independent properties. From ultrasonic contact scans, areas of interest on an image can be studied with regard to underlying raw waveforms and derived ultrasonic properties by simply selecting the point on the image. The system offers various modes of in-depth comparison between scan points. Up to five scan points can be selected for comparative analysis at once. The system was developed with Borland Delphi software (Visual Pascal) and is based on a SQL database. It is ideal for classification of material properties, or location of microstructure variations in materials.

Identification and immunophenotypic characterization of normal and pathological mast cells.

PubMed

Morgado, José Mário; Sánchez-Muñoz, Laura; Teodósio, Cristina; Escribano, Luís

2014-01-01

Mast cells (MCs) are secretory cells that are central players in human allergic disease and immune responses. With the exception of a few pathological situations, MCs are usually present at relatively low frequencies in most tissues. Since their first description, MCs in tissues were identified mostly using their morphological characteristics and their typical coloration when stained with aniline dyes. However, increasing availability of highly specific antibodies now permits the use of fluorescence-based flow cytometry as the method of choice for the quantification, characterization, and purification of cells in suspension. This technique allows for a rapid analysis of thousands of events and for the identification of cells present at frequencies as low as one event in 10(6) unwanted cells. This method also permits for simultaneous characterization of multiple antigens at a single-cell level, which is ideal in order to study rare populations of cells like MCs. Here we describe the basis of flow cytometry-based immunophenotyping applied to the study of MC. The protocol focuses on the study of human MCs present in body fluids (mainly bone marrow) but can easily be adapted to study MCs from other tissues and species.

Genotoxicity of TiO2 nanoparticles assessed by mini-gel comet assay and micronucleus scoring with flow cytometry.

PubMed

Di Bucchianico, Sebastiano; Cappellini, Francesca; Le Bihanic, Florane; Zhang, Yuning; Dreij, Kristian; Karlsson, Hanna L

2017-01-01

The widespread production and use of nanoparticles calls for faster and more reliable methods to assess their safety. The main aim of this study was to investigate the genotoxicity of three reference TiO 2 nanomaterials (NM) within the frame of the FP7-NANoREG project, with a particular focus on testing the applicability of mini-gel comet assay and micronucleus (MN) scoring by flow cytometry. BEAS-2B cells cultured under serum-free conditions were exposed to NM100 (anatase, 50-150nm), NM101 (anatase, 5-8nm) and NM103 (rutile, 20-28nm) for 3, 24 or 48h mainly at concentrations 1-30 μg/ml. In the mini-gel comet assay (eight gels per slide), we included analysis of (i) DNA strand breaks, (ii) oxidised bases (Fpg-sensitive sites) and (iii) light-induced DNA damage due to photocatalytic activity. Furthermore, MN assays were used and we compared the results of more high-throughput MN scoring with flow cytometry to that of cytokinesis-block MN cytome assay scored manually using a microscope. Various methods were used to assess cytotoxic effects and the results showed in general no or low effects at the doses tested. A weak genotoxic effect of the tested TiO 2 materials was observed with an induction of oxidised bases for all three materials of which NM100 was the most potent. When the comet slides were briefly exposed to lab light, a clear induction of DNA strand breaks was observed for the anatase materials, but not for the rutile. This highlights the risk of false positives when testing photocatalytically active materials if light is not properly avoided. A slight increase in MN formation for NM103 was observed in the different MN assays at the lower doses tested (1 and 5 μg/ml). We conclude that mini-gel comet assay and MN scoring using flow cytometry successfully can be used to efficiently study cytotoxic and genotoxic properties of nanoparticles. © The Author 2016. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society.

Genotoxicity of TiO2 nanoparticles assessed by mini-gel comet assay and micronucleus scoring with flow cytometry

PubMed Central

Di Bucchianico, Sebastiano; Cappellini, Francesca; Le Bihanic, Florane; Zhang, Yuning; Dreij, Kristian; Karlsson, Hanna L.

2017-01-01

The widespread production and use of nanoparticles calls for faster and more reliable methods to assess their safety. The main aim of this study was to investigate the genotoxicity of three reference TiO2 nanomaterials (NM) within the frame of the FP7-NANoREG project, with a particular focus on testing the applicability of mini-gel comet assay and micronucleus (MN) scoring by flow cytometry. BEAS-2B cells cultured under serum-free conditions were exposed to NM100 (anatase, 50–150nm), NM101 (anatase, 5–8nm) and NM103 (rutile, 20–28nm) for 3, 24 or 48h mainly at concentrations 1–30 μg/ml. In the mini-gel comet assay (eight gels per slide), we included analysis of (i) DNA strand breaks, (ii) oxidised bases (Fpg-sensitive sites) and (iii) light-induced DNA damage due to photocatalytic activity. Furthermore, MN assays were used and we compared the results of more high-throughput MN scoring with flow cytometry to that of cytokinesis-block MN cytome assay scored manually using a microscope. Various methods were used to assess cytotoxic effects and the results showed in general no or low effects at the doses tested. A weak genotoxic effect of the tested TiO2 materials was observed with an induction of oxidised bases for all three materials of which NM100 was the most potent. When the comet slides were briefly exposed to lab light, a clear induction of DNA strand breaks was observed for the anatase materials, but not for the rutile. This highlights the risk of false positives when testing photocatalytically active materials if light is not properly avoided. A slight increase in MN formation for NM103 was observed in the different MN assays at the lower doses tested (1 and 5 μg/ml). We conclude that mini-gel comet assay and MN scoring using flow cytometry successfully can be used to efficiently study cytotoxic and genotoxic properties of nanoparticles. PMID:27382040

Discriminating cellular heterogeneity using microwell-based RNA cytometry

PubMed Central

Dimov, Ivan K.; Lu, Rong; Lee, Eric P.; Seita, Jun; Sahoo, Debashis; Park, Seung-min; Weissman, Irving L.; Lee, Luke P.

2014-01-01

Discriminating cellular heterogeneity is important for understanding cellular physiology. However, it is limited by the technical difficulties of single-cell measurements. Here, we develop a two-stage system to determine cellular heterogeneity. In the first stage, we perform multiplex single-cell RNA-cytometry in a microwell array containing over 60,000 reaction chambers. In the second stage, we use the RNA-cytometry data to determine cellular heterogeneity by providing a heterogeneity likelihood score. Moreover, we use Monte-Carlo simulation and RNA-cytometry data to calculate the minimum number of cells required for detecting heterogeneity. We applied this system to characterize the RNA distributions of aging related genes in a highly purified mouse hematopoietic stem cell population. We identified genes that reveal novel heterogeneity of these cells. We also show that changes in expression of genes such as Birc6 during aging can be attributed to the shift of relative portions of cells in the high-expressing subgroup versus low-expressing subgroup. PMID:24667995

Flow cytometry enables identification of sporophytic eliciting stress treatments in gametic cells.

PubMed

Ribalta, F M; Croser, J S; Ochatt, S J

2012-01-01

Flow cytometry was used to quantify the effect of individual and combined stress treatments on elicitation of androgenesis by analyzing the relative nuclear DNA content of in vitro cultured microspores of Pisum sativum L. Differences in relative nuclear DNA content of microspores within anthers after stress treatments were clearly evident from the flow cytometry profiles, and permitted us to predict whether a combination of stresses were elicitors or enhancers of androgenesis. This is the first report to assess the effect of various stress treatments in a plant species based on relative nuclear DNA content and to use this information to categorize them as 'elicitors' or 'enhancers'. Flow cytometry represents a simple, quick and reliable way to analyze and discriminate the effect of various stress treatments on elicitation of androgenesis. These results form a solid basis for further efforts designed to enhance responses and to extend double haploid technology to other legumes. Copyright © 2011 Elsevier GmbH. All rights reserved.

Capture of Fluorescence Decay Times by Flow Cytometry

PubMed Central

Naivar, Mark A.; Jenkins, Patrick; Freyer, James P.

2012-01-01

In flow cytometry, the fluorescence decay time of an excitable species has been largely underutilized and is not likely found as a standard parameter on any imaging cytometer, sorting, or analyzing system. Most cytometers lack fluorescence lifetime hardware mainly owing to two central issues. Foremost, research and development with lifetime techniques has lacked proper exploitation of modern laser systems, data acquisition boards, and signal processing techniques. Secondly, a lack of enthusiasm for fluorescence lifetime applications in cells and with bead-based assays has persisted among the greater cytometry community. In this unit, we describe new approaches that address these issues and demonstrate the simplicity of digitally acquiring fluorescence relaxation rates in flow. The unit is divided into protocol and commentary sections in order to provide a most comprehensive discourse on acquiring the fluorescence lifetime with frequency-domain methods. The unit covers (i) standard fluorescence lifetime acquisition (protocol-based) with frequency-modulated laser excitation, (ii) digital frequency-domain cytometry analyses, and (iii) interfacing fluorescence lifetime measurements onto sorting systems. Within the unit is also a discussion on how digital methods are used for aliasing in order to harness higher frequency ranges. Also, a final discussion is provided on heterodyning and processing of waveforms for multi-exponential decay extraction. PMID:25419263

A high-throughput AO/PI-based cell concentration and viability detection method using the Celigo image cytometry.

PubMed

Chan, Leo Li-Ying; Smith, Tim; Kumph, Kendra A; Kuksin, Dmitry; Kessel, Sarah; Déry, Olivier; Cribbes, Scott; Lai, Ning; Qiu, Jean

2016-10-01

To ensure cell-based assays are performed properly, both cell concentration and viability have to be determined so that the data can be normalized to generate meaningful and comparable results. Cell-based assays performed in immuno-oncology, toxicology, or bioprocessing research often require measuring of multiple samples and conditions, thus the current automated cell counter that uses single disposable counting slides is not practical for high-throughput screening assays. In the recent years, a plate-based image cytometry system has been developed for high-throughput biomolecular screening assays. In this work, we demonstrate a high-throughput AO/PI-based cell concentration and viability method using the Celigo image cytometer. First, we validate the method by comparing directly to Cellometer automated cell counter. Next, cell concentration dynamic range, viability dynamic range, and consistency are determined. The high-throughput AO/PI method described here allows for 96-well to 384-well plate samples to be analyzed in less than 7 min, which greatly reduces the time required for the single sample-based automated cell counter. In addition, this method can improve the efficiency for high-throughput screening assays, where multiple cell counts and viability measurements are needed prior to performing assays such as flow cytometry, ELISA, or simply plating cells for cell culture.

Autoblocker: a system for detecting and blocking of network scanning based on analysis of netflow data

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bobyshev, A.; Lamore, D.; Demar, P.

2004-12-01

In a large campus network, such at Fermilab, with tens of thousands of nodes, scanning initiated from either outside of or within the campus network raises security concerns. This scanning may have very serious impact on network performance, and even disrupt normal operation of many services. In this paper we introduce a system for detecting and automatic blocking excessive traffic of different kinds of scanning, DoS attacks, virus infected computers. The system, called AutoBlocker, is a distributed computing system based on quasi-real time analysis of network flow data collected from the border router and core switches. AutoBlocker also has anmore » interface to accept alerts from IDS systems (e.g. BRO, SNORT) that are based on other technologies. The system has multiple configurable alert levels for the detection of anomalous behavior and configurable trigger criteria for automated blocking of scans at the core or border routers. It has been in use at Fermilab for about 2 years, and has become a very valuable tool to curtail scan activity within the Fermilab campus network.« less

Light-scattering flow cytometry for identification and characterization of blood microparticles

NASA Astrophysics Data System (ADS)

Konokhova, Anastasiya I.; Yurkin, Maxim A.; Moskalensky, Alexander E.; Chernyshev, Andrei V.; Tsvetovskaya, Galina A.; Chikova, Elena D.; Maltsev, Valeri P.

2012-05-01

We describe a novel approach to study blood microparticles using the scanning flow cytometer, which measures light scattering patterns (LSPs) of individual particles. Starting from platelet-rich plasma, we separated spherical microparticles from non-spherical plasma constituents, such as platelets and cell debris, based on similarity of their LSP to that of sphere. This provides a label-free method for identification (detection) of microparticles, including those larger than 1 μm. Next, we rigorously characterized each measured particle, determining its size and refractive index including errors of these estimates. Finally, we employed a deconvolution algorithm to determine size and refractive index distributions of the whole population of microparticles, accounting for largely different reliability of individual measurements. Developed methods were tested on a blood sample of a healthy donor, resulting in good agreement with literature data. The only limitation of this approach is size detection limit, which is currently about 0.5 μm due to used laser wavelength of 0.66 μm.

FuGEFlow: data model and markup language for flow cytometry

PubMed Central

Qian, Yu; Tchuvatkina, Olga; Spidlen, Josef; Wilkinson, Peter; Gasparetto, Maura; Jones, Andrew R; Manion, Frank J; Scheuermann, Richard H; Sekaly, Rafick-Pierre; Brinkman, Ryan R

2009-01-01

Background Flow cytometry technology is widely used in both health care and research. The rapid expansion of flow cytometry applications has outpaced the development of data storage and analysis tools. Collaborative efforts being taken to eliminate this gap include building common vocabularies and ontologies, designing generic data models, and defining data exchange formats. The Minimum Information about a Flow Cytometry Experiment (MIFlowCyt) standard was recently adopted by the International Society for Advancement of Cytometry. This standard guides researchers on the information that should be included in peer reviewed publications, but it is insufficient for data exchange and integration between computational systems. The Functional Genomics Experiment (FuGE) formalizes common aspects of comprehensive and high throughput experiments across different biological technologies. We have extended FuGE object model to accommodate flow cytometry data and metadata. Methods We used the MagicDraw modelling tool to design a UML model (Flow-OM) according to the FuGE extension guidelines and the AndroMDA toolkit to transform the model to a markup language (Flow-ML). We mapped each MIFlowCyt term to either an existing FuGE class or to a new FuGEFlow class. The development environment was validated by comparing the official FuGE XSD to the schema we generated from the FuGE object model using our configuration. After the Flow-OM model was completed, the final version of the Flow-ML was generated and validated against an example MIFlowCyt compliant experiment description. Results The extension of FuGE for flow cytometry has resulted in a generic FuGE-compliant data model (FuGEFlow), which accommodates and links together all information required by MIFlowCyt. The FuGEFlow model can be used to build software and databases using FuGE software toolkits to facilitate automated exchange and manipulation of potentially large flow cytometry experimental data sets. Additional project documentation, including reusable design patterns and a guide for setting up a development environment, was contributed back to the FuGE project. Conclusion We have shown that an extension of FuGE can be used to transform minimum information requirements in natural language to markup language in XML. Extending FuGE required significant effort, but in our experiences the benefits outweighed the costs. The FuGEFlow is expected to play a central role in describing flow cytometry experiments and ultimately facilitating data exchange including public flow cytometry repositories currently under development. PMID:19531228

Genome-Wide Nucleic Acid/Protein Interaction in Breast Cancer

DTIC Science & Technology

2005-04-01

Chen W, Zhang J et al: Large-scale genotyping of complex DNA. Nat Biotechnol 2003, 21(10):1233-1237. 25. Bolstad BM, Irizarry RA, Astrand M, Speed TP...2124836802660188/sup8.xls 235 Analysis of RNA-protein interactions by flow cytometry Alexander S Brodsky’*, Angus PR Johnston 2, Matt Trau 2 & Pamela A Silver1...Natl Acad Sci USA (2001) 98(23):12954-12959. genotyping using the Qbead system: A quantum dot-encoded microsphere-based assay. Nucleic Acids Res (2003

Annual Progress Report FY-91. Volume 1 and 2.

DTIC Science & Technology

1992-03-12

Pulmonary Med Tech 11 0644 GS Berger, TA Allergy Microbiologist 12 0403 GS Billups, L Flow Cytom Chemist 12 1320 GS Vacant Pulmonary Kyle Metabolic Unit...Reactions 11 (11/89) 3349 Salata, Kalman PhD. Mitogen-Inducible T Suppressor Cell 12 Assay by Flow Cytometry (12/89) 3350 Salata, Kalman PhD. Flow ...3/90) 3354 Salata, Kalman PhD. Two Way Mixed Lymphocyte Culture: 17 Analysis by Two Color Flow Cytometry (4/90) 3355 Salata, Kalman PhD. Effect of

Effect of cryopreservation on delineation of immune cell subpopulations in tumor specimens as determinated by multiparametric single cell mass cytometry analysis.

PubMed

Kadić, Elma; Moniz, Raymond J; Huo, Ying; Chi, An; Kariv, Ilona

2017-02-02

Comprehensive understanding of cellular immune subsets involved in regulation of tumor progression is central to the development of cancer immunotherapies. Single cell immunophenotyping has historically been accomplished by flow cytometry (FC) analysis, enabling the analysis of up to 18 markers. Recent advancements in mass cytometry (MC) have facilitated detection of over 50 markers, utilizing high resolving power of mass spectrometry (MS). This study examined an analytical and operational feasibility of MC for an in-depth immunophenotyping analysis of the tumor microenvironment, using the commercial CyTOF™ instrument, and further interrogated challenges in managing the integrity of tumor specimens. Initial longitudinal studies with frozen peripheral blood mononuclear cells (PBMCs) showed minimal MC inter-assay variability over nine independent runs. In addition, detection of common leukocyte lineage markers using MC and FC detection confirmed that these methodologies are comparable in cell subset identification. An advanced multiparametric MC analysis of 39 total markers enabled a comprehensive evaluation of cell surface marker expression in fresh and cryopreserved tumor samples. This comparative analysis revealed significant reduction of expression levels of multiple markers upon cryopreservation. Most notably myeloid derived suppressor cells (MDSC), defined by co-expression of CD66b + and CD15 + , HLA-DR dim and CD14 - phenotype, were undetectable in frozen samples. These results suggest that optimization and evaluation of cryopreservation protocols is necessary for accurate biomarker discovery in frozen tumor specimens.

Combination of CD157 and FLAER to Detect Peripheral Blood Eosinophils by Multiparameter Flow Cytometry.

PubMed

Carulli, Giovanni; Marini, Alessandra; Sammuri, Paola; Domenichini, Cristiana; Ottaviano, Virginia; Pacini, Simone; Petrini, Mario

2015-01-01

The identification of eosinophils by flow cytometry is difficult because most of the surface antigens expressed by eosinophils are shared with neutrophils. Some methods have been proposed, generally based on differential light scatter properties, enhanced autofluorescence, lack of CD16 or selective positivity of CD52. Such methods, however, show several limitations. In the present study we report a novel method based on the analysis of glycosylphosphatidylinositol (GPI)-linked molecules. The combination of CD157 and FLAER was used, since FLAER recognizes all GPI-linked molecules, while CD157 is absent on the membrane of eosinophils and expressed by neutrophils. Peripheral blood samples from normal subjects and patients with variable percentages of eosinophils (n = 31), and without any evidence for circulating immature myeloid cells, were stained with the combination of FLAER-Alexa Fluor and CD157-PE. A FascCanto II cytometer was used. Granulocytes were gated after CD33 staining and eosinophils were identified as CD157(-)/FLAER(+) events. Neutrophils were identified as CD157(+)/FLAER(+) events. The percentages of eosinophils detected by this method showed a very significant correlation both with automated counting and with manual counting (r = 0.981 and 0.989, respectively). Sorting assays were carried out by a S3 Cell Sorter: cytospins obtained from CD157(-)/FLAER(+) events consisted of 100% eosinophils, while samples from CD157(+)/FLAER(+) events were represented only by neutrophils. In conclusion, this method shows high sensitivity and specificity in order to distinguish eosinophils from neutrophils by flow cytometry. However, since CD157 is gradually up-regulated throughout bone marrow myeloid maturation, our method cannot be applied to cases characterized by immature myeloid cells.

Computed tomographic-based quantification of emphysema and correlation to pulmonary function and mechanics.

PubMed

Washko, George R; Criner, Gerald J; Mohsenifar, Zab; Sciurba, Frank C; Sharafkhaneh, Amir; Make, Barry J; Hoffman, Eric A; Reilly, John J

2008-06-01

Computed tomographic based indices of emphysematous lung destruction may highlight differences in disease pathogenesis and further enable the classification of subjects with Chronic Obstructive Pulmonary Disease. While there are multiple techniques that can be utilized for such radiographic analysis, there is very little published information comparing the performance of these methods in a clinical case series. Our objective was to examine several quantitative and semi-quantitative methods for the assessment of the burden of emphysema apparent on computed tomographic scans and compare their ability to predict lung mechanics and function. Automated densitometric analysis was performed on 1094 computed tomographic scans collected upon enrollment into the National Emphysema Treatment Trial. Trained radiologists performed an additional visual grading of emphysema on high resolution CT scans. Full pulmonary function test results were available for correlation, with a subset of subjects having additional measurements of lung static recoil. There was a wide range of emphysematous lung destruction apparent on the CT scans and univariate correlations to measures of lung function were of modest strength. No single method of CT scan analysis clearly outperformed the rest of the group. Quantification of the burden of emphysematous lung destruction apparent on CT scan is a weak predictor of lung function and mechanics in severe COPD with no uniformly superior method found to perform this analysis. The CT based quantification of emphysema may augment pulmonary function testing in the characterization of COPD by providing complementary phenotypic information.

Computational methods for evaluation of cell-based data assessment--Bioconductor.

PubMed

Le Meur, Nolwenn

2013-02-01

Recent advances in miniaturization and automation of technologies have enabled cell-based assay high-throughput screening, bringing along new challenges in data analysis. Automation, standardization, reproducibility have become requirements for qualitative research. The Bioconductor community has worked in that direction proposing several R packages to handle high-throughput data including flow cytometry (FCM) experiment. Altogether, these packages cover the main steps of a FCM analysis workflow, that is, data management, quality assessment, normalization, outlier detection, automated gating, cluster labeling, and feature extraction. Additionally, the open-source philosophy of R and Bioconductor, which offers room for new development, continuously drives research and improvement of theses analysis methods, especially in the field of clustering and data mining. This review presents the principal FCM packages currently available in R and Bioconductor, their advantages and their limits. Copyright © 2012 Elsevier Ltd. All rights reserved.

Flow Cytometry Technician | Center for Cancer Research

Cancer.gov

PROGRAM DESCRIPTION The Basic Science Program (BSP) pursues independent, multidisciplinary research in basic and applied molecular biology, immunology, retrovirology, cancer biology, and human genetics. Research efforts and support are an integral part of the Center for Cancer Research (CCR) at the Frederick National Laboratory for Cancer Research (FNLCR). KEY ROLES/RESPONSIBILITIES The Flow Cytometry Core (Flow Core) of the Cancer and Inflammation Program (CIP) is a service core which supports the research efforts of the CCR by providing expertise in the field of flow cytometry (using analyzers and sorters) with the goal of gaining a more thorough understanding of the biology of cancer and cancer cells. The Flow Core provides service to 12-15 CIP laboratories and more than 22 non-CIP laboratories. Flow core staff provide technical advice on the experimental design of applications, which include immunological phenotyping, cell function assays, and cell cycle analysis. Work is performed per customer requirements, and no independent research is involved. The Flow Cytometry Technician will be responsible for: Monitor performance of and maintain high dimensional flow cytometer analyzers and cell sorters Operate high dimensional flow cytometer analyzers and cell sorters Monitoring lab supply levels and order lab supplies, perform various record keeping responsibilities Assist in the training of scientific end users on the use of flow cytometry in their research, as well as how to operate and troubleshoot the bench-top analyzer instruments Experience with sterile technique and tissue culture

Role of receptor occupancy assays by flow cytometry in drug development.

PubMed

Stewart, Jennifer J; Green, Cherie L; Jones, Nicholas; Liang, Meina; Xu, Yuanxin; Wilkins, Danice E C; Moulard, Maxime; Czechowska, Kamila; Lanham, David; McCloskey, Thomas W; Ferbas, John; van der Strate, Barry W A; Högerkorp, Carl-Magnus; Wyant, Timothy; Lackey, Alan; Litwin, Virginia

2016-03-01

The measurement of the binding of a biotherapeutic to its cellular target, receptor occupancy (RO), is increasingly important in development of biologically-based therapeutic agents. Receptor occupancy (RO) assays by flow cytometry describe the qualitative and/or quantitative assessment of the binding of a therapeutic agent to its cell surface target. Such RO assays can be as simple as measuring the number of cell surface receptors bound by an antireceptor therapeutic agent or can be designed to address more complicated scenarios such as internalization or shedding events once a receptor engages the administered therapeutic agent. Data generated from RO assays can also be used to model whether given doses of an experimental therapeutic agent and their administration schedules lead to predicted levels of receptor occupancy and whether the receptor is modulated (up or down) on cells engaged by the therapeutic agent. There are a variety of approaches that can be used when undertaking RO assays and with the ability to measure distinct subsets in heterogeneous populations, flow cytometry is ideally suited to RO measurements. This article highlights the importance of RO assays on the flow cytometric platform in the development of biotherapeutic agents. © 2016 The Authors Cytometry Part B: Clinical Cytometry Published by Wiley Periodicals, Inc.

Targeted detection of murine colonic dysplasia in vivo with flexible multispectral scanning fiber endoscopy

NASA Astrophysics Data System (ADS)

Joshi, Bishnu P.; Miller, Sharon J.; Lee, Cameron; Gustad, Adam; Seibel, Eric J.; Wang, Thomas D.

2012-02-01

We demonstrate a multi-spectral scanning fiber endoscope (SFE) that collects fluorescence images in vivo from three target peptides that bind specifically to murine colonic adenomas. This ultrathin endoscope was demonstrated in a genetically engineered mouse model of spontaneous colorectal adenomas based on somatic Apc (adenomatous polyposis coli) gene inactivation. The SFE delivers excitation at 440, 532, 635 nm with <2 mW per channel. The target 7-mer peptides were conjugated to visible organic dyes, including 7-Diethylaminocoumarin-3-carboxylic acid (DEAC) (λex=432 nm, λem=472 nm), 5-Carboxytetramethylrhodamine (5-TAMRA) (λex=535 nm, λem=568 nm), and CF-633 (λex=633 nm, λem=650 nm). Target peptides were first validated using techniques of pfu counting, flow cytometry and previously established methods of fluorescence endoscopy. Peptides were applied individually or in combination and detected with fluorescence imaging. The ability to image multiple channels of fluorescence concurrently was successful for all three channels in vitro, while two channels were resolved simultaneously in vivo. Selective binding of the peptide was evident to adenomas and not to adjacent normal-appearing mucosa. Multispectral wide-field fluorescence detection using the SFE is achievable, and this technology has potential to advance early cancer detection and image-guided therapy in human patients by simultaneously visualizing multiple over expressed molecular targets unique to dysplasia.

The Means: Cytometry and Mass Spectrometry Converge in a Single Cell Deep Profiling Platform

PubMed Central

Weis-Garcia, Frances; Bandura, Dmitry; Baranov, Vladimir; Ornatsky, Olga; Tanner, Scott

2013-01-01

Inductively Coupled Plasma Mass Spectrometry (ICP-MS) is a distinct flavor of mass spectrometry that has had little association with cell biology: it remains the state of the art for the determination of the atomic composition of materials. Unrelatedly, flow cytometry is the superior method for distinguishing the heterogeneity of cells through the determination of antigen signatures using tagged antibodies. Simply replacing fluorophore tags with stable isotopes of the heavy metals, and measuring these cell-by-cell with ICP-MS, dramatically increases the number of probes that can be simultaneously measured in cytometry and enables a transformative increase in the resolution of rare cell populations in complex biological samples. While this can be thought of as a novel incarnation of single-cell targeted proteomics, the metal-labeling reagents, ICP-MS of single cells, and accompanying informatics comprise a new field of technology termed Mass Cytometry. While the conception of mass cytometry is simple the embodiment to address the issues of multi-parameter flow cytometry has been far more challenging. There are many elements, and many more stable isotopes of those elements, that might be used as distinct reporter tags. Still, there are many approaches to conjugating metals to antibodies (or other affinity reagents) and work in this area along with developing new applications is ongoing. The mass resolution and linear (quantitative) dynamic range of ICP-MS allows those many stable isotopes to be measured simultaneously and without the spectral overlap issues that limit fluorescence assay. However, the adaptation of ICP-MS to allow high-speed simultaneous measurement with single cell distinction at high throughput required innovation of the cell introduction system, ion optics (sampling, transmission and beam-shaping), mass analysis, and signal handling and processing. An overview of “the nuts and bolts” of Mass Cytometry is presented.

Cardiac Hypertrophy is Positively Regulated by MicroRNA-24 in Rats

PubMed Central

Gao, Juan; Zhu, Min; Liu, Rui-Feng; Zhang, Jian-Shu; Xu, Ming

2018-01-01

Background: MicroRNA-24 (miR-24) plays an important role in heart failure by reducing the efficiency of myocardial excitation-contraction coupling. Prolonged cardiac hypertrophy may lead to heart failure, but little is known about the role of miR-24 in cardiac hypertrophy. This study aimed to preliminarily investigate the function of miR-24 and its mechanisms in cardiac hypertrophy. Methods: Twelve Sprague-Dawley rats with a body weight of 50 ± 5 g were recruited and randomly divided into two groups: a transverse aortic constriction (TAC) group and a sham surgery group. Hypertrophy index was measured and calculated by echocardiography and hematoxylin and eosin staining. TargetScans algorithm-based prediction was used to search for the targets of miR-24, which was subsequently confirmed by a real-time polymerase chain reaction and luciferase assay. Immunofluorescence labeling was used to measure the cell surface area, and 3H-leucine incorporation was used to detect the synthesis of total protein in neonatal rat cardiac myocytes (NRCMs) with the overexpression of miR-24. In addition, flow cytometry was performed to observe the alteration in the cell cycle. Statistical analysis was carried out with GraphPad Prism v5.0 and SPSS 19.0. A two-sided P < 0.05 was considered as the threshold for significance. Results: The expression of miR-24 was abnormally increased in TAC rat cardiac tissue (t = −2.938, P < 0.05). TargetScans algorithm-based prediction demonstrated that CDKN1B (p27, Kip1), a cell cycle regulator, was a putative target of miR-24, and was confirmed by luciferase assay. The expression of p27 was decreased in TAC rat cardiac tissue (t = 2.896, P < 0.05). The overexpression of miR-24 in NRCMs led to the decreased expression of p27 (t = 4.400, P < 0.01), and decreased G0/G1 arrest in cell cycle and cardiomyocyte hypertrophy. Conclusion: MiR-24 promotes cardiac hypertrophy partly by affecting the cell cycle through down-regulation of p27 expression. PMID:29786048

Cardiac Hypertrophy is Positively Regulated by MicroRNA‑24 in Rats

PubMed

Gao, Juan; Zhu, Min; Liu, Rui-Feng; Zhang, Jian-Shu; Xu, Ming

2018-06-05

MicroRNA-24 (miR-24) plays an important role in heart failure by reducing the efficiency of myocardial excitation-contraction coupling. Prolonged cardiac hypertrophy may lead to heart failure, but little is known about the role of miR-24 in cardiac hypertrophy. This study aimed to preliminarily investigate the function of miR-24 and its mechanisms in cardiac hypertrophy. Twelve Sprague-Dawley rats with a body weight of 50 ± 5 g were recruited and randomly divided into two groups: a transverse aortic constriction (TAC) group and a sham surgery group. Hypertrophy index was measured and calculated by echocardiography and hematoxylin and eosin staining. TargetScans algorithm-based prediction was used to search for the targets of miR-24, which was subsequently confirmed by a real-time polymerase chain reaction and luciferase assay. Immunofluorescence labeling was used to measure the cell surface area, and 3 H-leucine incorporation was used to detect the synthesis of total protein in neonatal rat cardiac myocytes (NRCMs) with the overexpression of miR-24. In addition, flow cytometry was performed to observe the alteration in the cell cycle. Statistical analysis was carried out with GraphPad Prism v5.0 and SPSS 19.0. A two-sided P < 0.05 was considered as the threshold for significance. The expression of miR-24 was abnormally increased in TAC rat cardiac tissue (t = -2.938, P < 0.05). TargetScans algorithm-based prediction demonstrated that CDKN1B (p27, Kip1), a cell cycle regulator, was a putative target of miR-24, and was confirmed by luciferase assay. The expression of p27 was decreased in TAC rat cardiac tissue (t = 2.896, P < 0.05). The overexpression of miR-24 in NRCMs led to the decreased expression of p27 (t = 4.400, P < 0.01), and decreased G0/G1 arrest in cell cycle and cardiomyocyte hypertrophy. MiR-24 promotes cardiac hypertrophy partly by affecting the cell cycle through down-regulation of p27 expression.

Label-free in vivo flow cytometry in zebrafish using two-photon autofluorescence imaging.

PubMed

Zeng, Yan; Xu, Jin; Li, Dong; Li, Li; Wen, Zilong; Qu, Jianan Y

2012-07-01

We demonstrate a label-free in vivo flow cytometry in zebrafish blood vessels based on two-photon excited autofluorescence imaging. The major discovery in this work is the strong autofluorescence emission from the plasma in zebrafish blood. The plasma autofluorescence provides excellent contrast for visualizing blood vessels and counting blood cells. In addition, the cellular nicotinamide adenine dinucleotide autofluorescence enables in vivo imaging and counting of white blood cells (neutrophils).

Flow Cytometry Techniques in Radiation Biology

DTIC Science & Technology

1988-06-01

Henidtopoietic stem cells SUMMARY Hematopoietic stem cells ( HSC ) are present in the marrow at a concentration of approximately 2-3 HSC per 1000 nucleated marrow...cells. In the past, only clonogenic assays requiring 8-13 days and ten irradiated recipient rodents were available for assaying HSC . Because of the...importance of HSC in the postirradiation syndrome, we have developed a new rapid method based on flow cytometry not only to assay but also to purify and

Development of a Flow Cytometry-Based Method for Rapid Detection of Escherichia coli and Shigella Spp. Using an Oligonucleotide Probe

PubMed Central

Xue, Yong; Wilkes, Jon G.; Moskal, Ted J.; Williams, Anna J.; Cooper, Willie M.; Nayak, Rajesh; Rafii, Fatemeh; Buzatu, Dan A.

2016-01-01

Standard methods to detect Escherichia coli contamination in food use the polymerase chain reaction (PCR) and agar culture plates. These methods require multiple incubation steps and take a long time to results. An improved rapid flow-cytometry based detection method was developed, using a fluorescence-labeled oligonucleotide probe specifically binding a16S rRNA sequence. The method positively detected 51 E. coli isolates as well as 4 Shigella species. All 27 non-E. coli strains tested gave negative results. Comparison of the new genetic assay with a total plate count (TPC) assay and agar plate counting indicated similar sensitivity, agreement between cytometry cell and colony counts. This method can detect a small number of E.coli cells in the presence of large numbers of other bacteria. This method can be used for rapid, economical, and stable detection of E. coli and Shigella contamination in the food industry and other contexts. PMID:26913737

Development of a Flow Cytometry-Based Method for Rapid Detection of Escherichia coli and Shigella Spp. Using an Oligonucleotide Probe.

PubMed

Xue, Yong; Wilkes, Jon G; Moskal, Ted J; Williams, Anna J; Cooper, Willie M; Nayak, Rajesh; Rafii, Fatemeh; Buzatu, Dan A

2016-01-01

Standard methods to detect Escherichia coli contamination in food use the polymerase chain reaction (PCR) and agar culture plates. These methods require multiple incubation steps and take a long time to results. An improved rapid flow-cytometry based detection method was developed, using a fluorescence-labeled oligonucleotide probe specifically binding a16S rRNA sequence. The method positively detected 51 E. coli isolates as well as 4 Shigella species. All 27 non-E. coli strains tested gave negative results. Comparison of the new genetic assay with a total plate count (TPC) assay and agar plate counting indicated similar sensitivity, agreement between cytometry cell and colony counts. This method can detect a small number of E.coli cells in the presence of large numbers of other bacteria. This method can be used for rapid, economical, and stable detection of E. coli and Shigella contamination in the food industry and other contexts.

Microflow1, a sheathless fiber-optic flow cytometry biomedical platform: demonstration onboard the international space station.

PubMed

Dubeau-Laramée, Geneviève; Rivière, Christophe; Jean, Isabelle; Mermut, Ozzy; Cohen, Luchino Y

2014-04-01

A fiber-optic based flow cytometry platform was designed to build a portable and robust instrument for space applications. At the core of the Microflow1 is a unique fiber-optic flow cell fitted to a fluidic system and fiber coupled to the source and detection channels. A Microflow1 engineering unit was first tested and benchmarked against a commercial flow cytometer as a reference in a standard laboratory environment. Testing in parabolic flight campaigns was performed to establish Microflow1's performance in weightlessness, before operating the new platform on the International Space Station. Microflow1 had comparable performances to commercial systems, and operated remarkably and robustly in weightlessness (microgravity). Microflow1 supported immunophenotyping as well as microbead-based multiplexed cytokine assays in the space environment and independently of gravity levels. Results presented here provide evidence that this fiber-optic cytometer technology is inherently compatible with the space environment with negligible compromise to analytical performance. © 2013 International Society for Advancement of Cytometry.

Ultrasonic Data Display and Analysis System Developed (Including Fuzzy Logic Analysis) for the Windows-Based PC

NASA Technical Reports Server (NTRS)

Lovelace, Jeffrey J.; Cios, Kryzsztof J.; Roth, Don J.; cAO, wEI n.

2001-01-01

Post-Scan Interactive Data Display (PSIDD) III is a user-oriented Windows-based system that facilitates the display and comparison of ultrasonic contact measurement data obtained at NASA Glenn Research Center's Ultrasonic Nondestructive Evaluation measurement facility. The system is optimized to compare ultrasonic measurements made at different locations within a material or at different stages of material degradation. PSIDD III provides complete analysis of the primary waveforms in the time and frequency domains along with the calculation of several frequency-dependent properties including phase velocity and attenuation coefficient and several frequency-independent properties, like the cross correlation velocity. The system allows image generation on all the frequency-dependent properties at any available frequency (limited by the bandwidth used in the scans) and on any of the frequency-independent properties. From ultrasonic contact scans, areas of interest on an image can be studied with regard to underlying raw waveforms and derived ultrasonic properties by simply selecting the point on the image. The system offers various modes of indepth comparison between scan points. Up to five scan points can be selected for comparative analysis at once. The system was developed with Borland Delphi software (Visual Pascal) and is based on an SQL data base. It is ideal for the classification of material properties or the location of microstructure variations in materials. Along with the ultrasonic contact measurement software that it is partnered with, this system is technology ready and can be transferred to users worldwide.

Utility of peripheral blood immunophenotyping by flow cytometry in the diagnosis of pediatric acute leukemia.

PubMed

Metrock, Laura K; Summers, Ryan J; Park, Sunita; Gillespie, Scott; Castellino, Sharon; Lew, Glen; Keller, Frank G

2017-10-01

Childhood acute leukemia is traditionally diagnosed from a bone marrow aspirate (BMA). New-onset acute leukemia patients do not always have visible circulating blasts in the peripheral blood (PB) at diagnosis. While the role of bone marrow flow cytometry for the diagnosis of acute leukemia is well established, the utility of PB flow cytometry (PBFC) is unknown. We performed a single-institution retrospective analysis to compare PBFC versus BMA in establishing or excluding a diagnosis of childhood acute leukemia. We retrospectively identified 485 PBFC samples with concurrent BMA from 2008 to 2013. Results of four-color flow cytometry for immunophenotypic characterization of leukemic versus nonclonal disease were characterized. Sensitivity and specificity were calculated among patients without a known diagnosis or prior therapy. Among 485 samples eligible for analysis, 120 had negative PBFC and BMA, 359 had positive PBFC and BMA, 3 had negative PBFC and positive BMA, and 3 had positive PBFC and negative BMA. There were small but significant differences in sensitivity (100 vs. 93.8%; P = 0.002) and positive predictive value (100 vs. 93.8%; P = 0.002) favoring BMA over PBFC among those demonstrating absence of circulating morphologic blasts. PBFC has high sensitivity and specificity for the diagnosis of childhood acute leukemia. The predictive value of PBFC remains high for patients without visible circulating blasts and may enhance the diagnostic process for determining the indications for marrow testing. © 2017 Wiley Periodicals, Inc.

Impulse excitation scanning acoustic microscopy for local quantification of Rayleigh surface wave velocity using B-scan analysis

NASA Astrophysics Data System (ADS)

Cherry, M.; Dierken, J.; Boehnlein, T.; Pilchak, A.; Sathish, S.; Grandhi, R.

2018-01-01

A new technique for performing quantitative scanning acoustic microscopy imaging of Rayleigh surface wave (RSW) velocity was developed based on b-scan processing. In this technique, the focused acoustic beam is moved through many defocus distances over the sample and excited with an impulse excitation, and advanced algorithms based on frequency filtering and the Hilbert transform are used to post-process the b-scans to estimate the Rayleigh surface wave velocity. The new method was used to estimate the RSW velocity on an optically flat E6 glass sample, and the velocity was measured at ±2 m/s and the scanning time per point was on the order of 1.0 s, which are both improvement from the previous two-point defocus method. The new method was also applied to the analysis of two titanium samples, and the velocity was estimated with very low standard deviation in certain large grains on the sample. A new behavior was observed with the b-scan analysis technique where the amplitude of the surface wave decayed dramatically on certain crystallographic orientations. The new technique was also compared with previous results, and the new technique has been found to be much more reliable and to have higher contrast than previously possible with impulse excitation.

Biotechnology Apprenticeship for Secondary-Level Students: Teaching Advanced Cell Culture Techniques for Research

PubMed Central

Lewis, Jennifer R.; Kotur, Mark S.; Butt, Omar; Kulcarni, Sumant; Riley, Alyssa A.; Ferrell, Nick; Sullivan, Kathryn D.; Ferrari, Mauro

2002-01-01

The purpose of this article is to discuss small-group apprenticeships (SGAs) as a method to instruct cell culture techniques to high school participants. The study aimed to teach cell culture practices and to introduce advanced imaging techniques to solve various biomedical engineering problems. Participants designed and completed experiments using both flow cytometry and laser scanning cytometry during the 1-month summer apprenticeship. In addition to effectively and efficiently teaching cell biology laboratory techniques, this course design provided an opportunity for research training, career exploration, and mentoring. Students participated in active research projects, working with a skilled interdisciplinary team of researchers in a large research institution with access to state-of-the-art instrumentation. The instructors, composed of graduate students, laboratory managers, and principal investigators, worked well together to present a real and worthwhile research experience. The students enjoyed learning cell culture techniques while contributing to active research projects. The institution's researchers were equally enthusiastic to instruct and serve as mentors. In this article, we clarify and illuminate the value of small-group laboratory apprenticeships to the institution and the students by presenting the results and experiences of seven middle and high school participants and their instructors. PMID:12587031

Biotechnology apprenticeship for secondary-level students: teaching advanced cell culture techniques for research.

PubMed

Lewis, Jennifer R; Kotur, Mark S; Butt, Omar; Kulcarni, Sumant; Riley, Alyssa A; Ferrell, Nick; Sullivan, Kathryn D; Ferrari, Mauro

2002-01-01

The purpose of this article is to discuss small-group apprenticeships (SGAs) as a method to instruct cell culture techniques to high school participants. The study aimed to teach cell culture practices and to introduce advanced imaging techniques to solve various biomedical engineering problems. Participants designed and completed experiments using both flow cytometry and laser scanning cytometry during the 1-month summer apprenticeship. In addition to effectively and efficiently teaching cell biology laboratory techniques, this course design provided an opportunity for research training, career exploration, and mentoring. Students participated in active research projects, working with a skilled interdisciplinary team of researchers in a large research institution with access to state-of-the-art instrumentation. The instructors, composed of graduate students, laboratory managers, and principal investigators, worked well together to present a real and worthwhile research experience. The students enjoyed learning cell culture techniques while contributing to active research projects. The institution's researchers were equally enthusiastic to instruct and serve as mentors. In this article, we clarify and illuminate the value of small-group laboratory apprenticeships to the institution and the students by presenting the results and experiences of seven middle and high school participants and their instructors.

Near infrared lasers in flow cytometry.

PubMed

Telford, William G

2015-07-01

Technology development in flow cytometry has closely tracked laser technology, the light source that flow cytometers almost exclusively use to excite fluorescent probes. The original flow cytometers from the 1970s and 1980s used large water-cooled lasers to produce only one or two laser lines at a time. Modern cytometers can take advantage of the revolution in solid state laser technology to use almost any laser wavelength ranging from the ultraviolet to the near infrared. Commercial cytometers can now be equipped with many small solid state lasers, providing almost any wavelength needed for cellular analysis. Flow cytometers are now equipped to analyze 20 or more fluorescent probes simultaneously, requiring multiple laser wavelengths. Instrument developers are now trying to increase this number by designing fluorescent probes that can be excited by laser wavelength at the "edges" of the visible light range, in the near ultraviolet and near-infrared region. A variety of fluorescent probes have been developed that excite with violet and long wavelength ultraviolet light; however, the near-infrared range (660-800 nm) has yet seen only exploitation in flow cytometry. Fortunately, near-infrared laser diodes and other solid state laser technologies appropriate for flow cytometry have been in existence for some time, and can be readily incorporated into flow cytometers to accelerate fluorescent probe development. The near infrared region represents one of the last "frontiers" to maximize the number of fluorescent probes that can be analyzed by flow cytometry. In addition, near infrared fluorescent probes used in biomedical tracking and imaging could also be employed for flow cytometry with the correct laser wavelengths. This review describes the available technology, including lasers, fluorescent probes and detector technology optimal for near infrared signal detection. Published by Elsevier Inc.

Toxic Effects of Ethyl Cinnamate on the Photosynthesis and Physiological Characteristics of Chlorella vulgaris Based on Chlorophyll Fluorescence and Flow Cytometry Analysis

PubMed Central

Jiao, Yang; Ouyang, Hui-Ling; Jiang, Yu-Jiao; Kong, Xiang-Zhen; He, Wei; Liu, Wen-Xiu; Yang, Bin; Xu, Fu-Liu

2015-01-01

The toxic effects of ethyl cinnamate on the photosynthetic and physiological characteristics of Chlorella vulgaris were studied based on chlorophyll fluorescence and flow cytometry analysis. Parameters, including biomass, F v/F m (maximal photochemical efficiency of PSII), ФPSII (actual photochemical efficiency of PSII in the light), FDA, and PI staining fluorescence, were measured. The results showed the following: (1) The inhibition on biomass increased as the exposure concentration increased. 1 mg/L ethyl cinnamate was sufficient to reduce the total biomass of C. vulgaris. The 48-h and 72-h EC50 values were 2.07 mg/L (1.94–2.20) and 1.89 mg/L (1.82–1.97). (2) After 24 h of exposure to 2–4 mg/L ethyl cinnamate, the photosynthesis of C. vulgaris almost ceased, manifesting in ФPSII being close to zero. After 72 h of exposure to 4 mg/L ethyl cinnamate, the F v/F m of C. vulgaris dropped to zero. (3) Ethyl cinnamate also affected the cellular physiology of C. vulgaris, but these effects resulted in the inhibition of cell yield rather than cell death. Exposure to ethyl cinnamate resulted in decreased esterase activities in C. vulgaris, increased average cell size, and altered intensities of chlorophyll a fluorescence. Overall, esterase activity was the most sensitive variable. PMID:26101784

Page layout analysis and classification for complex scanned documents

NASA Astrophysics Data System (ADS)

Erkilinc, M. Sezer; Jaber, Mustafa; Saber, Eli; Bauer, Peter; Depalov, Dejan

2011-09-01

A framework for region/zone classification in color and gray-scale scanned documents is proposed in this paper. The algorithm includes modules for extracting text, photo, and strong edge/line regions. Firstly, a text detection module which is based on wavelet analysis and Run Length Encoding (RLE) technique is employed. Local and global energy maps in high frequency bands of the wavelet domain are generated and used as initial text maps. Further analysis using RLE yields a final text map. The second module is developed to detect image/photo and pictorial regions in the input document. A block-based classifier using basis vector projections is employed to identify photo candidate regions. Then, a final photo map is obtained by applying probabilistic model based on Markov random field (MRF) based maximum a posteriori (MAP) optimization with iterated conditional mode (ICM). The final module detects lines and strong edges using Hough transform and edge-linkages analysis, respectively. The text, photo, and strong edge/line maps are combined to generate a page layout classification of the scanned target document. Experimental results and objective evaluation show that the proposed technique has a very effective performance on variety of simple and complex scanned document types obtained from MediaTeam Oulu document database. The proposed page layout classifier can be used in systems for efficient document storage, content based document retrieval, optical character recognition, mobile phone imagery, and augmented reality.

CymeR: cytometry analysis using KNIME, docker and R

PubMed Central

Muchmore, B.; Alarcón-Riquelme, M.E.

2017-01-01

Abstract Summary: Here we present open-source software for the analysis of high-dimensional cytometry data using state of the art algorithms. Importantly, use of the software requires no programming ability, and output files can either be interrogated directly in CymeR or they can be used downstream with any other cytometric data analysis platform. Also, because we use Docker to integrate the multitude of components that form the basis of CymeR, we have additionally developed a proof-of-concept of how future open-source bioinformatic programs with graphical user interfaces could be developed. Availability and Implementation: CymeR is open-source software that ties several components into a single program that is perhaps best thought of as a self-contained data analysis operating system. Please see https://github.com/bmuchmore/CymeR/wiki for detailed installation instructions. Contact: brian.muchmore@genyo.es or marta.alarcon@genyo.es PMID:27998935

CymeR: cytometry analysis using KNIME, docker and R.

PubMed

Muchmore, B; Alarcón-Riquelme, M E

2017-03-01

Here we present open-source software for the analysis of high-dimensional cytometry data using state of the art algorithms. Importantly, use of the software requires no programming ability, and output files can either be interrogated directly in CymeR or they can be used downstream with any other cytometric data analysis platform. Also, because we use Docker to integrate the multitude of components that form the basis of CymeR, we have additionally developed a proof-of-concept of how future open-source bioinformatic programs with graphical user interfaces could be developed. CymeR is open-source software that ties several components into a single program that is perhaps best thought of as a self-contained data analysis operating system. Please see https://github.com/bmuchmore/CymeR/wiki for detailed installation instructions. brian.muchmore@genyo.es or marta.alarcon@genyo.es. © The Author 2016. Published by Oxford University Press.

Point-based and model-based geolocation analysis of airborne laser scanning data

NASA Astrophysics Data System (ADS)

Sefercik, Umut Gunes; Buyuksalih, Gurcan; Jacobsen, Karsten; Alkan, Mehmet

2017-01-01

Airborne laser scanning (ALS) is one of the most effective remote sensing technologies providing precise three-dimensional (3-D) dense point clouds. A large-size ALS digital surface model (DSM) covering the whole Istanbul province was analyzed by point-based and model-based comprehensive statistical approaches. Point-based analysis was performed using checkpoints on flat areas. Model-based approaches were implemented in two steps as strip to strip comparing overlapping ALS DSMs individually in three subareas and comparing the merged ALS DSMs with terrestrial laser scanning (TLS) DSMs in four other subareas. In the model-based approach, the standard deviation of height and normalized median absolute deviation were used as the accuracy indicators combined with the dependency of terrain inclination. The results demonstrate that terrain roughness has a strong impact on the vertical accuracy of ALS DSMs. From the relative horizontal shifts determined and partially improved by merging the overlapping strips and comparison of the ALS, and the TLS, data were found not to be negligible. The analysis of ALS DSM in relation to TLS DSM allowed us to determine the characteristics of the DSM in detail.

Frequencies of micronucleated reticulocytes, a dosimeter of DNA double-strand breaks, in infants receiving computed tomography or cardiac catheterization.

PubMed

Khattab, Mona; Walker, Dale M; Albertini, Richard J; Nicklas, Janice A; Lundblad, Lennart K A; Vacek, Pamela M; Walker, Vernon E

2017-08-01

The use of computed tomography (CT scans) has increased dramatically in recent decades, raising questions about the long-term safety of CT-emitted x-rays especially in infants who are more sensitive to radiation-induced effects. Cancer risk estimates for CT scans typically are extrapolated from models; therefore, new approaches measuring actual DNA damage are needed for improved estimations. Hence, changes in a dosimeter of DNA double-strand breaks, micronucleated reticulocytes (MN-RETs) measured by flow cytometry, were investigated in mice and infants exposed to CT scans. In male C57BL/6N mice (6-8 weeks-of-age), there was a dose-related increase in MN-RETs in blood samples collected 48h after CT scans delivering targeted exposures of 1-130 cGy x-rays (n=5-10/group, r=0.994, p=0.01), with significant increases occurring at exposure levels as low as 0.83 cGy x-rays compared to control mice (p=0.002). In paired blood specimens from infants with no history of a prior CT scan, there was no difference in MN-RET frequencies found 2h before (mean, 0.10±0.07%) versus 48h after (mean, 0.11±0.05%) a scheduled CT scan/cardiac catheterization. However, in infants having prior CT scan(s), MN-RET frequencies measured at 48h after a scheduled CT scan (mean=0.22±0.12%) were significantly higher than paired baseline values (mean, 0.17±0.07%; p=0.032). Increases in baseline (r=0.722, p<0.001) and 48-h post exposure (r=0.682, p<0.001) levels of MN-RETs in infants with a history of prior CT scans were significantly correlated with the number of previous CT scans. These preliminary findings suggest that prior CT scans increase the cellular responses to subsequent CT exposures. Thus, further investigation is needed to characterize the potential cancer risk from single versus repeated CT scans or cardiac catheterizations in infants. Copyright © 2017 Elsevier B.V. All rights reserved.

Screening Nylon-3 Polymers, a New Class of Cationic Amphiphiles, for siRNA Delivery

PubMed Central

2015-01-01

Amphiphilic nucleic acid carriers have attracted strong interest. Three groups of nylon-3 copolymers (poly-β-peptides) possessing different cationic/hydrophobic content were evaluated as siRNA delivery agents in this study. Their ability to condense siRNA was determined in SYBR Gold assays. Their cytotoxicity was tested by MTT assays, their efficiency of delivering Alexa Fluor-488-labeled siRNA intracellularly in the presence and absence of uptake inhibitors was assessed by flow cytometry, and their transfection efficacies were studied by luciferase knockdown in a cell line stably expressing luciferase (H1299/Luc). Endosomal release was determined by confocal laser scanning microscopy and colocalization with lysotracker. All polymers efficiently condensed siRNA at nitrogen-to-phosphate (N/P) ratios of 5 or lower, as reflected in hydrodynamic diameters smaller than that at N/P 1. Although several formulations had negative zeta potentials at N/P 1, G2C and G2D polyplexes yielded >80% uptake in H1299/Luc cells, as determined by flow cytometry. Luciferase knockdown (20–65%) was observed after transfection with polyplexes made of the high molecular weight polymers that were the most hydrophobic. The ability of nylon-3 polymers to deliver siRNA intracellularly even at negative zeta potential implies that they mediate transport across cell membranes based on their amphiphilicity. The cellular uptake route was determined to strongly depend on the presence of cholesterol in the cell membrane. These polymers are, therefore, very promising for siRNA delivery at reduced surface charge and toxicity. Our study identified nylon-3 formulations at low N/P ratios for effective gene knockdown, indicating that nylon-3 polymers are a new, promising type of gene delivery agent. PMID:25437915

Screening nylon-3 polymers, a new class of cationic amphiphiles, for siRNA delivery.

PubMed

Nadithe, Venkatareddy; Liu, Runhui; Killinger, Bryan A; Movassaghian, Sara; Kim, Na Hyung; Moszczynska, Anna B; Masters, Kristyn S; Gellman, Samuel H; Merkel, Olivia M

2015-02-02

Amphiphilic nucleic acid carriers have attracted strong interest. Three groups of nylon-3 copolymers (poly-β-peptides) possessing different cationic/hydrophobic content were evaluated as siRNA delivery agents in this study. Their ability to condense siRNA was determined in SYBR Gold assays. Their cytotoxicity was tested by MTT assays, their efficiency of delivering Alexa Fluor-488-labeled siRNA intracellularly in the presence and absence of uptake inhibitors was assessed by flow cytometry, and their transfection efficacies were studied by luciferase knockdown in a cell line stably expressing luciferase (H1299/Luc). Endosomal release was determined by confocal laser scanning microscopy and colocalization with lysotracker. All polymers efficiently condensed siRNA at nitrogen-to-phosphate (N/P) ratios of 5 or lower, as reflected in hydrodynamic diameters smaller than that at N/P 1. Although several formulations had negative zeta potentials at N/P 1, G2C and G2D polyplexes yielded >80% uptake in H1299/Luc cells, as determined by flow cytometry. Luciferase knockdown (20-65%) was observed after transfection with polyplexes made of the high molecular weight polymers that were the most hydrophobic. The ability of nylon-3 polymers to deliver siRNA intracellularly even at negative zeta potential implies that they mediate transport across cell membranes based on their amphiphilicity. The cellular uptake route was determined to strongly depend on the presence of cholesterol in the cell membrane. These polymers are, therefore, very promising for siRNA delivery at reduced surface charge and toxicity. Our study identified nylon-3 formulations at low N/P ratios for effective gene knockdown, indicating that nylon-3 polymers are a new, promising type of gene delivery agent.

Flow cytometry of sputum: assessing inflammation and immune response elements in the bronchial airways

PubMed Central

Lay, John C.; Peden, David B.; Alexis, Neil E.

2012-01-01

Background The evaluation of sputum leukocytes by flow cytometry is an opportunity to assess characteristics of cells residing in the central airways, yet it is hampered by certain inherent properties of sputum including mucus and large amounts of contaminating cells and debris. Objective To develop a gating strategy based on specific antibody panels in combination with light scatter properties for flow cytometric evaluation of sputum cells. Methods Healthy and mild asthmatic volunteers underwent sputum induction. Manually selected mucus “plug” material was treated with dithiothrietol, filtered and total leukocytes acquired. Multicolor flow cytometry was performed using specific gating strategies based on light scatter properties, differential expression of CD45 and cell lineage markers to discriminate leukocytes from squamous epithelial cells and debris. Results The combination of forward scatter and CD45 expression reliably segregated sputum leukocytes from contaminating squamous epithelial cells and debris. Overlap of major leukocyte populations (neutrophils, macrophages/monocytes) required the use of specific antibodies (e.g. CD16, CD64, CD14, HLA-DR) that differentiated granulocytes from monocytes and macrophages. These gating strategies allowed identification of small populations of eosinophils, CD11c+ myeloid dendritic cells, B cells and NK cells. Conclusions Multicolor flow cytometry can be successfully applied to sputum samples to identify and characterize leukocyte populations residing on the surfaces of the central airways. PMID:21639708

Rapid and Sensitive Quantification of Vibrio cholerae and Vibrio mimicus Cells in Water Samples by Use of Catalyzed Reporter Deposition Fluorescence In Situ Hybridization Combined with Solid-Phase Cytometry

PubMed Central

Schauer, Sonja; Sommer, Regina; Farnleitner, Andreas H.

2012-01-01

A new protocol for rapid, specific, and sensitive cell-based quantification of Vibrio cholerae/Vibrio mimicus in water samples was developed. The protocol is based on catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) in combination with solid-phase cytometry. For pure cultures, we were able to quantify down to 6 V. cholerae cells on one membrane with a relative precision of 39% and down to 12 cells with a relative precision of 17% after hybridization with the horseradish peroxidase (HRP)-labeled probe Vchomim1276 (specific for V. cholerae and V. mimicus) and signal amplification. The corresponding position of the probe on the 16S rRNA is highly accessible even when labeled with HRP. For the first time, we were also able to successfully quantify V. cholerae/V. mimicus via solid-phase cytometry in extremely turbid environmental water samples collected in Austria. Cell numbers ranged from 4.5 × 101 cells ml−1 in the large saline lake Neusiedler See to 5.6 × 104 cells ml−1 in an extremely turbid shallow soda lake situated nearby. We therefore suggest CARD-FISH in combination with solid-phase cytometry as a powerful tool to quantify V. cholerae/V. mimicus in ecological studies as well as for risk assessment and monitoring programs. PMID:22885749

Automated sample area definition for high-throughput microscopy.

PubMed

Zeder, M; Ellrott, A; Amann, R

2011-04-01

High-throughput screening platforms based on epifluorescence microscopy are powerful tools in a variety of scientific fields. Although some applications are based on imaging geometrically defined samples such as microtiter plates, multiwell slides, or spotted gene arrays, others need to cope with inhomogeneously located samples on glass slides. The analysis of microbial communities in aquatic systems by sample filtration on membrane filters followed by multiple fluorescent staining, or the investigation of tissue sections are examples. Therefore, we developed a strategy for flexible and fast definition of sample locations by the acquisition of whole slide overview images and automated sample recognition by image analysis. Our approach was tested on different microscopes and the computer programs are freely available (http://www.technobiology.ch). Copyright © 2011 International Society for Advancement of Cytometry.

Aptamer-based microspheres for highly sensitive protein detection using fluorescently-labeled DNA nanostructures.

PubMed

Han, Daehoon; Hong, Jinkee; Kim, Hyun Cheol; Sung, Jong Hwan; Lee, Jong Bum

2013-11-01

Many highly sensitive protein detection techniques have been developed and have played an important role in the analysis of proteins. Herein, we report a novel technique that can detect proteins sensitively and effectively using aptamer-based DNA nanostructures. Thrombin was used as a target protein and aptamer was used to capture fluorescent dye-labeled DNA nanobarcodes or thrombin on a microsphere. The captured DNA nanobarcodes were replaced by a thrombin and aptamer interaction. The detection ability of this approach was confirmed by flow cytometry with different concentrations of thrombin. Our detection method has great potential for rapid and simple protein detection with a variety of aptamers.

Multiparametric flow cytometry in the diagnosis and characterization of low-grade pulmonary mucosa-associated lymphoid tissue lymphomas.

PubMed

Zaer, F S; Braylan, R C; Zander, D S; Iturraspe, J A; Almasri, N M

1998-06-01

Primary mucosa associated lymphoid tissue (MALT) lymphomas are rare neoplasms that seem to have a better prognosis than nodal lymphomas. Morphologic diagnosis of these lesions may be difficult because of features that overlap with those of benign lymphoid infiltrates. In this study, we assessed the contribution of multi-parametric flow cytometry in demonstrating clonality and further characterizing pulmonary MALT lymphomas. Based on a clinical or pathologic suspicion of MALT-lymphoma, 3 transbronchial biopsies, 4 fine needle aspirates, 1 core needle biopsy, and 13 wedge excisions of lung were submitted fresh (unfixed) to our laboratory for evaluation. Among the 13 cases diagnosed as MALT lymphomas, B-cell monoclonality was established by identifying expression of a single immunoglobulin light chain on CD20 or CD19-positive cells in 12 cases. One case lacked expression of both light chains on B-cells. Of 11 lymphoma cases in which CD5 and CD10 surface antigens were assessed, no cases expressed CD10, and 1 case demonstrated weak CD5 expression. Nine of 10 cases studied were diploid and 1 case was hyperdiploid. All of the lymphomas displayed low (< or = 3%) S-phase fractions consistent with low grade processes. In 10 patients with short follow-up, none died of their disease and the majority had no evidence of lymphoma dissemination. In seven of the remaining eight cases, B-cells were polyclonal consistent with reactive processes. In one morphologically reactive case, flow cytometric analysis was unsuccessful because of poor cell viability. The pulmonary MALT lymphomas in this study represent a group of B-cell tumors with distinctive morphologic, immunophenotypic, and cell kinetic characteristics. Multi-parametric flow cytometry is useful for confirming B-cell monoclonality and illustrating an antigenic profile compatible with this diagnosis. Flow cytometry can be particularly helpful when working with small biopsies and cytologic samples with limited diagnostic material and may abrogate the need for more aggressive surgical procedures.

Ultraviolet 320 nm laser excitation for flow cytometry.

PubMed

Telford, William; Stickland, Lynn; Koschorreck, Marco

2017-04-01

Although multiple lasers and high-dimensional analysis capability are now standard on advanced flow cytometers, ultraviolet (UV) lasers (usually 325-365 nm) remain an uncommon excitation source for cytometry. This is primarily due to their cost, and the small number of applications that require this wavelength. The development of the Brilliant Ultraviolet (BUV fluorochromes, however, has increased the importance of this formerly niche excitation wavelength. Historically, UV excitation was usually provided by water-cooled argon- and krypton-ion lasers. Modern flow cytometers primary rely on diode pumped solid state lasers emitting at 355 nm. While useful for all UV-excited applications, DPSS UV lasers are still large by modern solid state laser standards, and remain very expensive. Smaller and cheaper near UV laser diodes (NUVLDs) emitting at 375 nm make adequate substitutes for 355 nm sources in many situations, but do not work as well with very short wavelength probes like the fluorescent calcium chelator indo-1. In this study, we evaluate a newly available UV 320 nm laser for flow cytometry. While shorter in wavelength that conventional UV lasers, 320 is close to the 325 nm helium-cadmium wavelength used in the past on early benchtop cytometers. A UV 320 nm laser was found to excite almost all Brilliant Ultraviolet dyes to nearly the same level as 355 nm sources. Both 320 nm and 355 nm sources worked equally well for Hoechst and DyeCycle Violet side population analysis of stem cells in mouse hematopoetic tissue. The shorter wavelength UV source also showed excellent excitation of indo-1, a probe that is not compatible with NUVLD 375 nm sources. In summary, a 320 nm laser module made a suitable substitute for conventional 355 nm sources. This laser technology is available in a smaller form factor than current 355 nm units, making it useful for small cytometers with space constraints. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

Hydrostatic Pressure Enhances Vital Staining with Carboxyfluorescein or Carboxydichlorofluorescein in Saccharomyces cerevisiae: Efficient Detection of Labeled Yeasts by Flow Cytometry

PubMed Central

Abe, Fumiyoshi

1998-01-01

The extent of intracellular accumulation of the fluorescent dye carboxyfluorescein or carboxydichlorofluorescein (CDCF) in Saccharomyces cerevisiae was found to be increased 5- to 10-fold under a nonlethal hydrostatic pressure of 30 to 50 MPa. This observation was confirmed by analysis of individual labeled cells by flow cytometry. The pressure-induced enhancement of staining with CDCF required d-glucose and was markedly inhibited by 2-deoxy-d-glucose, suggesting that glucose metabolism has a role in the process. PMID:9501452

Assessment of Severity of Ovine Smoke Inhalation Injury by Analysis of Computed Tomographic Scans

DTIC Science & Technology

2003-09-01

Computerized analysis of three- dimensional reconstructed scans was also performed, based on Hounsfield unit ranges: hyperinflated, 1,000 to 900; normal...the interactive segmentation function of the software. The pulmonary parenchyma was separated into four regions based on the Hounsfield unit (HU...SII) severity. Methods: Twenty anesthetized sheep underwent graded SII: group I, no smoke; group II, 5 smoke units ; group III, 10 units ; and group IV

Assessment of cell concentration and viability of isolated hepatocytes using flow cytometry.

PubMed

Wigg, Alan J; Phillips, John W; Wheatland, Loretta; Berry, Michael N

2003-06-01

The assessment of cell concentration and viability of freshly isolated hepatocyte preparations has been traditionally performed using manual counting with a Neubauer counting chamber and staining for trypan blue exclusion. Despite the simple and rapid nature of this assessment, concerns about the accuracy of these methods exist. Simple flow cytometry techniques which determine cell concentration and viability are available yet surprisingly have not been extensively used or validated with isolated hepatocyte preparations. We therefore investigated the use of flow cytometry using TRUCOUNT Tubes and propidium iodide staining to measure cell concentration and viability of isolated rat hepatocytes in suspension. Analysis using TRUCOUNT Tubes provided more accurate and reproducible measurement of cell concentration than manual cell counting. Hepatocyte viability, assessed using propidium iodide, correlated more closely than did trypan blue exclusion with all indicators of hepatocyte integrity and function measured (lactate dehydrogenase leakage, cytochrome p450 content, cellular ATP concentration, ammonia and lactate removal, urea and albumin synthesis). We conclude that flow cytometry techniques can be used to measure cell concentration and viability of isolated hepatocyte preparations. The techniques are simple, rapid, and more accurate than manual cell counting and trypan blue staining and the results are not affected by protein-containing media.

Barcoding of live human PBMC for multiplexed mass cytometry*

PubMed Central

Mei, Henrik E.; Leipold, Michael D.; Schulz, Axel Ronald; Chester, Cariad; Maecker, Holden T.

2014-01-01

Mass cytometry is developing as a means of multiparametric single cell analysis. Here, we present an approach to barcoding separate live human PBMC samples for combined preparation and acquisition on a CyTOF® instrument. Using six different anti-CD45 antibody (Ab) conjugates labeled with Pd104, Pd106, Pd108, Pd110, In113, and In115, respectively, we barcoded up to 20 samples with unique combinations of exactly three different CD45 Ab tags. Cell events carrying more than or less than three different tags were excluded from analyses during Boolean data deconvolution, allowing for precise sample assignment and the electronic removal of cell aggregates. Data from barcoded samples matched data from corresponding individually stained and acquired samples, at cell event recoveries similar to individual sample analyses. The approach greatly reduced technical noise and minimizes unwanted cell doublet events in mass cytometry data, and reduces wet work and antibody consumption. It also eliminates sample-to-sample carryover and the requirement of instrument cleaning between samples, thereby effectively reducing overall instrument runtime. Hence, CD45-barcoding facilitates accuracy of mass cytometric immunophenotyping studies, thus supporting biomarker discovery efforts, and should be applicable to fluorescence flow cytometry as well. PMID:25609839

Toward development of a comprehensive external quality assurance program for polyfunctional intracellular cytokine staining assays

PubMed Central

Staats, Janet S.; Enzor, Jennifer H.; Sanchez, Ana M.; Rountree, Wes; Chan, Cliburn; Jaimes, Maria; Chan, Ray Chun-Fai; Gaur, Amitabh; Denny, Thomas N.; Weinhold, Kent J.

2014-01-01

The External Quality Assurance Program Oversight Laboratory (EQAPOL) Flow Cytometry Program assesses the proficiency of NIH/NIAID/DAIDS-supported and potentially other interested research laboratories in performing Intracellular Cytokine Staining (ICS) assays. The goal of the EQAPOL Flow Cytometry External Quality Assurance Program (EQAP) is to provide proficiency testing and remediation for participating sites. The program is not punitive; rather, EQAPOL aims to help sites identify areas for improvement. EQAPOL utilizes a highly standardized ICS assay to minimize variability and readily identify those sites experiencing technical difficulties with their assays. Here, we report the results of External Proficiency 3 (EP3) where participating sites performed a 7-color ICS assay. On average, sites perform well in the Flow Cytometry EQAP (median score is “Good”). The most common technical issues identified by the program involve protocol adherence and data analysis; these areas have been the focus of site remediation. The EQAPOL Flow Cytometry team is now in the process of expanding the program to 8-color ICS assays. Evaluating polyfunctional ICS responses would align the program with assays currently being performed in support of HIV immune monitoring assays. PMID:24968072

Avoiding false positive antigen detection by flow cytometry on blood cell derived microparticles: the importance of an appropriate negative control.

PubMed

Crompot, Emerence; Van Damme, Michael; Duvillier, Hugues; Pieters, Karlien; Vermeesch, Marjorie; Perez-Morga, David; Meuleman, Nathalie; Mineur, Philippe; Bron, Dominique; Lagneaux, Laurence; Stamatopoulos, Basile

2015-01-01

Microparticles (MPs), also called microvesicles (MVs) are plasma membrane-derived fragments with sizes ranging from 0.1 to 1μm. Characterization of these MPs is often performed by flow cytometry but there is no consensus on the appropriate negative control to use that can lead to false positive results. We analyzed MPs from platelets, B-cells, T-cells, NK-cells, monocytes, and chronic lymphocytic leukemia (CLL) B-cells. Cells were purified by positive magnetic-separation and cultured for 48h. Cells and MPs were characterized using the following monoclonal antibodies (CD19,20 for B-cells, CD3,8,5,27 for T-cells, CD16,56 for NK-cells, CD14,11c for monocytes, CD41,61 for platelets). Isolated MPs were stained with annexin-V-FITC and gated between 300nm and 900nm. The latex bead technique was then performed for easy detection of MPs. Samples were analyzed by Transmission (TEM) and Scanning Electron microscopy (SEM). Annexin-V positive events within a gate of 300-900nm were detected and defined as MPs. Our results confirmed that the characteristic antigens CD41/CD61 were found on platelet-derived-MPs validating our technique. However, for MPs derived from other cell types, we were unable to detect any antigen, although they were clearly expressed on the MP-producing cells in the contrary of several data published in the literature. Using the latex bead technique, we confirmed detection of CD41,61. However, the apparent expression of other antigens (already deemed positive in several studies) was determined to be false positive, indicated by negative controls (same labeling was used on MPs from different origins). We observed that mother cell antigens were not always detected on corresponding MPs by direct flow cytometry or latex bead cytometry. Our data highlighted that false positive results could be generated due to antibody aspecificity and that phenotypic characterization of MPs is a difficult field requiring the use of several negative controls.

Avoiding False Positive Antigen Detection by Flow Cytometry on Blood Cell Derived Microparticles: The Importance of an Appropriate Negative Control

PubMed Central

Crompot, Emerence; Van Damme, Michael; Duvillier, Hugues; Pieters, Karlien; Vermeesch, Marjorie; Perez-Morga, David; Meuleman, Nathalie; Mineur, Philippe; Bron, Dominique; Lagneaux, Laurence; Stamatopoulos, Basile

2015-01-01

Background Microparticles (MPs), also called microvesicles (MVs) are plasma membrane-derived fragments with sizes ranging from 0.1 to 1μm. Characterization of these MPs is often performed by flow cytometry but there is no consensus on the appropriate negative control to use that can lead to false positive results. Materials and Methods We analyzed MPs from platelets, B-cells, T-cells, NK-cells, monocytes, and chronic lymphocytic leukemia (CLL) B-cells. Cells were purified by positive magnetic-separation and cultured for 48h. Cells and MPs were characterized using the following monoclonal antibodies (CD19,20 for B-cells, CD3,8,5,27 for T-cells, CD16,56 for NK-cells, CD14,11c for monocytes, CD41,61 for platelets). Isolated MPs were stained with annexin-V-FITC and gated between 300nm and 900nm. The latex bead technique was then performed for easy detection of MPs. Samples were analyzed by Transmission (TEM) and Scanning Electron microscopy (SEM). Results Annexin-V positive events within a gate of 300-900nm were detected and defined as MPs. Our results confirmed that the characteristic antigens CD41/CD61 were found on platelet-derived-MPs validating our technique. However, for MPs derived from other cell types, we were unable to detect any antigen, although they were clearly expressed on the MP-producing cells in the contrary of several data published in the literature. Using the latex bead technique, we confirmed detection of CD41,61. However, the apparent expression of other antigens (already deemed positive in several studies) was determined to be false positive, indicated by negative controls (same labeling was used on MPs from different origins). Conclusion We observed that mother cell antigens were not always detected on corresponding MPs by direct flow cytometry or latex bead cytometry. Our data highlighted that false positive results could be generated due to antibody aspecificity and that phenotypic characterization of MPs is a difficult field requiring the use of several negative controls. PMID:25978814

Plasma membrane characterization, by scanning electron microscopy, of multipotent myoblasts-derived populations sorted using dielectrophoresis.

PubMed

Muratore, Massimo; Mitchell, Steve; Waterfall, Martin

2013-09-06

Multipotent progenitor cells have shown promise for use in biomedical applications and regenerative medicine. The implementation of such cells for clinical application requires a synchronized, phenotypically and/or genotypically, homogenous cell population. Here we have demonstrated the implementation of a biological tag-free dielectrophoretic device used for discrimination of multipotent myoblastic C2C12 model. The multipotent capabilities in differentiation, for these cells, diminishes with higher passage number, so for cultures above 70 passages only a small percentage of cells is able to differentiate into terminal myotubes. In this work we demonstrated that we could recover, above 96% purity, specific cell types from a mixed population of cells at high passage number without any biological tag using dielectrophoresis. The purity of the samples was confirmed by cytometric analysis using the cell specific marker embryonic myosin. To further investigate the dielectric properties of the cell plasma membrane we co-culture C2C12 with similar size, when in suspension, GFP-positive fibroblast as feeder layer. The level of separation between the cell types was above 98% purity which was confirmed by flow cytometry. These levels of separation are assumed to account for cell size and for the plasma membrane morphological differences between C2C12 and fibroblast unrelated to the stages of the cell cycle which was assessed by immunofluorescence staining. Plasma membrane conformational differences were further confirmed by scanning electron microscopy. Copyright © 2013 Elsevier Inc. All rights reserved.

Cell cycle arrest and induction of apoptosis by cajanin stilbene acid from Cajanus cajan in breast cancer cells.

PubMed

Fu, Yujie; Kadioglu, Onat; Wiench, Benjamin; Wei, Zuofu; Gao, Chang; Luo, Meng; Gu, Chengbo; Zu, Yuangang; Efferth, Thomas

2015-04-15

The low abundant cajanin stilbene acid (CSA) from Pigeon Pea (Cajanus cajan) has been shown to kill estrogen receptor α positive cancer cells in vitro and in vivo. Downstream effects such as cell cycle and apoptosis-related mechanisms have not been analyzed yet. We analyzed the activity of CSA by means of flow cytometry (cell cycle distribution, mitochondrial membrane potential, MMP), confocal laser scanning microscopy (MMP), DNA fragmentation assay (apoptosis), Western blotting (Bax and Bcl-2 expression, caspase-3 activation) as well as mRNA microarray hybridization and Ingenuity pathway analysis. CSA induced G2/M arrest and apoptosis in a concentration-dependent manner from 8.88 to 14.79 µM. The MMP broke down, Bax was upregulated, Bcl-2 downregulated and caspase-3 activated. Microarray profiling revealed that CSA affected BRCA-related DNA damage response and cell cycle-regulated chromosomal replication pathways. CSA inhibited breast cancer cells by DNA damage and cell cycle-related signaling pathways leading to cell cycle arrest and apoptosis. Copyright © 2015 Elsevier GmbH. All rights reserved.

Biological activity of Tat (47-58) peptide on human pathogenic fungi

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jung, Hyun Jun; Park, Yoonkyung; Department of Biotechnology, Chosun University, 375 Seosuk-dong, Kwangju 501-750

2006-06-23

Tat (47-58) peptide, a positively charged Arginine-rich peptide derived from HIV-1 regulatory protein Tat, is known for a peptidic delivery factor as a cell-penetrating peptide on mammalian cells. In this study, antifungal effect and its mode of action of Tat peptide were investigated on fungal cells. The results indicate that Tat peptide exhibits antifungal activity against pathogenic fungal cells without hemolytic effect on human erythrocytes. To understand the mechanism(s) of Tat peptide, the cellular distribution of the peptide was investigated. Tat peptide internalized in the fungal cells without any damage to cell membrane when examined using an artificial liposome (PC/cholesterol;more » 10:1, w/w). Moreover, flow cytometry analysis exhibited the uptake of Tat peptide by energy- and salt-independent pathway, and confocal scanning microscopy displayed that this peptide accumulated in the nucleus of fungal cells rapidly without any impediment by time or temperature, which generally influence on the viral infections. After penetration into the nuclear, the peptide affected the process of cell cycle of Candida albicans through the arrest at G1 phase.« less

Biological activity of Tat (47-58) peptide on human pathogenic fungi.

PubMed

Jung, Hyun Jun; Park, Yoonkyung; Hahm, Kyung-Soo; Lee, Dong Gun

2006-06-23

Tat (47-58) peptide, a positively charged Arginine-rich peptide derived from HIV-1 regulatory protein Tat, is known for a peptidic delivery factor as a cell-penetrating peptide on mammalian cells. In this study, antifungal effect and its mode of action of Tat peptide were investigated on fungal cells. The results indicate that Tat peptide exhibits antifungal activity against pathogenic fungal cells without hemolytic effect on human erythrocytes. To understand the mechanism(s) of Tat peptide, the cellular distribution of the peptide was investigated. Tat peptide internalized in the fungal cells without any damage to cell membrane when examined using an artificial liposome (PC/cholesterol; 10:1, w/w). Moreover, flow cytometry analysis exhibited the uptake of Tat peptide by energy- and salt-independent pathway, and confocal scanning microscopy displayed that this peptide accumulated in the nucleus of fungal cells rapidly without any impediment by time or temperature, which generally influence on the viral infections. After penetration into the nuclear, the peptide affected the process of cell cycle of Candida albicans through the arrest at G1 phase.

Flow Cytometry: Evolution of Microbiological Methods for Probiotics Enumeration.

PubMed

Pane, Marco; Allesina, Serena; Amoruso, Angela; Nicola, Stefania; Deidda, Francesca; Mogna, Luca

2018-05-14

The purpose of this trial was to verify that the analytical method ISO 19344:2015 (E)-IDF 232:2015 (E) is valid and reliable for quantifying the concentration of the probiotic Lactobacillus rhamnosus GG (ATCC 53103) in a finished product formulation. Flow cytometry assay is emerging as an alternative rapid method for microbial detection, enumeration, and population profiling. The use of flow cytometry not only permits the determination of viable cell counts but also allows for enumeration of damaged and dead cell subpopulations. Results are expressed as TFU (Total Fluorescent Units) and AFU (Active Fluorescent Units). In December 2015, the International Standard ISO 19344-IDF 232 "Milk and milk products-Starter cultures, probiotics and fermented products-Quantification of lactic acid bacteria by flow cytometry" was published. This particular ISO can be applied universally and regardless of the species of interest. Analytical method validation was conducted on 3 different industrial batches of L. rhamnosus GG according to USP39<1225>/ICH Q2R1 in term of: accuracy, precision (repeatability), intermediate precision (ruggedness), specificity, limit of quantification, linearity, range, robustness. The data obtained on the 3 batches of finished product have significantly demonstrated the validity and robustness of the cytofluorimetric analysis. On the basis of the results obtained, the ISO 19344:2015 (E)-IDF 232:2015 (E) "Quantification of lactic acid bacteria by flow cytometry" can be used for the enumeration of L. rhamnosus GG in a finished product formulation.

Kinetics of T-cell receptor-dependent antigen recognition determined in vivo by multi-spectral normalized epifluorescence laser scanning

NASA Astrophysics Data System (ADS)

Favicchio, Rosy; Zacharakis, Giannis; Oikonomaki, Katerina; Zacharopoulos, Athanasios; Mamalaki, Clio; Ripoll, Jorge

2012-07-01

Detection of multiple fluorophores in conditions of low signal represents a limiting factor for the application of in vivo optical imaging techniques in immunology where fluorescent labels report for different functional characteristics. A noninvasive in vivo Multi-Spectral Normalized Epifluorescence Laser scanning (M-SNELS) method was developed for the simultaneous and quantitative detection of multiple fluorophores in low signal to noise ratios and used to follow T-cell activation and clonal expansion. Colocalized DsRed- and GFP-labeled T cells were followed in tandem during the mounting of an immune response. Spectral unmixing was used to distinguish the overlapping fluorescent emissions representative of the two distinct cell populations and longitudinal data reported the discrete pattern of antigen-driven proliferation. Retrieved values were validated both in vitro and in vivo with flow cytometry and significant correlation between all methodologies was achieved. Noninvasive M-SNELS successfully quantified two colocalized fluorescent populations and provides a valid alternative imaging approach to traditional invasive methods for detecting T cell dynamics.

Imaging cytometry in a plastic ultra-mobile system

NASA Astrophysics Data System (ADS)

Martínez Vázquez, R.; Trotta, G.; Paturzo, M.; Volpe, A.; Bernava, G.; Basile, V.; Ancona, A.; Ferraro, P.; Fassi, I.; Osellame, R.

2017-03-01

We present a cost-effective and highly-portable plastic prototype that can be interfaced with a cell phone to implement an optofluidic imaging cytometry platform. It is based on a PMMA microfluidic chip that fits inside an opto-mechanical platform fabricated by a 3D printer. The fluorescence excitation and imaging is performed using the LED and the CMOS from the cell phone increasing the compactness of the system. A custom developed application is used to analyze the images and provide a value of particle concentration.

Image Quality Analysis and Optical Performance Requirement for Micromirror-Based Lissajous Scanning Displays

PubMed Central

Du, Weiqi; Zhang, Gaofei; Ye, Liangchen

2016-01-01

Micromirror-based scanning displays have been the focus of a variety of applications. Lissajous scanning displays have advantages in terms of power consumption; however, the image quality is not good enough. The main reason for this is the varying size and the contrast ratio of pixels at different positions of the image. In this paper, the Lissajous scanning trajectory is analyzed and a new method based on the diamond pixel is introduced to Lissajous displays. The optical performance of micromirrors is discussed. A display system demonstrator is built, and tests of resolution and contrast ratio are conducted. The test results show that the new Lissajous scanning method can be used in displays by using diamond pixels and image quality remains stable at different positions. PMID:27187390

Image Quality Analysis and Optical Performance Requirement for Micromirror-Based Lissajous Scanning Displays.

PubMed

Du, Weiqi; Zhang, Gaofei; Ye, Liangchen

2016-05-11

Micromirror-based scanning displays have been the focus of a variety of applications. Lissajous scanning displays have advantages in terms of power consumption; however, the image quality is not good enough. The main reason for this is the varying size and the contrast ratio of pixels at different positions of the image. In this paper, the Lissajous scanning trajectory is analyzed and a new method based on the diamond pixel is introduced to Lissajous displays. The optical performance of micromirrors is discussed. A display system demonstrator is built, and tests of resolution and contrast ratio are conducted. The test results show that the new Lissajous scanning method can be used in displays by using diamond pixels and image quality remains stable at different positions.

Fluorescent-Antibody Measurement Of Cancer-Cell Urokinase

NASA Technical Reports Server (NTRS)

Morrison, Dennis R.

1993-01-01

Combination of laboratory techniques provides measurements of amounts of urokinase in and between normal and cancer cells. Includes use of fluorescent antibodies specific against different forms of urokinase-type plasminogen activator, (uPA), fluorescence microscopy, quantitative analysis of images of sections of tumor tissue, and flow cytometry of different uPA's and deoxyribonucleic acid (DNA) found in suspended-tumor-cell preparations. Measurements provide statistical method for indicating or predicting metastatic potentials of some invasive tumors. Assessments of metastatic potentials based on such measurements used in determining appropriate follow-up procedures after surgical removal of tumors.

Automatic cytometric device using multiple wavelength excitations

NASA Astrophysics Data System (ADS)

Rongeat, Nelly; Ledroit, Sylvain; Chauvet, Laurence; Cremien, Didier; Urankar, Alexandra; Couderc, Vincent; Nérin, Philippe

2011-05-01

Precise identification of eosinophils, basophils, and specific subpopulations of blood cells (B lymphocytes) in an unconventional automatic hematology analyzer is demonstrated. Our specific apparatus mixes two excitation radiations by means of an acousto-optics tunable filter to properly control fluorescence emission of phycoerythrin cyanin 5 (PC5) conjugated to antibodies (anti-CD20 or anti-CRTH2) and Thiazole Orange. This way our analyzer combining techniques of hematology analysis and flow cytometry based on multiple fluorescence detection, drastically improves the signal to noise ratio and decreases the spectral overlaps impact coming from multiple fluorescence emissions.

Software ion scan functions in analysis of glycomic and lipidomic MS/MS datasets.

PubMed

Haramija, Marko

2018-03-01

Hardware ion scan functions unique to tandem mass spectrometry (MS/MS) mode of data acquisition, such as precursor ion scan (PIS) and neutral loss scan (NLS), are important for selective extraction of key structural data from complex MS/MS spectra. However, their software counterparts, software ion scan (SIS) functions, are still not regularly available. Software ion scan functions can be easily coded for additional functionalities, such as software multiple precursor ion scan, software no ion scan, and software variable ion scan functions. These are often necessary, since they allow more efficient analysis of complex MS/MS datasets, often encountered in glycomics and lipidomics. Software ion scan functions can be easily coded by using modern script languages and can be independent of instrument manufacturer. Here we demonstrate the utility of SIS functions on a medium-size glycomic MS/MS dataset. Knowledge of sample properties, as well as of diagnostic and conditional diagnostic ions crucial for data analysis, was needed. Based on the tables constructed with the output data from the SIS functions performed, a detailed analysis of a complex MS/MS glycomic dataset could be carried out in a quick, accurate, and efficient manner. Glycomic research is progressing slowly, and with respect to the MS experiments, one of the key obstacles for moving forward is the lack of appropriate bioinformatic tools necessary for fast analysis of glycomic MS/MS datasets. Adding novel SIS functionalities to the glycomic MS/MS toolbox has a potential to significantly speed up the glycomic data analysis process. Similar tools are useful for analysis of lipidomic MS/MS datasets as well, as will be discussed briefly. Copyright © 2017 John Wiley & Sons, Ltd.

Removal of batch effects using distribution-matching residual networks.

PubMed

Shaham, Uri; Stanton, Kelly P; Zhao, Jun; Li, Huamin; Raddassi, Khadir; Montgomery, Ruth; Kluger, Yuval

2017-08-15

Sources of variability in experimentally derived data include measurement error in addition to the physical phenomena of interest. This measurement error is a combination of systematic components, originating from the measuring instrument and random measurement errors. Several novel biological technologies, such as mass cytometry and single-cell RNA-seq (scRNA-seq), are plagued with systematic errors that may severely affect statistical analysis if the data are not properly calibrated. We propose a novel deep learning approach for removing systematic batch effects. Our method is based on a residual neural network, trained to minimize the Maximum Mean Discrepancy between the multivariate distributions of two replicates, measured in different batches. We apply our method to mass cytometry and scRNA-seq datasets, and demonstrate that it effectively attenuates batch effects. our codes and data are publicly available at https://github.com/ushaham/BatchEffectRemoval.git. yuval.kluger@yale.edu. Supplementary data are available at Bioinformatics online. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Proteomics Analysis of Bladder Cancer Exosomes*

PubMed Central

Welton, Joanne L.; Khanna, Sanjay; Giles, Peter J.; Brennan, Paul; Brewis, Ian A.; Staffurth, John; Mason, Malcolm D.; Clayton, Aled

2010-01-01

Exosomes are nanometer-sized vesicles, secreted by various cell types, present in biological fluids that are particularly rich in membrane proteins. Ex vivo analysis of exosomes may provide biomarker discovery platforms and form non-invasive tools for disease diagnosis and monitoring. These vesicles have never before been studied in the context of bladder cancer, a major malignancy of the urological tract. We present the first proteomics analysis of bladder cancer cell exosomes. Using ultracentrifugation on a sucrose cushion, exosomes were highly purified from cultured HT1376 bladder cancer cells and verified as low in contaminants by Western blotting and flow cytometry of exosome-coated beads. Solubilization in a buffer containing SDS and DTT was essential for achieving proteomics analysis using an LC-MALDI-TOF/TOF MS approach. We report 353 high quality identifications with 72 proteins not previously identified by other human exosome proteomics studies. Overrepresentation analysis to compare this data set with previous exosome proteomics studies (using the ExoCarta database) revealed that the proteome was consistent with that of various exosomes with particular overlap with exosomes of carcinoma origin. Interrogating the Gene Ontology database highlighted a strong association of this proteome with carcinoma of bladder and other sites. The data also highlighted how homology among human leukocyte antigen haplotypes may confound MASCOT designation of major histocompatability complex Class I nomenclature, requiring data from PCR-based human leukocyte antigen haplotyping to clarify anomalous identifications. Validation of 18 MS protein identifications (including basigin, galectin-3, trophoblast glycoprotein (5T4), and others) was performed by a combination of Western blotting, flotation on linear sucrose gradients, and flow cytometry, confirming their exosomal expression. Some were confirmed positive on urinary exosomes from a bladder cancer patient. In summary, the exosome proteomics data set presented is of unrivaled quality. The data will aid in the development of urine exosome-based clinical tools for monitoring disease and will inform follow-up studies into varied aspects of exosome manufacture and function. PMID:20224111

Flow cytometry as a method for the evaluation of raw material, product and process in the dairy industry.

PubMed

Ruszczyńska, A; Szteyn, J; Wiszniewska-Laszczych, A

2007-01-01

Producing dairy products which are safe for consumers requires the constant monitoring of the microbiological quality of raw material, the production process itself and the end product. Traditional methods, still a "gold standard", require a specialized laboratory working on recognized and validated methods. Obtaining results is time- and labor-consuming and do not allow rapid evaluation. Hence, there is a need for a rapid, precise method enabling the real-time monitoring of microbiological quality, and flow cytometry serves this function well. It is based on labeling cells suspended in a solution with fluorescent dyes and pumping them into a measurement zone where they are exposed to a precisely focused laser beam. This paper is aimed at presenting the possibilities of applying flow cytometry in the dairy industry.

Control electronics for a multi-laser/multi-detector scanning system

NASA Technical Reports Server (NTRS)

Kennedy, W.

1980-01-01

The Mars Rover Laser Scanning system uses a precision laser pointing mechanism, a photodetector array, and the concept of triangulation to perform three dimensional scene analysis. The system is used for real time terrain sensing and vision. The Multi-Laser/Multi-Detector laser scanning system is controlled by a digital device called the ML/MD controller. A next generation laser scanning system, based on the Level 2 controller, is microprocessor based. The new controller capabilities far exceed those of the ML/MD device. The first draft circuit details and general software structure are presented.

Development of a control algorithm for the ultrasound scanning robot (NCCUSR) using ultrasound image and force feedback.

PubMed

Kim, Yeoun Jae; Seo, Jong Hyun; Kim, Hong Rae; Kim, Kwang Gi

2017-06-01

Clinicians who frequently perform ultrasound scanning procedures often suffer from musculoskeletal disorders, arthritis, and myalgias. To minimize their occurrence and to assist clinicians, ultrasound scanning robots have been developed worldwide. Although, to date, there is still no commercially available ultrasound scanning robot, many control methods have been suggested and researched. These control algorithms are either image based or force based. If the ultrasound scanning robot control algorithm was a combination of the two algorithms, it could benefit from the advantage of each one. However, there are no existing control methods for ultrasound scanning robots that combine force control and image analysis. Therefore, in this work, a control algorithm is developed for an ultrasound scanning robot using force feedback and ultrasound image analysis. A manipulator-type ultrasound scanning robot named 'NCCUSR' is developed and a control algorithm for this robot is suggested and verified. First, conventional hybrid position-force control is implemented for the robot and the hybrid position-force control algorithm is combined with ultrasound image analysis to fully control the robot. The control method is verified using a thyroid phantom. It was found that the proposed algorithm can be applied to control the ultrasound scanning robot and experimental outcomes suggest that the images acquired using the proposed control method can yield a rating score that is equivalent to images acquired directly by the clinicians. The proposed control method can be applied to control the ultrasound scanning robot. However, more work must be completed to verify the proposed control method in order to become clinically feasible. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Scanning probe recognition microscopy investigation of tissue scaffold properties

PubMed Central

Fan, Yuan; Chen, Qian; Ayres, Virginia M; Baczewski, Andrew D; Udpa, Lalita; Kumar, Shiva

2007-01-01

Scanning probe recognition microscopy is a new scanning probe microscopy technique which enables selective scanning along individual nanofibers within a tissue scaffold. Statistically significant data for multiple properties can be collected by repetitively fine-scanning an identical region of interest. The results of a scanning probe recognition microscopy investigation of the surface roughness and elasticity of a series of tissue scaffolds are presented. Deconvolution and statistical methods were developed and used for data accuracy along curved nanofiber surfaces. Nanofiber features were also independently analyzed using transmission electron microscopy, with results that supported the scanning probe recognition microscopy-based analysis. PMID:18203431

Scanning probe recognition microscopy investigation of tissue scaffold properties.

PubMed

Fan, Yuan; Chen, Qian; Ayres, Virginia M; Baczewski, Andrew D; Udpa, Lalita; Kumar, Shiva

2007-01-01

Scanning probe recognition microscopy is a new scanning probe microscopy technique which enables selective scanning along individual nanofibers within a tissue scaffold. Statistically significant data for multiple properties can be collected by repetitively fine-scanning an identical region of interest. The results of a scanning probe recognition microscopy investigation of the surface roughness and elasticity of a series of tissue scaffolds are presented. Deconvolution and statistical methods were developed and used for data accuracy along curved nanofiber surfaces. Nanofiber features were also independently analyzed using transmission electron microscopy, with results that supported the scanning probe recognition microscopy-based analysis.

Identification and Characterization of an Acinetobacter baumannii Biofilm-Associated Protein▿

PubMed Central

Loehfelm, Thomas W.; Luke, Nicole R.; Campagnari, Anthony A.

2008-01-01

We have identified a homologue to the staphylococcal biofilm-associated protein (Bap) in a bloodstream isolate of Acinetobacter baumannii. The fully sequenced open reading frame is 25,863 bp and encodes a protein with a predicted molecular mass of 854 kDa. Analysis of the nucleotide sequence reveals a repetitive structure consistent with bacterial cell surface adhesins. Bap-specific monoclonal antibody (MAb) 6E3 was generated to an epitope conserved among 41% of A. baumannii strains isolated during a recent outbreak in the U.S. military health care system. Flow cytometry confirms that the MAb 6E3 epitope is surface exposed. Random transposon mutagenesis was used to generate A. baumannii bap1302::EZ-Tn5, a mutant negative for surface reactivity to MAb 6E3 in which the transposon disrupts the coding sequence of bap. Time course confocal laser scanning microscopy and three-dimensional image analysis of actively growing biofilms demonstrates that this mutant is unable to sustain biofilm thickness and volume, suggesting a role for Bap in supporting the development of the mature biofilm structure. This is the first identification of a specific cell surface protein directly involved in biofilm formation by A. baumannii and suggests that Bap is involved in intercellular adhesion within the mature biofilm. PMID:18024522

Smart Cancer Cell Targeting Imaging and Drug Delivery System by Systematically Engineering Periodic Mesoporous Organosilica Nanoparticles.

PubMed

Lu, Nan; Tian, Ying; Tian, Wei; Huang, Peng; Liu, Ying; Tang, Yuxia; Wang, Chunyan; Wang, Shouju; Su, Yunyan; Zhang, Yunlei; Pan, Jing; Teng, Zhaogang; Lu, Guangming

2016-02-10

The integration of diagnosis and therapy into one nanoplatform, known as theranostics, has attracted increasing attention in the biomedical areas. Herein, we first present a cancer cell targeting imaging and drug delivery system based on engineered thioether-bridged periodic mesoporous organosilica nanoparticles (PMOs). The PMOs are stably and selectively conjugated with near-infrared fluorescence (NIRF) dye Cyanine 5.5 (Cy5.5) and anti-Her2 affibody on the outer surfaces to endow them with excellent NIRF imaging and cancer targeting properties. Also, taking the advantage of the thioether-group-incorporated mesopores, the release of chemotherapy drug doxorubicin (DOX) loaded in the PMOs is responsive to the tumor-related molecule glutathione (GSH). The drug release percentage reaches 84.8% in 10 mM of GSH solution within 24 h, which is more than 2-fold higher than that without GSH. In addition, the drug release also exhibits pH-responsive, which reaches 53.6% at pH 5 and 31.7% at pH 7.4 within 24 h. Confocal laser scanning microscopy and flow cytometry analysis demonstrate that the PMOs-based theranostic platforms can efficiently target to and enter Her2 positive tumor cells. Thus, the smart imaging and drug delivery nanoplatforms induce high tumor cell growth inhibition. Meanwhile, the Cy5.5 conjugated PMOs perform great NIRF imaging ability, which could monitor the intracellular distribution, delivery and release of the chemotherapy drug. In addition, cell viability and histological assessments show the engineered PMOs have good biocompatibility, further encouraging the following biomedical applications. Over all, the systemically engineered PMOs can serve as a novel cancer cell targeting imaging and drug delivery platform with NIRF imaging, GSH and pH dual-responsive drug release, and high tumor cell targeting ability.

Design and analysis of a sub-aperture scanning machine for the transmittance measurements of large-aperture optical system

NASA Astrophysics Data System (ADS)

He, Yingwei; Li, Ping; Feng, Guojin; Cheng, Li; Wang, Yu; Wu, Houping; Liu, Zilong; Zheng, Chundi; Sha, Dingguo

2010-11-01

For measuring large-aperture optical system transmittance, a novel sub-aperture scanning machine with double-rotating arms (SSMDA) was designed to obtain sub-aperture beam spot. Optical system full-aperture transmittance measurements can be achieved by applying sub-aperture beam spot scanning technology. The mathematical model of the SSMDA based on a homogeneous coordinate transformation matrix is established to develop a detailed methodology for analyzing the beam spot scanning errors. The error analysis methodology considers two fundamental sources of scanning errors, namely (1) the length systematic errors and (2) the rotational systematic errors. As the systematic errors of the parameters are given beforehand, computational results of scanning errors are between -0.007~0.028mm while scanning radius is not lager than 400.000mm. The results offer theoretical and data basis to the research on transmission characteristics of large optical system.

In vivo flow cytometry of circulating clots using negative photothermal and photoacoustic contrasts.

PubMed

Galanzha, Ekaterina I; Sarimollaoglu, Mustafa; Nedosekin, Dmitry A; Keyrouz, Salah G; Mehta, Jawahar L; Zharov, Vladimir P

2011-10-01

Conventional photothermal (PT) and photoacousic (PA) imaging, spectroscopy, and cytometry are preferentially based on positive PT/PA effects, when signals are above background. Here, we introduce PT/PA technique based on detection of negative signals below background. Among various new applications, we propose label-free in vivo flow cytometry of circulating clots. No method has been developed for the early detection of clots of different compositions as a source of thromboembolism including ischemia at strokes and myocardial infarction. When a low-absorbing, platelet-rich clot passes a laser-irradiated vessel volume, a transient decrease in local absorption results in an ultrasharp negative PA hole in blood background. Using this phenomenon alone or in combination with positive contrasts, we demonstrated identification of white, red, and mixed clots on a mouse model of myocardial infarction and human blood. The concentration and size of clots were measured with threshold down to few clots in the entire circulation with size as low as 20 μm. This multiparameter diagnostic platform using portable personal high-speed flow cytometer with negative dynamic contrast mode has potential to real-time defining risk factors for cardiovascular diseases, and for prognosis and prevention of stroke or use clot count as a marker of therapy efficacy. Possibility for label-free detection of platelets, leukocytes, tumor cells or targeting themby negative PA probes (e.g., nonabsorbing beads or bubbles) is also highlighted. Copyright © 2011 International Society for Advancement of Cytometry.

Effective Immunological Guidance of Genetic Analyses Including Exome Sequencing in Patients Evaluated for Hemophagocytic Lymphohistiocytosis.

PubMed

Ammann, Sandra; Lehmberg, Kai; Zur Stadt, Udo; Klemann, Christian; Bode, Sebastian F N; Speckmann, Carsten; Janka, Gritta; Wustrau, Katharina; Rakhmanov, Mirzokhid; Fuchs, Ilka; Hennies, Hans C; Ehl, Stephan

2017-11-01

We report our experience in using flow cytometry-based immunological screening prospectively as a decision tool for the use of genetic studies in the diagnostic approach to patients with hemophagocytic lymphohistiocytosis (HLH). We restricted genetic analysis largely to patients with abnormal immunological screening, but included whole exome sequencing (WES) for those with normal findings upon Sanger sequencing. Among 290 children with suspected HLH analyzed between 2010 and 2014 (including 17 affected, but asymptomatic siblings), 87/162 patients with "full" HLH and 79/111 patients with "incomplete/atypical" HLH had normal immunological screening results. In 10 patients, degranulation could not be tested. Among the 166 patients with normal screening, genetic analysis was not performed in 107 (all with uneventful follow-up), while 154 single gene tests by Sanger sequencing in the remaining 59 patients only identified a single atypical CHS patient. Flow cytometry correctly predicted all 29 patients with FHL-2, XLP1 or 2. Among 85 patients with defective NK degranulation (including 13 asymptomatic siblings), 70 were Sanger sequenced resulting in a genetic diagnosis in 55 (79%). Eight patients underwent WES, revealing mutations in two known and one unknown cytotoxicity genes and one metabolic disease. FHL3 was the most frequent genetic diagnosis. Immunological screening provided an excellent decision tool for the need and depth of genetic analysis of HLH patients and provided functionally relevant information for rapid patient classification, contributing to a significant reduction in the time from diagnosis to transplantation in recent years.

Eight color immunophenotyping of T-, B- and NK-cell subpopulations for characterization of chronic immunodeficiencies.

PubMed

A, Boldt; S, Borte; S, Fricke; K, Kentouche; F, Emmrich; M, Borte; F, Kahlenberg; U, Sack

2014-01-16

Background: The heterogeneity of primary and secondary immunodeficiencies demands for the development of a comprehensive flow cytometric screening system, based on reference values that support a standardized immunophenotypic characterization of most lymphocyte subpopulations. Methods: Peripheral blood samples from healthy adult volunteers (n=25) were collected and split into eight panel fractions (100µl each). Subsequently, pre-mixed 8-color antibody cocktails were incubated per specific panel of whole blood to detect and differentiate cell subsets of: (i) a general lymphocyte overviews, (ii) B-cell subpopulations, (iii) CD4+ subpopulations, (iv) CD8+ subpopulations, (v) regulatory T-cells, (vi) recent thymic emigrants, (vii) NK-cell subpopulations, (viii) NK-cell activation markers. All samples were lysed, washed and measured by flow cytometry. FACS DIVA software was used for data analysis and calculation of quadrant statistics (mean values, standard error of mean, percentile ranges). Results: Whole blood staining of lymphocytes provided the analysis of: (i) CD3+, 4+, 8+, 19+, 16/56+, and activated CD4/8 cells; (ii) immature, naïve, non-switched/switched, memory, (activated) CD21 low , transitional B-cells, plasmablasts/plasmacells; (iii and iv) naïve, central memory, effector, effector memory, TH1/TH2/TH17-like and CCR5+CD8-cells; (v) CD25+, regulatory T-cells (naïve/memory, HLA-DR+); (vi) α/β- and γ/δ-T-cells, recent thymic emigrants in CD4/CD8 cells; (vii) immature/mature CD56 bright , CD94/NKG2D+ NK-cells; and (viii) Nkp30, 44, 46 and CD57+NK-cells. Clinical examples and quadrant statistics are provided. Conclusion: The present study represents a practical approach to standardize the immunophenotyping of most T-, B- and NK-cell subpopulations. That allows differentiating, whether abnormalities or developmental shifts observed in lymphocyte subpopulations originates either from primary or secondary immunological disturbance. © 2014 Clinical Cytometry Society. Copyright © 2014 Clinical Cytometry Society.

Candidiasis and the impact of flow cytometry on antifungal drug discovery.

PubMed

Ku, Tsun Sheng N; Bernardo, Stella; Walraven, Carla J; Lee, Samuel A

2017-11-01

Invasive candidiasis continues to be associated with significant morbidity and mortality as well as substantial health care costs nationally and globally. One of the contributing factors is the development of resistance to antifungal agents that are already in clinical use. Moreover, there are known treatment limitations with all of the available antifungal agents. Since traditional techniques in novel drug discovery are time consuming, high-throughput screening using flow cytometry presents as a potential tool to identify new antifungal agents that would be useful in the management of these patients. Areas covered: In this review, the authors discuss the use of automated high-throughput screening assays based upon flow cytometry to identify potential antifungals from a library comprised of a large number of bioactive compounds. They also review studies that employed the use of this research methodology that has identified compounds with antifungal activity. Expert opinion: High-throughput screening using flow cytometry has substantially decreased the processing time necessary for screening thousands of compounds, and has helped enhance our understanding of fungal pathogenesis. Indeed, the authors see this technology as a powerful tool to help scientists identify new antifungal agents that can be added to the clinician's arsenal in their fight against invasive candidiasis.

Characterization of subpopulations and immune-related parameters of hemocytes in the green-lipped mussel Perna viridis.

PubMed

Wang, Youji; Hu, Menghong; Chiang, M W L; Shin, P K S; Cheung, S G

2012-03-01

The green-lipped mussel Perna viridis is distributed widely in the estuarine and coastal areas of the Indo-Pacific region and extensively cultured as an inexpensive protein source. Morphology and immunological activities of hemocytes of P. viridis were investigated using flow cytometry and light and electron microscopy. Three major types of hemocytes were identified in the hemolymph, including dense-granulocyte, semi-granulocyte (small and large size) and hyalinocyte. Other hemocytes, which occurred in low numbers, included granulocytes with different electron-dense/lucent granules and hemoblast-like cells. Based on flow cytometry, two subpopulations were identified. Granulocytes were larger cells, and the more abundant, containing numerous granules in the cytoplasm, and hyalinocytes were the smaller and less abundant with the fewest granules. Flow cytometry revealed that the granulocytes were more active in cell phagocytosis, contained the higher lysosomal content, and showed higher esterase activity and reactive oxygen species (ROS) generation compared with hyalinocytes. Immune functions assessed by the flow cytometry indicated that the granulocytes were the main hemocytes involved in the cellular defence in P. viridis. Copyright © 2011. Published by Elsevier Ltd.

Guide to red fluorescent proteins and biosensors for flow cytometry.

PubMed

Piatkevich, Kiryl D; Verkhusha, Vladislav V

2011-01-01

Since the discovery of the first red fluorescent protein (RFP), named DsRed, 12 years ago, a wide pallet of red-shifted fluorescent proteins has been cloned and biotechnologically developed into monomeric fluorescent probes for optical microscopy. Several new types of monomeric RFPs that change the emission wavelength either with time, called fluorescent timers, or after a brief irradiation with violet light, known as photoactivatable proteins, have been also engineered. Moreover, RFPs with a large Stokes shift of fluorescence emission have been recently designed. Because of their distinctive excitation and fluorescence detection conditions developed specifically for microscopy, these fluorescent probes can be suboptimal for flow cytometry. Here, we have selected and summarized the advanced orange, red, and far-red fluorescent proteins with the properties specifically required for the flow cytometry applications. Their effective brightness was calculated for the laser sources available for the commercial flow cytometers and sorters. Compatibility of the fluorescent proteins of different colors in a multiparameter flow cytometry was determined. Novel FRET pairs, utilizing RFPs, RFP-based intracellular biosensors, and their application to a high-throughput screening, are also discussed. Copyright © 2011 Elsevier Inc. All rights reserved.

Application of different analytical methods for the characterization of non-spherical micro- and nanoparticles.

PubMed

Mathaes, Roman; Winter, Gerhard; Engert, Julia; Besheer, Ahmed

2013-09-10

Non-spherical micro- and nanoparticles have recently gained considerable attention due to their surprisingly different interaction with biological systems compared to their spherical counterparts, opening new opportunities for drug delivery and vaccination. Up till now, electron microscopy is the only method to quantitatively identify the critical quality attributes (CQAs) of non-spherical particles produced by film-stretching; namely size, morphology and the quality of non-spherical particles (degree of contamination with spherical ones). However, electron microscopy requires expensive instrumentation, demanding sample preparation and non-trivial image analysis. To circumvent these drawbacks, the ability of different particle analysis methods to quantitatively identify the CQA of spherical and non-spherical poly(1-phenylethene-1,2-diyl (polystyrene) particles over a wide size range (40 nm, 2 μm and 10 μm) was investigated. To this end, light obscuration, image-based analysis methods (Microflow imaging, MFI, and Vi-Cell XR Coulter Counter) and flow cytometry were used to study particles in the micron range, while asymmetric flow field fractionation (AF4) coupled to multi-angle laser scattering (MALS) and quasi elastic light scattering (QELS) was used for particles in the nanometer range, and all measurements were benchmarked against electron microscopy. Results show that MFI can reliably identify particle size and aspect ratios of the 10 μm particles, but not the 2 μm ones. Meanwhile, flow cytometry was able to differentiate between spherical and non-spherical 10 or 2 μm particles, and determine the amount of impurities in the sample. As for the nanoparticles, AF4 coupled to MALS and QELS allowed the measurement of the geometric (rg) and hydrodynamic (rh) radii of the particles, as well as their shape factors (rg/rh), confirming their morphology. While this study shows the utility of MFI, flow cytometry and AF4 for quantitative evaluation of the CQA of non-spherical particles over a wide size range, the limitations of the methods are discussed. The use of orthogonal characterization methods can provide a complete picture about the CQA of non-spherical particles over a wide size range. Copyright © 2013 Elsevier B.V. All rights reserved.

Comparative analysis of cell wall surface glycan expression in Candida albicans and Saccharomyces cerevisiae yeasts by flow cytometry.

PubMed

Martínez-Esparza, M; Sarazin, A; Jouy, N; Poulain, D; Jouault, T

2006-07-31

The yeast Candida albicans is an opportunistic pathogen, part of the normal human microbial flora that causes infections in immunocompromised individuals with a high morbidity and mortality levels. Recognition of yeasts by host cells is based on components of the yeast cell wall, which are considered part of its virulence attributes. Cell wall glycans play an important role in the continuous interchange that regulates the balance between saprophytism and parasitism, and also between resistance and infection. Some of these molecular entities are expressed both by the pathogenic yeast C. albicans and by Saccharomyces cerevisiae, a related non-pathogenic yeast, involving similar molecular mechanisms and receptors for recognition. In this work we have exploited flow cytometry methods for probing surface glycans of the yeasts. We compared glycan expression by C. albicans and by S. cerevisiae, and studied the effect of culture conditions. Our results show that the expression levels of alpha- and beta-linked mannosides as well as beta-glucans can be successfully evaluated by flow cytometry methods using different antibodies independent of agglutination reactions. We also found that the surface expression pattern of beta-mannosides detected by monoclonal or polyclonal antibodies are differently modulated during the growth course. These data indicate that the yeast beta-mannosides exposed on mannoproteins and/or phospholipomannan are increased in stationary phase, whereas those linked to mannan are not affected by the yeast growth phase. The cytometric method described here represents a useful tool to investigate to what extent C. albicans is able to regulate its glycan surface expression and therefore modify its virulence properties.

A single-platform approach using flow cytometry and microbeads to evaluate immune reconstitution in mice after bone marrow transplantation.

PubMed

Perruche, Sylvain; Kleinclauss, François; Lienard, Agnès; Robinet, Eric; Tiberghien, Pierre; Saas, Philippe

2004-11-01

The monitoring of immune reconstitution in murine models of HC transplantation, using accurate and automated methods, is necessary in view of the recent developments of hematopoietic cell (HC) transplantation (including reduced intensity conditioning regimens) as well as emerging immunological concepts (such as the involvement of dendritic cells or regulatory T cells). Here, we describe the use of a single-platform approach based on flow cytometry and tubes that contain a defined number of microbeads to evaluate absolute blood cell counts in mice. This method, previously used in humans to quantify CD34+ stem cells or CD4+ T cells in HIV infected patients, was adapted for mouse blood samples. A CD45 gating strategy in this "lyse no wash" protocol makes it possible to discriminate erythroblasts or red blood cell debris from CD45+ leukocytes, thus avoiding cell loss. Tubes contain a lyophilized brightly fluorescent microbead pellet permitting the acquisition of absolute counts of leukocytes after flow cytometric analysis. We compared this method to determine absolute counts of circulating cells with another method combining Unopette reservoir diluted blood samples, hemocytometer, microscopic examination and flow cytometry. The sensitivity of this single-platform approach was evaluated in different situations encountered in allogeneic HC transplantation, including immune cell depletion after different conditioning regimens, activation status of circulating cells after transplantation, evaluation of in vivo cell depletion and hematopoietic progenitor mobilization in the periphery. This single-platform flow cytometric assay can also be proposed to standardize murine (or other mammalian species) leukocyte count determination for physiological, pharmacological/toxicological and diagnostic applications in veterinary practice.

Variation compensation and analysis on diaphragm curvature analysis for emphysema quantification on whole lung CT scans

NASA Astrophysics Data System (ADS)

Keller, Brad M.; Reeves, Anthony P.; Barr, R. Graham; Yankelevitz, David F.; Henschke, Claudia I.

2010-03-01

CT scans allow for the quantitative evaluation of the anatomical bases of emphysema. Recently, a non-density based geometric measurement of lung diagphragm curvature has been proposed as a method for the quantification of emphysema from CT. This work analyzes variability of diaphragm curvature and evaluates the effectiveness of a compensation methodology for the reduction of this variability as compared to emphysema index. Using a dataset of 43 scan-pairs with less than a 100 day time-interval between scans, we find that the diaphragm curvature had a trend towards lower overall variability over emphysema index (95% CI:-9.7 to + 14.7 vs. -15.8 to +12.0), and that the variation of both measures was reduced after compensation. We conclude that the variation of the new measure can be considered comparable to the established measure and the compensation can reduce the apparent variation of quantitative measures successfully.

2006 Bethesda International Consensus recommendations on the immunophenotypic analysis of hematolymphoid neoplasia by flow cytometry: optimal reagents and reporting for the flow cytometric diagnosis of hematopoietic neoplasia.

PubMed

Wood, Brent L; Arroz, Maria; Barnett, David; DiGiuseppe, Joseph; Greig, Bruce; Kussick, Steven J; Oldaker, Teri; Shenkin, Mark; Stone, Elizabeth; Wallace, Paul

2007-01-01

Immunophenotyping by flow cytometry has become standard practice in the evaluation and monitoring of patients with hematopoietic neoplasia. However, despite its widespread use, considerable variability continues to exist in the reagents used for evaluation and the format in which results are reported. As part of the 2006 Bethesda Consensus conference, a committee was formed to attempt to define a consensus set of reagents suitable for general use in the diagnosis and monitoring of hematopoietic neoplasms. The committee included laboratory professionals from private, public, and university hospitals as well as large reference laboratories that routinely operate clinical flow cytometry laboratories with an emphasis on lymphoma and leukemia immunophenotyping. A survey of participants successfully identified the cell lineage(s) to be evaluated for each of a variety of specific medical indications and defined a set of consensus reagents suitable for the initial evaluation of each cell lineage. Elements to be included in the reporting of clinical flow cytometric results for leukemia and lymphoma evaluation were also refined and are comprehensively listed. The 2006 Bethesda Consensus conference represents the first successful attempt to define a set of consensus reagents suitable for the initial evaluation of hematopoietic neoplasia. Copyright 2007 Clinical Cytometry Society.

Alternative fluorescent labeling strategies for characterizing gram-positive pathogenic bacteria: Flow cytometry supported counting, sorting, and proteome analysis of Staphylococcus aureus retrieved from infected host cells.

PubMed

Hildebrandt, Petra; Surmann, Kristin; Salazar, Manuela Gesell; Normann, Nicole; Völker, Uwe; Schmidt, Frank

2016-10-01

Staphylococcus aureus is a Gram-positive opportunistic pathogen that is able to cause a broad range of infectious diseases in humans. Furthermore, S. aureus is able to survive inside nonprofessional phagocytic host cell which serve as a niche for the pathogen to hide from the immune system and antibiotics therapies. Modern OMICs technologies provide valuable tools to investigate host-pathogen interactions upon internalization. However, these experiments are often hampered by limited capabilities to retrieve bacteria from such an experimental setting. Thus, the aim of this study was to develop a labeling strategy allowing fast detection and quantitation of S. aureus in cell lysates or infected cell lines by flow cytometry for subsequent proteome analyses. Therefore, S. aureus cells were labeled with the DNA stain SYTO ® 9, or Vancomycin BODIPY ® FL (VMB), a glycopeptide antibiotic binding to most Gram-positive bacteria which was conjugated to a fluorescent dye. Staining of S. aureus HG001 with SYTO 9 allowed counting of bacteria from pure cultures but not in cell lysates from infection experiments. In contrast, with VMB it was feasible to stain bacteria from pure cultures as well as from samples of infection experiments. VMB can also be applied for histocytochemistry analysis of formaldehyde fixed cell layers grown on coverslips. Proteome analyses of S. aureus labeled with VMB revealed that the labeling procedure provoked only minor changes on proteome level and allowed cell sorting and analysis of S. aureus from infection settings with sensitivity similar to continuous gfp expression. Furthermore, VMB labeling allowed precise counting of internalized bacteria and can be employed for downstream analyses, e.g., proteomics, of strains not easily amendable to genetic manipulation such as clinical isolates. © 2016 International Society for Advancement of Cytometry. © 2016 International Society for Advancement of Cytometry.

Quantitative analysis of cellular glutathione by flow cytometry utilizing monochlorobimane: some applications to radiation and drug resistance in vitro and in vivo.

PubMed

Rice, G C; Bump, E A; Shrieve, D C; Lee, W; Kovacs, M

1986-12-01

An assay using a bimane derivative has been developed to detect free glutathione (GSH) in individual viable cells by flow cytometry. Monochlorobimane [syn-(ClCH2CH3)-1,5-diazabicycla[3.30]acta-3,6-diene-2,8-dio ne], itself nonfluorescent, reacts with GSH to form a highly fluorescent derivative. High pressure liquid chromatography analysis showed that, using specific staining conditions, the only low molecular weight fluorescent derivative formed in Chinese hamster ovary cells was that formed with GSH. Very little reaction with protein sulfhydryls was observed. Rates of GSH depletion in Chinese hamster ovary cells exposed to diethylmaleate were essentially the same, whether measured by relative fluorescence intensity, by flow cytometry or by enzymatic assay on cellular extracts. This method was shown to be useful for measurement of GSH resynthesis, uptake, and depletion by prolonged hypoxia and misonidazole treatment. Since measurements are made on individual cells, cell-to-cell variation and populational heterogeneity in GSH content are revealed by flow cytometry. Although under most conditions in vitro GSH content is relatively homogeneous, under certain circumstances, such as release from hypoxia, heterogeneity in populational GSH levels was observed. The significance of this heterogeneity is discussed in regard to the induction of gene amplification and drug resistance by transient hypoxia. Numerous subclones of Chinese hamster ovary cells selected by growth in Adriamycin or methotrexate-containing medium express elevated levels of GSH per cell. The method was extended to quantitate the GSH content of cells excised from EMT-6/SF mouse tumors that had been treated in vivo with L-buthionine-S-R-sulfoximine, an inhibitor of GSH synthesis. The bivariate analysis (forward angle light scatter versus monochlorobimane fluorescence) of cells derived from these tumors gave excellent resolution of normal and tumor cells and demonstrated extensive heterogeneity in the tumor cell population with respect to GSH content per cell.

Resonant-cavity apparatus for cytometry or particle analysis

DOEpatents

Gourley, Paul L.

1998-01-01

A resonant-cavity apparatus for cytometry or particle analysis. The apparatus comprises a resonant optical cavity having an analysis region within the cavity for containing one or more biological cells or dielectric particles to be analyzed. In the presence of a cell or particle, a light beam in the form of spontaneous emission or lasing is generated within the resonant optical cavity and is encoded with information about the cell or particle. An analysis means including a spectrometer and/or a pulse-height analyzer is provided within the apparatus for recovery of the information from the light beam to determine a size, shape, identification or other characteristics about the cells or particles being analyzed. The recovered information can be grouped in a multi-dimensional coordinate space for identification of particular types of cells or particles. In some embodiments of the apparatus, the resonant optical cavity can be formed, at least in part, from a vertical-cavity surface-emitting laser. The apparatus and method are particularly suited to the analysis of biological cells, including blood cells, and can further include processing means for manipulating, sorting, or eradicating cells after analysis thereof.

Flow Cytometry Scientist | Center for Cancer Research

Cancer.gov

PROGRAM DESCRIPTION The Basic Science Program (BSP) pursues independent, multidisciplinary research in basic and applied molecular biology, immunology, retrovirology, cancer biology, and human genetics. Research efforts and support are an integral part of the Center for Cancer Research (CCR) at the Frederick National Laboratory for Cancer Research (FNLCR). KEY ROLES/RESPONSIBILITIES The Flow Cytometry Core (Flow Core) in the Cancer and Inflammation Program (CIP) is a service core which supports the research efforts of the CCR by providing expertise in the field of flow cytometry (using analyzers and sorters) with the goal of gaining a more thorough understanding of the biology of the immune system, cancer, and inflammation processes. The Flow Core provides service to 12-15 CIP laboratories and more than 22 non-CIP laboratories. Flow core staff provide technical advice on the experimental design of applications, which include immunological phenotyping, cell function assays, and cell cycle analysis. Work is performed per customer requirements, and no independent research is involved. The Flow Cytometry Scientist will be responsible for: Daily management of the Flow Cytometry Core, to include the supervision and guidance of technical staff members Monitor performance of and maintain high dimensional flow cytometer analyzers and cell sorters Operate high dimensional flow cytometer analyzers and cell sorters Provide scientific expertise to the user community and facilitate the development of cutting edge technologies Interact with Flow Core users and customers, and provide technical and scientific advice, and guidance regarding their experiments, including possible collaborations Train staff and scientific end users on the use of flow cytometry in their research, as well as teach them how to operate and troubleshoot the bench-top analyzer instruments Prepare and deliver lectures, as well as one-on-one training sessions, with customers/users Ensure that protocols are up-to-date, and appropriately adhered to Experience with sterile technique and tissue culture

Impaired coordination between signaling pathways is revealed in human colorectal cancer using single-cell mass cytometry of archival tissue blocks

PubMed Central

Simmons, Alan J.; Scurrah, Cherie’ R.; McKinley, Eliot T.; Herring, Charles A.; Irish, Jonathan M.; Washington, Mary K.; Coffey, Robert J.; Lau, Ken S.

2016-01-01

Cellular heterogeneity poses a significant challenge to understanding tissue level phenotypes and confounds conventional bulk analyses. To facilitate the analysis of signaling at the single-cell level in human tissues, we applied mass cytometry using CyTOF (Cytometry Time-of-Flight) to formalin-fixed paraffin-embedded (FFPE) normal and diseased intestinal specimens. We developed and validated a technique called FFPE-DISSECT (Disaggregation for Intracellular Signaling in Single Epithelial Cells from Tissue), a single-cell approach for characterizing native signaling states from embedded solid tissue samples. We applied FFPE-DISSECT coupled to mass cytometry and found differential signaling by tumor necrosis factor α (TNF-α) in intestinal enterocytes, goblet cells and enteroendocrine cells, implicating the role of the downstream RAS-RAF-MEK-ERK signaling pathway in dictating goblet cell identity. In addition, application of FFPE-DISSECT, mass cytometry, and data-driven computational analyses to human colon specimens confirmed reduced differentiation in colorectal cancer (CRC) compared to normal colon, and revealed quantitative increases in inter- and intra-tissue heterogeneity in CRC with regards to the modular regulation of signaling pathways. Specifically, modular co-regulation of the kinases P38 and ERK, the translation regulator 4EBP1, and the transcription factor CREB in the proliferative compartment of the normal colon was loss in CRC, as evidenced by their impaired coordination over samplings of single cells in tissue. Our data suggest that this single-cell approach, applied in conjunction with genomic annotation, such as microsatellite instability and mutations in KRAS and BRAF, allows rapid and detailed characterization of cellular heterogeneity from clinical repositories of embedded human tissues. FFPE-DISSECT coupled of mass cytometry can be used for deriving cellular landscapes from archived patient samples, beyond CRC, and as a high resolution tool for disease characterization and subtyping. PMID:27729552

Polyethylene glycol-conjugated chondroitin sulfate A derivative nanoparticles for tumor-targeted delivery of anticancer drugs.

PubMed

Lee, Jae-Young; Park, Ju-Hwan; Lee, Jeong-Jun; Lee, Song Yi; Chung, Suk-Jae; Cho, Hyun-Jong; Kim, Dae-Duk

2016-10-20

Polyethylene glycol (PEG)-decorated chondroitin sulfate A-deoxycholic acid (CSD) nanoparticles (NPs) were fabricated for the selective delivery of doxorubicin (DOX) to ovarian cancer. CSD-PEG was synthesized via amide bond formation between the NH2 group of methoxypolyethylene glycol amine and the COOH group of CSD. CSD-PEG/DOX NPs with a 247nm mean diameter, negative zeta potential, and >90% drug encapsulation efficiency were prepared. Sustained and pH-dependent DOX release profiles from CSD-PEG NPs were observed in dissolution tests. Endocytosis of NPs by SKOV-3 cells (CD44 receptor-positive human ovarian cancer cells), based on the CSA-CD44 receptor interaction, was determined by flow cytometry and confocal laser scanning microscopy (CLSM) studies. PEGylation of NPs also resulted in reduced drug clearance (CL) in vivo and improved relative bioavailability, compared to non-PEGylated NPs, as determined by the pharmacokinetic study performed after intravenous administration in rats. Developed CSD-PEG NPs can be a promising delivery vehicle for the therapy of CD44 receptor-expressing ovarian cancers. Copyright © 2016 Elsevier Ltd. All rights reserved.

Preparation of Deep Sea Fish Oil-Based Nanostructured Lipid Carriers with Enhanced Cellular Uptake.

PubMed

Zhu, Qiu-Yun; Guissi, Fida; Yang, Ru-Ya; Wang, Qian; Wang, Ke; Chen, Dan; Han, Zhi-Hao; Ma, Yi; Zhang, Min; Gu, Yue-Qing

2015-12-01

Nanostructured lipid carriers (NLC) are a promising pharmaceutical delivery system with mean diameter less than 200 nm which are dispersed in an aqueous phase containing emulsifier(s), to increase the water solubility, stability and bioavailability of oil compounds. Herein we prepared a promising NLC with glyceryl monostearate (GMS) as the solid lipid template and deep sea fish oil as the liquid lipid template using melted-ultrasonic method. Fish oil-NLC had a mean size of 84.7 ± 2.6 nm and a zeta potential that ranged from -17.87 mV to -32.91 mV. The nanoparticles exhibited good stability for four weeks with a high encapsulation efficiency of 87.5 ± 5.2%. Afterwards, confocal laser scanning microscopy (CLSM) and flow cytometry (FCM) were used to investigate the contribution of Fish oil-NLC in enhancing fluorescein isothiocyanate (FITC) cellular uptake in comparison with free FITC. The results of this study indicated the possibility of this carrier to overcome the shortcomings of deep sea fish oil and to provide a novel bifunctional carrier with nutritional potential and drug delivery ability.

Intracellular drug delivery nanocarriers of glutathione-responsive degradable block copolymers having pendant disulfide linkages.

PubMed

Khorsand, Behnoush; Lapointe, Gabriel; Brett, Christopher; Oh, Jung Kwon

2013-06-10

Self-assembled micelles of amphiphilic block copolymers (ABPs) with stimuli-responsive degradation (SRD) properties have a great promise as nanotherapeutics exhibiting enhanced release of encapsulated therapeutics into targeted cells. Here, thiol-responsive degradable micelles based on a new ABP consisting of a pendant disulfide-labeled methacrylate polymer block (PHMssEt) and a hydrophilic poly(ethylene oxide) (PEO) block were investigated as effective intracellular nanocarriers of anticancer drugs. In response to glutathione (GSH) as a cellular trigger, the cleavage of pendant disulfide linkages in hydrophobic PHMssEt blocks of micellar cores caused the destabilization of self-assembled micelles due to change in hydrophobic/hydrophilic balance. Such GSH-triggered micellar destabilization changed their size distribution with an appearance of large aggregates and led to enhanced release of encapsulated anticancer drugs. Cell culture results from flow cytometry and confocal laser scanning microscopy for cellular uptake as well as cell viability measurements for high anticancer efficacy suggest that new GSH-responsive degradable PEO-b-PHMssEt micelles offer versatility in multifunctional drug delivery applications.

Influence of particle geometry and PEGylation on phagocytosis of particulate carriers.

PubMed

Mathaes, Roman; Winter, Gerhard; Besheer, Ahmed; Engert, Julia

2014-04-25

Particle geometry of micro- and nanoparticles has been identified as an important design parameter to influence the interaction with cells such as macrophages. A head to head comparison of elongated, non-spherical and spherical micro- and nanoparticles with and without PEGylation was carried out to benchmark two phagocytosis inhibiting techniques. J774.A1 macrophages were incubated with fluorescently labeled PLGA micro- and nanoparticles and analyzed by confocal laser scanning microscope (CLSM) and flow cytometry (FACS). Particle uptake into macrophages was significantly reduced upon PEGylation or elongated particle geometry. A combination of both, an elongated shape and PEGylation, had the strongest phagocytosis inhibiting effect for nanoparticles. Copyright © 2014 Elsevier B.V. All rights reserved.

Automatic Classification of Cellular Expression by Nonlinear Stochastic Embedding (ACCENSE).

PubMed

Shekhar, Karthik; Brodin, Petter; Davis, Mark M; Chakraborty, Arup K

2014-01-07

Mass cytometry enables an unprecedented number of parameters to be measured in individual cells at a high throughput, but the large dimensionality of the resulting data severely limits approaches relying on manual "gating." Clustering cells based on phenotypic similarity comes at a loss of single-cell resolution and often the number of subpopulations is unknown a priori. Here we describe ACCENSE, a tool that combines nonlinear dimensionality reduction with density-based partitioning, and displays multivariate cellular phenotypes on a 2D plot. We apply ACCENSE to 35-parameter mass cytometry data from CD8(+) T cells derived from specific pathogen-free and germ-free mice, and stratify cells into phenotypic subpopulations. Our results show significant heterogeneity within the known CD8(+) T-cell subpopulations, and of particular note is that we find a large novel subpopulation in both specific pathogen-free and germ-free mice that has not been described previously. This subpopulation possesses a phenotypic signature that is distinct from conventional naive and memory subpopulations when analyzed by ACCENSE, but is not distinguishable on a biaxial plot of standard markers. We are able to automatically identify cellular subpopulations based on all proteins analyzed, thus aiding the full utilization of powerful new single-cell technologies such as mass cytometry.

A Rare Case of Pure Erythroid Sarcoma in a Pediatric Patient: Case Report and Literature Review

PubMed Central

Tarín, Fabián; Niveiro, María; Tasso, María; Alda, Olga; Verdú, José J.; De Paz, Francisco; López, Silvia; Del Cañizo, María; Such, Esperanza; Barragán, Eva; Martirena, Fernanda

2017-01-01

We describe an exceptional case of erythroid sarcoma in a pediatric patient as a growing orbital mass with no evidence of morphologic bone marrow involvement, who was finally diagnosed of pure erythroid sarcoma based on histopathology and flow cytometry criteria. We discuss the contribution of standardized eight-color flow cytometry as a rapid and reliable diagnostic method. The use of normal bone marrow databases allowed us to identify small aberrant populations in bone marrow and later confirm the diagnosis in the neoplastic tissue. PMID:29261159

A Rare Case of Pure Erythroid Sarcoma in a Pediatric Patient: Case Report and Literature Review.

PubMed

Manresa, Pablo; Tarín, Fabián; Niveiro, María; Tasso, María; Alda, Olga; López, Francisco; Sarmiento, Héctor; Verdú, José J; De Paz, Francisco; López, Silvia; Del Cañizo, María; Such, Esperanza; Barragán, Eva; Martirena, Fernanda

2017-12-20

We describe an exceptional case of erythroid sarcoma in a pediatric patient as a growing orbital mass with no evidence of morphologic bone marrow involvement, who was finally diagnosed of pure erythroid sarcoma based on histopathology and flow cytometry criteria. We discuss the contribution of standardized eight-color flow cytometry as a rapid and reliable diagnostic method. The use of normal bone marrow databases allowed us to identify small aberrant populations in bone marrow and later confirm the diagnosis in the neoplastic tissue.

Microfluidics and photonics for Bio-System-on-a-Chip: a review of advancements in technology towards a microfluidic flow cytometry chip.

PubMed

Godin, Jessica; Chen, Chun-Hao; Cho, Sung Hwan; Qiao, Wen; Tsai, Frank; Lo, Yu-Hwa

2008-10-01

Microfluidics and photonics come together to form a field commonly referred to as 'optofluidics'. Flow cytometry provides the field with a technology base from which both microfluidic and photonic components be developed and integrated into a useful device. This article reviews some of the more recent developments to familiarize a reader with the current state of the technologies and also highlights the requirements of the device and how researchers are working to meet these needs.

Developments in label-free microfluidic methods for single-cell analysis and sorting.

PubMed

Carey, Thomas R; Cotner, Kristen L; Li, Brian; Sohn, Lydia L

2018-04-24

Advancements in microfluidic technologies have led to the development of many new tools for both the characterization and sorting of single cells without the need for exogenous labels. Label-free microfluidics reduce the preparation time, reagents needed, and cost of conventional methods based on fluorescent or magnetic labels. Furthermore, these devices enable analysis of cell properties such as mechanical phenotype and dielectric parameters that cannot be characterized with traditional labels. Some of the most promising technologies for current and future development toward label-free, single-cell analysis and sorting include electronic sensors such as Coulter counters and electrical impedance cytometry; deformation analysis using optical traps and deformation cytometry; hydrodynamic sorting such as deterministic lateral displacement, inertial focusing, and microvortex trapping; and acoustic sorting using traveling or standing surface acoustic waves. These label-free microfluidic methods have been used to screen, sort, and analyze cells for a wide range of biomedical and clinical applications, including cell cycle monitoring, rapid complete blood counts, cancer diagnosis, metastatic progression monitoring, HIV and parasite detection, circulating tumor cell isolation, and point-of-care diagnostics. Because of the versatility of label-free methods for characterization and sorting, the low-cost nature of microfluidics, and the rapid prototyping capabilities of modern microfabrication, we expect this class of technology to continue to be an area of high research interest going forward. New developments in this field will contribute to the ongoing paradigm shift in cell analysis and sorting technologies toward label-free microfluidic devices, enabling new capabilities in biomedical research tools as well as clinical diagnostics. This article is categorized under: Diagnostic Tools > Biosensing Diagnostic Tools > Diagnostic Nanodevices. © 2018 Wiley Periodicals, Inc.

Flow cytometric DNA analysis of cirrhotic liver cells in patients with hepatocellular carcinoma can provide a new prognostic factor.

PubMed

Ruà, S; Comino, A; Fruttero, A; Torchio, P; Bouzari, H; Taraglio, S; Torchio, B; Capussotti, L

1996-09-15

DNA flow cytometry of hepatocellular carcinoma (HCC) cells has been investigated in many studies, but, to the best of our knowledge, there are no data on DNA analysis of cirrhotic parenchyma around the HCC. In this study, cell kinetics and ploidy of parenchymal cells around HCC were performed to ascertain if this would predict the possibility of recurrence in the cirrhotic areas. The DNA content of 93 cases of HCC and of cirrhotic liver around the tumor nodules was analyzed by flow cytometry. Ploidy and proliferative index of HCC and cirrhotic liver were compared with macroscopic, histologic, and clinical features of each case and linked with the behavior of these tumors. Survival curves were assessed according to the Kaplan-Meier method. A multivariate analysis based on Cox proportional hazards regression model was performed on cases of diploid cirrhosis cells in which the S-phase fraction was evaluable. The univariate analysis of survival suggested significant roles for age, number of intrahepatic nodules, Edmondson-Steiner's classification, portal invasion, vascular invasion, presence of necrosis, hepatitis B surface antigen, alpha-feto-protein, Child's score, ploidy, and S-phase fraction of HCC cells. The DNA analysis of the cirrhotic cells showed that polyploidy was dramatically reduced in patients with HCC, compared with normal hepatocytes, and aneuploid clones were present among diploid cells. High S-phase fraction of cirrhotic cells and Child-Pugh classification were the strongest independent parameters affecting the tumor behavior in this study. The results of this study suggest that S-phase fraction of cirrhotic liver parenchyma may be employed as a new parameter in the prognostic evaluation of HCC patients.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Srivastava, Nimisha; Singh, Anup K

Microfluidic devices and methods for flow cytometry are described. In described examples, various sample handling and preparation steps may be carried out within a same microfluidic device as flow cytometry steps. A combination of imaging and flow cytometry is described. In some examples, spiral microchannels serve as incubation chambers. Examples of automated sample handling and flow cytometry are described.

Separating the signal from the noise: Expanding flow cytometry into the sub-micron range.

EPA Science Inventory

Cytometry Part A Special Section: Separating the signal from the noise: Expanding flow cytometry into the sub-micron range. The current Cytometry Part A Special Section presents three studies that utilize cytometers to study sub-micron particles. The three studies involve the 1...

Assessing basophil activation by using flow cytometry and mass cytometry in blood stored 24 hours before analysis.

PubMed

Mukai, Kaori; Gaudenzio, Nicolas; Gupta, Sheena; Vivanco, Nora; Bendall, Sean C; Maecker, Holden T; Chinthrajah, Rebecca S; Tsai, Mindy; Nadeau, Kari C; Galli, Stephen J

2017-03-01

Basophil activation tests (BATs) have promise for research and for clinical monitoring of patients with allergies. However, BAT protocols vary in blood anticoagulant used and temperature and time of storage before testing, complicating comparisons of results from various studies. We attempted to establish a BAT protocol that would permit analysis of blood within 24 hours of obtaining the sample. Blood from 46 healthy donors and 120 patients with peanut allergy was collected into EDTA or heparin tubes, and samples were stored at 4°C or room temperature for 4 or 24 hours before performing BATs. Stimulation with anti-IgE or IL-3 resulted in strong upregulation of basophil CD203c in samples collected in EDTA or heparin, stored at 4°C, and analyzed 24 hours after sample collection. However, a CD63 hi population of basophils was not observed in any conditions in EDTA-treated samples unless exogenous calcium/magnesium was added at the time of anti-IgE stimulation. By contrast, blood samples collected in heparin tubes were adequate for quantification of upregulation of basophil CD203c and identification of a population of CD63 hi basophils, irrespective of whether the specimens were analyzed by means of conventional flow cytometry or cytometry by time-of-flight mass spectrometry, and such tests could be performed after blood was stored for 24 hours at 4°C. BATs to measure upregulation of basophil CD203c and induction of a CD63 hi basophil population can be conducted with blood obtained in heparin tubes and stored at 4°C for 24 hours. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Laboratory Tests of Multiplex Detection of PCR Amplicons Using the Luminex 100 Flow Analyzer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Venkateswaran, K.S.; Nasarabadi, S.; Langlois, R.G.

2000-05-05

Lawrence Livermore National Laboratory (LLNL) demonstrated the power of flow cytometry in detecting the biological agents simulants at JFT III. LLNL pioneered in the development of advanced nucleic acid analyzer (ANM) for portable real time identification. Recent advances in flow cytometry provide a means for multiplexed nucleic acid detection and immunoassay of pathogenic microorganisms. We are presently developing multiplexed immunoassays for the simultaneous detection of different simulants. Our goal is to build an integrated instrument for both nucleic acid analysis and immuno detection. In this study we evaluated the Luminex LX 100 for concurrent identification of more than one PCRmore » amplified product. ANAA has real-time Taqman fluorescent detection capability for rapid identification of field samples. However, its multiplexing ability is limited by the combination of available fluorescent labels. Hence integration of ANAA with flow cytometry can give the rapidity of ANAA amplification and the multiplex capability of flow cytometry. Multiplexed flow cytometric analysis is made possible using a set of fluorescent latex microsphere that are individually identified by their red and infrared fluorescence. A green fluorochrome is used as the assay signal. Methods were developed for the identification of specific nucleic acid sequences from Bacillus globigii (Bg), Bacillus thuringensis (Bt) and Erwinia herbicola (Eh). Detection sensitivity using different reporter fluorochromes was tested with the LX 100, and also different assay formats were evaluated for their suitability for rapid testing. A blind laboratory trial was carried out December 22-27, 1999 to evaluate bead assays for multiplex identification of Bg and Bt PCR products. This report summarizes the assay development, fluorochrome comparisons, and the results of the blind trial conducted at LLNL for the laboratory evaluation of the LX 100 flow analyzer.« less

ZAP-70 staining in chronic lymphocytic leukemia.

PubMed

Villamor, Neus

2005-05-01

Chronic lymphocytic leukemia (CLL) is the most common chronic leukemia in Western countries. The disease has an extremely variable clinical course, and several prognostic features have been identified to assess individual risk. The configuration of the immunoglobulin variable heavy-chain gene (IgV(H)) is a strong predictor of the outcome. CLL patients with unmutated IgV(H) status have an aggressive clinical course and a short survival. Unfortunately, analysis of IgV(H) gene configuration is not available in most clinical laboratories. A small number of genes are differentially expressed between unmutated IgV(H) and mutated IgV(H) clinical forms of CLL. One of these genes is ZAP-70, which is detected in leukemic cells from patients with the unmutated IgV(H) form of CLL. Flow cytometry presents advantages over other methods to detect ZAP-70, and its quantification by flow cytometry has proved its predictive value. This unit focuses on protocols to quantify ZAP-70 by flow cytometry in CLL.

Principles and applications of flow cytometry and cell sorting in companion animal medicine.

PubMed

Wilkerson, Melinda J

2012-01-01

Flow cytometry measures multiple characteristic of single cells using light scatter properties and fluorescence properties of fluorescent probes with specificity to cellular constituents. The use of flow cytometry in the veterinary clinical laboratory has become more routine in veterinary diagnostic laboratories and institutions (http://www.vet.k-state.edu/depts/dmp/service/immunology/index.htm), and reference laboratories. The most common applications in small animal medicine includes quantitation of erythrocytes and leukocytes in automated hematology instruments, detection of antibodies to erythrocytes and platelets in cases of immune-mediated diseases, immunophenotyping of leukocytes and lymphocytes in immunodeficiency syndromes, or leukemias and lymphomas. DNA content analysis to identify aneuploidy or replicating cells in tumor preparations has not gained routine acceptance because of the variability of prognostic results. Other applications including cell sorting and multiplexing using microspheres are potential assays of the future once they become validated and the instrumentation footprint becomes more and more compact, less expensive, and easier to use.

Fluorescence-based detection and quantification of features of cellular senescence.

PubMed

Cho, Sohee; Hwang, Eun Seong

2011-01-01

Cellular senescence is a spontaneous organismal defense mechanism against tumor progression which is raised upon the activation of oncoproteins or other cellular environmental stresses that must be circumvented for tumorigenesis to occur. It involves growth-arrest state of normal cells after a number of active divisions. There are multiple experimental routes that can drive cells into a state of senescence. Normal somatic cells and cancer cells enter a state of senescence upon overexpression of oncogenic Ras or Raf protein or by imposing certain kinds of stress such as cellular tumor suppressor function. Both flow cytometry and confocal imaging analysis techniques are very useful in quantitative analysis of cellular senescence phenomenon. They allow quantitative estimates of multiple different phenotypes expressed in multiple cell populations simultaneously. Here we review the various types of fluorescence methodologies including confocal imaging and flow cytometry that are frequently utilized to study a variety of senescence. First, we discuss key cell biological changes occurring during senescence and review the current understanding on the mechanisms of these changes with the goal of improving existing protocols and further developing new ones. Next, we list specific senescence phenotypes associated with each cellular trait along with the principles of their assay methods and the significance of the assay outcomes. We conclude by selecting appropriate references that demonstrate a typical example of each method. Copyright © 2011 Elsevier Inc. All rights reserved.

Minimal residual disease evaluation by flow cytometry is a complementary tool to cytogenetics for treatment decisions in acute myeloid leukaemia.

PubMed

Vidriales, María-Belén; Pérez-López, Estefanía; Pegenaute, Carlota; Castellanos, Marta; Pérez, José-Juan; Chandía, Mauricio; Díaz-Mediavilla, Joaquín; Rayón, Consuelo; de Las Heras, Natalia; Fernández-Abellán, Pascual; Cabezudo, Miguel; de Coca, Alfonso García; Alonso, Jose M; Olivier, Carmen; Hernández-Rivas, Jesús M; Montesinos, Pau; Fernández, Rosa; García-Suárez, Julio; García, Magdalena; Sayas, María-José; Paiva, Bruno; González, Marcos; Orfao, Alberto; San Miguel, Jesús F

2016-01-01

The clinical utility of minimal residual disease (MRD) analysis in acute myeloid leukaemia (AML) is not yet defined. We analysed the prognostic impact of MRD level at complete remision after induction therapy using multiparameter flow cytometry in 306 non-APL AML patients. First, we validated the prognostic value of MRD-thresholds we have previously proposed (≥ 0.1%; ≥ 0.01-0.1%; and <0.01), with a 5-year RFS of 38%, 50% and 71%, respectively (p=0.002). Cytogenetics is the most relevant prognosis factor in AML, however intermediate risk cytogenetics represent a grey zone that require other biomarkers for risk stratification, and we show that MRD evaluation discriminate three prognostic subgroups (p=0.03). Also, MRD assessments yielded relevant information on favourable and adverse cytogenetics, since patients with favourable cytogenetics and high MRD levels have poor prognosis and patients with adverse cytogenetics but undetectable MRD overcomes the adverse prognosis. Interestingly, in patients with intermediate or high MRD levels, intensification with transplant improved the outcome as compared with chemotherapy, while the type of intensification therapy did not influenced the outcome of patients with low MRD levels. Multivariate analysis revealed age, MRD and cytogenetics as independent variables. Moreover, a scoring system, easy in clinical practice, was generated based on MRD level and cytogenetics. Copyright © 2015 Elsevier Ltd. All rights reserved.

Chip-Based Dynamic Real-Time Quantification of Drug-Induced Cytotoxicity in Human Tumor Cells

PubMed Central

Wlodkowic, Donald; Skommer, Joanna; McGuinness, Dagmara; Faley, Shannon; Kolch, Walter; Darzynkiewicz, Zbigniew; Cooper, Jonathan M.

2013-01-01

Cell cytotoxicity tests are among the most common bioassays using flow cytometry and fluorescence imaging analysis. The permeability of plasma membranes to charged fluorescent probes serves, in these assays, as a marker distinguishing live from dead cells. Since it is generally assumed that probes, such as propidium iodide (PI) or 7-amino-actinomycin D (7-AAD), are themselves cytotoxic, they are currently generally used only as the end-point markers of assays for live versus dead cells. In the current study, we provide novel insights into potential applications of these classical plasma membrane integrity markers in the dynamic tracking of drug-induced cytotoxicity. We show that treatment of a number of different human tumor cell lines in cultures for up to 72 h with the PI, 7-AAD, SYTOX Green (SY-G), SYTOX Red (SYR), TO-PRO, and YO-PRO had no effect on cell viability assessed by the integrity of plasma membrane, cell cycle progression, and rate of proliferation. We subsequently explore the potential of dynamic labeling with these markers in real-time analysis, by comparing results from both conventional cytometry and microfluidic chips. Considering the simplicity of the staining protocols and their low cost combined with the potential for real-time data collection, we show how that real-time fluorescent imaging and Lab-on-a-Chip platforms have the potential to be used for automated drug screening routines. PMID:19572560

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kovtun, Oleg; Ross, Emily J.; Tomlinson, Ian D.

Here we present the development and validation of a flow cytometry-based dopamine transporter (DAT) binding assay that uses antagonist-conjugated quantum dots (QDs). Our anticipation is that our QD-based assay is of immediate value to the high throughput screening of novel DAT modulators.

QUALITY ASSESSMENT OF CONFOCAL MICROSCOPY SLIDE-BASED SYSTEMS: INSTABLITY

EPA Science Inventory

Background: All slide-based fluorescence cytometry detections systems basically include an excitation light source, intermediate optics, and a detection device (CCD or PMT). Occasionally, this equipment becomes unstable, generating unreliable and inferior data. Methods: A num...