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Sample records for high-throughput flow cytometric

  1. High-throughput multi-parameter flow-cytometric analysis from micro-quantities of plasmodium-infected blood.

    PubMed

    Apte, Simon H; Groves, Penny L; Roddick, Joanne S; P da Hora, Vanusa; Doolan, Denise L

    2011-10-01

    Despite significant technological and conceptual advances over the last century, evaluation of the efficacy of anti-malarial vaccines or drugs continues to rely principally on direct microscopic visualisation of parasites on thick and/or thin Giemsa-stained blood smears. This requires technical expertise of the microscopist, is highly subjective and error-prone, and does not account for aberrations such as anaemia. Many published methods have shown that flow cytometric analysis of blood is a highly versatile method that can readily detect nucleic acid-stained parasitised red blood cells within cultured cell populations and in ex-vivo samples. However several impediments, including the difficulty in distinguishing reticulocytes from infected red blood cells and the fickle nature of red blood cells, have precluded the development and universal adoption of flow-cytometric based assays for ex-vivo sample analysis. We have developed a novel high-throughput assay for the flow cytometric assessment of blood that overcomes these impediments by utilising the unique properties of the nucleic acid stain DAPI to differentially stain RNA and DNA, combined with novel fixation and analysis protocols. The assay allows the rapid and reliable analysis of multiple parameters from micro-volumes of blood, including: parasitaemia, platelet count, reticulocyte count, normocyte count, white blood cell count and delineation of subsets and phenotypic markers including, but not limited to, CD4(+) and CD8(+) T cells, and the expression of phenotypic markers such as PD-L1 or intracellular cytokines. The assay requires less than one drop of blood and is therefore suitable for short interval time-course experiments and allows the progression of infection and immune responses to be closely monitored in the laboratory or cytometer-equipped field locations. Herein, we describe the technique and demonstrate its application in vaccinology and with a range of rodent and human parasite species

  2. A novel tool for high-throughput screening of granulocyte-specific antibodies using the automated flow cytometric granulocyte immunofluorescence test (Flow-GIFT).

    PubMed

    Nguyen, Xuan Duc; Dengler, Thomas; Schulz-Linkholt, Monika; Klüter, Harald

    2011-02-03

    Transfusion-related acute lung injury (TRALI) is a severe complication related with blood transfusion. TRALI has usually been associated with antibodies against leukocytes. The flow cytometric granulocyte immunofluorescence test (Flow-GIFT) has been introduced for routine use when investigating patients and healthy blood donors. Here we describe a novel tool in the automation of the Flow-GIFT that enables a rapid screening of blood donations. We analyzed 440 sera from healthy female blood donors for the presence of granulocyte antibodies. As positive controls, 12 sera with known antibodies against anti-HNA-1a, -b, -2a; and -3a were additionally investigated. Whole-blood samples from HNA-typed donors were collected and the test cells isolated using cell sedimentation in a Ficoll density gradient. Subsequently, leukocytes were incubated with the respective serum and binding of antibodies was detected using FITC-conjugated antihuman antibody. 7-AAD was used to exclude dead cells. Pipetting steps were automated using the Biomek NXp Multichannel Automation Workstation. All samples were prepared in the 96-deep well plates and analyzed by flow cytometry. The standard granulocyte immunofluorescence test (GIFT) and granulocyte agglutination test (GAT) were also performed as reference methods. Sixteen sera were positive in the automated Flow-GIFT, while five of these sera were negative in the standard GIFT (anti-HNA 3a, n = 3; anti-HNA-1b, n = 1) and GAT (anti-HNA-2a, n = 1). The automated Flow-GIFT was able to detect all granulocyte antibodies, which could be only detected in GIFT in combination with GAT. In serial dilution tests, the automated Flow-GIFT detected the antibodies at higher dilutions than the reference methods GIFT and GAT. The Flow-GIFT proved to be feasible for automation. This novel high-throughput system allows an effective antigranulocyte antibody detection in a large donor population in order to prevent TRALI due to transfusion of blood products.

  3. High-Throughput Contact Flow Lithography.

    PubMed

    Le Goff, Gaelle C; Lee, Jiseok; Gupta, Ankur; Hill, William Adam; Doyle, Patrick S

    2015-10-01

    High-throughput fabrication of graphically encoded hydrogel microparticles is achieved by combining flow contact lithography in a multichannel microfluidic device and a high capacity 25 mm LED UV source. Production rates of chemically homogeneous particles are improved by two orders of magnitude. Additionally, the custom-built contact lithography instrument provides an affordable solution for patterning complex microstructures on surfaces.

  4. Ultrasensitive flow cytometric analyses

    SciTech Connect

    Jett, J.H.; Cram, L.S.; Keller, R.A.; Martin, J.C.; Saunders, G.C.; Sklar, L.A.; Steinkamp, J.A.

    1993-01-01

    New techniques and approaches to cellular analysis being developed at the Los Alamos National Flow Cytometry Resource can be divided into those that improve sensitivity and those that move the technology into new areas by refining existing approaches. An example of the first category is a flow cytometric system capable of measuring the phase shift of fluorescence emitted by fluorophors bound to cells is being assembled. This phase sensitive cytometer is be capable of quantifying fluorescence life time on a cell-by-cell basis as well as using the phase sensitive detection to separate fluorescence emissions that overlap spectrally but have different lifetimes. A Fourier transform flow cytometer capable of measuring the fluorescence emission spectrum of individual labeled cells at rates approaching several hundred per second is also in the new technology category. The current implementation is capable of resolving the visible region of the spectrum into 8 bands. With this instrument, it is possible to resolve the contributions of fluorophors with overlapping emission spectra and to determine the emission spectra of dyes such as calcium concentration indicators that are sensitive to the physiological environment. Flow cytometric techniques have been refined to the point that it is possible to detect individual fluorescent molecules in solution as they flow past a laser beam. This capability has lead to a rapid DNA sequencing project. The goal of the project is to develop a technique that is capable of sequencing long strands of DNA (40,000 kb) at a rate of between 100 and 1,000 bases per second.

  5. Computational analysis of high-throughput flow cytometry data

    PubMed Central

    Robinson, J Paul; Rajwa, Bartek; Patsekin, Valery; Davisson, Vincent Jo

    2015-01-01

    Introduction Flow cytometry has been around for over 40 years, but only recently has the opportunity arisen to move into the high-throughput domain. The technology is now available and is highly competitive with imaging tools under the right conditions. Flow cytometry has, however, been a technology that has focused on its unique ability to study single cells and appropriate analytical tools are readily available to handle this traditional role of the technology. Areas covered Expansion of flow cytometry to a high-throughput (HT) and high-content technology requires both advances in hardware and analytical tools. The historical perspective of flow cytometry operation as well as how the field has changed and what the key changes have been discussed. The authors provide a background and compelling arguments for moving toward HT flow, where there are many innovative opportunities. With alternative approaches now available for flow cytometry, there will be a considerable number of new applications. These opportunities show strong capability for drug screening and functional studies with cells in suspension. Expert opinion There is no doubt that HT flow is a rich technology awaiting acceptance by the pharmaceutical community. It can provide a powerful phenotypic analytical toolset that has the capacity to change many current approaches to HT screening. The previous restrictions on the technology, based on its reduced capacity for sample throughput, are no longer a major issue. Overcoming this barrier has transformed a mature technology into one that can focus on systems biology questions not previously considered possible. PMID:22708834

  6. Flow Cytometric Analysis of Extracellular Vesicles.

    PubMed

    Morales-Kastresana, Aizea; Jones, Jennifer C

    2017-01-01

    To analyze EVs with conventional flow cytometers, most researchers will find it necessary to bind EVs to beads that are large enough to be individually resolved on the flow cytometer available in their lab or facility. Although high-resolution flow cytometers are available and are being used for EV analysis, the use of these instruments for studying EVs requires careful use and validation by experienced small-particle flow cytometrists, beyond the scope of this chapter. Shown here is a method for using streptavidin-coated beads to capture biotinylated antibodies, and stain the bead-bound EVs with directly conjugated antibodies. We find that this method is a useful tool not only on its own, without further high resolution flow cytometric analysis, but also as a means for optimizing staining methods and testing new labels for later use in high resolution, single EV flow cytometric studies. The end of the chapter includes sphere-packing calculations to quantify aspects of EV- and bead-surface geometry, as a reference for use as readers of this chapter optimize their own flow cytometry assays with EVs.

  7. High throughput analysis of samples in flowing liquid

    DOEpatents

    Ambrose, W. Patrick; Grace, W. Kevin; Goodwin, Peter M.; Jett, James H.; Orden, Alan Van; Keller, Richard A.

    2001-01-01

    Apparatus and method enable imaging multiple fluorescent sample particles in a single flow channel. A flow channel defines a flow direction for samples in a flow stream and has a viewing plane perpendicular to the flow direction. A laser beam is formed as a ribbon having a width effective to cover the viewing plane. Imaging optics are arranged to view the viewing plane to form an image of the fluorescent sample particles in the flow stream, and a camera records the image formed by the imaging optics.

  8. Flow cytometric allergy diagnosis: basophil activation techniques.

    PubMed

    Bridts, Chris H; Sabato, Vito; Mertens, Christel; Hagendorens, Margo M; De Clerck, Luc S; Ebo, Didier G

    2014-01-01

    The basis of flow cytometric allergy diagnosis is quantification of changes in expression of basophilic surface membrane markers (Ebo et al., Clin Exp Allergy 34: 332-339, 2004). Upon encountering specific allergens recognized by surface receptor FcεRI-bound IgE, basophils not only secrete and generate quantifiable bioactive mediators but also up-regulate the expression of different markers (e.g., CD63, CD203c) which can be detected by multicolor flow cytometry using specific monoclonal antibodies (Ebo et al., Cytometry B Clin Cytom 74: 201-210, 2008). Here, we describe two flow cytometry-based protocols which allow detection of surface marker activation (Method 1) and changes in intragranular histamine (Method 2), both reflecting different facets of basophil activation.

  9. 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.

  10. High-throughput flow alignment of barcoded hydrogel microparticles†

    PubMed Central

    Chapin, Stephen C.; Pregibon, Daniel C.

    2010-01-01

    Suspension (particle-based) arrays offer several advantages over conventional planar arrays in the detection and quantification of biomolecules, including the use of smaller sample volumes, more favorable probe-target binding kinetics, and rapid probe-set modification. We present a microfluidic system for the rapid alignment of multifunctional hydrogel microparticles designed to bear one or several biomolecule probe regions, as well as a graphical code to identify the embedded probes. Using high-speed imaging, we have developed and optimized a flow-through system that (1) allows for a high particle throughput, (2) ensures proper particle alignment for decoding and target quantification, and (3) can be reliably operated continuously without clogging. A tapered channel flanked by side focusing streams is used to orient the flexible, tablet-shaped particles into a well-ordered flow in the center of the channel. The effects of channel geometry, particle geometry, particle composition, particle loading density, and barcode design are explored to determine the best combination for eventual use in biological assays. Particles in the optimized system move at velocities of ~50 cm s−1 and with throughputs of ~40 particles s−1. Simple physical models and CFD simulations have been used to investigate flow behavior in the device. PMID:19823726

  11. Flow cytometric enumeration of Plasmodium berghei-infected red blood cells stained with SYBR Green I.

    PubMed

    Somsak, Voravuth; Srichairatanakool, Somdet; Yuthavong, Yongyuth; Kamchonwongpaisan, Sumalee; Uthaipibull, Chairat

    2012-04-01

    High-throughput methods for evaluation of in vivo efficacy of candidate compounds against Plasmodium parasites are necessary during the antimalarial drug development process. It is essential that enumeration of parasitemia in the infected blood from experimental host animals is accurate and reliable. Flow cytometric enumeration of parasitized cells stained with fluorescent dye is a rapid alternative method to conventional microscopic counting. In this study, a protocol for flow cytometric enumeration of rodent malaria parasite Plasmodium berghei-infected red blood cells (RBC) stained with SYBR Green I was developed. The optimal concentration of SYBR Green I used to stain infected RBC was 4× for 30 min. This SYBR Green I staining protocol in combination with the bi-dimensional FL-1(530)/FL-3(620) detection method accurately detects parasitemia above 0.02%. The dye is stable during the prolonged incubation period necessary for accurate enumeration of parasitemia, with no loss of fluorescent signal over a period of hours. This protocol was validated in an antimalarial assay and the result was comparable to that obtained from conventional microscopic counting. The SYBR Green I flow cytometric protocol is thus a rapid and precise tool for high-throughput in vivo antimalarial drug screening.

  12. A scalable approach for high throughput branch flow filtration.

    PubMed

    Inglis, David W; Herman, Nick

    2013-05-07

    Microfluidic continuous flow filtration methods have the potential for very high size resolution using minimum feature sizes that are larger than the separation size, thereby circumventing the problem of clogging. Branch flow filtration is particularly promising because it has an unlimited dynamic range (ratio of largest passable particle to the smallest separated particle) but suffers from very poor volume throughput because when many branches are used, they cannot be identical if each is to have the same size cut-off. We describe a new iterative approach to the design of branch filtration devices able to overcome this limitation without large dead volumes. This is demonstrated by numerical modelling, fabrication and testing of devices with 20 branches, with dynamic ranges up to 6.9, and high filtration ratios (14-29%) on beads and fungal spores. The filters have a sharp size cutoff (10× depletion for 12% size difference), with large particle rejection equivalent to a 20th order Butterworth low pass filter. The devices are fully scalable, enabling higher throughput and smaller cutoff sizes and they are compatible with ultra low cost fabrication.

  13. Flow cytometric and laser scanning microscopic approaches in epigenetics research.

    PubMed

    Szekvolgyi, Lorant; Imre, Laszlo; Minh, Doan Xuan Quang; Hegedus, Eva; Bacso, Zsolt; Szabo, Gabor

    2009-01-01

    Our understanding of epigenetics has been transformed in recent years by the advance of technological possibilities based primarily on a powerful tool, chromatin immunoprecipitation (ChIP). However, in many cases, the detection of epigenetic changes requires methods providing a high-throughput (HTP) platform. Cytometry has opened a novel approach for the quantitative measurement of molecules, including PCR products, anchored to appropriately addressed microbeads (Pataki et al. 2005. Cytometry 68, 45-52). Here we show selected examples for the utility of two different cytometry-based platforms of epigenetic analysis: ChIP-on-beads, a flow-cytometric test of local histone modifications (Szekvolgyi et al. 2006. Cytometry 69, 1086-1091), and the laser scanning cytometry-based measurement of global epigenetic modifications that might help predict clinical behavior in different pathological conditions. We anticipate that such alternative tools may shortly become indispensable in clinical practice, translating the systematic screening of epigenetic tags from basic research into routine diagnostics of HTP demand.

  14. Microfluidic impedance flow cytometry enabling high-throughput single-cell electrical property characterization.

    PubMed

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

    2015-04-29

    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.

  15. 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

  16. High-throughput flowing upstream sperm sorting in a retarding flow field for human semen analysis.

    PubMed

    Wu, Jen-Kuei; Chen, Peng-Chun; Lin, Yu-Nan; Wang, Chia-Woei; Pan, Li-Chern; Tseng, Fan-Gang

    2017-03-13

    In this paper, we propose a microfluidic device capable of generating a retarding flow field for the sorting and separation of human motile sperm in a high-throughput manner. The proposed sorting/separation process begins with a rapid flow field in a straight-flow zone to carry sperm into a sorting zone to maintain the sperm's mobility. The sorting zone consists of a diffuser-type sperm sorter to differentiate sperm with different motilities based on the flowing upstream nature of human sperm in a retarding flow field. The dead sperm will then be separated from the live ones by passing through a dumbbell flow field to the outlet for disposal. The proposed flowing upstream sperm sorter (FUSS) is designed to imitate the selection mechanism found in the female body when sperm swim into the uterus. The experimental results demonstrate the utility of this device with regard to throughput (approximately 200 000 sperm per minute and a maximum of 200 million cells per mL), efficiency (90% of selected sperm are mobile), and the ability to select sperm with high motility (∼20% of sperm with a velocity exceeding 120 μm s(-1)). The proposed device is suitable for intrauterine insemination as well as in vitro fertilization thanks to the highly efficient sorting process not interfering with the natural function and energy resource of human sperm.

  17. Flow cytometric determination of quantitative immunophenotypes

    NASA Astrophysics Data System (ADS)

    Redelman, Douglas; Ensign, Wayne; Roberts, Don

    2001-05-01

    Immunofluorescent flow cytometric analysis of peripheral blood leucocytes is most commonly used to identify and enumerate cells defined by one or more clusters of differentiation (CD) antigens. Although less widely employed, quantitative tests that measure the amounts of CD antigens expressed per cell are used in some situations such as the characterization of lymphomas and leukocytes or the measurement of CD38 on CD3plu8pluT cells in HIV infected individuals. The CD antigens used to identify leukocyte populations are functionally important molecules and it is known that under- or over-expression of some CD antigens can affect cellular responses. For example, high or low expression of CD19 on B cells is associated with autoimmune conditions or depressed antibody responses, respectively. In the current studies, the quantitative expression of CD antigens on T cells, B cells and monocytes was determined in a group of age and sex-matched Marines at several times before and after training exercises. There was substantial variation among these individuals in the quantitative expression of CD antigens and in the number of cells in various populations. However, there was relatively little variation within individuals during the two months they were examined. Thus, the number of cells in leukocyte sub-populations and the amount of CD antigens expressed per cell appear to comprise a characteristic quantitative immunophenotype.

  18. Development of an automated analysis system for data from flow cytometric intracellular cytokine staining assays from clinical vaccine trials

    PubMed Central

    Shulman, Nick; Bellew, Matthew; Snelling, George; Carter, Donald; Huang, Yunda; Li, Hongli; Self, Steven G.; McElrath, M. Juliana; De Rosa, Stephen C.

    2008-01-01

    Background Intracellular cytokine staining (ICS) by multiparameter flow cytometry is one of the primary methods for determining T cell immunogenicity in HIV-1 clinical vaccine trials. Data analysis requires considerable expertise and time. The amount of data is quickly increasing as more and larger trials are performed, and thus there is a critical need for high throughput methods of data analysis. Methods A web based flow cytometric analysis system, LabKey Flow, was developed for analyses of data from standardized ICS assays. A gating template was created manually in commercially-available flow cytometric analysis software. Using this template, the system automatically compensated and analyzed all data sets. Quality control queries were designed to identify potentially incorrect sample collections. Results Comparison of the semi-automated analysis performed by LabKey Flow and the manual analysis performed using FlowJo software demonstrated excellent concordance (concordance correlation coefficient >0.990). Manual inspection of the analyses performed by LabKey Flow for 8-color ICS data files from several clinical vaccine trials indicates that template gates can appropriately be used for most data sets. Conclusions The semi-automated LabKey Flow analysis system can analyze accurately large ICS data files. Routine use of the system does not require specialized expertise. This high-throughput analysis will provide great utility for rapid evaluation of complex multiparameter flow cytometric measurements collected from large clinical trials. PMID:18615598

  19. High Throughput Label Free Measurement of Cancer Cell Adhesion Kinetics Under Hemodynamic Flow.

    PubMed

    Spencer, Adrianne; Baker, Aaron B

    2016-01-27

    The kinetics of receptor-mediated cell adhesion to extracellular matrix and adherent cell monolayers plays a key role in many physiological and pathological processes including cancer metastasis. Within this process the presence of fluidic shear forces is a key regulator of binding equilibrium and kinetics of cell adhesion. Current techniques to examine the kinetics of cell adhesion are either performed in the absence of flow or are low throughput, limiting their application to pharmacological compound screening or the high throughput investigation of biological mechanisms. We developed a high throughput flow device that applies flow in a multi-well format and interfaced this system with electric cell-substrate impedance sensing (ECIS) system to allow label free detection of cell adhesion. We demonstrate that this combined system is capable of making real time measurements of cancer cell adhesion to extracellular matrix and immobilized platelets. In addition, we examined the dependence of the kinetics of binding of cancer cells on the level of shear stress and in the presence of small molecule inhibitors to adhesion-related pathways. This versatile system is broadly adaptable to the high throughput study of cell adhesion kinetics for many applications including drug screening and the investigation of the mechanisms of cancer metastasis.

  20. High Throughput Label Free Measurement of Cancer Cell Adhesion Kinetics Under Hemodynamic Flow

    PubMed Central

    Spencer, Adrianne; Baker, Aaron B.

    2016-01-01

    The kinetics of receptor-mediated cell adhesion to extracellular matrix and adherent cell monolayers plays a key role in many physiological and pathological processes including cancer metastasis. Within this process the presence of fluidic shear forces is a key regulator of binding equilibrium and kinetics of cell adhesion. Current techniques to examine the kinetics of cell adhesion are either performed in the absence of flow or are low throughput, limiting their application to pharmacological compound screening or the high throughput investigation of biological mechanisms. We developed a high throughput flow device that applies flow in a multi-well format and interfaced this system with electric cell-substrate impedance sensing (ECIS) system to allow label free detection of cell adhesion. We demonstrate that this combined system is capable of making real time measurements of cancer cell adhesion to extracellular matrix and immobilized platelets. In addition, we examined the dependence of the kinetics of binding of cancer cells on the level of shear stress and in the presence of small molecule inhibitors to adhesion-related pathways. This versatile system is broadly adaptable to the high throughput study of cell adhesion kinetics for many applications including drug screening and the investigation of the mechanisms of cancer metastasis. PMID:26816215

  1. Flow cytometric immunophenotyping in posttransplant lymphoproliferative disorders.

    PubMed

    Dunphy, Cherie H; Gardner, Laura J; Grosso, Leonard E; Evans, H Lance

    2002-01-01

    We studied the flow cytometric immunophenotyping (FCI) and genotypic data of 11 specimens from 10 transplant recipients and categorized them based on a scheme for posttransplant lymphoproliferative disorders (PTLDs). Specimens had been analyzed by polymerase chain reaction and/or Southern blot for T-cell and B-cell (immunoglobulin heavy chain and light chain genes) gene rearrangements (BGR). The categories for PTLDs were as follows: 1, 1; 2, 6; and 3, 4. The plasmacytic and polymorphic B-cell hyperplasias (PBCHs) revealed no monoclonal/aberrant cells by FCI or genotypic studies (GS). Three of 4 polymorphic B-cell lymphomas (PBCLs) revealed monoclonal or aberrant (no surface light chain) B cells by FCI; 1 of 3 revealed a BGR. However, the 1 case with no monoclonal/aberrant B cells by FCI revealed a BGR. Both immunoblastic lymphomas revealed monoclonal or aberrant B cells by FCI; 1 revealed a BGR. Both multiple myelomas revealed monoclonal plasma cells by FCI; 1 revealed a BGR. In the 4 PTLDs with monoclonal/aberrant B cells by FCI and no clonality detected by GS, the GS were performed on fresh and paraffin-embedded tissue samples. FCI of the plasmacytic and PBCHs supported no clonal process by GS. FCI defined a clonal process in 2 PBCLs, I immunoblastic lymphoma, and 1 multiple myeloma that were negative by GS. However, 1 PBCL that was polyclonal by FCI was monoclonal by GS. Thus, FCI is useful for identifying a clonal process in PTLDs with negative results by GS; FCI and GS should be performed routinely in PTLDs to detect a clonal process.

  2. High-throughput continuous flow synthesis of nickel nanoparticles for the catalytic hydrodeoxygenation of guaiacol

    SciTech Connect

    Roberts, Emily J.; Habas, Susan E.; Wang, Lu; Ruddy, Daniel A.; White, Erick A.; Baddour, Frederick G.; Griffin, Michael B.; Schaidle, Joshua A.; Malmstadt, Noah; Brutchey, Richard L.

    2016-11-07

    The translation of batch chemistries to high-throughput continuous flow methods dresses scaling, automation, and reproducibility concerns associated with the implementation of colloidally prepared nanoparticle (NP) catalysts for industrial catalytic processes. Nickel NPs were synthesized by the high-temperature amine reduction of a Ni2+ precursor using a continuous millifluidic (mF) flow method, achieving yields greater than 60%. The resulting Ni NP catalysts were compared against catalysts prepared in a batch reaction under conditions analogous to the continuous flow conditions with respect to total reaction volume, time, and temperature and by traditional incipient wetness (IW) impregnation for the hydrodeoxygenation (HDO) of guaiacol under ex situ catalytic fast pyrolysis conditions. Compared to the IW method, the colloidally prepared NPs displayed increased morphological control and narrowed size distributions, and the NPs prepared by both methods showed similar size, shape, and crystallinity. The Ni NP catalyst synthesized by the continuous flow method exhibited similar H-adsorption site densities, site-time yields, and selectivities towards deoxygenated products as compared to the analogous batch reaction, and outperformed the IW catalyst with respect to higher selectivity to lower oxygen content products and a 6.9-fold slower deactivation rate. These results demonstrate the utility of synthesizing colloidal Ni NP catalysts using continuous flow methods while maintaining the catalytic properties displayed by the batch equivalent. Finally, this methodology can be extended to other catalytically relevant base metals for the high-throughput synthesis of metal NPs for the catalytic production of biofuels.

  3. State of the Art High-Throughput Approaches to Genotoxicity: Flow Micronucleus, Ames II, GreenScreen and Comet

    EPA Pesticide Factsheets

    State of the Art High-Throughput Approaches to Genotoxicity: Flow Micronucleus, Ames II, GreenScreen and Comet (Presented by Dr. Marilyn J. Aardema, Chief Scientific Advisor, Toxicology, Dr. Leon Stankowski, et. al. (6/28/2012)

  4. High-throughput continuous flow femtosecond laser-assisted cell optoporation and transfection.

    PubMed

    Breunig, Hans Georg; Uchugonova, Aisada; Batista, Ana; König, Karsten

    2014-12-01

    We present a femtosecond-laser based nanoprocessing system for transient optical cell membrane poration to allow the introduction of foreign molecules into the interior of a cell with very high throughput. In the setup, cells flow through a micro-flow tube for spatial confinement and are simultaneously targeted by fs laser radiation. Beam-shaping generates a focal geometry along a line which is scanned across the micro-flow cell to increase the number of reachable cells. Successful cell membrane poration was observed indirectly by cell transfection even with cell-light interaction times in the millisecond range. The system was characterized by experiments with Chinese hamster ovary cells regarding cell viability, the uptake of extrinsic molecules and cell transfection efficiency. The continuous flow of cells enables a tremendous increase of cell throughput compared to previous nonflow approaches by treating millions of cells, although with only limited efficiency. The setup opens the possibility to realize a completely automated high-throughput laser-assisted cell-poration system which could be integrated in lab-on-a-chip devices.

  5. Massively Parrell Rogue Cell Detection Using Serial Time-Encoded Amplified Microscopy of Inertially Ordered Cells in High Throughput Flow

    DTIC Science & Technology

    2012-08-01

    To show the utility of the STEAM flow analyzer, we used it to demonstrate high-throughput screening of Saccharomyces cerevisiae , commonly known as...protein therapeutics. Growth in yeast can be studied and optimized by flow cytometry or microscopy – both of which possess specific limitations...and provides poor characterization of the asymmetric growth of yeast in comparison with imaging. A high-throughput microscopy technique such as our

  6. Continuous segmented-flow polymerase chain reaction for high-throughput miniaturized DNA amplification.

    PubMed

    Curcio, Mario; Roeraade, Johan

    2003-01-01

    A continuous segmented-flow method for sequential DNA amplification is described in order to provide a basis for high-throughput genetic analysis. The approach allows an immediate distinction between amplified and nonamplified products. A mixture of sample and reagents are loaded in the form of small segments one after another in a 15-m-long narrow-bore Teflon tube, coiled such as to be repeatedly exposed to three different temperature zones. After having passed the heated zones, the samples are mixed with an intercalating dye by flow injection and sequentially detected on-line by laser-induced fluorescence. The aqueous samples travel as separate segments in a continuous flow of an immiscible, organic liquid. Perfluorodecalin was shown to be particularly suitable due to its hydrophobicity and inert properties. To reduce carryover between samples, an intermediate water plug between two consecutive samples was required. Selected regions from human genomic DNA were successfully amplified in 300-nL volumes after 30 passes through the heated zones. The total reaction time was approximately 45 min, and the detection interval between individual samples was 1 min. Automation and the possibility to further reduce sample volumes, as well as to employ many reaction columns simultaneously, should provide a platform for an extremely high throughput.

  7. Two-step protocol for preparing adherent cells for high-throughput flow cytometry.

    PubMed

    Kaur, Mandeep; Esau, Luke

    2015-09-01

    We have developed a simple, cost-effective, and labor-efficient two-step protocol for preparing adherent cells for high-throughput flow cytometry. Adherent cells were grown on microplates, detached with 2.9 mM EDTA (pH 6.14) added directly to wells containing cell culture medium, stained, and then analyzed on a flow cytometer. This protocol bypasses washing, centrifugation, and transfer between plates, reducing the cell loss that occurs in standard multistep protocols. The method has been validated using six adherent cell lines, four commercially available dyes, and two antibodies; the results have been confirmed using two different flow cytometry (FC) instruments. Our approach has been used for estimating apoptosis, mitochondrial membrane potential, reactive oxygen species, and autophagy in response to exposure to pure compounds as well as plant and bacterial extracts.

  8. Microfluidic chip integrating high throughput continuous-flow PCR and DNA hybridization for bacteria analysis.

    PubMed

    Jiang, Xiran; Shao, Ning; Jing, Wenwen; Tao, Shengce; Liu, Sixiu; Sui, Guodong

    2014-05-01

    Rapid identification of clinical pathogens is the initial and essential step for antimicrobial therapy. Herein, we successfully developed a microfluidic device which combines high-throughput continuous-flow PCR and DNA hybridization for the detection of various bacterial pathogens. Universal primers were designed based on the conserved regions of bacterial 16S ribosomal DNA (16S rDNA), and specific probes were designed from a variable region of 16S rDNA within the amplicon sequences. In the chip operation, after the continuous flow PCR was achieved in the first microfluidic chip, the product was directly introduced into a hybridization chip integrated with microarray containing the immobilized DNA probes. The target-probe hybridization was completed within 1h at 55 °C, and fluorescence signals were obtained as the readout. The presented device is simple, versatile and with less sample consumption compared with traditional instruments. It can perform high-throughput bacteria detections continuously in a single assay, which makes it a promising platform for clinical bacteria identifications.

  9. Continuous-flow high pressure hydrogenation reactor for optimization and high-throughput synthesis.

    PubMed

    Jones, Richard V; Godorhazy, Lajos; Varga, Norbert; Szalay, Daniel; Urge, Laszlo; Darvas, Ferenc

    2006-01-01

    This paper reports on a novel continuous-flow hydrogenation reactor and its integration with a liquid handler to generate a fully automated high-throughput hydrogenation system for library synthesis. The reactor, named the H-Cube, combines endogenous hydrogen generation from the electrolysis of water with a continuous flow-through system. The system makes significant advances over current batch hydrogenation reactors in terms of safety, reaction validation efficiency, and rates of reaction. The hydrogenation process is described along with a detailed description of the device's main parts. The reduction of a series of functional groups, varying in difficulty up to 70 degrees C and 70 bar are also described. The paper concludes with the integration of the device into an automated liquid handler followed by the reduction of a nitro compound in a high throughput manner. The system is fully automated and can conduct 5 reactions in the time it takes to perform and workup one reaction manually on a standard batch reactor.

  10. High-throughput continuous flow synthesis of nickel nanoparticles for the catalytic hydrodeoxygenation of guaiacol

    DOE PAGES

    Roberts, Emily J.; Habas, Susan E.; Wang, Lu; ...

    2016-11-07

    The translation of batch chemistries to high-throughput continuous flow methods dresses scaling, automation, and reproducibility concerns associated with the implementation of colloidally prepared nanoparticle (NP) catalysts for industrial catalytic processes. Nickel NPs were synthesized by the high-temperature amine reduction of a Ni2+ precursor using a continuous millifluidic (mF) flow method, achieving yields greater than 60%. The resulting Ni NP catalysts were compared against catalysts prepared in a batch reaction under conditions analogous to the continuous flow conditions with respect to total reaction volume, time, and temperature and by traditional incipient wetness (IW) impregnation for the hydrodeoxygenation (HDO) of guaiacol undermore » ex situ catalytic fast pyrolysis conditions. Compared to the IW method, the colloidally prepared NPs displayed increased morphological control and narrowed size distributions, and the NPs prepared by both methods showed similar size, shape, and crystallinity. The Ni NP catalyst synthesized by the continuous flow method exhibited similar H-adsorption site densities, site-time yields, and selectivities towards deoxygenated products as compared to the analogous batch reaction, and outperformed the IW catalyst with respect to higher selectivity to lower oxygen content products and a 6.9-fold slower deactivation rate. These results demonstrate the utility of synthesizing colloidal Ni NP catalysts using continuous flow methods while maintaining the catalytic properties displayed by the batch equivalent. Finally, this methodology can be extended to other catalytically relevant base metals for the high-throughput synthesis of metal NPs for the catalytic production of biofuels.« less

  11. Titer plate formatted continuous flow thermal reactors for high throughput applications: fabrication and testing

    NASA Astrophysics Data System (ADS)

    Sang-Won Park, Daniel; Chen, Pin-Chuan; You, Byoung Hee; Kim, Namwon; Park, Taehyun; Lee, Tae Yoon; Datta, Proyag; Desta, Yohannes; Soper, Steven A.; Nikitopoulos, Dimitris E.; Murphy, Michael C.

    2010-05-01

    A high throughput, multi-well (96) polymerase chain reaction (PCR) platform, based on a continuous flow (CF) mode of operation, was developed. Each CFPCR device was confined to a footprint of 8 × 8 mm2, matching the footprint of a well on a standard micro-titer plate. While several CFPCR devices have been demonstrated, this is the first example of a high-throughput multi-well continuous flow thermal reactor configuration. Verification of the feasibility of the multi-well CFPCR device was carried out at each stage of development from manufacturing to demonstrating sample amplification. The multi-well CFPCR devices were fabricated by micro-replication in polymers, polycarbonate to accommodate the peak temperatures during thermal cycling in this case, using double-sided hot embossing. One side of the substrate contained the thermal reactors and the opposite side was patterned with structures to enhance thermal isolation of the closely packed constant temperature zones. A 99 bp target from a λ-DNA template was successfully amplified in a prototype multi-well CFPCR device with a total reaction time as low as ~5 min at a flow velocity of 3 mm s-1 (15.3 s cycle-1) and a relatively low amplification efficiency compared to a bench-top thermal cycler for a 20-cycle device; reducing the flow velocity to 1 mm s-1 (46.2 s cycle-1) gave a seven-fold improvement in amplification efficiency. Amplification efficiencies increased at all flow velocities for 25-cycle devices with the same configuration.

  12. New device for high-throughput viability screening of flow biofilms.

    PubMed

    Benoit, Michael R; Conant, Carolyn G; Ionescu-Zanetti, Cristian; Schwartz, Michael; Matin, A

    2010-07-01

    Control of biofilms requires rapid methods to identify compounds effective against them and to isolate resistance-compromised mutants for identifying genes involved in enhanced biofilm resistance. While rapid screening methods for microtiter plate well ("static") biofilms are available, there are no methods for such screening of continuous flow biofilms ("flow biofilms"). Since the latter biofilms more closely approximate natural biofilms, development of a high-throughput (HTP) method for screening them is desirable. We describe here a new method using a device comprised of microfluidic channels and a distributed pneumatic pump (BioFlux) that provides fluid flow to 96 individual biofilms. This device allows fine control of continuous or intermittent fluid flow over a broad range of flow rates, and the use of a standard well plate format provides compatibility with plate readers. We show that use of green fluorescent protein (GFP)-expressing bacteria, staining with propidium iodide, and measurement of fluorescence with a plate reader permit rapid and accurate determination of biofilm viability. The biofilm viability measured with the plate reader agreed with that determined using plate counts, as well as with the results of fluorescence microscope image analysis. Using BioFlux and the plate reader, we were able to rapidly screen the effects of several antimicrobials on the viability of Pseudomonas aeruginosa PAO1 flow biofilms.

  13. Online Flowing Colloidosomes for Sequential Multi-Analyte High-Throughput SERS Analysis.

    PubMed

    Phan-Quang, Gia Chuong; Wee, Elizabeth Hui Zi; Yang, Fengling; Lee, Hiang Kwee; Phang, In Yee; Feng, Xiaotong; Alvarez-Puebla, Ramón A; Ling, Xing Yi

    2017-04-11

    3D plasmonic colloidosomes are superior SERS sensors owing to their high sensitivity and excellent tolerance to laser misalignment. Herein, we incorporate plasmonic colloidosomes in a microfluidic channel for online SERS detection. Our method resolves the poor signal reproducibility and inter-sample contamination in the existing online SERS platforms. Our flow system offers rapid and continuous online detection of 20 samples in less than 5 min with excellent signal reproducibility. The isolated colloidosomes prevent cross-sample and channel contamination, allowing accurate quantification of samples over a concentration range of five orders of magnitude. Our system demonstrates high-resolution multiplex detection with fully preserved signal and Raman features of individual analytes in a mixture. High-throughput multi-assay analysis is performed, which highlights that our system is capable of rapid identification and quantification of a sequence of samples containing various analytes and concentrations.

  14. Pinched flow coupled shear-modulated inertial microfluidics for high-throughput rare blood cell separation.

    PubMed

    Bhagat, Ali Asgar S; Hou, Han Wei; Li, Leon D; Lim, Chwee Teck; Han, Jongyoon

    2011-06-07

    Blood is a highly complex bio-fluid with cellular components making up >40% of the total volume, thus making its analysis challenging and time-consuming. In this work, we introduce a high-throughput size-based separation method for processing diluted blood using inertial microfluidics. The technique takes advantage of the preferential cell focusing in high aspect-ratio microchannels coupled with pinched flow dynamics for isolating low abundance cells from blood. As an application of the developed technique, we demonstrate the isolation of cancer cells (circulating tumor cells (CTCs)) spiked in blood by exploiting the difference in size between CTCs and hematologic cells. The microchannel dimensions and processing parameters were optimized to enable high throughput and high resolution separation, comparable to existing CTC isolation technologies. Results from experiments conducted with MCF-7 cells spiked into whole blood indicate >80% cell recovery with an impressive 3.25 × 10(5) fold enrichment over red blood cells (RBCs) and 1.2 × 10(4) fold enrichment over peripheral blood leukocytes (PBL). In spite of a 20× sample dilution, the fast operating flow rate allows the processing of ∼10(8) cells min(-1) through a single microfluidic device. The device design can be easily customized for isolating other rare cells from blood including peripheral blood leukocytes and fetal nucleated red blood cells by simply varying the 'pinching' width. The advantage of simple label-free separation, combined with the ability to retrieve viable cells post enrichment and minimal sample pre-processing presents numerous applications for use in clinical diagnosis and conducting fundamental studies.

  15. High-throughput DNA Stretching in Continuous Elongational Flow for Genome Sequence Scanning

    NASA Astrophysics Data System (ADS)

    Meltzer, Robert; Griffis, Joshua; Safranovitch, Mikhail; Malkin, Gene; Cameron, Douglas

    2014-03-01

    Genome Sequence Scanning (GSS) identifies and compares bacterial genomes by stretching long (60 - 300 kb) genomic DNA restriction fragments and scanning for site-selective fluorescent probes. Practical application of GSS requires: 1) high throughput data acquisition, 2) efficient DNA stretching, 3) reproducible DNA elasticity in the presence of intercalating fluorescent dyes. GSS utilizes a pseudo-two-dimensional micron-scale funnel with convergent sheathing flows to stretch one molecule at a time in continuous elongational flow and center the DNA stream over diffraction-limited confocal laser excitation spots. Funnel geometry has been optimized to maximize throughput of DNA within the desired length range (>10 million nucleobases per second). A constant-strain detection channel maximizes stretching efficiency by applying a constant parabolic tension profile to each molecule, minimizing relaxation and flow-induced tumbling. The effect of intercalator on DNA elasticity is experimentally controlled by reacting one molecule of DNA at a time in convergent sheathing flows of the dye. Derivations of accelerating flow and non-linear tension distribution permit alignment of detected fluorescence traces to theoretical templates derived from whole-genome sequence data.

  16. Emerging flow injection mass spectrometry methods for high-throughput quantitative analysis.

    PubMed

    Nanita, Sergio C; Kaldon, Laura G

    2016-01-01

    Where does flow injection analysis mass spectrometry (FIA-MS) stand relative to ambient mass spectrometry (MS) and chromatography-MS? Improvements in FIA-MS methods have resulted in fast-expanding uses of this technique. Key advantages of FIA-MS over chromatography-MS are fast analysis (typical run time <60 s) and method simplicity, and FIA-MS offers high-throughput without compromising sensitivity, precision and accuracy as much as ambient MS techniques. Consequently, FIA-MS is increasingly becoming recognized as a suitable technique for applications where quantitative screening of chemicals needs to be performed rapidly and reliably. The FIA-MS methods discussed herein have demonstrated quantitation of diverse analytes, including pharmaceuticals, pesticides, environmental contaminants, and endogenous compounds, at levels ranging from parts-per-billion (ppb) to parts-per-million (ppm) in very complex matrices (such as blood, urine, and a variety of foods of plant and animal origin), allowing successful applications of the technique in clinical diagnostics, metabolomics, environmental sciences, toxicology, and detection of adulterated/counterfeited goods. The recent boom in applications of FIA-MS for high-throughput quantitative analysis has been driven in part by (1) the continuous improvements in sensitivity and selectivity of MS instrumentation, (2) the introduction of novel sample preparation procedures compatible with standalone mass spectrometric analysis such as salting out assisted liquid-liquid extraction (SALLE) with volatile solutes and NH4(+) QuEChERS, and (3) the need to improve efficiency of laboratories to satisfy increasing analytical demand while lowering operational cost. The advantages and drawbacks of quantitative analysis by FIA-MS are discussed in comparison to chromatography-MS and ambient MS (e.g., DESI, LAESI, DART). Generally, FIA-MS sits 'in the middle' between ambient MS and chromatography-MS, offering a balance between analytical

  17. High Throughput Flow Cytometry Bead-based Multiplex Assay for Identification of Rho GTPase Inhibitors

    PubMed Central

    Surviladze, Zurab; Young, Susan M; Sklar, Larry A

    2015-01-01

    Summary Rho family GTPases and their effector proteins regulate a wide range of cell signaling pathways. In normal physiological conditions their activity is tightly controlled and it is not surprising that their aberrant activation contributes to tumorigenesis or other diseases. For this reason, the identification of small, cell permeable molecules capable of inhibition of Rho GTPases can be extraordinarily useful, particularly if they are specific and act reversibly. Herein we describe a flow cytometric assay, which allows us to measure the activity of six small GTPases simultaneously. GST-tagged small GTPases are bound to six glutathione bead sets each set having a different intensity of red fluorescence at a fixed wavelength. The coated bead sets were washed, combined, and dispensed into 384-well plates with test compounds, and fluorescent-GTP binding was used as the read-out. This multiplex bead-based assay was successfully used for to identify both general and selective inhibitors of Rho family GTPases. PMID:22144280

  18. 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.

  19. Flow Cytometric Analysis and Sorting of Heterodera glycines Eggs

    PubMed Central

    Tylka, G. L.; Niblack, T. L.; Walk, T. C.; Harkins, K. R.; Barnett, L.; Baker, N. K.

    1993-01-01

    A nondestructive technique was developed to characterize and separate eggs of soybean cyst nematode, Heterodera glycines, by developmental stage using flow cytometry. Eggs from cysts cultured on susceptible soybean roots were suspended in 0.1% xanthan gum or 59% sucrose and loaded into either a Coulter EPICS 752 or EPICS 753 flow cytometer. Eggs were analyzed and sorted according to forward angle and 90° light scatter, flow cytometric parameters that are relative measures of object size and granularity, respectively. Mature eggs containing vermiform juveniles were less granular and slightly larger than eggs in earlier stages of embryogeny, allowing for separation of mature eggs from immature eggs. The effectiveness of flow cytometric sorting was evaluated by comparing the developmental stages of subpopulations of unsorted and sorted eggs. Of a subpopulation of unsorted eggs, 62% contained vermiform juveniles, whereas 85 to 95% of sorted subpopulations of larger, less granular eggs contained vermiform juveniles. Suspending H. glycines eggs in 0.1% xanthan gum or 59% sucrose for flow cytometric analysis had no effect on subsequent egg hatch in vitro. This technique is an efficient and effective means to collect large, relatively homogeneous quantities of H. glycines eggs in early or late embryogeny, and would likely be useful for analyzing and sorting eggs of other nematode species for use in developmental, genetic, or physiological research, or for identification and collection of parasitized eggs. PMID:19279815

  20. High-throughput magnetic flow sorting of human cells selected on the basis of magnetophoretic mobility

    NASA Astrophysics Data System (ADS)

    Reece, Lisa M.; Sanders, Lehanna; Kennedy, David; Guernsey, Byron; Todd, Paul; Leary, James F.

    2010-02-01

    We have shown the potential of a new method for optimizing the separation of human stem cell subsets from peripheral blood based on a novel cell labeling technique that leverages the capabilities of a new commercially available high speed magnetic cell sorting system (IKOTECH LLC, New Albany, IN). This new system sorts cells in a continuously flowing manner using a Quadrupole Magnetic cell Sorter (QMS). The sorting mechanism is based upon the magnetophoretic mobility of the cells, a property related to the relative binding distributions of magnetic particles per cell, as determined by the utilization of a Magnetic Cell Tracking Velocimeter (MCTV). KG-1 cells were competitively labeled with anti-CD34 magnetic beads and anti-CD34 FITC to obtain an optimal level of magnetophoretic mobility as visualized by the MCTV for high throughput sort recovery in the QMS. In QMS sorting, the concept of split-flow thin channel (SPLITT) separation technology is applied by having a sample stream enter a vertical annular flow channel near the channel's interior wall followed by another sheath flow entering near the exterior wall. The two flows are initially separated by a flow splitter. They pass through the bore of a Halbach permanent quadrupole magnet assembly, which draws magnetized cells outward and deflects them into a positive outflow, while negative cells continue straight out via the inner flow lamina. QMS sorts cells based upon their magnetophoretic mobility, or the velocity of a cell per unit ponderomotive force, the counterpart of fluorescence intensity in flow cytometry. The magnetophoretic mobility distribution of a cell population, measured by automated MCTV, is used as input data for the algorithmic control of sample, sheath, and outlet flow velocities of the QMS. In this study, the relative binding distributions of magnetic particles per cell were determined by MCTV using novel sorting and sizing algorithms. The resulting mobility histograms were used to set the QMS

  1. Flow cytometric immunofluorescence of rat anterior pituitary cells

    NASA Technical Reports Server (NTRS)

    Hatfield, J. Michael; Hymer, W. C.

    1985-01-01

    A flow cytometric immunofluorescence technique was developed for the quantification of growth hormone, prolactin, and luteinizing hormone producing cells. The procedure is based on indirect-immunofluorescence of intracellular hormone using an EPICS V cell sorter and can objectively count 50,000 cells in about 3 minutes. It can be used to study the dynamics of pituitary cell populations under various physiological and pharmacological conditions.

  2. Flow cytometric immunofluorescence of rat anterior pituitary cells

    NASA Technical Reports Server (NTRS)

    Hatfield, J. Michael; Hymer, W. C.

    1985-01-01

    A flow cytometric immunofluorescence technique was developed for the quantification of growth hormone, prolactin, and luteinizing hormone producing cells. The procedure is based on indirect-immunofluorescence of intracellular hormone using an EPICS V cell sorter and can objectively count 50,000 cells in about 3 minutes. It can be used to study the dynamics of pituitary cell populations under various physiological and pharmacological conditions.

  3. High-throughput quantification of stabilizers in polymeric materials by flow injection tandem mass spectrometry.

    PubMed

    Beißmann, Susanne; Reisinger, Michael; Reimann, Andreas; Klampfl, Christian W; Buchberger, Wolfgang

    2014-04-30

    High-throughput methods for identification and quantification of stabilizers in plastic materials are of significant importance in order to evaluate the suitability of materials of unknown origin for specific application areas, to clarify reasons for failure of materials, or for comparison of materials from different sources. In the present study, a highly sensitive and rapid flow injection method coupled to selected reaction monitoring mass spectrometry (MS) for comprehensive analysis of 21 polymer stabilizers in polyolefins is demonstrated. A critical factor for this approach is the choice of ionization mode, as no separation was performed prior to MS detection. Differences between several ionization techniques regarding matrix effects are reported. Atmospheric pressure chemical ionization was found to be the most suitable ionization technique, with no significant matrix effects observed. The developed method has a linear dynamic range over two to three orders of magnitude with correlation coefficients better than 0.99 for all studied analytes. Following a multistep sample preparation protocol, the method allowed quantification down to minimum values of between 0.0001 and 0.04 wt% depending on the type of stabilizer. Results were compared to an established chromatographic approach and showed very good correlation (bias below 7.5%). The applicability of the optimized method could be demonstrated for both the qualitative and quantitative determination of polymer stabilizers in polyolefins. Furthermore, the described approach yields a complete analysis in a much shorter time than can be achieved with commonly applied chromatographic methods. Copyright © 2014 John Wiley & Sons, Ltd.

  4. The Application of Imaging Flow Cytometry to High-Throughput Biodosimetry

    PubMed Central

    Wilkins, Ruth C.; Rodrigues, Matthew A.; Beaton-Green, Lindsay A.

    2017-01-01

    Biodosimetry methods, including the dicentric chromosome assay, the cytokinesis-block micronucleus assay and the γH2AX marker of DNA damage are used to determine the dose of ionizing radiation. These techniques are particularly useful when physical dosimetry is absent or questioned. While these assays can be very sensitive and specific, the standard methods need to be adapted to increase sample throughput in the case of a large-scale radiological/nuclear event. Recent modifications to the microscope-based assays have resulted in some increased throughput, and a number of biodosimetry networks have been, and continue to be, established and strengthened. As the imaging flow cytometer (IFC) is a technology that can automatically image and analyze processed blood samples for markers of radiation damage, the microscope-based biodosimetry techniques can be modified for the IFC for high-throughput biological dosimetry. Furthermore, the analysis templates can be easily shared between networked biodosimetry laboratories for increased capacity and improved standardization. This review describes recent advances in IFC methodology and their application to biodosimetry. PMID:28250914

  5. An Improved Method for High-throughput Discrimination and Enumeration of Sedimentary Cells Using Flow Cytometry

    NASA Astrophysics Data System (ADS)

    Morono, Y.; Kallmeyer, J.; Terada, T.; Inagaki, F.; IODP Expedition 329 Shipboard Science Party

    2011-12-01

    Detection and enumeration of microbial life in marine subsurface environments provides primary information on the extent and habitability of the Earth's biosphere. Flow cytometry (FCM) is a powerful tool for identifying and enumerating fluorescence-stained cells with high throughput, using fluorescent intensity, range of wavelength, and cell size. FCM is widely used in medical sciences and aquatic microbial ecology. However, mineral grains and difficulties in distinguishing between life cells and non-specific background fluorescence prevented FCM to be applied for counting microbial cells in sediment or rock samples. SYBR Green I-stained cells can be distinguished from non-biological background signals based on differences in their fluorescence spectra. Here we extended this technique to FCM analysis by modifying the cell detachment protocol using a density gradient method, and then standardized an FCM cell counting method for various types of marine subsurface sediments. Microbial cells in sediment samples could effectively be detached and analyzed discriminatively with FCM. The high capacity of FCM to count particles (up to 10,000 cells/sec) and its high sensitivity will provide information about microbial cell abundance at high spatial resolution and with unprecedented accuracy. This improved cell count method will be useful to evaluate samples with high depth resolution, including narrow geochemical and geological interfaces as potential specific microbial niches, and may even help to asses very low population densities at the fringe of the biosphere.

  6. Discovery of Regulators of Receptor Internalization with High-Throughput Flow Cytometry

    PubMed Central

    Tapia, Phillip H.; Fisher, Gregory W.; Simons, Peter C.; Strouse, J. Jacob; Foutz, Terry; Waggoner, Alan S.; Jarvik, Jonathan; Sklar, Larry A.

    2012-01-01

    We developed a platform combining fluorogen-activating protein (FAP) technology with high-throughput flow cytometry to detect real-time protein trafficking to and from the plasma membrane in living cells. The hybrid platform facilitates drug discovery for trafficking receptors such as G protein-coupled receptors and was validated with the β2-adrenergic receptor (β2AR) system. When a chemical library containing ∼1200 off-patent drugs was screened against cells expressing FAP-tagged β2ARs, all 33 known β2AR-active ligands in the library were successfully identified, together with a number of compounds that might regulate receptor internalization in a nontraditional manner. Results indicated that the platform identified ligands of target proteins regardless of the associated signaling pathway; therefore, this approach presents opportunities to search for biased receptor modulators and is suitable for screening of multiplexed targets for improved efficiency. The results revealed that ligands may be biased with respect to the rate or duration of receptor internalization and that receptor internalization may be independent of activation of the mitogen-activated protein kinase pathway. PMID:22767611

  7. High-throughput microcoil NMR of compound libraries using zero-dispersion segmented flow analysis.

    PubMed

    Kautz, Roger A; Goetzinger, Wolfgang K; Karger, Barry L

    2005-01-01

    An automated system for loading samples into a microcoil NMR probe has been developed using segmented flow analysis. This approach enhanced 2-fold the throughput of the published direct injection and flow injection methods, improved sample utilization 3-fold, and was applicable to high-field NMR facilities with long transfer lines between the sample handler and NMR magnet. Sample volumes of 2 microL (10-30 mM, approximately 10 microg) were drawn from a 96-well microtiter plate by a sample handler, then pumped to a 0.5-microL microcoil NMR probe as a queue of closely spaced "plugs" separated by an immiscible fluorocarbon fluid. Individual sample plugs were detected by their NMR signal and automatically positioned for stopped-flow data acquisition. The sample in the NMR coil could be changed within 35 s by advancing the queue. The fluorocarbon liquid wetted the wall of the Teflon transfer line, preventing the DMSO samples from contacting the capillary wall and thus reducing sample losses to below 5% after passage through the 3-m transfer line. With a wash plug of solvent between samples, sample-to-sample carryover was <1%. Significantly, the samples did not disperse into the carrier liquid during loading or during acquisitions of several days for trace analysis. For automated high-throughput analysis using a 16-second acquisition time, spectra were recorded at a rate of 1.5 min/sample and total deuterated solvent consumption was <0.5 mL (1 US dollar) per 96-well plate.

  8. Flow cytometric studies of human osteosarcoma.

    PubMed

    Mankin, H J; Gebhardt, M C; Springfield, D S; Litwak, G J; Kusazaki, K; Rosenberg, A E

    1991-09-01

    A number of recent studies have emphasized the potential value of flow cytometry as a "marker" to assess the malignity and therefore to help predict the biologic behavior of neoplasms, including bone tumors. Using propidium iodide and a home-built flow cytometer, the authors have studied the DNA distribution in 95 patients with osteosarcoma and determined the percentage of cells in diploidy, S-phase, tetraploidy, and aneuploidy. Using these values and a derived one, mean DNA concentration, it was possible to demonstrate the extent of the abnormalities observed in this group of neoplasms and show their severity as compared with the normal pattern. When the data are compared against disease-free survival and total survival, correlations were noted that, although weak, suggested that some patterns were predictive of increased risk of metastasis and death. The effect of treatment could also be assessed by evaluating the pattern before and after chemotherapy and correlating these with survival. It seems likely that with some improvement in technology, flow cytometry will be of value in the future in assessing the prognosis for osteosarcoma and predicting whether treatment has been effective.

  9. Flow cytometric detection method for DNA samples

    DOEpatents

    Nasarabadi, Shanavaz [Livermore, CA; Langlois, Richard G [Livermore, CA; Venkateswaran, Kodumudi S [Round Rock, TX

    2011-07-05

    Disclosed herein are two methods for rapid multiplex analysis to determine the presence and identity of target DNA sequences within a DNA sample. Both methods use reporting DNA sequences, e.g., modified conventional Taqman.RTM. probes, to combine multiplex PCR amplification with microsphere-based hybridization using flow cytometry means of detection. Real-time PCR detection can also be incorporated. The first method uses a cyanine dye, such as, Cy3.TM., as the reporter linked to the 5' end of a reporting DNA sequence. The second method positions a reporter dye, e.g., FAM.TM. on the 3' end of the reporting DNA sequence and a quencher dye, e.g., TAMRA.TM., on the 5' end.

  10. Flow cytometric detection method for DNA samples

    DOEpatents

    Nasarabadi, Shanavaz; Langlois, Richard G.; Venkateswaran, Kodumudi S.

    2006-08-01

    Disclosed herein are two methods for rapid multiplex analysis to determine the presence and identity of target DNA sequences within a DNA sample. Both methods use reporting DNA sequences, e.g., modified conventional Taqman.RTM. probes, to combine multiplex PCR amplification with microsphere-based hybridization using flow cytometry means of detection. Real-time PCR detection can also be incorporated. The first method uses a cyanine dye, such as, Cy3.TM., as the reporter linked to the 5' end of a reporting DNA sequence. The second method positions a reporter dye, e.g., FAM, on the 3' end of the reporting DNA sequence and a quencher dye, e.g., TAMRA, on the 5' end.

  11. A flow cytometric approach to quantify biofilms.

    PubMed

    Kerstens, Monique; Boulet, Gaëlle; Van Kerckhoven, Marian; Clais, Sofie; Lanckacker, Ellen; Delputte, Peter; Maes, Louis; Cos, Paul

    2015-07-01

    Since biofilms are important in many clinical, industrial, and environmental settings, reliable methods to quantify these sessile microbial populations are crucial. Most of the currently available techniques do not allow the enumeration of the viable cell fraction within the biofilm and are often time consuming. This paper proposes flow cytometry (FCM) using the single-stain viability dye TO-PRO(®)-3 iodide as a fast and precise alternative. Mature biofilms of Candida albicans and Escherichia coli were used to optimize biofilm removal and dissociation, as a single-cell suspension is needed for accurate FCM enumeration. To assess the feasibility of FCM quantification of biofilms, E. coli and C. albicans biofilms were analyzed using FCM and crystal violet staining at different time points. A combination of scraping and rinsing proved to be the most efficient technique for biofilm removal. Sonicating for 10 min eliminated the remaining aggregates, resulting in a single-cell suspension. Repeated FCM measurements of biofilm samples revealed a good intraday precision of approximately 5 %. FCM quantification and the crystal violet assay yielded similar biofilm growth curves for both microorganisms, confirming the applicability of our technique. These results show that FCM using TO-PRO(®)-3 iodide as a single-stain viability dye is a valid fast alternative for the quantification of viable cells in a biofilm.

  12. A flow cytometric method for platelet counting in platelet concentrates.

    PubMed

    van der Meer, Pieter F; Karssing-van Leeuwen, Willy; Kurtz, Jim; Spengler, Hans-Peter; Blair, AbbeJane; Devine, Dana; Harrison, Paul; Lambrecht, Bernd; VandenBroeke, Tania; de Wildt, Janny; de Korte, Dirk

    2012-01-01

    The platelets (PLTs) in PLT concentrates are counted with hematology analyzers, but varying results among different hematology analyzers are observed, making comparisons very difficult. Due to the absence of red blood cells in PLT concentrates, the International Council for Standardization in Hematology (ICSH) reference method was modified to be used for PLT concentrates and validated in an international comparative study. Five PLT samples were shipped to eight participating centers of the Biomedical Excellence for Safer Transfusion (BEST) Collaborative and counted on the same day. PLTs were stained with fluorescein isothiocyanate-labeled anti-CD41a in tubes (TruCount, BD Biosciences), measured on a flow cytometer, and analyzed with a uniform template. These samples were also counted on 15 hematology analyzers. The ICSH method and newly developed BEST method yielded PLT counting results with less than 1% difference (not significant). The intercenter coefficient of variation (CV) of the BEST method was on average 6.3% versus 7.6% on average for hematology analyzers. The CV of individual hematology analyzers was on average 0.9%, which was considerably lower than for the flow cytometers with a mean of 3.7%. The BEST flow cytometric method has a smaller intercenter CV and a smaller center-to-center deviation from the group mean compared to hematology analyzers. Conversely, individual hematology analyzers are more precise than the flow cytometric method. Thus, the flow cytometric method provides a calibration tool to allow comparisons between centers, but there is no need to replace routine counting with hematology analyzers. © 2011 American Association of Blood Banks.

  13. Flow cytometric scoring of micronucleated erythrocytes: an efficient platform for assessing in vivo cytogenetic damage.

    PubMed

    Dertinger, Stephen D; Torous, Dorothea K; Hayashi, Makoto; MacGregor, James T

    2011-01-01

    The relative simplicity of the micronucleated erythrocyte endpoint has made it amenable to automated scoring approaches. Flow cytometry is one such scoring platform that has been employed successfully. This review describes the evolution and properties of flow cytometry-based scoring of micronucleated erythrocytes. The methodology has become widely applied to rodent blood specimens and the high throughput nature of the technology provides a number of advantages over manual microscopic scoring. For instance, the ability to efficiently survey many dose levels and many more cells per specimen relative to microscopy benefits studies that are designed to identify no observable effect levels or lowest observable effect levels. Furthermore, flow cytometry makes it practical to study species with low spontaneous reticulocyte (RET) counts and micronucleus (MN) frequencies, thereby facilitating integration of blood-based micronucleated reticulocyte (MN-RET) frequency measurements into experiments conducted across species of toxicological interest. This capability enhances genotoxicity assessments that have historically been made in dedicated MN tests performed in one species. Importantly, the feasibility of using MN-RET frequencies in blood from humans as an index of genetic damage in bone marrow opens a critical area of application that had not been practical previously. We conclude with recommendations for additional work that is needed to more fully realise the potential of flow cytometric in vivo MN scoring.

  14. A complementary role of multiparameter flow cytometry and high-throughput sequencing for minimal residual disease detection in chronic lymphocytic leukemia: an European Research Initiative on CLL study

    PubMed Central

    Rawstron, A C; Fazi, C; Agathangelidis, A; Villamor, N; Letestu, R; Nomdedeu, J; Palacio, C; Stehlikova, O; Kreuzer, K-A; Liptrot, S; O'Brien, D; de Tute, R M; Marinov, I; Hauwel, M; Spacek, M; Dobber, J; Kater, A P; Gambell, P; Soosapilla, A; Lozanski, G; Brachtl, G; Lin, K; Boysen, J; Hanson, C; Jorgensen, J L; Stetler-Stevenson, M; Yuan, C; Broome, H E; Rassenti, L; Craig, F; Delgado, J; Moreno, C; Bosch, F; Egle, A; Doubek, M; Pospisilova, S; Mulligan, S; Westerman, D; Sanders, C M; Emerson, R; Robins, H S; Kirsch, I; Shanafelt, T; Pettitt, A; Kipps, T J; Wierda, W G; Cymbalista, F; Hallek, M; Hillmen, P; Montserrat, E; Ghia, P

    2016-01-01

    In chronic lymphocytic leukemia (CLL) the level of minimal residual disease (MRD) after therapy is an independent predictor of outcome. Given the increasing number of new agents being explored for CLL therapy, using MRD as a surrogate could greatly reduce the time necessary to assess their efficacy. In this European Research Initiative on CLL (ERIC) project we have identified and validated a flow-cytometric approach to reliably quantitate CLL cells to the level of 0.0010% (10−5). The assay comprises a core panel of six markers (i.e. CD19, CD20, CD5, CD43, CD79b and CD81) with a component specification independent of instrument and reagents, which can be locally re-validated using normal peripheral blood. This method is directly comparable to previous ERIC-designed assays and also provides a backbone for investigation of new markers. A parallel analysis of high-throughput sequencing using the ClonoSEQ assay showed good concordance with flow cytometry results at the 0.010% (10−4) level, the MRD threshold defined in the 2008 International Workshop on CLL guidelines, but it also provides good linearity to a detection limit of 1 in a million (10−6). The combination of both technologies would permit a highly sensitive approach to MRD detection while providing a reproducible and broadly accessible method to quantify residual disease and optimize treatment in CLL. PMID:26639181

  15. Flow cytometric measurement of total DNA and incorporated halodeoxyuridine

    DOEpatents

    Dolbeare, Frank A.; Gray, Joe W.

    1986-01-01

    A method for the simultaneous flow cytometric measurement of the total DNA content and the level of DNA synthesis in normal and malignant cells is disclosed. The sensitivity of the method allows a study of cell cycle traverse rates for large scale cell populations as well as single cell measurements. A DNA stain such as propidium iodide is used as the probe for the measurement of total DNA content and a monoclonal antibody reactive with a DNA precursor such as bromodeoxyuridine (BrdU) is used as a probe for the measurement of BrdU uptake by the cells as a measure of DNA synthesis.

  16. Method for flow cytometric detection of Listeria monocytogenes in milk.

    PubMed Central

    Donnelly, C W; Baigent, G J

    1986-01-01

    This report describes a method for the detection of Listeria monocytogenes in raw milk by flow cytometric analysis of fluorescently labeled bacterial populations. The use of immunofluorescence in combination with measures of DNA content by propidium iodide labeling and size by light scattering enabled specific identification of L. monocytogenes from Streptococcus faecalis, Streptococcus agalactiae, Streptococcus uberis, Staphylococcus epidermidis, and Staphylococcus hyicus. Additional specific resolution of L. monocytogenes populations was achieved through selective enrichment of raw milk in Listeria enrichment broth. These procedures should permit the rapid screening of milk and other food samples for L. monocytogenes and eliminate many of the short-comings associated with conventional fluorescent-antibody procedures. PMID:3096202

  17. Massively Parallel Rogue Cell Detection Using Serial Time-Encoded Amplified Microscopy of Inertially Ordered Cells in High Throughput Flow

    DTIC Science & Technology

    2011-08-01

    limited by the microfluidic device’s tolerance to high pressures caused by high flow rates, not the STEAM camera’s image acquisition speed as well...further chemical analysis of the cells. While in our proof-of-concept demonstration, we showed high- throughput screening of budding yeast and... ethanol for 15 minutes. It was then rinsed with ethanol and heated to 65°C for 30 minutes to prevent swelling. PDMS was then cast on the PDMS replica

  18. Automated High-Dimensional Flow Cytometric Data Analysis

    NASA Astrophysics Data System (ADS)

    Pyne, Saumyadipta; Hu, Xinli; Wang, Kui; Rossin, Elizabeth; Lin, Tsung-I.; Maier, Lisa; Baecher-Allan, Clare; McLachlan, Geoffrey; Tamayo, Pablo; Hafler, David; de Jager, Philip; Mesirov, Jill

    Flow cytometry is widely used for single cell interrogation of surface and intracellular protein expression by measuring fluorescence intensity of fluorophore-conjugated reagents. We focus on the recently developed procedure of Pyne et al. (2009, Proceedings of the National Academy of Sciences USA 106, 8519-8524) for automated high- dimensional flow cytometric analysis called FLAME (FLow analysis with Automated Multivariate Estimation). It introduced novel finite mixture models of heavy-tailed and asymmetric distributions to identify and model cell populations in a flow cytometric sample. This approach robustly addresses the complexities of flow data without the need for transformation or projection to lower dimensions. It also addresses the critical task of matching cell populations across samples that enables downstream analysis. It thus facilitates application of flow cytometry to new biological and clinical problems. To facilitate pipelining with standard bioinformatic applications such as high-dimensional visualization, subject classification or outcome prediction, FLAME has been incorporated with the GenePattern package of the Broad Institute. Thereby analysis of flow data can be approached similarly as other genomic platforms. We also consider some new work that proposes a rigorous and robust solution to the registration problem by a multi-level approach that allows us to model and register cell populations simultaneously across a cohort of high-dimensional flow samples. This new approach is called JCM (Joint Clustering and Matching). It enables direct and rigorous comparisons across different time points or phenotypes in a complex biological study as well as for classification of new patient samples in a more clinical setting.

  19. Duplex High Throughput Flow Cytometry Screen Identifies Two Novel Formylpeptide Receptor Family Probes1

    PubMed Central

    Young, Susan M.; Bologa, Cristian M.; Fara, Dan; Bryant, Bj K.; Strouse, J. Jacob; Arterburn, Jeffrey B.; Ye, Richard D.; Oprea, Tudor I.; Prossnitz, Eric R.; Sklar, Larry A.; Edwards, Bruce S.

    2008-01-01

    Introduction Of recent clinical interest have been two related human G-protein coupled receptors: formylpeptide receptor (FPR), linked to anti-bacterial inflammation and malignant glioma cell metastasis; and formylpeptide receptor like-1 (FPRL1), linked to chronic inflammation in systemic amyloidosis, Alzheimer’s disease and prion diseases. In association with the National Institutes of Health (NIH) Molecular Library Screening Network, we implemented a flow cytometry based high throughput screening (HTS) approach for identifying selective small molecule FPR and FPRL1 ligands. Methods The screening assay measured the ability of test compounds to competitively displace a high-affinity, fluorescein-labeled peptide ligand from FPR, FPRL1 or both. U937 cells expressing FPR and RBL cells expressing FPRL1 were tested together in a “duplex” format. The U937 cells were color-coded with red fluorescent dye allowing their distinction during analysis. Compounds, cells and fluorescent ligand were sequentially combined (no-wash) in 15 μL assay volumes in 384-well plates. Throughput averaged ∼11 min per plate to analyze ∼4000 cells (∼2000/receptor) in a 2 μL aspirate from each well. Results/Conclusions In primary single concentration HTS of 24,304 NIH Small Molecule Repository compounds, 253 resulted in inhibition >30% (181 for FPR, 72 for FPRL1) of which 40 had selective binding inhibition constants (Ki) ≤ 4 μM (34 for FPR and 6 for FPRL1). An additional 1,446 candidate compounds were selected by structure-activity -relationship analysis of the hits and screened to identify novel ligands for FPR (3570-0208, Ki= 95 ± 10 nM) and FPRL1 (BB-V-115, Ki= 270 ± 51 nM). Each was a selective antagonist in calcium response assays and the most potent small molecule antagonist reported for its respective receptor to date. The duplex assay format reduced assay time, minimized reagent requirements, and provided selectivity information at every screening stage, thus proving

  20. Duplex high-throughput flow cytometry screen identifies two novel formylpeptide receptor family probes.

    PubMed

    Young, Susan M; Bologa, Cristian M; Fara, Dan; Bryant, Bj K; Strouse, Juan Jacob; Arterburn, Jeffrey B; Ye, Richard D; Oprea, Tudor I; Prossnitz, Eric R; Sklar, Larry A; Edwards, Bruce S

    2009-03-01

    Of recent, clinical interest have been two related human G-protein coupled receptors: formylpeptide receptor (FPR), linked to antibacterial inflammation and malignant glioma cell metastasis; and FPR like-1 (FPRL1), linked to chronic inflammation in systemic amyloidosis, Alzheimer's disease, and prion diseases. In association with the National Institutes of Health (NIH) Molecular Library Screening Network, we implemented a flow-cytometry-based high-throughput screening (HTS) approach for identifying selective small molecule FPR and FPRL1 ligands. The screening assay measured the ability of test compounds to competitively displace a high-affinity, fluorescein- labeled peptide ligand from FPR, FPRL1, or both. U937 cells expressing FPR and rat basophil leukemia (RBL) cells expressing FPRL1 were tested together in a "duplex" format. The U937 cells were color coded with red-fluorescent dye allowing their distinction during analysis. Compounds, cells, and fluorescent ligand were sequentially combined (no wash) in 15 microl assay volumes in 384-well plates. Throughput averaged approximately 11 min per plate to analyze approximately 4,000 cells ( approximately 2,000/receptor) in a 2 microl aspirate from each well. In primary single concentration HTS of 24,304 NIH Small Molecule Repository compounds, 253 resulted in inhibition >30% (181 for FPR, 72 for FPRL1) of which 40 had selective binding inhibition constants (K(i)) < or = 4 microM (34 for FPR and 6 for FPRL1). An additional 1,446 candidate compounds were selected by structure-activity-relationship analysis of the hits and screened to identify novel ligands for FPR (3570-0208, K(i) = 95 +/- 10 nM) and FPRL1 (BB-V-115, K(i) = 270 +/- 51 nM). Each was a selective antagonist in calcium response assays and the most potent small molecule antagonist reported for its respective receptor to date. The duplex assay format reduced assay time, minimized reagent requirements, and provided selectivity information at every screening

  1. Flow cytometric analysis of inflammatory cells in ischemic rat brain.

    PubMed

    Campanella, Marilena; Sciorati, Clara; Tarozzo, Glauco; Beltramo, Massimiliano

    2002-02-01

    Inflammation plays a key role in cerebral ischemia through activation of microglia and infiltration by leukocytes. Flow cytometry is a well-established method for quantitative and qualitative analysis of inflammatory cells. However, this technique has not been applied to the study of cerebral ischemia inflammation. The aim of this study was to establish a flow cytometric method to measure inflammatory cells in ischemic brain. To perform flow cytometry on brain tissue, we developed 2 cell-isolation methods based on different mechanical dissociation and Percoll gradient separation techniques. The methods were tested on a rat model of permanent middle cerebral artery occlusion. Morphological and immunophenotypic analyses, with the use of anti-CD11b, anti-CD45, and alphabeta T-cell receptor antibodies, were employed to identify and quantify inflammatory cells. Both methods gave consistent results in terms of yield and reproducibility. The cell suspension contained granulocytes, macrophages, lymphocytes, and neural cells. Morphological and immunophenotypic analyses enabled the identification of a cell-scatter gate (R1a) enriched in inflammatory cells. With both methods, a higher number of events in R1a were recorded in the ischemic hemisphere than in the nonischemic hemisphere (P< or =0.001). CD11b, CD45, and alphabeta T-cell receptor staining confirmed that this augmentation was a reflection of the increase in the number of granulocytes, cells of the monocytic lineage, and lymphocytes. Quantitative flow cytometric analysis of ischemic rat brain is feasible and provides a reliable and rapid assay to assess neuroinflammation in experimental models of brain ischemia.

  2. A Radial Flow Microfluidic Device for Ultra-high-throughput Affinity-based Isolation of Circulating Tumor Cells

    PubMed Central

    Murlidhar, Vasudha; Zeinali, Mina; Grabauskiene, Svetlana; Ghannad-Rezaie, Mostafa; Wicha, Max S.; Simeone, Diane M.; Ramnath, Nithya; Reddy, Rishindra M.

    2015-01-01

    Circulating tumor cells (CTCs) are believed to play an important role in metastasis, a process responsible for the majority of cancer-related deaths. But their rarity in the bloodstream makes microfluidic isolation complex and time-consuming. Additionally the low processing speeds can be a hindrance to obtaining higher yields of CTCs, limiting their potential use as biomarkers for early diagnosis. Here we report a high throughput microfluidic technology, the OncoBean Chip, employing radial flow that introduces a varying shear profile across the device enabling efficient cell capture by affinity at high flow rates. The recovery from whole blood was validated with cancer cell lines H1650 and MCF7, achieving a mean efficiency >80% at a throughput of 10 mL hr−1 in contrast to a flow rate of 1 mL hr−1 standardly reported with other microfluidic devices. Cells were recovered with a viability rate of 93% at these high speeds, increasing the ability to use captured CTCs for downstream analysis. Broad clinical application was demonstrated using comparable flow rates from blood specimens obtained from breast, pancreatic and lung cancer patients. Comparable CTC numbers were recovered in all the samples at the two flow rates demonstrating the ability of the technology to perform at high-throughputs. PMID:25074448

  3. A radial flow microfluidic device for ultra-high-throughput affinity-based isolation of circulating tumor cells.

    PubMed

    Murlidhar, Vasudha; Zeinali, Mina; Grabauskiene, Svetlana; Ghannad-Rezaie, Mostafa; Wicha, Max S; Simeone, Diane M; Ramnath, Nithya; Reddy, Rishindra M; Nagrath, Sunitha

    2014-12-10

    Circulating tumor cells (CTCs) are believed to play an important role in metastasis, a process responsible for the majority of cancer-related deaths. But their rarity in the bloodstream makes microfluidic isolation complex and time-consuming. Additionally the low processing speeds can be a hindrance to obtaining higher yields of CTCs, limiting their potential use as biomarkers for early diagnosis. Here, a high throughput microfluidic technology, the OncoBean Chip, is reported. It employs radial flow that introduces a varying shear profile across the device, enabling efficient cell capture by affinity at high flow rates. The recovery from whole blood is validated with cancer cell lines H1650 and MCF7, achieving a mean efficiency >80% at a throughput of 10 mL h(-1) in contrast to a flow rate of 1 mL h(-1) standardly reported with other microfluidic devices. Cells are recovered with a viability rate of 93% at these high speeds, increasing the ability to use captured CTCs for downstream analysis. Broad clinical application is demonstrated using comparable flow rates from blood specimens obtained from breast, pancreatic, and lung cancer patients. Comparable CTC numbers are recovered in all the samples at the two flow rates, demonstrating the ability of the technology to perform at high throughputs. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Flow cytometric measurement of total DNA and incorporated halodeoxyuridine

    DOEpatents

    Dolbeare, Frank A.; Gray, Joe W.

    1988-01-01

    A method for the simultaneous flow cytometric measurement of the total DNA content and the level of DNA synthesis in normal and malignant cells is disclosed. The sensitivity of the method allows a study of cell cycle traverse rates for large scale cell populations as well as single cell measurements. A DNA stain such as propidium iodide or Hoechst 33258 is used as the probe for the measurement of total DNA content and a monoclonal antibody reactive with a DNA precursor such as halodeoxy-uridine (HdU), more specifically bromodeoxyuridine (BrdU) is used as a probe for the measurement of HdU or BrdU uptake by the cells as a measure of DNA synthesis.

  5. A novel flow cytometric method to assess inflammasome formation.

    PubMed

    Sester, David P; Thygesen, Sara J; Sagulenko, Vitaliya; Vajjhala, Parimala R; Cridland, Jasmyn A; Vitak, Nazarii; Chen, Kaiwen W; Osborne, Geoffrey W; Schroder, Kate; Stacey, Katryn J

    2015-01-01

    Inflammasomes are large protein complexes induced by a wide range of microbial, stress, and environmental stimuli that function to induce cell death and inflammatory cytokine processing. Formation of an inflammasome involves dramatic relocalization of the inflammasome adapter protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) into a single speck. We have developed a flow cytometric assay for inflammasome formation, time of flight inflammasome evaluation, which detects the change in ASC distribution within the cell. The transit of ASC into the speck is detected by a decreased width or increased height of the pulse of emitted fluorescence. This assay can be used to quantify native inflammasome formation in subsets of mixed cell populations ex vivo. It can also provide a rapid and sensitive technique for investigating molecular interactions in inflammasome formation, by comparison of wild-type and mutant proteins in inflammasome reconstitution experiments.

  6. Oxidative product formation in irradiated neutrophils. A flow cytometric analysis

    SciTech Connect

    Wolber, R.A.; Duque, R.E.; Robinson, J.P.; Oberman, H.A.

    1987-03-01

    The effect of irradiation on neutrophil oxidative function was evaluated using a flow cytometric assay of intracellular hydrogen peroxide (H/sub 2/O/sub 2/) production. This assay quantitates the H/sub 2/O/sub 2/-dependent conversion of the nonfluorescent compound, 2'-7'-dichlorofluorescein (DCFH), into fluorescent 2'-7'-dichlorofluorescein (DCF) on a single-cell basis. Intracellular H/sub 2/O/sub 2/ production in response to stimulation with phorbol myristate acetate was not affected by neutrophil irradiation at doses up to 2500 rad. In addition, irradiation of intracellular DCFH and aqueous 2'-7'-dichlorofluorescein diacetate (DCFH-DA) resulted in DCF production, which suggested that oxidative molecules produced by aqueous radiolysis were detected by this assay. This study indicates that radiation doses of 1500 to 2500 rad, which are sufficient to prevent induction of graft-versus-host disease by transfused blood components, are not deleterious to neutrophil oxidative metabolism.

  7. Comparative flow cytometric analysis of immunofunctionalized nanowire and nanoparticle signatures.

    PubMed

    Prina-Mello, Adriele; Whelan, Aine M; Atzberger, Ann; McCarthy, Joseph E; Byrne, Fiona; Davies, Gemma-Louise; Coey, J M D; Volkov, Yuri; Gun'ko, Yurii K

    2010-01-01

    Flow cytometry is one of the gold-standard techniques used in clinical medicine for quantitative immunoassaying. The continuous development of its probes, commonly fluorescent nanoparticles, is important. Lately, the introduction of quantitative multiplexed immunoassay has challenged the use of nanoparticles as probes. Functionalized fluorescent silica-based magnetic nanowires are investigated under flow cytometry as a novel probe category. The preparation and full characterization of these multimodal nanowires is reported and compared to those of silica-based magnetic nanoparticles by flow cytometry. Full characterization includes transmission electron microscopy and fluorescence microscopy imaging, flow cytometric assaying, superconducting quantum interference device (SQUID) magnetization, and Mössbauer spectroscopy measurements. This work shows that loaded silica nanowires have intrinsic geometrical advantages when compared to similar spherical particles due to their unique "flow cytometry fingerprint" when utilized as magnetic carriers for immunodetection applications. These advantages account for a 17% yield in detecting the functional binding between THP-1 and ICAM-1, by utilizing a much lower concentration than that required for the nanoparticles.

  8. The cytometric future: it ain't necessarily flow!

    PubMed

    Shapiro, Howard M

    2011-01-01

    Initial approaches to cytometry for classifying and characterizing cells were based on microscopy; it was necessary to collect relatively high-resolution images of cells because only a few specific reagents usable for cell identification were available. Although flow cytometry, now the dominant cytometric technology, typically utilizes lenses similar to microscope lenses for light collection, improved, more quantitative reagents allow the necessary information to be acquired in the form of whole-cell measurements of the intensities of light transmission, scattering, and/or fluorescence.Much of the cost and complexity of both automated microscopes and flow cytometers arises from the necessity for them to measure one cell at a time. Recent developments in digital camera technology now offer an alternative in which one or more low-magnification, low-resolution images are made of a wide field containing many cells, using inexpensive light-emitting diodes (LEDs) for illumination. Minimalist widefield imaging cytometers can provide a smaller, less complex, and substantially less expensive alternative to flow cytometry, critical in systems intended for in resource-poor areas. Minimalism is, likewise, a good philosophy in developing instrumentation and methodology for both clinical and large-scale research use; it simplifies quality assurance and compliance with regulatory requirements, as well as reduces capital outlays, material costs, and personnel training requirements. Also, importantly, it yields "greener" technology.

  9. Rapid flow cytometric susceptibility testing of Candida albicans.

    PubMed Central

    Ramani, R; Ramani, A; Wong, S J

    1997-01-01

    A rapid flow cytometric assay for in vitro antifungal drug susceptibility testing was developed by adapting the proposed reference method for broth macrodilution testing of yeasts. Membrane permeability changes caused by the antifungal agent were measured by flow cytometry using propidium iodide, a nucleic acid-binding fluorochrome largely excluded by the intact cell membrane. We determined the in vitro susceptibility of 31 Candida albicans isolates and two quality control strains (Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258) to amphotericin B and fluconazole. Amphotericin B MICs ranged from 0.03 to 2.0 microg/ml, while fluconazole MICs ranged from 0.125 to 128 microg/ml. This method results in clear-cut endpoints that were reproducible. Four-hour incubation was required for fluconazole, whereas a 2-h incubation was sufficient for amphotericin B to provide MICs comparable to the reference macrodilution method developed by the National Committee for Clinical Laboratory Standards Subcommittee on Antifungal Susceptibility Tests. Results of these studies show that flow cytometry provides a rapid and sensitive in vitro method for antifungal susceptibility testing of C. albicans. PMID:9276410

  10. High-throughput nanoliter sample introduction microfluidic chip-based flow injection analysis system with gravity-driven flows.

    PubMed

    Du, Wen-Bin; Fang, Qun; He, Qiao-Hong; Fang, Zhao-Lun

    2005-03-01

    In this work, a simple, robust, and automated microfluidic chip-based FIA system with gravity-driven flows and liquid-core waveguide (LCW) spectrometric detection was developed. The high-throughput sample introduction system was composed of a capillary sampling probe and an array of horizontally positioned microsample vials with a slot fabricated on the bottom of each vial. FI sample loading and injection were performed by linearly moving the array of vials filled alternately with 50-microL samples and carrier, allowing the probe inlet to enter the solutions in the vials through the slots sequentially and the sample and carrier solution to be introduced into the chip driven by gravity. The performance of the system was demonstrated using the complexation of o-phenanthroline with Fe(II) as a model reaction. A 20-mm-long Teflon AF 2400 capillary (50-microm i.d., 375-microm o.d.) was connected to the chip to function as a LCW detection flow cell with a cell volume of 40 nL and effective path length of 1.7 cm. Linear absorbance response was obtained in the range of 1.0-100 microM Fe(II) (r2=0.9967), and a good reproducibility of 0.6% RSD (n=18) was achieved. The sensitivity was comparable with that obtained using conventional FIA systems, which typically consume 10,000-fold more sample. The highest sampling throughput of 1000 h-1 was obtained by using injection times of 0.08 and 3.4 s for sample and carrier solution, respectively, with a sample consumption of only 0.6 nL for each cycle.

  11. Flow-injection electrospray ionization mass spectrometry of crude cell extracts for high-throughput bacterial identification.

    PubMed

    Vaidyanathan, Seetharaman; Kell, Douglas B; Goodacre, Royston

    2002-02-01

    Flow-injection electrospray ionization mass spectrometry (FI-ESI-MS) of unfractionated cell-free extracts obtained from bacterial cells suspended in a solvent mixture was investigated as a rapid analytical method for reproducible, high-throughput bacterial identification. Five bacterial strains (two Escherichia coli, two Bacillus spp. and one Brevibacillus laterosporus) were studied in this investigation. Axenically grown bacterial cells were suspended in an acidic organic solvent and the cell-free extract was sequentially injected into a solvent flow stream that was sprayed into the ionization chamber of the ESI-MS. The spectra produced contained reproducible information, which was useful for discriminating between the bacteria. Tandem mass spectrometry was used to characterize further the peaks, and at least three classes of macromolecules, namely phospholipids, glycolipids, and proteins, were found to contribute most to the spectral information. Bacterial extracts stored under different conditions gave very similar mass spectra for each of the five bacterial strains, indicating that the extracts were stable even at room temperature for up to 24 h, with no loss of information content, which has obvious implications for automated high-throughput analysis. An analysis of the components of the extracting solvent mixture and their effects on the spectral information showed that acetonitrile contributes most significantly to the extraction process and hence to the information content of the spectra.

  12. Cell Surface Profiling Using High-Throughput Flow Cytometry: A Platform for Biomarker Discovery and Analysis of Cellular Heterogeneity

    PubMed Central

    Gedye, Craig A.; Hussain, Ali; Paterson, Joshua; Smrke, Alannah; Saini, Harleen; Sirskyj, Danylo; Pereira, Keira; Lobo, Nazleen; Stewart, Jocelyn; Go, Christopher; Ho, Jenny; Medrano, Mauricio; Hyatt, Elzbieta; Yuan, Julie; Lauriault, Stevan; Kondratyev, Maria; van den Beucken, Twan; Jewett, Michael; Dirks, Peter; Guidos, Cynthia J.; Danska, Jayne; Wang, Jean; Wouters, Bradly; Neel, Benjamin; Rottapel, Robert; Ailles, Laurie E.

    2014-01-01

    Cell surface proteins have a wide range of biological functions, and are often used as lineage-specific markers. Antibodies that recognize cell surface antigens are widely used as research tools, diagnostic markers, and even therapeutic agents. The ability to obtain broad cell surface protein profiles would thus be of great value in a wide range of fields. There are however currently few available methods for high-throughput analysis of large numbers of cell surface proteins. We describe here a high-throughput flow cytometry (HT-FC) platform for rapid analysis of 363 cell surface antigens. Here we demonstrate that HT-FC provides reproducible results, and use the platform to identify cell surface antigens that are influenced by common cell preparation methods. We show that multiple populations within complex samples such as primary tumors can be simultaneously analyzed by co-staining of cells with lineage-specific antibodies, allowing unprecedented depth of analysis of heterogeneous cell populations. Furthermore, standard informatics methods can be used to visualize, cluster and downsample HT-FC data to reveal novel signatures and biomarkers. We show that the cell surface profile provides sufficient molecular information to classify samples from different cancers and tissue types into biologically relevant clusters using unsupervised hierarchical clustering. Finally, we describe the identification of a candidate lineage marker and its subsequent validation. In summary, HT-FC combines the advantages of a high-throughput screen with a detection method that is sensitive, quantitative, highly reproducible, and allows in-depth analysis of heterogeneous samples. The use of commercially available antibodies means that high quality reagents are immediately available for follow-up studies. HT-FC has a wide range of applications, including biomarker discovery, molecular classification of cancers, or identification of novel lineage specific or stem cell markers. PMID:25170899

  13. Flow cytometric and morphological analyses of Pinus pinaster somatic embryogenesis.

    PubMed

    Marum, Liliana; Loureiro, João; Rodriguez, Eleazar; Santos, Conceição; Oliveira, M Margarida; Miguel, Célia

    2009-09-25

    An approach combining morphological profiling and flow cytometric analysis was used to assess genetic stability during the several steps of somatic embryogenesis in Pinus pinaster. Embryogenic cell lines of P. pinaster were established from immature zygotic embryos excised from seeds obtained from open-pollinated trees. During the maturation stage, phenotype of somatic embryos was characterized as being either normal or abnormal. Based upon the prevalent morphological traits, different types of abnormal embryos underwent further classification and quantification. Nuclear DNA content of maritime pine using the zygotic embryos was estimated to be 57.04 pg/2C, using propidium iodide flow cytometry. According to the same methodology, no significant differences (P< or =0.01) in DNA ploidy were detected among the most frequently observed abnormal phenotypes, embryogenic cell lines, zygotic and normal somatic embryos, and somatic embryogenesis-derived plantlets. Although the differences in DNA ploidy level do not exclude the occurrence of a low level of aneuploidy, the results obtained point to the absence of major changes in ploidy level during the somatic embryogenesis process of this economically important species. Therefore, our primary goal of true-to-typeness was assured at this level.

  14. Qualitative flow cytometric analysis of Malaysian myelodysplastic syndromes (MDS) patients.

    PubMed

    Mohadese Hashem, Broojerdi; Rajesh, Ramasamy; Sabariah, M Noor; Zainina, Binti Seman

    2012-02-01

    Myelodysplastic syndromes (MDS) are a group of haematological malignancies categorized by ineffective hematopoiesis that result in dysplasia. Although morphological diagnosis is a traditional and standard technique that is used for the diagnosis of MDS, the heterogeneous blood and bone marrow characteristics of MDS patients can potentially obscure the right diagnosis. Thus, we have utilized flow cytometric immunophenotyping as a supportive mechanism to obtain a more accurate and faster method for detection of abnormal markers in MDS. Flow cytometry was used for analyzing bone marrow samples from newly diagnosed MDS patients to investigate the abnormal antigen expression patterns in granulocytic, monocytic, erythroid, lymphoid lineages and myeloid precursors. The results were compared with those obtained from cases that had Idiopathic Thrombocytopenic Purpura (ITP) as a control. The most common abnormality found in the granulocytic lineage was the decrease of CD10. Low expressions of CD13 were the most frequent abnormality in the monocytic lineage. The erythroid lineage was found to have low expression of CD235A+/CD71+, reduce of CD71 and decreased CD235a. In conclusion, this method is useful for confirming cases in which it is difficult to make a diagnosis by morphology.

  15. Flow cytometric detection of anti-gliadin antibodies.

    PubMed

    Presani, G; Perticarari, S; Mangiarotti, M A

    1989-05-12

    A very sensitive solid-phase fluorescent immunoassay to detect anti-alpha-gliadin IgA class antibodies is described. The solid phase consisted of polystyrene carboxylated microspheres, of 5 microns diameter, coated with alpha-gliadin. Serum-specific antibodies bound to the alpha-gliadin were measured by flow cytometry using fluorescein-conjugated anti-human IgA. 41 samples were tested and the results compared with those obtained by a standard method: an enzyme-linked immunosorbent assay (ELISA). A good correlation was found between the two techniques (r = 0.96). The sera of untreated coeliac children showed significantly higher antibody values than the sera of children on a gluten-free diet or healthy control groups. The flow cytometric method was more sensitive when the Kolgomorov/Smirnov test was used to analyse the histograms. This method provides an alternative screening test for coeliac disease and may also be used to confirm borderline results obtained in the ELISA test.

  16. Subtle Mitsunobu couplings under super-heating: the role of high-throughput continuous flow and microwave strategies.

    PubMed

    Manvar, Atul; Shah, Anamik

    2014-11-07

    Non-conventional heating techniques, high-throughput microwave-assisted synthesis and continuous flow penetrate almost every scientific field. Mitsunobu coupling is a ubiquitous choice for the dehydrative redox condensation of primary or secondary alcohols with (pro)nucleophiles. The aim of this review is to showcase the ease of subtle Mitsunobu coupling under super-heating. Surprisingly, this strategy is rather non-trivial; considering the sensitivity of reagents, Mitsunobu chemistry is typically performed at lower temperatures or under ambient conditions. In view of the absence of any previous work focusing on this topic, the current review considers the utility of super-heating in fragile Mitsunobu reactions. Therefore, we anticipate that this review will also bridge some of the apparent gaps in the extant literature by specifically describing the advances made by non-conventional heating assisted by microwave or continuous flow in one of the most powerful stereochemical transformations.

  17. High-Throughput Isolation of Giant Viruses in Liquid Medium Using Automated Flow Cytometry and Fluorescence Staining.

    PubMed

    Khalil, Jacques Y B; Robert, Stephane; Reteno, Dorine G; Andreani, Julien; Raoult, Didier; La Scola, Bernard

    2016-01-01

    The isolation of giant viruses using amoeba co-culture is tedious and fastidious. Recently, the procedure was successfully associated with a method that detects amoebal lysis on agar plates. However, the procedure remains time-consuming and is limited to protozoa growing on agar. We present here advances for the isolation of giant viruses. A high-throughput automated method based on flow cytometry and fluorescent staining was used to detect the presence of giant viruses in liquid medium. Development was carried out with the Acanthamoeba polyphaga strain widely used in past and current co-culture experiments. The proof of concept was validated with virus suspensions: artificially contaminated samples but also environmental samples from which viruses were previously isolated. After validating the technique, and fortuitously isolating a new Mimivirus, we automated the technique on 96-well plates and tested it on clinical and environmental samples using other protozoa. This allowed us to detect more than 10 strains of previously known species of giant viruses and seven new strains of a new virus lineage. This automated high-throughput method demonstrated significant time saving, and higher sensitivity than older techniques. It thus creates the means to isolate giant viruses at high speed.

  18. High-Throughput Isolation of Giant Viruses in Liquid Medium Using Automated Flow Cytometry and Fluorescence Staining

    PubMed Central

    Khalil, Jacques Y. B.; Robert, Stephane; Reteno, Dorine G.; Andreani, Julien; Raoult, Didier; La Scola, Bernard

    2016-01-01

    The isolation of giant viruses using amoeba co-culture is tedious and fastidious. Recently, the procedure was successfully associated with a method that detects amoebal lysis on agar plates. However, the procedure remains time-consuming and is limited to protozoa growing on agar. We present here advances for the isolation of giant viruses. A high-throughput automated method based on flow cytometry and fluorescent staining was used to detect the presence of giant viruses in liquid medium. Development was carried out with the Acanthamoeba polyphaga strain widely used in past and current co-culture experiments. The proof of concept was validated with virus suspensions: artificially contaminated samples but also environmental samples from which viruses were previously isolated. After validating the technique, and fortuitously isolating a new Mimivirus, we automated the technique on 96-well plates and tested it on clinical and environmental samples using other protozoa. This allowed us to detect more than 10 strains of previously known species of giant viruses and seven new strains of a new virus lineage. This automated high-throughput method demonstrated significant time saving, and higher sensitivity than older techniques. It thus creates the means to isolate giant viruses at high speed. PMID:26858703

  19. Evaluation of polymeric gene delivery nanoparticles by nanoparticle tracking analysis and high-throughput flow cytometry.

    PubMed

    Shmueli, Ron B; Bhise, Nupura S; Green, Jordan J

    2013-03-01

    Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases(1) and as a technology for regenerative medicine(2). Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery(1,3) . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers(4) that are hydrolytically degradable(5,6) and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)(7), mouse mammary epithelial cells(8), human brain cancer cells(9) and macrovascular (human umbilical vein, HUVECs) endothelial cells(10). A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained(11). In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi

  20. Novel statistical approach for evaluating flow cytometric in vitro micronucleus data.

    PubMed

    Wojciechowski, J P; Gleason, C R; Roberts, D J; Custer, L L

    2016-10-01

    Statistical methods currently recommended for analysis of in vitro micronucleus data are based on small sample sizes. The tests are designed to evaluate linear trends and differences between treated and control samples. When using flow cytometric analysis, >5 times the number of cells are easily evaluated, and the variance estimates from these large samples are small. Application of these recommended tests to large samples resulted in statistically significant outcomes which were not considered to be biologically meaningful. Alternative statistical methods for testing trends and differences among treatments that were either widely used, or sample-size independent, were investigated. Using data from 95 experiments (from 2011-2013) where 19% of the experiments were considered positive, results for the various statistical methods were compared. When using either the recommended or alternate methods, 42-68% of the experiments resulted in statistically significant results (p < 0.05). A new concept was then tested using the same data sets: the "z' factor", designed to identify 'hits' during high throughput screening. Using this simple-to-compute statistic the number of significant calls was reduced to 27%. Then, when combined with a biological criterion based on historical vehicle control data, there was restoration of the original positive frequency (19%). Given the larger sample sizes evaluated using flow cytometry, we have demonstrated that traditional statistical tests may be overly sensitive to small changes in micronucleus induction, and that a simple-to-compute index of separation (z') may be a better tool for analysis, provided that the response is first determined to be biologically meaningful. Environ. Mol. Mutagen. 57:589-604, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  1. Detection of circulating immune complexes by Raji cell assay: comparison of flow cytometric and radiometric methods

    SciTech Connect

    Kingsmore, S.F.; Crockard, A.D.; Fay, A.C.; McNeill, T.A.; Roberts, S.D.; Thompson, J.M.

    1988-01-01

    Several flow cytometric methods for the measurement of circulating immune complexes (CIC) have recently become available. We report a Raji cell flow cytometric assay (FCMA) that uses aggregated human globulin (AHG) as primary calibrator. Technical advantages of the Raji cell flow cytometric assay are discussed, and its clinical usefulness is evaluated in a method comparison study with the widely used Raji cell immunoradiometric assay. FCMA is more precise and has greater analytic sensitivity for AHG. Diagnostic sensitivity by the flow cytometric method is superior in systemic lupus erythematosus (SLE), rheumatoid arthritis, and vasculitis patients: however, diagnostic specificity is similar for both assays, but the reference interval of FCMA is narrower. Significant correlations were found between CIC levels obtained with both methods in SLE, rheumatoid arthritis, and vasculitis patients and in longitudinal studies of two patients with cerebral SLE. The Raji cell FCMA is recommended for measurement of CIC levels to clinical laboratories with access to a flow cytometer.

  2. Flow cytometric analysis of the cell cycle in chronic gastritis.

    PubMed

    Guerci, A; Chambre, J F; Franck, P; Floquet, J; Gaucher, P; Guerci, O

    1992-09-01

    Flow cytometric cell cycle analysis was recorded in gastric biopsy specimens from patients with normal gastric mucosa (GM), superficial gastritis (SG) and chronic atrophic gastritis (CAG). Cell-cycle analysis showed significantly higher percentages of cells in S- and S+G2/M-phase in CAG than in SG and normal GM (P < 0.0001). Moreover, CAG with severe or moderate atrophy showed significantly higher percentages of cells in S-phase (P < 0.05) and S+G2/M-phase (P < 0.02) than CAG with mild atrophy in antrum. In fundus, even if this increase was observed, it did not reach statistical significance. Consideration of concomitant pathologic findings such as oesophagite, gastric or duodenal ulcer, duodenite or benign polyp allowed a better differentiation of CAG both in antrum and in fundus. Significantly higher S-phase was observed in CAG with severe or moderate atrophy than in CAG with mild atrophy (P < 0.05). No statistically significant results were observed in patients with normal gastric mucosa or chronic gastritis and a concomitant pathologic finding.

  3. Flow cytometric immunophenotypic analysis of 306 cases of multiple myeloma.

    PubMed

    Lin, Pei; Owens, Rebecca; Tricot, Guido; Wilson, Carla S

    2004-04-01

    Bone marrow aspirates from 306 patients with multiple myeloma were analyzed by flow cytometric immunophenotyping. The plasma cells (PCs) were identified by their characteristic light scatter distribution and reactivity patterns to CD138, CD38, and CD45. Monoclonality was confirmed by immunoglobulin light chain analysis. The immunophenotypic profile of the PCs was determined with a panel of antibodies. Moderate to bright expression of CD56, CD117, CD20, CD45, and CD52 was detected in 71.7%, 17.8%, 9.3%, 8.8%, and 5.2% of cases, respectively. These antigens were expressed by a distinct subpopulation of the PCs in 6.3%, 2.2%, 3.7%, 2.9%, and 2.6% of additional cases. CD19 was negative in more than 99% of cases. The combination of CD38 and CD138 was superior to CD38 alone for identifying CD45+ myeloma and separating CD20+ myeloma from B-cell lymphoma. PC immunophenotyping might be useful for detecting minimal residual disease in cases with aberrant antigen expression and for selection of therapeutic agents that have specific membrane targets.

  4. Flow cytometric life cycle analysis in cellular radiation biology

    SciTech Connect

    Wood, J.C.S.

    1982-01-01

    Three approaches to flow cytometric histogram analysis were developed: (1) differential histogram analysis, (2) DNA histogram analysis, and (3) multiparameter data analysis. These techniques were applied to an important unresolved problem in radiation biology. The initial responses to irradiation of a mammalian cell which occur during the first two cell cycles following the irradiation are of considerable interest to the radiation biologist. During the first two post-irradiation cell cycles, cells which ultimately will survive repair radiation-induced damage, while some cells begin to express some of the radiation-induced nuclear and chomatin damage. Caffeine- and thymidine-treated, and untreated gamma-irradiated cell populations were studied with respect to the radiation-induced G2 delay, deficient DNA synthesis, and the appearance of cells with abnormal DNA contents. It is hypothesized that the measured deficiency in DNA synthesis observed in the first post-irradiation cell cycle may be a result of daughter cells from abnormal first post-irradiation mitoses.

  5. Flow cytometric determination of FcgammaRIIa (CD32) polymorphism.

    PubMed

    van Royen-Kerkhof, Annet; Sanders, Elisabeth A M; Wijngaarden, Siska; van Roon, Joel A G; Voorhorst-Ogink, Marleen; Walraven, Vanessa; Gerritsen, Arnout; van Dijk, Marc A; Kuis, W; Rijkers, Ger T; Keler, Tibor; Leusen, Jeanette H W; van de Winkel, Jan G J

    2004-11-01

    A guanine to adenine point mutation results in an arginine (R) to histidine (H) substitution in FcgammaRIIa at residue 131 that strongly impacts receptor function. This FcgammaRIIa polymorphism is mostly typed by allele-specific polymerase chain reactions (PCR) or in functional assays, dependent on ligand binding. Both types of methods are laborious, time consuming, and not readily available in routine laboratories. We generated a panel of human antibodies against FcgammaRII, and one of them, MDE-9, selectively recognized the FcgammaRIIa-H131 allotype. MDE-9 was applicable to detect FcgammaRIIa-H131 in both flow cytometry and immunohistochemistry. MDE-9 was used to develop an FcgammaRIIa allotyping method based on flow cytometry. In a "single-tube assay", FITC-labeled MDE-9 (specific for FcgammaRIIa-H131) and Cy3-labeled mAb 41H16 (specific for FcgammaRIIa-R131) were added to 50 mul samples of whole blood. The results of flow cytometric FcgammaRIIa allotyping correlated completely with PCR genotyping. This novel allotyping assay should facilitate the screening of patients in a routine diagnostic setting. In addition, a combination of MDE-9 and 41H16 can be used in FcgammaRIIa-H/H131 homozygous individuals to detect FcgammaRIIa and FcgammaRIIb surface expression on monocytes. This is an important application of these antibodies because, to this day, no antibodies were available to specifically study the surface expression of FcgammaRIIb.

  6. High-Throughput Screen for the Chemical Inhibitors of Antiapoptotic Bcl-2 Family Proteins by Multiplex Flow Cytometry

    PubMed Central

    Curpan, Ramona F.; Simons, Peter C.; Zhai, Dayong; Young, Susan M.; Carter, Mark B.; Bologa, Cristian G.; Oprea, Tudor I.; Satterthwait, Arnold C.; Reed, John C.; Edwards, Bruce S.

    2011-01-01

    Abstract The human Bcl-2 family includes six antiapoptotic members (Bcl-2, Bcl-B, Bcl-W, Bcl-XL, Bfl-1, and Mcl-1) and many proapoptotic members, wherein a balance between the two determines cell life or death in many physiological and disease contexts. Elevated expression of various antiapoptotic Bcl-2 members is commonly observed in cancers, and chemical inhibitors of these proteins have been shown to promote apoptosis of malignant cells in culture, in animal models, and in human clinical trials. All six antiapoptotic members bind a helix from the proapoptotic family member Bim, thus quenching Bim's apoptotic signal. Here, we describe the use of a multiplex, high-throughput flow cytometry assay for the discovery of small molecule modulators that disrupt the interaction between the antiapoptotic members of the Bcl-2 family and Bim. The six antiapoptotic Bcl-2 family members were expressed as glutathione-S-transferase fusion proteins and bound individually to six glutathione bead sets, with each set having a different intensity of red fluorescence. A fluorescein-conjugated Bcl-2 homology region 3 (BH3) peptide from Bim was employed as a universal ligand. Flow cytometry measured the amount of green peptide bound to each bead set in a given well, with inhibitory compounds resulting in a decrease of green fluorescence on one or more bead set(s). Hits and cheminformatically selected analogs were retested in a dose–response series, resulting in three “active” compounds for Bcl-B. These three compounds were validated by fluorescence polarization and isothermal titration calorimetry. We discuss some of the lessons learned about screening a chemical library provided by the National Institutes of Health Small Molecule Repository (∼195,000 compounds) using high-throughput flow cytometry. PMID:21561376

  7. High-throughput screen for the chemical inhibitors of antiapoptotic bcl-2 family proteins by multiplex flow cytometry.

    PubMed

    Curpan, Ramona F; Simons, Peter C; Zhai, Dayong; Young, Susan M; Carter, Mark B; Bologa, Cristian G; Oprea, Tudor I; Satterthwait, Arnold C; Reed, John C; Edwards, Bruce S; Sklar, Larry A

    2011-10-01

    The human Bcl-2 family includes six antiapoptotic members (Bcl-2, Bcl-B, Bcl-W, Bcl-X(L), Bfl-1, and Mcl-1) and many proapoptotic members, wherein a balance between the two determines cell life or death in many physiological and disease contexts. Elevated expression of various antiapoptotic Bcl-2 members is commonly observed in cancers, and chemical inhibitors of these proteins have been shown to promote apoptosis of malignant cells in culture, in animal models, and in human clinical trials. All six antiapoptotic members bind a helix from the proapoptotic family member Bim, thus quenching Bim's apoptotic signal. Here, we describe the use of a multiplex, high-throughput flow cytometry assay for the discovery of small molecule modulators that disrupt the interaction between the antiapoptotic members of the Bcl-2 family and Bim. The six antiapoptotic Bcl-2 family members were expressed as glutathione-S-transferase fusion proteins and bound individually to six glutathione bead sets, with each set having a different intensity of red fluorescence. A fluorescein-conjugated Bcl-2 homology region 3 (BH3) peptide from Bim was employed as a universal ligand. Flow cytometry measured the amount of green peptide bound to each bead set in a given well, with inhibitory compounds resulting in a decrease of green fluorescence on one or more bead set(s). Hits and cheminformatically selected analogs were retested in a dose-response series, resulting in three "active" compounds for Bcl-B. These three compounds were validated by fluorescence polarization and isothermal titration calorimetry. We discuss some of the lessons learned about screening a chemical library provided by the National Institutes of Health Small Molecule Repository (∼195,000 compounds) using high-throughput flow cytometry.

  8. Design of a high-throughput flow perfusion bioreactor system for tissue engineering.

    PubMed

    Dahlin, Rebecca L; Meretoja, Ville V; Ni, Mengwei; Kasper, F Kurtis; Mikos, Antonios G

    2012-10-01

    Flow perfusion culture is used in many areas of tissue engineering and offers several key advantages. However, one challenge to these cultures is the relatively low-throughput nature of perfusion bioreactors. Here, a flow perfusion bioreactor with increased throughput was designed and built for tissue engineering. This design uses an integrated medium reservoir and flow chamber in order to increase the throughput, limit the volume of medium required to operate the system, and simplify the assembly and operation.

  9. Flow cytometric analysis of crayfish haemocytes activated by lipopolysaccharides

    USGS Publications Warehouse

    Cardenas, W.; Dankert, J.R.; Jenkins, J.A.

    2004-01-01

    Lipopolysaccharides (LPS) from Gram-negative bacteria are strong stimulators of white river crayfish, Procambarus zonangulus, haemocytes in vitro. Following haemocyte treatment with LPS and with LPS from rough mutant R5 (LPS Rc) from Salmonella minnesota, flow cytometric analysis revealed a conspicuous and reproducible decrease in cell size as compared to control haemocytes. These LPS molecules also caused a reduction in haemocyte viability as assessed by flow cytometry with the fluorescent dyes calcein-AM and ethidium homodimer. The onset of cell size reduction was gradual and occurred prior to cell death. Haemocytes treated with LPS from S. minnesota without the Lipid A moiety (detoxified LPS) decreased in size without a reduction of viability. The action of LPS on crayfish haemocytes appeared to be related to the activation of the prophenoloxidase system because phenoloxidase (PO)-specific activity in the supernatants from control and detoxified LPS-treated cells was significantly lower than that from LPS and LPS-Rc treated cells (P < 0.05). Furthermore, addition of trypsin inhibitor to the LPS treatments caused noticeable delays in cell size and viability changes. These patterns of cellular activation by LPS formulations indicated that crayfish haemocytes react differently to the polysaccharide and lipid A moieties of LPS, where lipid A is cytotoxic and the polysaccharide portion is stimulatory. These effects concur with the general pattern of mammalian cell activation by LPS, thereby indicting commone innate immune recognition mechanisms to bacterial antigens between cells from mammals and invertebrates. These definitive molecular approaches used to verify and identify mechanisms of invertbrate haemocyte responses to LPS could be applied with other glycoconjugates, soluble mediators, or xenobiotic compounds.

  10. Use of flow cytometry for high-throughput cell population estimates in brain tissue

    PubMed Central

    Young, Nicole A.; Flaherty, David K.; Airey, David C.; Varlan, Peter; Aworunse, Feyi; Kaas, Jon H.; Collins, Christine E.

    2012-01-01

    The large size of primate brains is an impediment to obtaining high-resolution cell number maps of the cortex in humans and non-human primates. We present a rapid, flow cytometry-based cell counting method that can be used to estimate cell numbers from homogenized brain tissue samples comprising the entire cortical sheet. The new method, called the flow fractionator, is based on the isotropic fractionator (IF) method (Herculano-Houzel and Lent, 2005), but substitutes flow cytometry analysis for manual, microscope analysis using a Neubauer counting chamber. We show that our flow cytometry-based method for total cell estimation in homogenized brain tissue provides comparable data to that obtained using a counting chamber on a microscope. The advantages of the flow fractionator over existing methods are improved precision of cell number estimates and improved speed of analysis. PMID:22798947

  11. Use of flow cytometry for high-throughput cell population estimates in brain tissue.

    PubMed

    Young, Nicole A; Flaherty, David K; Airey, David C; Varlan, Peter; Aworunse, Feyi; Kaas, Jon H; Collins, Christine E

    2012-01-01

    The large size of primate brains is an impediment to obtaining high-resolution cell number maps of the cortex in humans and non-human primates. We present a rapid, flow cytometry-based cell counting method that can be used to estimate cell numbers from homogenized brain tissue samples comprising the entire cortical sheet. The new method, called the flow fractionator, is based on the isotropic fractionator (IF) method (Herculano-Houzel and Lent, 2005), but substitutes flow cytometry analysis for manual, microscope analysis using a Neubauer counting chamber. We show that our flow cytometry-based method for total cell estimation in homogenized brain tissue provides comparable data to that obtained using a counting chamber on a microscope. The advantages of the flow fractionator over existing methods are improved precision of cell number estimates and improved speed of analysis.

  12. Segmented continuous-flow multiplex polymerase chain reaction microfluidics for high-throughput and rapid foodborne pathogen detection.

    PubMed

    Shu, Bowen; Zhang, Chunsun; Xing, Da

    2014-05-15

    High-throughput and rapid identification of multiple foodborne bacterial pathogens is vital in global public health and food industry. To fulfill this need, we propose a segmented continuous-flow multiplex polymerase chain reaction (SCF-MPCR) on a spiral-channel microfluidic device. The device consists of a disposable polytetrafluoroethylene (PTFE) capillary microchannel coiled on three isothermal blocks. Within the channel, n segmented flow regimes are sequentially generated, and m-plex PCR is individually performed in each regime when each mixture is driven to pass three temperature zones, thus providing a rapid analysis throughput of m×n. To characterize the performance of the microfluidic device, continuous-flow multiplex PCR in a single segmented flow has been evaluated by investigating the effect of key reaction parameters, including annealing temperatures, flow rates, polymerase concentration and amount of input DNA. With the optimized parameters, the genomic DNAs from Salmonella enterica, Listeria monocytogenes, Escherichia coli O157:H7 and Staphylococcus aureus could be amplified simultaneously in 19min, and the limit of detection was low, down to 10(2) copiesμL(-1). As proof of principle, the spiral-channel SCF-MPCR was applied to sequentially amplify four different bacterial pathogens from banana, milk, and sausage, displaying a throughput of 4×3 with no detectable cross-contamination.

  13. High-throughput chemical residue analysis by fast extraction and dilution flow injection mass spectrometry.

    PubMed

    Nanita, Sergio C

    2011-01-21

    Fast extraction and dilution flow injection mass spectrometry (FED-FI-MS) is presented as a technique to increase throughput in quantitative multiresidue screening in complex matrices, while meeting current analytical method quality requirements.

  14. Clinical behavior of acoustic tumors. A flow cytometric analysis.

    PubMed

    Kesterson, L; Shelton, C; Dressler, L; Berliner, K I

    1993-03-01

    Recently, nonsurgical treatment of acoustic tumors has been advocated as an alternative to surgical resection. Because of the relatively short follow-up in reported series of radiation-treated acoustic tumors, the lack of growth of some tumors may merely reflect the variable biologic growth potential of these tumors and not the result of treatment. DNA flow cytometry has been used to predict biologic activity in other solid tumors. It is applied in this study to assess the variability of growth potential in a typical acoustic tumor population and to determine whether relationships exist between flow cytometric data and clinical characteristics of acoustic tumors. DNA flow cytometry techniques were used to evaluate formalin-fixed, paraffin-embedded tissue previously obtained from patients who were surgically treated for acoustic neuromas. Relationships between flow cytometry data and historical data were also statistically evaluated. Tissue samples were from patients of a large private otologic practice. Subjects were a convenience sample of 49 patients (26 female and 23 male) with a mean age of 59 years who had undergone surgical removal of an acoustic neuroma. None of the patients had other stigmata of neurofibromatosis or tumor recurrence. All tissue specimens were pathologically confirmed acoustic neurons, with a range in tumor size from 1 to 6 cm. The measures included DNA ploidy and S-phase fraction. Historical data included age, sex, size of tumor, presenting symptom, and symptom duration. All 49 tumors showed a diploid distribution, with S-phase values ranging from 1.07% to 20.74% (mean +/- SD, 6.30 +/- 4.24). The ploidy and S-phase data compare favorably with previously published data in which fresh tissue was used. There were no statistically significant relationships between S-phase value and historical data. The wide range of S-phase values is consistent with a large variation in tumor growth potential and suggests caution in interpreting the results of

  15. Automated high-throughput flow-through real-time diagnostic system

    DOEpatents

    Regan, John Frederick

    2012-10-30

    An automated real-time flow-through system capable of processing multiple samples in an asynchronous, simultaneous, and parallel fashion for nucleic acid extraction and purification, followed by assay assembly, genetic amplification, multiplex detection, analysis, and decontamination. The system is able to hold and access an unlimited number of fluorescent reagents that may be used to screen samples for the presence of specific sequences. The apparatus works by associating extracted and purified sample with a series of reagent plugs that have been formed in a flow channel and delivered to a flow-through real-time amplification detector that has a multiplicity of optical windows, to which the sample-reagent plugs are placed in an operative position. The diagnostic apparatus includes sample multi-position valves, a master sample multi-position valve, a master reagent multi-position valve, reagent multi-position valves, and an optical amplification/detection system.

  16. High-throughput pesticide residue quantitative analysis achieved by tandem mass spectrometry with automated flow injection.

    PubMed

    Nanita, Sergio C; Pentz, Anne M; Bramble, Frederick Q

    2009-04-15

    The use of automated flow injection with MS/MS detection for fast quantitation of agrochemicals in food and water samples was demonstrated in this study. Active ingredients from the sulfonylurea herbicide and carbamate insecticide classes were selected as model systems. Samples were prepared using typical procedures from residue methods, placed in an autosampler, and injected directly into a triple quadrupole instrument without chromatographic separation. The technique allows data acquisition in 15 s per injection, with samples being injected every 65 s, representing a significant improvement from the 15-30 min needed in typical HPLC/MS/MS methods. The availability of HPLC systems is an advantage since they can be used in flow-injection mode (bypassing the column compartment). Adequate accuracy, linearity, and precision (R(2) > 0.99 and RSD < 20%) were obtained using external standards prepared in each control matrix. The limit of quantitation (LOQ) achieved for all analytes was 0.01 mg/kg in food samples and 0.1 ng/mL in water; while limits of detection (LOD) were estimated to be about 0.003 mg/kg and 0.03 ng/mL in food and water, respectively. The advantages and limitations of flow injection MS/MS for ultratrace-level quantitative analysis in complex matrixes are discussed.

  17. Method for flow cytometric monitoring of Renibacterium salmoninarum inactivation

    USGS Publications Warehouse

    Pascho, R.J.; Ongerth, J.E.

    2000-01-01

    The slow growth of Renibacterium salmoninarum limits the usefulness of culture as a research tool. Development of a 2-color flow cytometric assay to quantify the proportions of live and dead R. salmoninarum in a test population is described. Bacteria were simultaneously stained with fluorescein isothiocyanate-conjugated immunoglobulin and exposed to the exclusion dye propidium iodide. Propidium iodide red fluorescence profiles of control groups of untreated and killed R. salmoninarum were compared with those for bacteria exposed to chlorine. Bacterial inactivation was based on mean red fluorescence intensity, and analyzed by high-red fluorescence intensity (HRFI) and curve subtraction (CS) analyses. When the concentration of R. salmoninarum was 8.65 x 106 bacteria ml-1 and the bacteria exposed to chlorine at 1 mg l-1 for periods from 1 to 20 min (high-Rs assessment), the mean red fluorescence intensity of the profile for each chlorine-exposure group was higher than that for the untreated control (p < 0.0001). When the concentration of R. salmoninarum was reduced to 1.76 x 106 bacteria ml-1 and exposed to 0.8 mg l-1 free chlorine level for periods from 20 s to 5 min (reduced-Rs assessment), the mean red fluorescence intensities of the exposure groups were higher than that for the untreated control only when the R. salmoninarum was exposed to chlorine for at least 1 min (p ??? 0.01). On the basis of red fluorescence intensity, the proportion of dead cells generally increased with the duration of chlorine exposure. Whereas the rates of inactivation derived from the HRFI and CS analyses did not correlate with the duration of exposure in the high-Rs assessment (r2 ??? 0.27), there was a correlation between these estimates and the duration of exposure in the reduced-Rs assessment (r2 ??? 0.92). Because of the rapid loss of culturable R. salmoninarum in both assessments following chlorine exposure, neither the duration of exposure nor the inactivation estimates correlated

  18. High-throughput quantitation of intracellular trafficking and organelle disruption by flow cytometry.

    PubMed

    Chia, Pei Zhi Cheryl; Ramdzan, Yasmin M; Houghton, Fiona J; Hatters, Danny M; Gleeson, Paul A

    2014-05-01

    Current methods for the quantitation of membrane protein trafficking rely heavily on microscopy, which has limited quantitative capacity for analyses of cell populations and is cumbersome to perform. Here we describe a simple flow cytometry-based method that circumvents these limitations. The method utilizes fluorescent pulse-width measurements as a highly sensitive indicator to monitor the changes in intracellular distributions of a fluorescently labelled molecule in a cell. Pulse-width analysis enabled us to discriminate cells with target proteins in different intracellular locations including Golgi, lyso-endosomal network and the plasma membrane, as well as detecting morphological changes in organelles such as Golgi perturbation. The movement of endogenous and exogenous retrograde cargo was tracked from the plasma membrane-to-endosomes-to-Golgi, by decreasing pulse-width values. A block in transport upon RNAi-mediated ablation of transport machinery was readily quantified, demonstrating the versatility of this technique to identify pathway inhibitors. We also showed that pulse-width can be exploited to sort and recover cells based on different intracellular staining patterns, e.g. early endosomes and Golgi, opening up novel downstream applications. Overall, the method provides new capabilities for viewing membrane transport in thousands of cells per minute, unbiased analysis of the trafficking of cargo, and the potential for rapid screening of inhibitors of trafficking pathways. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  19. A flow-free droplet-based device for high throughput polymorphic crystallization.

    PubMed

    Yang, Shih-Mo; Zhang, Dapeng; Chen, Wang; Chen, Shih-Chi

    2015-06-21

    Crystallization is one of the most crucial steps in the process of pharmaceutical formulation. In recent years, emulsion-based platforms have been developed and broadly adopted to generate high quality products. However, these conventional approaches such as stirring are still limited in several aspects, e.g., unstable crystallization conditions and broad size distribution; besides, only simple crystal forms can be produced. In this paper, we present a new flow-free droplet-based formation process for producing highly controlled crystallization with two examples: (1) NaCl crystallization reveals the ability to package saturated solution into nanoliter droplets, and (2) glycine crystallization demonstrates the ability to produce polymorphic crystallization forms by controlling the droplet size and temperature. In our process, the saturated solution automatically fills the microwell array powered by degassed bulk PDMS. A critical oil covering step is then introduced to isolate the saturated solution and control the water dissolution rate. Utilizing surface tension, the solution is uniformly packaged in the form of thousands of isolating droplets at the bottom of each microwell of 50-300 μm diameter. After water dissolution, individual crystal structures are automatically formed inside the microwell array. This approach facilitates the study of different glycine growth processes: α-form generated inside the droplets and γ-form generated at the edge of the droplets. With precise temperature control over nanoliter-sized droplets, the growth of ellipsoidal crystalline agglomerates of glycine was achieved for the first time. Optical and SEM images illustrate that the ellipsoidal agglomerates consist of 2-5 μm glycine clusters with inner spiral structures of ~35 μm screw pitch. Lastly, the size distribution of spherical crystalline agglomerates (SAs) produced from microwells of different sizes was measured to have a coefficient variation (CV) of less than 5%, showing

  20. Webcam-based flow cytometer using wide-field imaging for low cell number detection at high throughput.

    PubMed

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

    2014-09-07

    Here we describe a novel low-cost flow cytometer based on a webcam capable of low cell number detection in a large volume which may overcome the limitations of current flow cytometry. Several key elements have been combined to yield both high throughput and high sensitivity. The first element is a commercially available webcam capable of 187 frames per second video capture at a resolution of 320 × 240 pixels. The second element in this design is a 1 W 450 nm laser module for area-excitation, which combined with the webcam allows for rapid interrogation of a flow field. The final element is a 2D flow-cell which overcomes the flow limitation of hydrodynamic focusing and allows for higher sample throughput in a wider flow field. This cell allows for the linear velocity of target cells to be lower than in a conventional "1D" hydrodynamic focusing flow-cells typically used in cytometry at similar volumetric flow rates. It also allows cells to be imaged at the full frame rate of the webcam. Using this webcam-based flow cytometer with wide-field imaging, it was confirmed that the detection of fluorescently tagged 5 μm polystyrene beads in "1D" hydrodynamic focusing flow-cells was not practical for low cell number detection due to streaking from the motion of the beads, which did not occur with the 2D flow-cell design. The sensitivity and throughput of this webcam-based flow cytometer was then investigated using THP-1 human monocytes stained with SYTO-9 florescent dye in the 2D flow-cell. The flow cytometer was found to be capable of detecting fluorescently tagged cells at concentrations as low as 1 cell per mL at flow rates of 500 μL min(-1) in buffer and in blood. The effectiveness of detection was concentration dependent: at 100 cells per mL 84% of the cells were detected compared to microscopy, 10 cells per mL 79% detected and 1 cell per mL 59% of the cells were detected. With the blood samples spiked to 100 cells per mL, the average concentration for all samples

  1. Multichannel oscillatory-flow multiplex PCR microfluidics for high-throughput and fast detection of foodborne bacterial pathogens.

    PubMed

    Zhang, Chunsun; Wang, Haiying; Xing, Da

    2011-10-01

    In the field of continuous-flow PCR, the amplification throughput in a single reaction solution is low and the single-plex PCR is often used. In this work, we reported a flow-based multiplex PCR microfluidic system capable of performing high-throughput and fast DNA amplification for detection of foodborne bacterial pathogens. As a demonstration, the mixture of DNA targets associated with three different foodborne pathogens was included in a single PCR solution. Then, the solution flowed through microchannels incorporated onto three temperature zones in an oscillatory manner. The effect factors of this oscillatory-flow multiplex PCR thermocycling have been demonstrated, including effects of polymerase concentration, cycling times, number of cycles, and DNA template concentration. The experimental results have shown that the oscillatory-flow multiplex PCR, with a volume of only 5 μl, could be completed in about 13 min after 35 cycles (25 cycles) at 100 μl/min (70 μl/min), which is about one-sixth of the time required on the conventional machine (70 min). By using the presently designed DNA sample model, the minimum target concentration that could be detected at 30 μl/min was 9.8 × 10(-2) ng/μl (278-bp, S. enterica), 11.2 × 10(-2) ng/μl (168-bp, E. coli O157: H7), and 2.88 × 10(-2) ng/μl (106-bp, L. monocytogenes), which corresponds to approximately 3.72 × 10(4) copies/μl, 3.58 × 10(4) copies/μl, and 1.79 × 10(4) copies/μl, respectively. This level of speed and sensitivity is comparable to that achievable in most other continuous-flow PCR systems. In addition, the four individual channels were used to achieve multi-target PCR analysis of three different DNA samples from different food sources in parallel, thereby achieving another level of multiplexing.

  2. Bivariate flow cytometric analysis of murine intestinal epithelial cells for cytokinetic studies

    SciTech Connect

    Pallavicini, M.G.; Ng, C.R.; Gray, J.W.

    1984-01-01

    The heterogeneous nature of the small intestine and the lack of methods to obtain pure crypt populations has, in the past, limited the application of standard flow cytometric analysis for cytokinetic studies of the proliferating crypts. The authors describe a flow cytometric technique to discriminate crypt and villus cells in an epithelial cell suspension on the basis of cell length, and to measure the DNA content of the discriminated subpopulations. These data indicate that bivariate analysis of a mixed epithelial cell suspension can be used to distinguish mature villus cells, G/sub 1/ crypt cells, and S-phase crypt cells. In addition, continuous labeling studies suggest that the position of a cell on the cell length axis reflects epithelial cell maturity. The authors applied this flow cytometric technique to determine the cytokinetic nature of epithelial cells obtained by sequential digestion of the small intestine. 22 references, 4 figures, 2 tables.

  3. Multicenter evaluation of two flow cytometric methods for counting low levels of white blood cells.

    PubMed

    Dijkstra-Tiekstra, Margriet J; van der Meer, Pieter F; Pietersz, Ruby N I; de Wildt-Eggen, Janny

    2004-09-01

    Flow cytometric methods can be used to count residual white blood cells (WBCs) in WBC-reduced blood products, which should contain fewer than 1 x 10(6) WBCs per unit (approximately 3.3 WBCs/ microL). In this study two flow cytometric methods for counting WBCs under routine conditions in nine laboratories were evaluated. Panels of red blood cells (RBCs), platelets (PLTs), and plasma were prepared containing 33.3, 10.0, 3.3, 1.0, and 0.3 WBCs per microL and counted with flow cytometric methods (either LeucoCOUNT, BD Biosciences, four laboratories; or LeukoSure, Beckman Coulter, five laboratories). Requirements were that at the level of 3.3 WBCs per microL, coefficient of variation was < or =20 percent and accuracy was > or =80 percent. Routine flow cytometric quality control (QC) data of WBC-reduced blood products from two laboratories were analyzed. At the level of 3.3 WBCs per microL, none of the laboratories met the requirements for all three blood products. The LeucoCOUNT method met requirements at more laboratories than the LeukoSure method for RBCs and PLTs, but the opposite was true for plasma. Routine QC data showed that >99 percent of the flow cytometric measurements for WBC-reduced products was below the 95 percent prediction interval at 3.3 WBCs per microL. None of the laboratories met the requirements for accuracy and precision for all three blood products. Nevertheless, routine results showed that in >99 percent of the products, WBC counts were below guideline limits. Therefore, both flow cytometric methods are suitable for QC with pass-fail criterion.

  4. Identification of inhibitors of vacuolar proton-translocating ATPase pumps in yeast by high-throughput screening flow cytometry.

    PubMed

    Johnson, Rebecca M; Allen, Chris; Melman, Sandra D; Waller, Anna; Young, Susan M; Sklar, Larry A; Parra, Karlett J

    2010-03-15

    Fluorescence intensity of the pH-sensitive carboxyfluorescein derivative 2,7-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) was monitored by high-throughput flow cytometry in living yeast cells. We measured fluorescence intensity of BCECF trapped in yeast vacuoles, acidic compartments equivalent to lysosomes where vacuolar proton-translocating ATPases (V-ATPases) are abundant. Because V-ATPases maintain a low pH in the vacuolar lumen, V-ATPase inhibition by concanamycin A alkalinized the vacuole and increased BCECF fluorescence. Likewise, V-ATPase-deficient mutant cells had greater fluorescence intensity than wild-type cells. Thus, we detected an increase of fluorescence intensity after short- and long-term inhibition of V-ATPase function. We used yeast cells loaded with BCECF to screen a small chemical library of structurally diverse compounds to identify V-ATPase inhibitors. One compound, disulfiram, enhanced BCECF fluorescence intensity (although to a degree beyond that anticipated for pH changes alone in the mutant cells). Once confirmed by dose-response assays (EC(50)=26 microM), we verified V-ATPase inhibition by disulfiram in secondary assays that measured ATP hydrolysis in vacuolar membranes. The inhibitory action of disulfiram against V-ATPase pumps revealed a novel effect previously unknown for this compound. Because V-ATPases are highly conserved, new inhibitors identified could be used as research and therapeutic tools in cancer, viral infections, and other diseases where V-ATPases are involved. Copyright (c) 2009 Elsevier Inc. All rights reserved.

  5. Quantifying Distribution of Flow Cytometric TCR-Vβ Usage with Economic Statistics.

    PubMed

    van der Geest, Kornelis S M; Abdulahad, Wayel H; Horst, Gerda; Lorencetti, Pedro G; Bijzet, Johan; Arends, Suzanne; van der Heiden, Marieke; Buisman, Anne-Marie; Kroesen, Bart-Jan; Brouwer, Elisabeth; Boots, Annemieke M H

    2015-01-01

    Measuring changes of the T cell receptor (TCR) repertoire is important to many fields of medicine. Flow cytometry is a popular technique to study the TCR repertoire, as it quickly provides insight into the TCR-Vβ usage among well-defined populations of T cells. However, the interpretation of the flow cytometric data remains difficult, and subtle TCR repertoire changes may go undetected. Here, we introduce a novel means for analyzing the flow cytometric data on TCR-Vβ usage. By applying economic statistics, we calculated the Gini-TCR skewing index from the flow cytometric TCR-Vβ analysis. The Gini-TCR skewing index, which is a direct measure of TCR-Vβ distribution among T cells, allowed us to track subtle changes of the TCR repertoire among distinct populations of T cells. Application of the Gini-TCR skewing index to the flow cytometric TCR-Vβ analysis will greatly help to gain better understanding of the TCR repertoire in health and disease.

  6. Quantifying Distribution of Flow Cytometric TCR-Vβ Usage with Economic Statistics

    PubMed Central

    van der Geest, Kornelis S. M.; Abdulahad, Wayel H.; Horst, Gerda; Lorencetti, Pedro G.; Bijzet, Johan; Arends, Suzanne; van der Heiden, Marieke; Buisman, Anne-Marie; Kroesen, Bart-Jan; Brouwer, Elisabeth; Boots, Annemieke M. H.

    2015-01-01

    Measuring changes of the T cell receptor (TCR) repertoire is important to many fields of medicine. Flow cytometry is a popular technique to study the TCR repertoire, as it quickly provides insight into the TCR-Vβ usage among well-defined populations of T cells. However, the interpretation of the flow cytometric data remains difficult, and subtle TCR repertoire changes may go undetected. Here, we introduce a novel means for analyzing the flow cytometric data on TCR-Vβ usage. By applying economic statistics, we calculated the Gini-TCR skewing index from the flow cytometric TCR-Vβ analysis. The Gini-TCR skewing index, which is a direct measure of TCR-Vβ distribution among T cells, allowed us to track subtle changes of the TCR repertoire among distinct populations of T cells. Application of the Gini-TCR skewing index to the flow cytometric TCR-Vβ analysis will greatly help to gain better understanding of the TCR repertoire in health and disease. PMID:25923356

  7. Identification of unsuspected PNH-type cells in flow cytometric immunophenotypic analysis of peripheral blood and bone marrow.

    PubMed

    Thomason, Ronald W; Papiez, Joseph; Lee, Ronald V; Szczarkowski, Wlodek

    2004-07-01

    In this report, the flow cytometric expression patterns for CD14 on monocytic cells and CD16 on granulocytic cells in peripheral blood or bone marrow specimens are illustratedfor 15 patients proven to have a paroxysmal nocturnal hemoglobinuria (PNH) phenotype by flow cytometric analysis for CD55 and CD59. The varied clinical manifestations of PNH and its rarity may make it difficult to recognize clinically. As a result, blood or bone marrow samples may be submitted for flow cytometric analysis to exclude bone marrow neoplasia or dysplasia in patients with cytopenias rather than to exclude PNH. This was true in 5 of 15 study cases. Unlike CD55 and CD59, CD14 and/or CD16 are assessed routinely in the flow cytometric analysis of blood and bone marrow samples. Recognition of abnormal patterns of CD14 and CD16 expression might permit the identification of clinically unsuspected PNH by routine flow cytometric analysis.

  8. A flow-cytometric method to evaluate eosinophil-mediated uptake of probiotic Lactobacillus reuteri.

    PubMed

    Kraemer, Laura S; Brenner, Todd A; Krumholz, Julia O; Rosenberg, Helene F

    2017-06-01

    Eosinophils are resident leukocytes of gut mucosa. Here we present a combined flow cytometric-antibiotic protection assay to identify mouse eosinophils capable of bacterial uptake, specifically, Gram-positive Lactobacillus reuteri, in studies performed ex vivo. The assay may be adapted for use in vivo. Published by Elsevier B.V.

  9. Flow cytometric detection of bovine viral diarrhea virus in peripheral blood leukocytes of persistently infected cattle.

    PubMed Central

    Qvist, P; Aasted, B; Bloch, B; Meyling, A; Rønsholt, L; Houe, H

    1990-01-01

    Flow cytometry was investigated for detection of bovine viral diarrhea virus (BVDV) in peripheral blood mononuclear leukocytes of persistently infected cattle. The mononuclear leukocytes were purified by sedimentation in a gradient of Ficoll-Paque, fixed, permeabilized, and then labelled by indirect immunofluorescence using biotinylated immunoglobulins from a porcine antiserum to BVDV. Flow cytometric analysis of blood samples obtained from persistently infected cattle revealed virus in 3.0-21.0% (mean +/- SD, 11.2% +/- 6.4%) of the mononuclear leukocytes. Fluorescent cells were not observed in controls. Flow cytometric detection of BVDV in blood cells of persistently infected bovines is a rapid and objective technique which does not require cell culture facilities. PMID:2174298

  10. High throughput screening informatics.

    PubMed

    Ling, Xuefeng Bruce

    2008-03-01

    High throughput screening (HTS), an industrial effort to leverage developments in the areas of modern robotics, data analysis and control software, liquid handling devices, and sensitive detectors, has played a pivotal role in the drug discovery process, allowing researchers to efficiently screen millions of compounds to identify tractable small molecule modulators of a given biological process or disease state and advance them into high quality leads. As HTS throughput has significantly increased the volume, complexity, and information content of datasets, lead discovery research demands a clear corporate strategy for scientific computing and subsequent establishment of robust enterprise-wide (usually global) informatics platforms, which enable complicated HTS work flows, facilitate HTS data mining, and drive effective decision-making. The purpose of this review is, from the data analysis and handling perspective, to examine key elements in HTS operations and some essential data-related activities supporting or interfacing the screening process, and outline properties that various enabling software should have. Additionally, some general advice for corporate managers with system procurement responsibilities is offered.

  11. Howard University Flow Cytometric Sorter For Research and Education

    DTIC Science & Technology

    2015-08-04

    26/3/R02 (2005). 4. Brown, M. & Wittwer, C. Flow cytometry: principles and clinical applications in hematology . Clinical chemistry 46, 1221- 1229...2005). 4. Brown, M. & Wittwer, C. Flow cytometry: principles and clinical applications in hematology . Clinical chemistry 46, 1221-1229 (2000). 5

  12. High speed flow cytometric separation of viable cells

    DOEpatents

    Sasaki, Dennis T.; Van den Engh, Gerrit J.; Buckie, Anne-Marie

    1995-01-01

    Hematopoietic cell populations are separated to provide cell sets and subsets as viable cells with high purity and high yields, based on the number of original cells present in the mixture. High-speed flow cytometry is employed using light characteristics of the cells to separate the cells, where high flow speeds are used to reduce the sorting time.

  13. High speed flow cytometric separation of viable cells

    DOEpatents

    Sasaki, D.T.; Van den Engh, G.J.; Buckie, A.M.

    1995-11-14

    Hematopoietic cell populations are separated to provide cell sets and subsets as viable cells with high purity and high yields, based on the number of original cells present in the mixture. High-speed flow cytometry is employed using light characteristics of the cells to separate the cells, where high flow speeds are used to reduce the sorting time.

  14. Rapid flow cytometric method for measuring senescence associated beta-galactosidase activity in human fibroblasts.

    PubMed

    Noppe, Gerard; Dekker, Pim; de Koning-Treurniet, Corine; Blom, Joke; van Heemst, Diana; Dirks, Roeland W; Tanke, Hans J; Westendorp, Rudi G J; Maier, Andrea B

    2009-11-01

    Senescence associated-beta-galactosidase (SA-beta-gal) activity is a widely used marker for cellular senenescence. SA-beta-gal activity is routinely detected cytochemically, manually discriminating negative from positive cells. This method is time-consuming, subjective and therefore prone to operator-error. We aimed to optimize a flow cytometric method described by other workers using endothelial cells to better differentiate between populations of fibroblasts in degrees of SA-beta-gal activity. Skin fibroblasts were isolated from young (mean age +/- SD: 25.5 +/- 1.8) and very old (age 90.2 +/- 0.3) subjects. Different pH modulators were tested for toxicity. To induce stress-induced senescence, fibroblasts were exposed to rotenone. Senescence was assessed measuring SA-beta-gal activity by cytochemistry (X-gal) and by flow cytometry (C(12)FDG). The pH modulator Bafilomycin A1 (Baf A1) was found to be least toxic for fibroblasts and to differentiate best between nonstressed and stressed fibroblast populations. Under nonstressed conditions, fibroblasts from very old subjects showed higher SA-beta-gal activity than fibroblasts from young subjects. This difference was found for both the flow cytometric and cytochemical methods (P = 0.013 and P = 0.056 respectively). Under stress-induced conditions the flow cytometric method but not the cytochemical method revealed significant higher SA-beta-gal activity in fibroblasts from very old compared to young subjects (P = 0.004 and P = 0.635 respectively). We found the modified flow cytometric method measuring SA-beta-gal activity superior in discriminating between degrees of senescence in different populations of fibroblasts. Copyright 2009 International Society for Advancement of Cytometry.

  15. New optical configuration for flow cytometric sorting of aspherical cells

    NASA Astrophysics Data System (ADS)

    Sharpe, John C.; Schaare, Peter N.; Kuennemeyer, Rainer

    1997-05-01

    The orthogonal axes of illumination, flow, and detection in conventional sorting flow cytometers can limit accuracy or throughput when making fluorescence measurements on a spherical cells. A new radially symmetric optical configuration has been designed to overcome these problems. Both illumination and fluorescence collection are performed by a single optical element which encircles the sample stream flow axis. Unlike existing epi-illumination flow cytometer designs, these optics are compatible with electrostatic sorting. The resolution of this system is currently being evaluated for DNA chromosome content measurement with an ultimate goal of separation of X- and Y- chromosome-bearing mammalian spermatozoa. We describe the new optical configuration and present preliminary results of instrument performance. Comparison with a conventional orthogonal optical geometry is made using fluorescent microspheres, chicken red blood cells and chinchilla sperm.

  16. The utility of peripheral blood smear review for identifying specimens for flow cytometric immunophenotyping.

    PubMed

    Craig, F E

    2017-05-01

    Laboratory professionals are in an ideal situation to identify CBC and peripheral blood smear findings that raise the possibility of a hematolymphoid neoplasm, and based on this information make recommendations for additional studies, such as flow cytometric immunophenotyping. In some circumstances a definitive diagnosis can be established from the combined peripheral blood morphologic and immunophenotypic findings obviating the need for bone marrow evaluation, such as for chronic lymphocytic leukemia. Occasionally flow cytometric studies are superior to morphologic assessment, such as in screening for hairy cell leukemia or identifying lymphocytic-variant hypereosinophilia. However, there is increasing recognition of small immunophenotypically unusual or abnormal populations of peripheral blood cells, particularly in older patients, which are of uncertain clinical significance, such as monoclonal B lymphocytosis and T-cell clonopathy. Therefore, it is important to integrate peripheral blood smear review findings with the clinical and other information before recommending flow cytometry. In addition, it is important to recognize situations where the results of peripheral blood smear review and flow cytometric immunophenotyping do not explain the clinical findings. © 2017 John Wiley & Sons Ltd.

  17. Possible use of porphycenes as a membrane marker for flow cytometric detection of micronuclei

    SciTech Connect

    Wessels, J.M.; Nuesse, M. )

    1993-01-01

    For flow cytometric analysis of micronuclei induced by ionizing radiation or chemicals, debris plays an important role for the unambiguous detection of micronuclei. Several flow cytometric parameters have been used for a discrimination of micronuclei and debris (Schreiber et al., Cytometry 13:90-102, 1992). The lipophilic character of porphycenes can be used to selectively stain cellular and nuclear membranes. This chemical property of porphycenes requires the use of liposomes as a carrier. Porphycenes show high extinction coefficients at 360 nm and high fluorescence quantum yields between 600 nm and 750 nm. Due to their excellent photophysical properties they can therefore be used as a fluoroscent dye in combination with DNA specific dyes. Porphycenes (i.e. hydroxyethyl-tris(methoxyethyl)porphycene, HEPn) were used to either stain selectively the debris produced by membrane particles or to stain the membranes of nuclei and micronuclei for a better discrimination of debris and micronuclei.

  18. Recommendations for the evaluation of specimen stability for flow cytometric testing during drug development.

    PubMed

    Brown, Lynette; Green, Cherie L; Jones, Nicholas; Stewart, Jennifer J; Fraser, Stephanie; Howell, Kathy; Xu, Yuanxin; Hill, Carla G; Wiwi, Christopher A; White, Wendy I; O'Brien, Peter J; Litwin, Virginia

    2015-03-01

    The objective of this manuscript is to present an approach for evaluating specimen stability for flow cytometric methods used during drug development. While this approach specifically addresses stability assessment for assays to be used in clinical trials with centralized testing facilities, the concepts can be applied to any stability assessment for flow cytometric methods. The proposed approach is implemented during assay development and optimization, and includes suggestions for designing a stability assessment plan, data evaluation and acceptance criteria. Given that no single solution will be applicable in all scenarios, this manuscript offers the reader a roadmap for stability assessment and is intended to guide the investigator during both the method development phase and in the experimental design of the validation plan. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. An accurate and rapid flow cytometric diagnosis of BCR-ABL positive acute lymphoblastic leukemia

    PubMed Central

    Raponi, Sara; De Propris, Maria Stefania; Wai, Hobert; Intoppa, Stefania; Elia, Loredana; Diverio, Daniela; Vitale, Antonella; Foà, Robin; Guarini, Anna

    2009-01-01

    Tyrosine kinase inhibitors have profoundly modified the treatment and prognosis of chronic myeloid leukemia and Ph+ acute lymphoblastic leukemia. A rapid and accurate detection of the BCR-ABL fusion protein is paramount today for an optimal management of Ph+ acute lymphoblastic leukemia. We have utilized a recently described and commercialized immunoassay that identifies qualitatively the presence of the BCR-ABL protein in leukemic cell lysates. The BCR-ABL fusion protein is captured and detected by a cytometric bead assay and analyzed by flow cytometry. The assay was applied to 101 primary patient samples (94 acute leukemias and 7 chronic myeloid leukemia blast crisis) and the results of the immunoassay were concordant with those obtained by conventional molecular techniques. The method proved reliable, reproducible, of simple execution and it was successfully completed within four hours. This flow cytometric immunoassay has important implications for perfecting the management of Ph+ acute lymphoblastic leukemia patients worldwide. PMID:19608682

  20. Quantitative flow cytometric analysis of membrane antigen expression.

    PubMed

    D'hautcourt, Jean-Luc

    2002-11-01

    Immunological analysis for cell antigens has been performed by flow cytometry in a qualitative fashion for over thirty years. During that time it has become increasingly apparent that quantitative measurements such as number of antigens per cell provide unique and useful information. This unit on quantitative flow cytometry (QFCM) describes the most commonly used protocols, both direct and indirect, and the major methods of analysis for the number of antibody binding sites on a cell or particle. Practical applications include detection of antigen under- or overexpression in hematological malignancies, distinguishing between B cell lymphoproliferative disorders, and precise diagnosis of certain rare diseases.

  1. Repeatability of flow cytometric and classical measurement of phagocytosis and respiratory burst in bovine polymorphonuclear leukocytes.

    PubMed

    Kampen, Annette H; Tollersrud, Tore; Larsen, Stig; Roth, James A; Frank, Dagmar E; Lund, Arve

    2004-01-01

    Five methods for measurement of phagocytosis and respiratory burst activity of bovine blood polymorphonuclear leukocytes (PMNs) were evaluated. Eight cows were repeatedly sampled over a two week period and parallel samples tested in all five assays to assess the repeatability and stability of the methods. In the flow cytometric phagocytosis assay, ingestion of fluorescein labeled bacteria was measured, and in the flow cytometric assay for respiratory burst, oxidation of a dye by reactive oxygen species was recorded. In the classical assays, bactericidal effect on opsonized, live bacteria was quantified by the conversion of an indicator substance, superoxide anion production was assayed by the reduction of cytochrome c, whereas myeloperoxidase activity was determined with a radioactive iodination assay. The results showed that the Phagotest, Bursttest, cytochrome c and iodination assays gave repeatable results when samples were run in the same setup on the same day. Although day-to-day variability was significant in all assays, the described methods comprise a panel of useful tests for the evaluation of phagocytosis and respiratory burst activity in bovine PMNs. The flow cytometric methods represent a convenient alternative to the classical methods for measurement of phagocytosis and respiratory burst in bovine blood PMNs.

  2. Flow Cytometric Methods for Indirect Analysis and Quantification of Gametogenesis in Chlamydomonas reinhardtii (Chlorophyceae)

    PubMed Central

    Tomkins, Joseph L.

    2016-01-01

    Induction of sexual reproduction in the facultatively sexual Chlamydomonas reinhardtii is cued by depletion of nitrogen. We explore the capacity for indirect monitoring of population variation in the gametogenic process using flow cytometry. We describe a high-throughput method capable of identifying fluorescence, ploidy and scatter profiles that track vegetative cells entering and undergoing gametogenesis. We demonstrate for the first time, that very early and late growth phases reduce the capacity to distinguish putative gametes from vegetative cells based on scatter and fluorescence profiles, and that early/mid-logarithmic cultures show the optimal distinction between vegetative cells and gamete scatter profiles. We argue that early/mid logarithmic cultures are valuable in such high throughput comparative approaches when investigating optimisation or quantification of gametogenesis based on scatter and fluorescence profiles. This approach provides new insights into the impact of culture conditions on gametogenesis, while documenting novel scatter and fluorescence profile shifts which typify the process. This method has potential applications to; enabling quick high-throughput monitoring, uses in increasing efficiency in the quantification of gametogenesis, as a method of comparing the switch between vegetative and gametic states across treatments, and as criteria for enrichment of gametic phenotypes in cell sorting assays. PMID:27676075

  3. Flow cytometric MRD detection in selected mature B-cell malignancies.

    PubMed

    Böttcher, Sebastian; Ritgen, Matthias; Kneba, Michael

    2013-01-01

    The quantification of submicroscopic minimal residual disease (MRD) after therapy proved to have independent prognostic significance in many mature B-cell malignancies. With the advent of routine bench-top cytometers capable of simultaneously analyzing ≥ 4 colors and with improved standardization, flow cytometry has become the method of choice for MRD assessments in some lymphoma entities. Herein we describe general aspects of flow cytometric standardization. Using chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) as examples we explain in detail the application of flow cytometry for MRD detection.

  4. Flow Cytometric Measurement of Cellular Ionized Calcium Concentration

    DTIC Science & Technology

    1988-01-01

    membrane transport system for organic anions. J. man monocyles. J. cell. Physiol. 134: 131-136 Immun. 140: 915-920 (1988). (1988). 19 Finket, T.H...discuss the measurement of [Ca Ji using can function to transmit information from flow cytometry to analyze cells loaded with the l membrane to...Kim plasma membrane and across intracellular for technical assistance. calcium storage sites such as calctosomes, ability CodesAva and/ r- Dis t

  5. Flow cytometric analysis of glyoxalase-1 expression in human leukocytes.

    PubMed

    Skapare, Elina; Riekstina, Una; Liepinsh, Edgars; Konrade, Ilze; Makrecka, Marina; Maurina, Baiba; Dambrova, Maija

    2011-03-01

    Altered glyoxalase-1 (GLO-1) activity and expression is associated with the development of late diabetic complications, malignancy and oxidative stress- and aging-related diseases. In the present study, we developed a flow cytometry method for GLO-1 detection in human leukocytes isolated from peripheral blood samples to investigate GLO-1 expression in leukocyte subsets from type 1 and 2 diabetes mellitus patients (n = 11) and healthy subjects (n = 8). The flow cytometry analysis of GLO-1 in leukocytes showed that expression index of GLO-1-positive cells was slightly increased in mononuclear leukocytes from diabetic patients. This result correlated with the increase in GLO-1 activity in the whole blood samples of type 2 diabetes patients. In conclusion, the present study demonstrates that flow cytometry is suitable for the detection of the GLO-1 enzyme in human leukocytes and that this method could be used to investigate the fast adaptation of the glyoxalase system related to the pathogenesis of late complications of diabetes mellitus and other glycation stress-related disorders. Copyright © 2011 John Wiley & Sons, Ltd.

  6. The modified FACS calcein AM retention assay: A high throughput flow cytometer based method to measure cytotoxicity.

    PubMed

    Gillissen, M A; Yasuda, E; de Jong, G; Levie, S E; Go, D; Spits, H; van Helden, P M; Hazenberg, M D

    2016-07-01

    Current methods to determine cellular cytotoxicity in vitro are hampered by background signals that are caused by auto-fluorescent target and effector cells and by non-specific cell death. We combined and adjusted existing cell viability assays to develop a method that allows for highly reproducible, accurate, single cell analysis by high throughput FACS, in which non-specific cell death is corrected for. In this assay the number of living, calcein AM labeled cells that are green fluorescent are quantified by adding a fixed number of unlabeled calibration beads to the analysis. Using this modified FACS calcein AM retention method, we found EC50 values to be highly reproducible and considerably lower compared to EC50 values obtained by conventional assays, displaying the high sensitivity of this assay. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  7. Flow cytometric fluorescence lifetime analysis of DNA binding fluorochromes

    SciTech Connect

    Crissman, Harry A.; Cui, H. H.; Steinkamp, J. A.

    2002-01-01

    Most flow cytometry (FCM) applications monitor fluorescence intensity to quantitate the various cellular parameters; however, the fluorescence emission also contains information relative to the fluorescence lifetime. Recent developments in FCM (Pinsky et al., 1993; Steinkamp & Crissman, 1993; Steinkamp et al., 1993), provide for the measurement of fluorescence lifetime which is also commonly referred to as fluorescence decay, or the time interval in which a fluorochrome remains in the excited state. Many unbound fluorochromes have characteristic lifetime values that are determined by their molecular structure; however, when the probe becomes bound, the lifetime value is influenced by a number of factors that affect the probe interaction with a target molecule. Monitoring the changes in the lifetime of the probe yields information relating to the molecular conformation, the functional state or activity of the molecular target. In addition, the lifetime values can be used as signatures to resolve the emissions of multiple fluorochrome labels with overlapping emission spectra that cannot be resolved by conventional FCM methodology. Such strategies can increase the number of fluorochrome combinations used in a flow cytometer with a single excitation source. Our studies demonstrate various applications of lifetime measurements for the analysis of the binding of different fluorochromes to DNA in single cells. Data presented in this session will show the utility of lifetime measurements for monitoring changes in chromatin structure associated with cell cycle progression, cellular differentiation, or DNA damage, such as induced during apoptosis. Several studies show that dyes with specificity for nucleic acids display different lifetime values when bound to DNA or to dsRNA. The Phase Sensitive Flow Cytometer is a multiparameter instrument, capable of performing lifetime measurements in conjunction with all the conventional FCM measurements. Future modifications of this

  8. Rapid flow cytometric measurement of protein inclusions and nuclear trafficking

    PubMed Central

    Whiten, D. R.; San Gil, R.; McAlary, L.; Yerbury, J. J.; Ecroyd, H.; Wilson, M. R.

    2016-01-01

    Proteinaceous cytoplasmic inclusions are an indicator of dysfunction in normal cellular proteostasis and a hallmark of many neurodegenerative diseases. We describe a simple and rapid new flow cytometry-based method to enumerate, characterise and, if desired, physically recover protein inclusions from cells. This technique can analyse and resolve a broad variety of inclusions differing in both size and protein composition, making it applicable to essentially any model of intracellular protein aggregation. The method also allows rapid quantification of the nuclear trafficking of fluorescently labelled molecules. PMID:27516358

  9. A novel high-throughput multi-parameter flow cytometry based method for monitoring and rapid characterization of microbiome dynamics in anaerobic systems.

    PubMed

    Dhoble, Abhishek S; Bekal, Sadia; Dolatowski, William; Yanz, Connor; Lambert, Kris N; Bhalerao, Kaustubh D

    2016-11-01

    A novel multidimensional flow cytometry based method has been demonstrated to monitor and rapidly characterize the dynamics of the complex anaerobic microbiome associated with perturbations in external environmental factors. While community fingerprinting provides an estimate of the meta genomic structure, flow cytometry provides a fingerprint of the community morphology including its autofluorescence spectrum in a high-throughput manner. Using anaerobic microbial consortia perturbed with the controlled addition of various carbon sources, it is possible to quantitatively discriminate between divergent microbiome analogous to community fingerprinting techniques using automated ribosomal intergenic spacer analysis (ARISA). The utility of flow cytometry based method has also been demonstrated in a fully functional industry scale anaerobic digester to distinguish between microbiome composition caused by varying hydraulic retention time (HRT). This approach exploits the rich multidimensional information from flow cytometry for rapid characterization of the dynamics of microbial communities.

  10. Flow cytometric data analysis of circulating progenitor cell stability.

    PubMed

    Mahar, Ernestine A; Mou, Liping; Hayek, Salim S; Quyyumi, Arshed A; Waller, Edmund K

    2017-02-01

    A recent publication by Mekonnen et al. demonstrated that among women with non-obstructive coronary artery disease, higher levels of circulating progenitor cells in the blood (CPC), were associated with impaired coronary flow reserve [1]. We performed a quality control assessment of the stability of circulating blood progenitor cells in blood samples stored at 4 °C, to determine the time period during which blood samples can be analyzed and yield consistent data for progenitor cell content. Healthy volunteers (n=6) were recruited and underwent phlebotomy, and blood was stored in EDTA tubes at 4 °C. Flow cytometry was performed to quantitate progenitor cell subsets at 0-4 h, 24 h, and 48 h post phlebotomy. All processed samples were fixed with 1% Paraformaldehyde and 1,000,000 total data events were collected. We found no significant differences in PC data for both CD34+ (P=0.68 for one-way ANOVA) and CD34+/CD133+ (P=0.74 for one-way ANOVA).

  11. Flow cytometric measurement of pollutant stresses on algal cells

    SciTech Connect

    Berglund, D.L.; Eversman, S.

    1988-03-01

    The lichen Usnea fulvoreagens (Raes). Raes. was treated with four pH levels (5.5, 4.5, 3.5, and 2.5) of simulated acid rain (sulfuric acid, nitric acid, and a 1:1 combination of both) and automobile exhaust. The samples were dissociated and analyzed by a Becton-Dickinson FACS 440 flow cytometer. Analyses included measurement of chlorophyll autofluorescence and fluorescence due to uptake of fluorescein diacetate (FDA) and calcofluor white M2R (CFW). Cell parameters measured were esterase activity (FDA), membrane permeability (FDA, CFW), and intracellular pH (FDA). Mean fluorescence intensity from FDA staining and numbers of events were incorporated with autofluorescence information to produce a stress index of relative cell stress. Results indicated that highly stressed samples (lower pH treatments and greater exposure to exhaust) exhibited a low stress index of FDA fluorescence.Au

  12. Flow cytometric measurement of total DNA and incorporated halodeoxyuridine

    DOEpatents

    Dolbeare, F.A.; Gray, J.W.

    1983-10-18

    A method for the simultaneous flow cylometric measurement of total cellular DNA content and of the uptake of DNA precursors as a measure of DNA synthesis during various phases of the cell cycle in normal and malignant cells in vitro and in vivo is described. The method comprises reacting cells with labelled halodeoxyuridine (HdU), partially denaturing cellular DNA, adding to the reaction medium monoclonal antibodies (mabs) reactive with HdU, reacting the bound mabs with a second labelled antibody, incubating the mixture with a DNA stain, and measuring simultaneously the intensity of the DNA stain as a measure of the total cellular DNA and the HdU incorporated as a measure of DNA synthesis. (ACR)

  13. Flow cytometric analysis: Interdependence of healthy and infected udder quarters.

    PubMed

    Blagitz, M G; Souza, F N; Batista, C F; Diniz, S A; Azevedo, L F F; Silva, M X; Haddad, J P A; Heinemann, M B; Cerqueira, M M O P; Della Libera, A M M P

    2015-04-01

    An important question about intramammary infections that is still debated in the literature is the independence or interdependence of the quarters of dairy cows. The present study sought to explore milk neutrophil function and the milk lymphocyte profile of uninfected quarters from uninfected and infected (one infected quarter per cow) udders to evaluate interdependence of the quarters. Thus, 32 (8 cows) and 18 (6 cows) uninfected quarters from uninfected and infected udders were used, respectively. Using flow cytometry, we evaluated the percentage of milk neutrophils and their expression of adhesion molecules L-selectin (CD62L), β2-integrin (CD11b), and an endothelial-selectin ligand (CD44); levels of intracellular reactive oxygen species (ROS); phagocytosis of Staphylococcus aureus by milk neutrophils; and neutrophil viability. Furthermore, we assessed the percentage of B-cell (CD21(+)) and T-lymphocyte subsets (CD3(+)/CD4(+)/CD8(-), CD3(+)/CD8(+)/CD4(-), CD3(+)/CD4(+)/CD25(-), CD3(+)/CD4(+)/CD25(+), and CD3(+)/CD4(-)/CD25(-)) using flow cytometry with monoclonal antibodies. The infected quarter did not affect somatic cell count or the percentage of neutrophils in the neighboring uninfected quarters. Furthermore, the infected quarter did not influence neutrophil viability, intracellular reactive oxygen species production, or phagocytosis of S. aureus by milk neutrophils. Conversely, the expression of adhesion molecules CD11b, CD62L, and CD44 by milk neutrophils differed between uninfected quarters from infected versus uninfected udders. The lymphocyte subsets did not differ between groups, except for a higher percentage of B cells in uninfected quarters from infected udders than in those from uninfected udders. Thus, our study strongly supports the hypothesis of interdependence of quarters based on the influence of infection on both the percentage of B cells and the expression of adhesion molecules by milk neutrophils in the neighboring uninfected quarters

  14. Ultrastructural and flow cytometric analyses of lipid accumulation in microalgae

    SciTech Connect

    Solomon, J.A.; Hand, R.E. Jr.; Mann, R.C.

    1986-12-01

    Lipid accumulation in three species of microalgae was investigated with flow cytometry (FCM) and transmission electron microscopy (TEM). Previous studies using batch cultures of a algae have led to the assumption that lipid accumulation in microalgae is a gradual process requiring at least several days for completion. However, FCM reveals, through changes in the chlorophyll:lipid ratio, that the time span required for individual cells to change metabolic state is short. Simultaneous FCM measurements of chlorophyll and nile red (neutral lipid) fluorescence in individual cells of nitrogen-deficient Isochrysis populations revealed a bimodal population distribution as one stage in the lipid accumulation process. The fact that two discrete populations exist, with few cells in an intermediate stage, suggests rapid response to a liqid trigger. Interpretations of light and electron microscopic observations are consistent with this hypothesis. The time required for an entire population to achieve maximum lipid content is considerably longer than that required for a single cell, due to the variation in response time among cells. In this study high lipid cultures were sometimes obtained by using FCM to separate high lipid cells from the remainder of the population. FCM holds much promise for strain enhancement but considerable developmental work, directed at providing more consistent results, remains to be done. 8 refs., 35 figs.

  15. Flow cytometric study of in vitro neutrophil activation by biomaterials.

    PubMed

    Gorbet, M B; Yeo, E L; Sefton, M V

    1999-03-05

    Neutrophil activation for adherent and nonadherent cells, as measured by flow cytometry, was not strongly dependent on material surface chemistry. We had hypothesized that material-induced neutrophil activation was an important parameter associated with material failure. All materials tested [cellophane, an acrylonitrile copolymer (AN69), Pellethane, nylon, polyethylene terephthalate, low density polyethylene, and polydimethylsiloxane] activated isolated human neutrophils, which were resuspended in plasma or serum, to similar extents based on L-selectin shedding, CD11b upregulation, and stimulation of the oxidative burst after 30-min exposure. Inhibition of complement activation by sCR1 unexpectedly had little effect if any on nonadherent neutrophils. However, neutrophil adhesion, but not the level of activation of the adherent cells, was strongly dependent on complement activation. Pretreatment with albumin did not inhibit adhesion or reduce neutrophil activation, but plasma pretreatment resulted in increased activation for nonadherent and adherent cells. More adhesion and a higher level of activation of adherent cells was observed following pretreatment with fibrinogen, a ligand of CD11b. Taken together these results suggest that upon contact with a material, neutrophil activation may occur though mechanisms that are not mediated by complement. For example, the presence of plasma proteins such as fibrinogen at the interface may trigger activation and the release of other activating agents. Although the material differences are small, the extent of activation may be significant and warrant further study of the mechanism and consequences of that activation.

  16. DNA flow cytometric analysis in variable types of hydropic placentas

    PubMed Central

    Atabaki pasdar, Fatemeh; Khooei, Alireza; Fazel, Alireza; Rastin, Maryam; Tabasi, Nafise; Peirouvi, Tahmineh; Mahmoudi, Mahmoud

    2015-01-01

    Background: Differential diagnosis between complete hydatidiform mole, partial hydatidiform mole and hydropic abortion, known as hydropic placentas is still a challenge for pathologists but it is very important for patient management. Objective: We analyzed the nuclear DNA content of various types of hydropic placentas by flowcytometry. Materials and Methods: DNA ploidy analysis was performed in 20 non-molar (hydropic and non-hydropic spontaneous abortions) and 20 molar (complete and partial moles), formalin-fixed, paraffin-embedded tissue samples by flow cytometry. The criteria for selection were based on the histopathologic diagnosis. Results: Of 10 cases histologically diagnosed as complete hydatiform mole, 9 cases yielded diploid histograms, and 1 case was tetraploid. Of 10 partial hydatidiform moles, 8 were triploid and 2 were diploid. All of 20 cases diagnosed as spontaneous abortions (hydropic and non-hydropic) yielded diploid histograms. Conclusion: These findings signify the importance of the combined use of conventional histology and ploidy analysis in the differential diagnosis of complete hydatidiform mole, partial hydatidiform mole and hydropic abortion. PMID:26221125

  17. Flow cytometric techniques to characterise physiological states of Acinetobacter calcoaceticus.

    PubMed

    Müller, S; Ullrich, S; Lösche, A; Loffhagen, N; Babel, W

    2000-03-01

    Monitoring biotechnological processes involves acquiring information about key metabolic events and, ideally, single cell states should be determined to obtain comprehensive data on the physiological status of the surveyed population. In this paper, growth stages of the strain Acinetobacter calcoaceticus 69-V were characterised at the single cell level using flow cytometry. Four methods for analysing bacterial cellular characteristics by fluorescence were compared with respect to their sensitivity to changes in the physiological states induced by changing micro-environmental conditions. DNA analysis was confirmed to be highly informative with regard to the multiplication activity of the population. Measuring the membrane potential related fluorescence intensity (MPRFI) and the rRNA content were found to be useful for describing high-active cell states. A method for the measurement of the fluidity related fluorescence intensity (FRFI) was developed, since it allowed changes in the fluidity of the bacterial membrane to be detected, and thereby provided a valuable means of tracking adaptation of the population to micro-environmental deviations from optimal growth conditions.

  18. Flow cytometric DNA and 5'-nucleotide phosphodiesterase in endometrium.

    PubMed

    Tsou, K C; Hong, D H; Varello, M A; Wheeler, J E; Giuntoli, R; Mangan, C E; Mikuta, J J

    1985-11-01

    One hundred endometrium specimens have been studied with flow cytometry for DNA analysis (FCDA) and a proliferative enzyme marker, 5'-nucleotide phosphodiesterase (5'-NPD). FCDA data showed that aneuploidy was present in only 5 of 40 cancer specimens. However, with corrected histograms, a higher DNA value was observed in the G2/M (6%) of all cancer compared with noncancer specimens (4%). Thus, FCDA can be a useful diagnostic aid for endometrial cancer. The determination of 5'-NPD was done with a quenching method based on the use of 5'-(5-iodo-3-indoxyl)-thymidine phosphodiester as a substrate and 4',6-diamidino-2-phenylindole for DNA. This method could qualitatively define which population of the cell cycle had a higher enzyme level and also quantitatively gave the enzyme units per cell. It was found that 12.5% of all cancer specimens had 5'-NPD activity in the G0/G1 cells and 87.5% in the S and/or G2/M cells, whereas in the noncancer specimens 5'-NPD was found in 28.5% of the G0/G1 cells and 71.5% of the specimens had 5'-NPD in the S and/or G2/M cells. Furthermore, the concentration of 5'-NPD was found to be five times higher in the G2/M cells of the cancer specimens than that in the noncancer specimens. However, in the hyperplasia specimens, the activity was only two times higher in the same cell cycle fraction than in the normal specimens. The results of this investigation provided for the first time evidence that this exonuclease activity alters in the cell cycle fractions and that a decrease in the enzyme activity in G0/G1 cells and an increase in G2/M cells may be a useful marker for neoplastic development in human endometrial cancer.

  19. Application and commercialization of flow cytometrically sex-sorted semen.

    PubMed

    Rath, D; Johnson, L A

    2008-07-01

    The current technology to sort X and Y chromosome bearing sperm population requires individual identification and selection of spermatozoa in a modified high-speed flow cytometer. For farm animal species, the technology is capable of producing sexed sperm at greater than 90% purity. However, only in the bovine, the technology has reached a developmental level that allows its commercial application. Meanwhile, the demand for female calves has grown rapidly, which encourages the demand for sex-sorted semen from high genetic value bulls. The success of the technology will depend mainly on the fertilizing capacity of the sorted spermatozoa, as this is the most affecting and economically relevant factor. To date, fertility is still variable and is quite dependent on post-sort processing. New processing techniques are under investigation and will likely be able to improve the fertility rates after AI with sex-sorted semen. It is of great importance to select the right bulls and to test the sorted samples on a routine basis. In addition to the demand for sex-sorted semen by the cattle industry, there is also a significant demand expressed by pig farmers. However, it is still unknown if the use of sex-sorted semen through commercial pig AI will be economically feasible. For the pig, the combination of in vitro fertilization with sexed semen and non-surgical embryo transfer is an alternative that merits further scientific attention. Recent developments in ovine AI and ET will make it very likely that commercial sheep industry will adopt the sexing technology in their breeding concepts.

  20. Flow Cytometric Assessment of Viability of Lactic Acid Bacteria

    PubMed Central

    Bunthof, Christine J.; Bloemen, Karen; Breeuwer, Pieter; Rombouts, Frank M.; Abee, Tjakko

    2001-01-01

    The viability of lactic acid bacteria is crucial for their applications as dairy starters and as probiotics. We investigated the usefulness of flow cytometry (FCM) for viability assessment of lactic acid bacteria. The esterase substrate carboxyfluorescein diacetate (cFDA) and the dye exclusion DNA binding probes propidium iodide (PI) and TOTO-1 were tested for live/dead discrimination using a Lactococcus, a Streptococcus, three Lactobacillus, two Leuconostoc, an Enterococcus, and a Pediococcus species. Plate count experiments were performed to validate the results of the FCM assays. The results showed that cFDA was an accurate stain for live cells; in exponential-phase cultures almost all cells were labeled, while 70°C heat-killed cultures were left unstained. PI did not give clear live/dead discrimination for some of the species. TOTO-1, on the other hand, gave clear discrimination between live and dead cells. The combination of cFDA and TOTO-1 gave the best results. Well-separated subpopulations of live and dead cells could be detected with FCM. Cell sorting of the subpopulations and subsequent plating on agar medium provided direct evidence that cFDA labels the culturable subpopulation and that TOTO-1 labels the nonculturable subpopulation. Applied to cultures exposed to deconjugated bile salts or to acid, cFDA and TOTO-1 proved to be accurate indicators of culturability. Our experiments with lactic acid bacteria demonstrated that the combination of cFDA and TOTO-1 makes an excellent live/dead assay with versatile applications. PMID:11319119

  1. New flow cytometric assays for monitoring cell-mediated cytotoxicity

    PubMed Central

    Zaritskaya, Liubov; Shurin, Michael R; Sayers, Thomas J; Malyguine, Anatoli M

    2010-01-01

    The exact immunologic responses after vaccination that result in effective antitumor immunity have not yet been fully elucidated and the data from ex vivo T-cell assays have not yet defined adequate surrogate markers for clinical efficacy. A more detailed knowledge of the specific immune responses that correlate with positive clinical outcomes should help to develop better or novel strategies to effectively activate the immune system against tumors. Furthermore, clinically relevant material is often limited and, thus, precludes the ability to perform multiple assays. The two main assays currently used to monitor lymphocyte-mediated cytoxicity in cancer patients are the 51Cr-release assay and IFN-γ ELISpot assay. The former has a number of disadvantages, including low sensitivity, poor labeling and high spontaneous release of isotope from some tumor target cells. Additional problems with the 51Cr-release assay include difficulty in obtaining autologous tumor targets, and biohazard and disposal problems for the isotope. The ELISpot assays do not directly measure cytotoxic activity and are, therefore, a surrogate marker of cyotoxic capacity of effector T cells. Furthermore, they do not assess cytotoxicity mediated by the production of the TNF family of death ligands by the cytotoxic cells. Therefore, assays that allow for the simultaneous measurement of several parameters may be more advantageous for clinical monitoring. In this respect, multifactor flow cytometry-based assays are a valid addition to the currently available immunologic monitoring assays. Use of these assays will enable detection and enumeration of tumor-specific cytotoxic T lymphocytes and their specific effector functions and any correlations with clinical responses. Comprehensive, multifactor analysis of effector cell responses after vaccination may help to detect factors that determine the success or failure of a vaccine and its immunological potency. PMID:20518716

  2. New flow cytometric assays for monitoring cell-mediated cytotoxicity.

    PubMed

    Zaritskaya, Liubov; Shurin, Michael R; Sayers, Thomas J; Malyguine, Anatoli M

    2010-06-01

    The exact immunologic responses after vaccination that result in effective antitumor immunity have not yet been fully elucidated and the data from ex vivo T-cell assays have not yet defined adequate surrogate markers for clinical efficacy. A more detailed knowledge of the specific immune responses that correlate with positive clinical outcomes should help to develop better or novel strategies to effectively activate the immune system against tumors. Furthermore, clinically relevant material is often limited and, thus, precludes the ability to perform multiple assays. The two main assays currently used to monitor lymphocyte-mediated cytoxicity in cancer patients are the (51)Cr-release assay and IFN-gamma ELISpot assay. The former has a number of disadvantages, including low sensitivity, poor labeling and high spontaneous release of isotope from some tumor target cells. Additional problems with the (51)Cr-release assay include difficulty in obtaining autologous tumor targets, and biohazard and disposal problems for the isotope. The ELISpot assays do not directly measure cytotoxic activity and are, therefore, a surrogate marker of cyotoxic capacity of effector T cells. Furthermore, they do not assess cytotoxicity mediated by the production of the TNF family of death ligands by the cytotoxic cells. Therefore, assays that allow for the simultaneous measurement of several parameters may be more advantageous for clinical monitoring. In this respect, multifactor flow cytometry-based assays are a valid addition to the currently available immunologic monitoring assays. Use of these assays will enable detection and enumeration of tumor-specific cytotoxic T lymphocytes and their specific effector functions and any correlations with clinical responses. Comprehensive, multifactor analysis of effector cell responses after vaccination may help to detect factors that determine the success or failure of a vaccine and its immunological potency.

  3. Real-time detection of protein trafficking with high-throughput flow cytometry (HTFC) and fluorogen-activating protein (FAP) base biosensor.

    PubMed

    Wu, Yang; Tapia, Phillip H; Jarvik, Jonathan; Waggoner, Alan S; Sklar, Larry A

    2014-01-02

    We combined fluorogen-activating protein (FAP) technology with high-throughput flow cytometry to detect real-time protein trafficking to and from the plasma membrane in living cells. The hybrid platform allows drug discovery for trafficking receptors, such as G protein-coupled receptors, receptor tyrosine kinases, and ion channels, which were previously not suitable for high-throughput screening by flow cytometry. The system has been validated using the β2-adrenergic receptor (β2AR) system and extended to other GPCRs. When a chemical library containing ∼ 1200 off-patent drugs was screened against cells expressing FAP-tagged β2AR, all known β2AR active ligands in the library were successfully identified, together with a few compounds that were later confirmed to regulate receptor internalization in a nontraditional manner. The unexpected discovery of new ligands by this approach indicates the potential of using this protocol for GPCR de-orphanization. In addition, screens of multiplexed targets promise improved efficiency with minor protocol modification.

  4. A negative dielectrophoresis and gravity-driven flow-based high-throughput and high-efficiency cell-sorting system.

    PubMed

    Lee, Dongkyu; Kim, Dowon; Kim, Youngwoong; Park, Ki-Hyun; Oh, Eun-Jee; Kim, Yonggoo; Kim, Byungkyu

    2014-02-01

    We present a negative dielectrophoresis (n-DEP)-based cell separation system for high-throughput and high-efficiency cell separation. To achieve a high throughput, the proposed system comprises macro-sized channel and cantilever-type electrode (CE) arrays (L × W × H = 150 µm × 500 µm × 50 µm) to generate n-DEP force. For high efficiency, double separation modules, which have macro-sized channels and CE arrays in each separation module, are employed. In addition, flow regulators to precisely control the hydrodynamic force are allocated for each outlet. Because the hydrodynamic force and the n-DEP force acting on the target cell are the main determinants of the separation efficiency, we evaluate the theoretical amount of hydrodynamic force and n-DEP force acting on each target cell. Based on theoretical results, separation conditions are experimentally investigated. Finally, to demonstrate the separation performance, we performed the separation of target cells (live K562) from nontarget cells (dead K562) under conditions of low voltage (7Vp-p with 100 kHz) and a flow rate of 15 µL•min⁻¹, 6 µL•min⁻¹, and 8 µL•min⁻¹ in outlets 1, 2, and 3, respectively. The system can separate target cells with 95% separation efficiency in the case of the ratio of 5:1 (live K562:dead K562).

  5. How we assess adequacy of fine-needle aspiration materials intended for flow cytometric analysis.

    PubMed

    Brahimi, M; Arabi, A; Soltan, B E; Osmani, S; Benradouane, H; Bey, M; Yafour, N; Benzineb, B; Attaf, F; Seddiki, I; Rahal, S; Bekadja, M A

    2011-01-01

    Many articles have been published on the subject of FNAFNA, highlighting the usefulness of flow cytometry in the diagnosis and classification of lymphomas. But occasionally, flow cytometric evaluation fails to detect an abnormal population in a FNAFNA specimen involved by lymphoid neoplasm. Sampling errors (poor viability, peripheral blood contamination and hypocellular specimens) are the major reasons of this failure. In our laboratory we use a simple, fast and cost-effective approach to assess adequacy of FNAFNA materials and in this paper, we describe this procedure with giving some examples of interpretations of our results.

  6. An Automated High-Throughput Cell-Based Multiplexed Flow Cytometry Assay to Identify Novel Compounds to Target Candida albicans Virulence-Related Proteins

    PubMed Central

    Bernardo, Stella M.; Allen, Christopher P.; Waller, Anna; Young, Susan M.; Oprea, Tudor; Sklar, Larry A.; Lee, Samuel A.

    2014-01-01

    Although three major classes of systemic antifungal agents are clinically available, each is characterized by important limitations. Thus, there has been considerable ongoing effort to develop novel and repurposed agents for the therapy of invasive fungal infections. In an effort to address these needs, we developed a novel high-throughput, multiplexed screening method that utilizes small molecules to probe candidate drug targets in the opportunistic fungal pathogen Candida albicans. This method is amenable to high-throughput automated screening and is based upon detection of changes in GFP levels of individually tagged target proteins. We first selected four GFP-tagged membrane-bound proteins associated with virulence or antifungal drug resistance in C. albicans. We demonstrated proof-of-principle that modulation of fluorescence intensity can be used to assay the expression of specific GFP-tagged target proteins to inhibitors (and inducers), and this change is measurable within the HyperCyt automated flow cytometry sampling system. Next, we generated a multiplex of differentially color-coded C. albicans strains bearing C-terminal GFP-tags of each gene encoding candidate drug targets incubated in the presence of small molecules from the Prestwick Chemical Library in 384-well microtiter plate format. Following incubation, cells were sampled through the HyperCyt system and modulation of protein levels, as indicated by changes in GFP-levels of each strain, was used to identify compounds of interest. The hit rate for both inducers and inhibitors identified in the primary screen did not exceed 1% of the total number of compounds in the small-molecule library that was probed, as would be expected from a robust target-specific, high-throughput screening campaign. Secondary assays for virulence characteristics based on null mutant strains were then used to further validate specificity. In all, this study presents a method for the identification and verification of new

  7. Flow Cytometric Assays for Interrogating LAGLIDADG Homing Endonuclease DNA-Binding and Cleavage Properties

    PubMed Central

    Baxter, Sarah K.; Lambert, Abigail R.; Scharenberg, Andrew M.; Jarjour, Jordan

    2014-01-01

    A fast, easy, and scalable method to assess the properties of site-specific nucleases is crucial to understanding their in cellulo behavior in genome engineering or population-level gene drive applications. Here we describe an analytical platform that enables high-throughput, semiquantitative interrogation of the DNA-binding and catalytic properties of LAGLIDADG homing endonucleases (LHEs). Using this platform, natural or engineered LHEs are expressed on the surface of Saccharomyces cerevisiae yeast where they can be rapidly evaluated against synthetic DNA target sequences using flow cytometry. PMID:23423888

  8. An improved cell separation technique for marine subsurface sediments: applications for high-throughput analysis using flow cytometry and cell sorting

    PubMed Central

    Morono, Yuki; Terada, Takeshi; Kallmeyer, Jens; Inagaki, Fumio

    2013-01-01

    Summary Development of an improved technique for separating microbial cells from marine sediments and standardization of a high-throughput and discriminative cell enumeration method were conducted. We separated microbial cells from various types of marine sediment and then recovered the cells using multilayer density gradients of sodium polytungstate and/or Nycodenz, resulting in a notably higher percent recovery of cells than previous methods. The efficiency of cell extraction generally depends on the sediment depth; using the new technique we developed, more than 80% of the total cells were recovered from shallow sediment samples (down to 100 meters in depth), whereas ∼ 50% of cells were recovered from deep samples (100–365 m in depth). The separated cells could be rapidly enumerated using flow cytometry (FCM). The data were in good agreement with those obtained from manual microscopic direct counts over the range 104–108 cells cm−3. We also demonstrated that sedimentary microbial cells can be efficiently collected using a cell sorter. The combined use of our new cell separation and FCM/cell sorting techniques facilitates high-throughput and precise enumeration of microbial cells in sediments and is amenable to various types of single-cell analyses, thereby enhancing our understanding of microbial life in the largely uncharacterized deep subseafloor biosphere. PMID:23731283

  9. A Computational Framework to Emulate the Human Perspective in Flow Cytometric Data Analysis

    PubMed Central

    Ray, Surajit; Pyne, Saumyadipta

    2012-01-01

    Background In recent years, intense research efforts have focused on developing methods for automated flow cytometric data analysis. However, while designing such applications, little or no attention has been paid to the human perspective that is absolutely central to the manual gating process of identifying and characterizing cell populations. In particular, the assumption of many common techniques that cell populations could be modeled reliably with pre-specified distributions may not hold true in real-life samples, which can have populations of arbitrary shapes and considerable inter-sample variation. Results To address this, we developed a new framework flowScape for emulating certain key aspects of the human perspective in analyzing flow data, which we implemented in multiple steps. First, flowScape begins with creating a mathematically rigorous map of the high-dimensional flow data landscape based on dense and sparse regions defined by relative concentrations of events around modes. In the second step, these modal clusters are connected with a global hierarchical structure. This representation allows flowScape to perform ridgeline analysis for both traversing the landscape and isolating cell populations at different levels of resolution. Finally, we extended manual gating with a new capacity for constructing templates that can identify target populations in terms of their relative parameters, as opposed to the more commonly used absolute or physical parameters. This allows flowScape to apply such templates in batch mode for detecting the corresponding populations in a flexible, sample-specific manner. We also demonstrated different applications of our framework to flow data analysis and show its superiority over other analytical methods. Conclusions The human perspective, built on top of intuition and experience, is a very important component of flow cytometric data analysis. By emulating some of its approaches and extending these with automation and rigor

  10. High throughput continuous titration based on a flow ratiometry controlled with feedback-based variable triangular waves and subsequent fixed triangular waves.

    PubMed

    Tanaka, Hideji; Baba, Tsutomu

    2005-10-15

    We propose a new approach for high-throughput continuous titration based on a flow ratiometry. The method was applied to potentiometric titrations of acids and bases. A base solution, the flow rate F(B) of which was varied in response to controller output voltage V(c), was merged with an acid solution under constant total (acid+base) flow rate. Downstream, the pH of the mixed solution was measured with a flow-through glass electrode. Initially, V(c), and thus F(B) was increased linearly. At the instant the equivalence point (EP) was sensed, the ramp direction of V(c) was reversed from upward to downward. The direction was reversed to upward when EP was sensed again. Such the feedback-based operation gives a triangular waveform of V(c), because there is a delay corresponding mainly to the transit time of merged solutions to reach the sensor. The value of V(c) that gives EP composition, V(E), was estimated by averaging the most recent maximum and minimum values of V(c). Next, fixed triangular waves of V(c) was used to control F(B). The amplitude and the scan rate of the waves were fixed narrower and faster, respectively, than those in the feedback-based operation in order to improve the throughput rate. The EP can be located as long as the scan range covers V(E). These automated processes limited the titration to just the narrow range around EP, and thus realized extremely high throughput rate of maximally 17.6 titrations per minute (=3.4s per titration) at R.S.D.=0.35%.

  11. Flow cytometric analysis of microbial contamination in food industry technological lines--initial study.

    PubMed

    Józwa, Wojciech; Czaczyk, Katarzyna

    2012-04-02

    Flow cytometry constitutes an alternative for traditional methods of microorganisms identification and analysis, including methods requiring cultivation step. It enables the detection of pathogens and other microorganisms contaminants without the need to culture microbial cells meaning that the sample (water, waste or food e.g. milk, wine, beer) may be analysed directly. This leads to a significant reduction of time required for analysis allowing monitoring of production processes and immediate reaction in case of contamination or any disruption occurs. Apart from the analysis of raw materials or products on different stages of manufacturing process, the flow cytometry seems to constitute an ideal tool for the assessment of microbial contamination on the surface of technological lines. In the present work samples comprising smears from 3 different surfaces of technological lines from fruit and vegetable processing company from Greater Poland were analysed directly with flow cytometer. The measured parameters were forward and side scatter of laser light signals allowing the estimation of microbial cell contents in each sample. Flow cytometric analysis of the surface of food industry production lines enable the preliminary evaluation of microbial contamination within few minutes from the moment of sample arrival without the need of sample pretreatment. The presented method of fl ow cytometric initial evaluation of microbial state of food industry technological lines demonstrated its potential for developing a robust, routine method for the rapid and labor-saving detection of microbial contamination in food industry.

  12. Flow cytometric analysis and chromosome sorting of barley (hordeum vulgare L).

    PubMed

    Lee, J H; Arumuganathan, K; Chung, Y S; Kim, K Y; Chung, W B; Bae, K S; Kim, D H; Chung, D S; Kwon, O C

    2000-12-31

    Flow cytometric analysis was systematically performed to optimize the concentration and duration of hydroxyurea (DNA synthesis inhibitor) and trifluralin (metaphase blocking reagent) treatments for synchronizing the cell cycle and accumulating metaphase chromosomes in barley root tips. A high metaphase index (76.5% in the root tip meristematic area) was routinely achieved. Seedlings of about 1.0-cm length were treated with 1.25 mM hydroxyurea for 14 h to synchronize the root tip meristem cells at the S/G2 phase. After rinsing with hydroxyurea, the seedlings were incubated in a hydroxyurea-free solution for 2 h and were treated with 1 microM trifluralin for 4 h to accumulate mitotic cells in the metaphase. The consistent high metaphase index depended on the uniform germination of seeds prior to treatment. High-quality and high-quantity isolated metaphase chromosomes were suitable for flow cytometric analysis and sorting. Flow karyotypes of barley chromosomes were established via univariate and bivariate analysis. A variation of flow karyotypes was detected among barley lines. Two single chromosome types were identified and sorted. Bivariate analysis showed no variation among barley individual chromosomes in AT and GC content.

  13. Rapid detection of haemotropic mycoplasma infection of feline erythrocytes using a novel flow cytometric approach

    PubMed Central

    2013-01-01

    Background The haemotropic mycoplasmas Mycoplasma haemofelis and Candidatus Mycoplasma haemominutum cause feline infectious anaemia with infection rates in feline populations reflecting widespread subclinical infection. Clinically significant infections are much rarer but can be life-threatening. Current diagnosis is dependent upon visualising organisms in stained blood smears, PCR or quantitative PCR (qPCR). These procedures are labour-intensive and time-consuming. Furthermore, PCR-based approaches offer limited insight into the disease burden of the infected animal. Methods We have developed a novel and rapid flow cytometric system that permits diagnosis of haemotropic mycoplasma infections and quantitation of the percentage of erythrocytes that are parasitized. The method exploits the fact that mature mammalian erythrocytes, the host cell for haemoplasmas, are enucleated and thus lack nucleic acid. DRAQ5 is a synthetic anthrocycline dye which rapidly crosses cell membranes and binds to nucleic acids. The presence of exogenous bacterial DNA in mammalian erythrocytes can, therefore, be detected by DRAQ5 uptake and flow cytometric detection of DRAQ5 fluorescence. Results Here, we show that this system can detect epi-erythrocytic infection of companion felines by haemotropic mycoplasma. Due to their differences in size, and hence the quantity of DNA, the two major feline hemoplasmas M. haemofelis and Candidatus M. haemominutum can be distinguished according to DRAQ5 fluorescence. We have also shown the usefulness of DRAQ5 uptake in monitoring a cat infected with M. haemofelis sequentially during treatment with doxycycline. Conclusions The technique described is the first report of a flow cytometric method for detecting haemotropic mycoplasmas in any species and could be applied to widespread screening of animal populations to assess infection by these epi-erythrocytic parasites. PMID:23725366

  14. Flow cytometric detection and analysis of tailless sperm caused by sonication or a chemical agent.

    PubMed

    Yamamoto, T; Yoneyama, M; Imanishi, M; Takeuchi, M

    2000-02-01

    Flow cytometric analysis has been developed to detect tailless sperm with heads detached from the tails at the neck position. When isolated tailless sperm suspension was subjected to flow cytometry, a second sperm population appeared alongside the normal sperm population on light scatter-histogram. The percentage of this second sperm population (85.2%) was in good agreement with that for the tailless sperm (88.7%) determined microscopically, indicating that the second sperm population would correspond to tailless sperm population in the light scatter-histogram. Rates for tailless sperm determined by flow cytometry significantly correlated with those estimated microscopically following exposure of sperm to either sonication (r = 0.94, P < 0.01), or nitrobenzene (r = 0.80, P < 0.01). The results indicated the utility of the light scatter-histogram in flow cytometry as a simple and convenient procedure for the detection of tailless sperm induced by chemical compounds.

  15. Two and three-color fluorescence flow cytometric analysis of immunoidentified viable bacteria.

    PubMed

    Barbesti, S; Citterio, S; Labra, M; Baroni, M D; Neri, M G; Sgorbati, S

    2000-07-01

    Traditional culture methods well established in the past and still in use are not able to detect the environmental microorganisms that exist in a viable but not culturable state. A number of different fluorescence-based assays have been developed over the past decade to detect and identify viable bacteria in the environment. We have developed a simple and rapid method for measuring the number and viability of immunolabeled bacteria by means of a two/three color fluorescence flow cytometric analysis. After washing, cultured bacteria in suspension were labeled with a rabbit polyclonal antibody recognizing the wall lipopolysaccharide complex. A secondary biotinylated anti-rabbit polyclonal antibody was added allowing the cells to be labeled with the streptavidin R-phycoerythrin-Cyanine 5 (RPE-Cy5) fluorochrome. Before flow cytometric analysis, bacterial suspensions were stained with SYBR Green I and propidium iodide which stain all of the cells and the non viable ones, respectively. With the appropriate filter sets of both Bryte-HS (Bio-Rad, Hercules, CA) and FACScan (Becton Dickinson, San Jose, CA) flow cytometers, the measurement of separated green (SYBR Green I), orange-red (propidium iodide), and far red (RPE-Cy5) fluorescence was possible, allowing the enumeration of viable immunodetected bacteria. The entire protocol is completed in less than 3 h, offering numerous possibilities for rapid and precise analyses in sanitary, industrial, and environmental microbiology. Copyright 2000 Wiley-Liss, Inc.

  16. [Research advances in value of flow cytometric immunophenotyping in diagnosis and prognostic evaluation of myelodysplastic syndrome].

    PubMed

    Lu, Dan; Liu, Yan-Rong

    2013-06-01

    Myelodysplastic syndromes (MDS) are myeloid neoplasms characterized by dysplasia in one or more linages of cells and increased risk of development of acute myeloid leukemia (AML). Along with the deeply understanding of myelodysplastic syndrome, the diagnosis standards of this disease experienced a leap in essence: from a single standard of morphological test in FAB to multiple detecting means in WHO standard of 2008, flow cytometry has been proposed as an adjunctive diagnostic test in the 2007 Vienna standards and the 2008 WHO standards. Recently, A heterogeneous spectrum of immunophenotypic abnormalities have been reported in MDS, and some of which are of great significance to the diagnosis, classification, prognosis assessment, and treatment of the disease. In the year of 2003, a flow cytometric scoring system (FCSS) was built to evaluate the prognosis of MDS patients, which was able to qualify the phenotypic aberrancies in the myelomonocytic, erythroid, and megakaryocytic lineage. It filled the gap of the international prognostic scoring system (IPSS) and the WHO classification-based prognostic scoring system (WPSS), and was of great value to the clinical diagnosis and treatment of MDS. In this article, the value of MDS immunophenotyping in diagnosis and prognosis evaluation of MDS is reviewed in term of MDS immunophenotypic abnormalities and flow cytometric scoring system.

  17. Simultaneous flow cytometric evaluation of phagocytosis and oxidative burst in human polymorphonuclear cells.

    PubMed

    Horvathova, M; Wsolova, L; Jahnova, E

    2005-01-01

    Phagocytosis and oxidative burst (OXIBURST) activity of human polymorphonuclear cells (PMNs) has been simultaneously measured directly in whole blood samples. The ingestion of yeast was assessed by the phagocytosis activity (FA) and phagocytosis index (FI), and the respiratory burst of PMNs was determined as dihydroethidine (DHE) oxidation. We received comparable results in the ingestion of yeast cells by PMNs using either light microscopy (77.31+/-7.56) or flow cytometry detection method (78.26+/-5.14). The significant differences (p<0.05) in FI and OXIBURST activity were find in the patients (2.29+/-0.29 and 14.67+/-3.99, respectively) when compared to healthy donors (1.64+/-0.21 and 32.38+/-14.94, respectively). The two-color flow cytometric procedure permits measurement of two different functions of neutrophils in one step. This flow cytometric procedure is simple, rapid and has the potential to be an alternative assay to test leukocyte function. (Fig. 3, Ref: 30.)

  18. Six-flow operations for catalyst development in Fischer-Tropsch synthesis: Bridging the gap between high-throughput experimentation and extensive product evaluation

    SciTech Connect

    Sartipi, Sina E-mail: J.Gascon@tudelft.nl; Jansma, Harrie; Bosma, Duco; Boshuizen, Bart; Makkee, Michiel; Gascon, Jorge E-mail: J.Gascon@tudelft.nl; Kapteijn, Freek

    2013-12-15

    Design and operation of a “six-flow fixed-bed microreactor” setup for Fischer-Tropsch synthesis (FTS) is described. The unit consists of feed and mixing, flow division, reaction, separation, and analysis sections. The reactor system is made of five heating blocks with individual temperature controllers, assuring an identical isothermal zone of at least 10 cm along six fixed-bed microreactor inserts (4 mm inner diameter). Such a lab-scale setup allows running six experiments in parallel, under equal feed composition, reaction temperature, and conditions of separation and analysis equipment. It permits separate collection of wax and liquid samples (from each flow line), allowing operation with high productivities of C5+ hydrocarbons. The latter is crucial for a complete understanding of FTS product compositions and will represent an advantage over high-throughput setups with more than ten flows where such instrumental considerations lead to elevated equipment volume, cost, and operation complexity. The identical performance (of the six flows) under similar reaction conditions was assured by testing a same catalyst batch, loaded in all microreactors.

  19. The Clinical Value of Flow Cytometric DNA Content Analysis in Patients with Soft Tissue Sarcomas

    PubMed Central

    Samur, Mustafa; Pamir, Ali; Erekul, Selim; Sağlik, Yener; Yildiz, Yusuf; Dinçol, Dilek; Içli, Fikri

    1999-01-01

    Purpose. The purpose of this study was to evaluate: (1) the correlation between grade and ploidy or S-phase fraction (SPF), (2) the prognostic value of DNA flow cytometric study in soft tissue sarcomas. Patients /Methods. In all, 47 tissue samples from soft tissue sarcoma patients, surgically treated in the same center, were included. Flow cytometric analyses were performed according to a modified version of the original method of Hedley et al. Results. DNA ploidy status could be determined in 44 samples out of 47 (success rate 94%). Of these 44, S-phase fraction could be calculated in 34 samples (77%). In the study group as a whole, aneuploidy was significantly correlated with high grade. Survival analyses were carried out in 21 patients with soft tissue sarcoma, all surgically treated in the same center, without chemotherapy or radiotherapy. In univariate analyses, DNA ploidy was found to be a significant factor for overall survival (OAS) and metastasis-free survival MFS. Mean OAS for aneuploid tumors and diploid tumors were 35 and 65 months (p=0.034), and mean MFS 23 and 61 months, respectively (p=0.005) . Discussion.There is a relation between histological grade and ploidy in soft tissue sarcomas. It appears that low-grade tumors are generally diploid, whereas high-grade tumors tend to be aneuploid. In a subgroup of patients treated only with surgery, DNA ploidy was found to be an important factor for predicting OAS and MFS. PMID:18521281

  20. Flow Cytometric Methods to Investigate Culture Heterogeneities for Plant Metabolic Engineering

    PubMed Central

    Gaurav, Vishal; Kolewe, Martin E.; Roberts, Susan C.

    2014-01-01

    Plant cell cultures provide an important method for production and supply of a variety of natural products, where conditions can be easily controlled, manipulated and optimized. Development and optimization of plant cell culture processes require both bioprocess engineering and metabolic engineering approaches. Cultures are generally highly heterogeneous, with significant variability amongst cells in terms of growth, metabolism and productivity of key metabolites. Taxus cultures produce the important anti-cancer agent Taxol® (i.e., paclitaxel) and have demonstrated significant variability amongst cell populations in culture with regards to paclitaxel accumulation, cell cycle participation and protein synthesis. To fully understand the link between cellular metabolism and culture behavior and to enable targeted metabolic engineering approaches, cultures need to be studied at a single cell level. This chapter describes the application of plant cell flow cytometric techniques to investigate culture heterogeneity at the single cell level, in order to optimize culture performance through targeted metabolic engineering. Flow cytometric analytical methods are described to study Taxus single cells, protoplasts and nuclei suspensions with respect to secondary metabolite accumulation, DNA content, cell size and complexity. Reproducible methods to isolate these single particle suspensions from aggregated Taxus cultures are discussed. Methods to stain both fixed and live cells for a variety of biological markers are provided to enable characterization of cell phenotypes. Fluorescence-activated cell sorting (FACS) methods are also presented to facilitate isolation of certain plant cell culture populations for both analysis and propagation of superior cell lines for use in bioprocesses. PMID:20552456

  1. Flow cytometric DNA analysis of parathyroid glands. Relationship between nuclear DNA and pathologic classifications.

    PubMed Central

    Bowlby, L. S.; DeBault, L. E.; Abraham, S. R.

    1987-01-01

    Nuclear DNA contents of 95 paraffin-embedded parathyroid glands (2 carcinomas, 56 adenomas, 10 primary and 17 secondary chief cell hyperplasias, and 10 normal glands) were determined by flow cytometric analysis. All normal parathyroid glands and secondary hyperplasias, 80% of the primary hyperplasias, and 73% of the adenomas had diploid DNA patterns, with 15% or less tetraploid cells. Twenty-one percent of the adenomas, 29% of the primary hyperplasias, and all carcinomas had diploid and tetraploid DNA distribution patterns, with greater than 15% of the cells in the tetraploid region. One of the carcinomas (Figure 4B, Case 2) had an additional near-triploid aneuploid peak. Three of the adenomas (5.4%) had near-triploid aneuploid patterns. One of the patients with carcinoma (Figure 4A, Case 1) died, 32 months after initial diagnosis, of disease-related causes. The remaining patient with carcinoma (Case 2) had a 47-month disease-free interval. All of the patients with hyperplastic and adenomatous glands are free of disease, after a mean follow-up interval of 25 months. This study indicated that flow cytometric analysis of nuclear DNA content does not complement conventional pathologic methods in distinguishing between parathyroid gland chief cell hyperplasia, adenoma, or carcinoma; however, it did suggest the possibility that parathyroid adenomas and primary chief cell hyperplasias may contain a subset of tumors that could manifest biologic malignancy if allowed to progress untreated. PMID:3618729

  2. Flow cytometric methods to investigate culture heterogeneities for plant metabolic engineering.

    PubMed

    Gaurav, Vishal; Kolewe, Martin E; Roberts, Susan C

    2010-01-01

    Plant cell cultures provide an important method for production and supply of a variety of natural products, where conditions can be easily controlled, manipulated, and optimized. Development and optimization of plant cell culture processes require both bioprocess engineering and metabolic engineering approaches. Cultures are generally highly heterogeneous, with significant variability amongst cells in terms of growth, metabolism, and productivity of key metabolites. Taxus cultures produce the important anti-cancer agent Taxol((R)) (i.e., paclitaxel) and have demonstrated significant variability amongst cell populations in culture with regard to paclitaxel accumulation, cell cycle participation, and protein synthesis. To fully understand the link between cellular metabolism and culture behavior and to enable targeted metabolic engineering approaches, cultures need to be studied at a single cell level. This chapter describes the application of plant cell flow cytometric techniques to investigate culture heterogeneity at the single cell level, in order to optimize culture performance through targeted metabolic engineering. Flow cytometric analytical methods are described to study Taxus single cells, protoplasts, and nuclei suspensions with respect to secondary metabolite accumulation, DNA content, cell size, and complexity. Reproducible methods to isolate these single particle suspensions from aggregated Taxus cultures are discussed. Methods to stain both fixed and live cells for a variety of biological markers are provided to enable characterization of cell phenotypes. Fluorescence-activated cell sorting (FACS) methods are also presented to facilitate isolation of certain plant cell culture populations for both analysis and propagation of superior cell lines for use in bioprocesses.

  3. Flow cytometric techniques for detection of candidate cancer stem cell subpopulations in canine tumour models.

    PubMed

    Blacking, T M; Waterfall, M; Samuel, K; Argyle, D J

    2012-12-01

    The cancer stem cell (CSC) hypothesis proposes that tumour growth is maintained by a distinct subpopulation of 'CSC'. This study applied flow cytometric methods, reported to detect CSC in both primary and cultured cancer cells of other species, to identify candidate canine subpopulations. Cell lines representing diverse canine malignancies, and cells derived from spontaneous canine tumours, were evaluated for expression of stem cell-associated surface markers (CD34, CD44, CD117 and CD133) and functional properties [Hoecsht 33342 efflux, aldehyde dehydrogenase (ALDH) activity]. No discrete marker-defined subsets were identified within established cell lines; cells derived directly from spontaneous tumours demonstrated more heterogeneity, although this diminished upon in vitro culture. Functional assays produced variable results, suggesting context-dependency. Flow cytometric methods may be adopted to identify putative canine CSC. Whilst cell lines are valuable in assay development, primary cells may provide a more rewarding model for studying tumour heterogeneity in the context of CSC. However, it will be essential to fully characterize any candidate subpopulations to ensure that they meet CSC criteria.

  4. Factors affecting flow cytometric detection of apoptotic nuclei by DNA analysis

    SciTech Connect

    Elstein, K.H.; Thomas, D.J.; Zucker, R.M.

    1995-10-01

    Apoptotic thymocyte nuclei normally appear on a flow cytometric DNA histogram as a subdiploid peak. We observed that addition of a specific RNase A preparation to the detergent-based lysing buffer increased the fluorescence of toxicant-induced apoptotic nuclei to the level of untreated diploid nuclei. The chelating agent EDTA partially inhibited the RNase effect, suggesting contaminating divalent cations may have been involved. Moreover, spectrofluorometric analysis revealed that addition of RNase or divalent cations decreased the amount of DNA present in the lysate. This suggested that the upscale fluorescence shift was due to a decrease in the ability of the lysing buffer to extract DNA, possibly as a result of cation-induced chromatin condensation, rather than increased accessibility of fluorochrome binding sites due to apoptotic degeneration. Moreover, during a 16-h culture, we observed a similar, but time-dependent, upscale shift in the fluorescence of thymocytes undergoing apoptosis either spontaneously or as a result of exposure to 1 {mu}M tributyltin methoxide (TBT), 2% ethanol, 2% methanol, or 1 {mu}M dexamethasone phosphate (DEX). This commonality of effect suggests that a similar magnitude of chromatin reorganization occurs in apoptotic cells in prolonged culture regardless of the method of apoptotic induction. These findings should alert investigators to potential inaccuracies in the flow cytometric quantitation of apoptosis in vitro systems employing prolonged toxicant exposures or complex lysing cocktails that may contain active contaminants. 37 refs., 3 figs., 1 tab.

  5. Flow cytometric analysis of cell-surface and intracellular antigens in the diagnosis of acute leukemia.

    PubMed

    Paredes-Aguilera, R; Romero-Guzman, L; Lopez-Santiago, N; Burbano-Ceron, L; Camacho-Del Monte, O; Nieto-Martinez, S

    2001-10-01

    To evaluate the usefulness of flow cytometric detection of intracellular antigens (Ags) in establishing proper lineage affiliation and its contribution to the diagnosis of acute leukemia, we studied 100 consecutive patients in whom acute leukemia was diagnosed between January 1997 and July 1998. Immunological classification was assessed using a three-line panel of monoclonal antibodies for phenotypic characterization of leukemic blast cells as proposed at the First Latin American Consensus Conference for Flow Cytometric Immunophenotyping of Leukemia. We found 74 cases of B-cell lineage acute lymphoblastic leukemia (ALL), seven cases of T-cell ALL, and 19 cases of acute myeloid leukemia (AML). In this study cytoplasmic (cy) CD79a, cyCD22, cyCD3, and cyMPO were highly sensitive, specific B, T, and myeloid markers that were expressed in virtually all cases of B and T cell ALL and in all subtypes of AML. Applied in combination with immunophenotyping this knowledge led to improvement in diagnostic precision and refinement of immunological classification, ensuring the selection of the most appropriate therapy for the patients studied. In conclusion, intracellular Ags detection was of utmost importance in establishing correct lineage affiliation in cases lacking expression of B, T, or myeloid surface Ags or disclosing equivocal or ambiguous immunophenotypic features and in identifying biphenotypic acute leukemia. In combination with FAB morphology and immunophenotyping, we were able to reliably classify all patients with acute leukemia in this study.

  6. Vortex-dislodged cells from bone marrow trephine biopsy yield satisfactory results for flow cytometric immunophenotyping.

    PubMed

    Bommannan, K; Sachdeva, M U S; Gupta, M; Bose, P; Kumar, N; Sharma, P; Naseem, S; Ahluwalia, J; Das, R; Varma, N

    2016-10-01

    A good bone marrow (BM) sample is essential in evaluating many hematologic disorders. An unsuccessful BM aspiration (BMA) procedure precludes a successful flow cytometric immunophenotyping (FCI) in most hematologic malignancies. Apart from FCI, most ancillary diagnostic techniques in hematology are less informative. We describe the feasibility of FCI in vortex-dislodged cell preparation obtained from unfixed trephine biopsy (TB) specimens. In pancytopenic patients and dry tap cases, routine diagnostic BMA and TB samples were complemented by additional trephine biopsies. These supplementary cores were immediately transferred into sterile tubes filled with phosphate-buffered saline, vortexed, and centrifuged. The cell pellet obtained was used for flow cytometric immunophenotyping. Of 7955 BMAs performed in 42 months, 34 dry tap cases were eligible for the study. Vortexing rendered a cell pellet in 94% of the cases (32 of 34), and FCI rendered a rapid diagnosis in 100% of the cases (32 of 32) where cell pellets were available. We describe an efficient procedure which could be effectively utilized in resource-limited centers and reduce the frequency of repeat BMA procedures. © 2016 John Wiley & Sons Ltd.

  7. Flow cytometric determination of residual white blood cell levels in preserved samples from leukoreduced blood products.

    PubMed

    Palmer, Douglas S; Birch, Paul; O'Toole, Joan; Henderson, Deborah; Scalia, Vito

    2008-01-01

    In preparation for a proposed consolidated testing service, Canadian Blood Services undertook the evaluation of a commercial test kit for the enumeration by flow cytometry of residual white blood cells (rWBCs) present in preserved samples recovered from leukoreduced (LR) blood and platelet products. The stability of preserved WBCs, the equivalency of WBCs used for spiking, test method precision, specificity, reliability, accuracy, and sensitivity were investigated. For comparative purposes, WBC counts were also determined by Nageotte as well as by flow cytometry. WBCs were stable up to 4 weeks at room temperature for all components by either method. Within methods, no differences were observed due to the source of WBC used for spiking purposes. By either method, test precision was acceptable (<20% coefficient of variation) and of similar reliability at a target value of 10 +/- 5 WBCs per microL. The flow cytometric method was shown to be more specific and accurate than the Nageotte method. Sensitivity by either method was 0.1 WBCs per microL. On average, Nageotte counts were lower than those observed by flow cytometry. These results demonstrate that WBCs in WBC stabilizing solution-treated samples from LR blood components were stabilized up to 4 weeks at room temperature and that rWBC determinations made with a WBC enumeration kit by flow cytometry have the required precision, specificity, reliability, and accuracy in the relevant test range. This validated WBC stabilization and flow cytometric counting method is considered acceptable as part of a quality control program for leukoreduced blood products.

  8. High-throughput determination of cortisol, cortisone, and melatonin in oral fluid by on-line turbulent flow liquid chromatography interfaced with liquid chromatography/tandem mass spectrometry.

    PubMed

    Fustinoni, Silvia; Polledri, Elisa; Mercadante, Rosa

    2013-07-15

    Cortisol, cortisone, and melatonin (CORTol, CORTone, and MELA, respectively) are hormones related to stress and sleep disorders. Their detection is relevant to epidemiological studies aimed at investigating the effects of circadian cycle disruption. The aim of this study was to develop and evaluate a high-throughput assay for the detection of CORTol, CORTone, and MELA concentrations in non-invasively collected oral fluid samples. A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method to measure levels of CORTol, CORTone, and MELA in oral fluid samples in the presence of deuterated analogs was optimized and validated. A 50 μL aliquot of oral fluid sample, obtained by centrifugation of a chewed swab, was purified using on-line turbulent flow liquid chromatography. Analytes were then separated using C18 reversed-phase chromatography, subjected to positive ionization using an electrospray source, then quantitated using a triple quadrupole mass detector in the selected reaction monitoring mode. Limits of quantification and linear dynamic ranges were found to be 0.55 nmol/L, 5.5 nmol/L, and 0.004 nmol/L, and up to 28 nmol/L, 277 nmol/L, and 0.43 nmol/L for CORTol, CORTone, and MELA, respectively. Inter- and intra-run precisions as relative standard deviation values were <5%, and accuracies were within 95-106% of theoretical concentrations. An evaluation of matrix effects showed that the use of deuterated analogs controlled sources of bias. Furthermore, the total analysis time per sample was 13 min, resulting in a throughput of approximately 100 samples/day. To our knowledge, this is the first automated, high-throughput assay for the simultaneous quantification of CORTol, CORTone, and MELA in oral fluid specimens. Copyright © 2013 John Wiley & Sons, Ltd.

  9. Flow cytometric analysis of dengue virus-infected cells in peripheral blood.

    PubMed

    Baclig, Michael O; Gervacio, Leonora T S; Suarez, Lady-Anne C; Buerano, Corazon C; Matias, Ronald R; Kumatori, Atsushi; Inoue, Shingo; Morita, Kouichi; Natividad, Filipinas F; Hasebe, Futoshi

    2010-11-01

    With the development of permeabilization techniques in flow cytometry and the availability of various monoclonal antibodies (MAbs) that specifically bind with cell surface and intracellular antigens, it is now possible to use flow cytometric assay to identify dengue virus (DEN) infected cells in peripheral blood. Blood samples were analyzed using phycoerythrin (PE) labeled anti-CD3, anti-CD14, anti-CD16, and anti-CD19 antibodies and Alexa Fluor 488 labeled anti-flavivirus monoclonal antibody (MAb) 6B6C-1. The predominant DEN-infected cells were CD19+ in this study. There was dim partial to moderately bright partial expression of CD19 positive cells in the blood samples tested. Virus isolation and serotype-specific RT-PCR revealed the cells were infected with dengue serotype 3 (DEN3). Our results suggest B cells may play an important role in DEN1 and DEN3 replication, and dissemination in vivo.

  10. Procarbazine effects on spermatogenesis in golden hamster: a flow cytometric evaluation.

    PubMed

    Weissenberg, R; Golan, R; Shochat, L; Lewin, L M

    2002-01-01

    The response of hamster testis to the administration of 450mg/kg procarbazine (PCB) over a period of 4 weeks was evaluated. Flow cytometry was used to investigate changes in cell populations in testicular single cell suspensions and to correlate these changes with those observed in histological sections. PCB caused significant decrease in testicular and epididymal weight and a drastic reduction in haploid cells and spermatogenic arrest, demonstrating variation among the test animals. The results obtained confirm previous observations concerning detrimental effects of PCB upon spermatogenesis in species such as the rat and mouse, though its effect on hamster testis is milder and does not include the germinal stem cells. The histological evaluation of the testis showed a good correlation with flow cytometric evaluation, emphasizing the usefulness of this method in providing quantitative and rapid results.

  11. Time-Domain Microfluidic Fluorescence Lifetime Flow Cytometry for High-Throughput Förster Resonance Energy Transfer Screening

    PubMed Central

    Nedbal, Jakub; Visitkul, Viput; Ortiz-Zapater, Elena; Weitsman, Gregory; Chana, Prabhjoat; Matthews, Daniel R; Ng, Tony; Ameer-Beg, Simon M

    2015-01-01

    Sensing ion or ligand concentrations, physico-chemical conditions, and molecular dimerization or conformation change is possible by assays involving fluorescent lifetime imaging. The inherent low throughput of imaging impedes rigorous statistical data analysis on large cell numbers. We address this limitation by developing a fluorescence lifetime-measuring flow cytometer for fast fluorescence lifetime quantification in living or fixed cell populations. The instrument combines a time-correlated single photon counting epifluorescent microscope with microfluidics cell-handling system. The associated computer software performs burst integrated fluorescence lifetime analysis to assign fluorescence lifetime, intensity, and burst duration to each passing cell. The maximum safe throughput of the instrument reaches 3,000 particles per minute. Living cells expressing spectroscopic rulers of varying peptide lengths were distinguishable by Förster resonant energy transfer measured by donor fluorescence lifetime. An epidermal growth factor (EGF)-stimulation assay demonstrated the technique's capacity to selectively quantify EGF receptor phosphorylation in cells, which was impossible by measuring sensitized emission on a standard flow cytometer. Dual-color fluorescence lifetime detection and cell-specific chemical environment sensing were exemplified using di-4-ANEPPDHQ, a lipophilic environmentally sensitive dye that exhibits changes in its fluorescence lifetime as a function of membrane lipid order. To our knowledge, this instrument opens new applications in flow cytometry which were unavailable due to technological limitations of previously reported fluorescent lifetime flow cytometers. The presented technique is sensitive to lifetimes of most popular fluorophores in the 0.5–5 ns range including fluorescent proteins and is capable of detecting multi-exponential fluorescence lifetime decays. This instrument vastly enhances the throughput of experiments involving

  12. Time-domain microfluidic fluorescence lifetime flow cytometry for high-throughput Förster resonance energy transfer screening.

    PubMed

    Nedbal, Jakub; Visitkul, Viput; Ortiz-Zapater, Elena; Weitsman, Gregory; Chana, Prabhjoat; Matthews, Daniel R; Ng, Tony; Ameer-Beg, Simon M

    2015-02-01

    Sensing ion or ligand concentrations, physico-chemical conditions, and molecular dimerization or conformation change is possible by assays involving fluorescent lifetime imaging. The inherent low throughput of imaging impedes rigorous statistical data analysis on large cell numbers. We address this limitation by developing a fluorescence lifetime-measuring flow cytometer for fast fluorescence lifetime quantification in living or fixed cell populations. The instrument combines a time-correlated single photon counting epifluorescent microscope with microfluidics cell-handling system. The associated computer software performs burst integrated fluorescence lifetime analysis to assign fluorescence lifetime, intensity, and burst duration to each passing cell. The maximum safe throughput of the instrument reaches 3,000 particles per minute. Living cells expressing spectroscopic rulers of varying peptide lengths were distinguishable by Förster resonant energy transfer measured by donor fluorescence lifetime. An epidermal growth factor (EGF)-stimulation assay demonstrated the technique's capacity to selectively quantify EGF receptor phosphorylation in cells, which was impossible by measuring sensitized emission on a standard flow cytometer. Dual-color fluorescence lifetime detection and cell-specific chemical environment sensing were exemplified using di-4-ANEPPDHQ, a lipophilic environmentally sensitive dye that exhibits changes in its fluorescence lifetime as a function of membrane lipid order. To our knowledge, this instrument opens new applications in flow cytometry which were unavailable due to technological limitations of previously reported fluorescent lifetime flow cytometers. The presented technique is sensitive to lifetimes of most popular fluorophores in the 0.5-5 ns range including fluorescent proteins and is capable of detecting multi-exponential fluorescence lifetime decays. This instrument vastly enhances the throughput of experiments involving fluorescence

  13. Analysis of a High-Throughput Cone-and-Plate Apparatus for the Application of Defined Spatiotemporal Flow to Cultured Cells

    PubMed Central

    Spruell, Christopher

    2014-01-01

    The shear stresses derived from blood flow regulate many aspects of vascular and immunobiology. In vitro studies on the shear stress-mediated mechanobiology of endothelial cells have been carried out using systems analogous to the cone-and-plate viscometer in which a rotating, low-angle cone applies fluid shear stress to cells grown on an underlying, flat culture surface. We recently developed a device that could perform high-throughput studies on shear-mediated mechanobiology through the rotation of cone-tipped shafts in a standard 96-well culture plate. Here, we present a model of the three-dimensional flow within the culture wells with a rotating, cone-tipped shaft. Using this model we examined the effects of modifying the design parameters of the system to allow the device to create a variety of flow profiles. We first examined the case of steady-state flow with the shaft rotating at constant angular velocity. By varying the angular velocity and distance of the cone from the underlying plate we were able to create flow profiles with controlled shear stress gradients in the radial direction within the plate. These findings indicate that both linear and non-linear spatial distributions in shear stress can be created across the bottom of the culture plate. In the transition and “parallel shaft” regions of the system, the angular velocities needed to provide high levels of physiological shear stress (5 Pa) created intermediate Reynolds number Taylor-Couette flow. In some cases, this led to the development of a flow regime in which stable helical vortices were created within the well. We also examined the system under oscillatory and pulsatile motion of the shaft and demonstrated minimal time lag between the rotation of the cone and the shear stress on the cell culture surface. Biotechnol. Bioeng. 2013;110: 1782-1793. PMID:23280552

  14. Analysis of a high-throughput cone-and-plate apparatus for the application of defined spatiotemporal flow to cultured cells.

    PubMed

    Spruell, Christopher; Baker, Aaron B

    2013-06-01

    The shear stresses derived from blood flow regulate many aspects of vascular and immunobiology. In vitro studies on the shear stress-mediated mechanobiology of endothelial cells have been carried out using systems analogous to the cone-and-plate viscometer in which a rotating, low-angle cone applies fluid shear stress to cells grown on an underlying, flat culture surface. We recently developed a device that could perform high-throughput studies on shear-mediated mechanobiology through the rotation of cone-tipped shafts in a standard 96-well culture plate. Here, we present a model of the three-dimensional flow within the culture wells with a rotating, cone-tipped shaft. Using this model we examined the effects of modifying the design parameters of the system to allow the device to create a variety of flow profiles. We first examined the case of steady-state flow with the shaft rotating at constant angular velocity. By varying the angular velocity and distance of the cone from the underlying plate we were able to create flow profiles with controlled shear stress gradients in the radial direction within the plate. These findings indicate that both linear and non-linear spatial distributions in shear stress can be created across the bottom of the culture plate. In the transition and "parallel shaft" regions of the system, the angular velocities needed to provide high levels of physiological shear stress (5 Pa) created intermediate Reynolds number Taylor-Couette flow. In some cases, this led to the development of a flow regime in which stable helical vortices were created within the well. We also examined the system under oscillatory and pulsatile motion of the shaft and demonstrated minimal time lag between the rotation of the cone and the shear stress on the cell culture surface.

  15. Multiplexed labeling of viable cells for high-throughput analysis of glycine receptor function using flow cytometry.

    PubMed

    Gilbert, Daniel F; Wilson, John C; Nink, Virginia; Lynch, Joseph W; Osborne, Geoffrey W

    2009-05-01

    Flow cytometry is an important drug discovery tool because it permits high-content multiparameter analysis of individual cells. A new method dramatically enhanced screening throughput by multiplexing many discrete fixed cell populations; however, this method is not suited to assays requiring functional cellular responses. HEK293 cells were transfected with unique mutant glycine receptors. Mutant receptor expression was confirmed by coexpression of yellow fluorescent protein (YFP). Commercially available cell-permeant dyes were used to label each glycine receptor expressing mutant with a unique optical code. All encoded cell lines were combined in a single tube and analyzed on a flow cytometer simultaneously before and after the addition of glycine receptor agonist. We decoded multiplexed cells that expressed functionally distinct glycine receptor chloride channels and analyzed responses to glycine in terms of chloride-sensitive YFP expression. Here, data provided by flow cytometry can be used to discriminate between functional and nonfunctional mutations in the glycine receptor, a process accelerated by the use of multiplexing. Further, this data correlates to data generated using a microscopy-based technique. The present study demonstrates multiplexed labeling of live cells, to enable cell populations to be subject to further cell culture and experimentation, and compares the results with those obtained using live cell microscopy. (c) 2009 International Society for Advancement of Cytometry.

  16. Evaluation of flow-injection tandem mass spectrometry for rapid and high-throughput quantitative determination of B vitamins in nutritional supplements.

    PubMed

    Bhandari, Deepak; Van Berkel, Gary J

    2012-08-29

    The use of flow-injection electrospray ionization tandem mass spectrometry for rapid and high-throughput mass spectral analysis of selected B vitamins, viz., B1, B2, B3, B5, and B6, in nutritional formulations was demonstrated. A simple and rapid (~5 min) in-tube sample preparation was performed. Automated flow injection introduced 1 μL of the extracts directly into the mass spectrometer ion source without chromatographic separation. Sample-to-sample analysis time was 60 s. Quantitative capabilities of the flow-injection analysis were tested using the method of standard additions and SRM 3280. The quantity determined for each B vitamin in SRM 3280 was within the statistical range provided for the respective certified values. This approach was also applied to two different commercial vitamin supplement tablets and proved to be successful in the quantification of the selected B vitamins, as evidenced by an agreement with the label values and the results obtained using isotope dilution liquid chromatography/mass spectrometry.

  17. Evaluation of Flow-Injection Tandem Mass Spectrometry for Rapid and High-Throughput Quantitative Determination of B-Vitamins in Nutritional Supplements

    SciTech Connect

    Bhandari, Deepak; Van Berkel, Gary J

    2012-01-01

    The use of flow-injection electrospray ionization tandem mass spectrometry for rapid and high-throughput mass spectral analysis of selected B-vitamins, viz. B1, B2, B3, B5, and B6, in nutritional formulations was demonstrated. A simple and rapid (~5 min) in-tube sample preparation was performed by adding extraction solvent to a powdered sample aliquot followed by agitation, centrifugation, and filtration to recover an extract for analysis. Automated flow injection introduced 1 L of the extracts directly into the mass spectrometer ion source without chromatographic separation. Sample-to-sample analysis time was 60 s representing significant improvement over conventional liquid chromatography approaches which typically require 25-45 min, and often require more significant sample preparation procedures. Quantitative capabilities of the flow-injection analysis were tested using the method of standard additions and NIST standard reference material (SRM 3280) multivitamin/multielement tablets. The quantity determined for each B-vitamin in SRM 3280 was within the statistical range provided for the respective certified values. The same sample preparation and analysis approach was also applied to two different commercial vitamin supplement tablets and proved to be successful in the quantification of the selected B-vitamins as evidenced by an agreement with the labels values and the results obtained using isotope dilution liquid chromatography/mass spectrometry.

  18. Online turbulent flow extraction coupled with liquid chromatography-tandem mass spectrometry for high throughput screening of anabolic steroids in horse urine.

    PubMed

    Shin, Hyun Du; Suh, Joon Hyuk; Kim, Junghyun; Cho, Hyun-Deok; Lee, Su Duk; Han, Kwan Seok; Wang, Yu; Han, Sang Beom

    2017-10-25

    A high throughput method for simultaneous screening of anabolic steroids and their metabolites (4-esterendione, trenbolone, boldenone, oxandrolone, nandrolone, methandrostenolone, testosterone, 1-androstendione, ethisterone, normethandrolone, methyltestosterone, 16β-Hydroxystanozolol, epitestosterone, bolasterone, norethandrolone, danazol, stanozolol and androstadienone) in equine urine by online turbulent flow extraction coupled with liquid chromatography-tandem mass spectrometry was developed. The use of turbulent flow chromatography could simplify pretreatment of horse urine, which has complex matrices as well as high viscosity. The urine was extracted by mixed-mode cation exchange solid phase extraction, and hydrolyzed using β-glucuronidase/arylsulfatase. Then, the sample was automatically loaded on the TurboFlow Cyclone extraction column for removal of further matrix, followed by separation on a fused core C18 column before MS/MS detection. Optimization and validation of the method were discussed in detail. All analytes were rapidly detected within 10min with high sensitivity (picogram to nanogram per milliliter level), and no interference was observed. The linearity range was from 0.1-10ng/mL for nine steroids and 1.0-50ng/mL for the others, with correlation of coefficient values over 0.995. Precision and accuracy ranged from 0.1 to 14.5% and 1.7 to 12.4%, respectively. The developed method was successfully applied to the analysis of anabolic steroids in horse urine after administration of a model drug. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Establishment of a finite element model for extracting chemical reaction kinetics in a micro-flow injection system with high throughput sampling.

    PubMed

    Wu, Zeng-Qiang; Du, Wen-Bin; Li, Jin-Yi; Xia, Xing-Hua; Fang, Qun

    2015-08-01

    Numerical simulation can provide valuable insights for complex microfluidic phenomena coupling mixing and diffusion processes. Herein, a novel finite element model (FEM) has been established to extract chemical reaction kinetics in a microfluidic flow injection analysis (micro-FIA) system using high throughput sample introduction. To reduce the computation burden, the finite element mesh generation is performed with different scales based on the different geometric sizes of micro-FIA. In order to study the contribution of chemical reaction kinetics under non-equilibrium condition, a pseudo-first-order chemical kinetics equation is adopted in the numerical simulations. The effect of reactants diffusion on reaction products is evaluated, and the results demonstrate that the Taylor dispersion plays a determining role in the micro-FIA system. In addition, the effects of flow velocity and injection volume on the reaction product are also simulated. The simulated results agree well with the ones from experiments. Although gravity driven flow is used to the numerical model in the present study, the FEM model also can be applied into the systems with other driving forces such as pressure. Therefore, the established FEM model will facilitate the understanding of reaction mechanism in micro-FIA systems and help us to optimize the manifold of micro-FIA systems. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Measurement of platelet aggregation, independently of patient platelet count: a flow-cytometric approach.

    PubMed

    Vinholt, P J; Frederiksen, H; Hvas, A-M; Sprogøe, U; Nielsen, C

    2017-06-01

    Essentials Platelet function may influence bleeding risk in thrombocytopenia, but useful tests are needed. A flow cytometric platelet aggregation test independent of the patient platelet count was made. Platelet aggregation was reduced in thrombocytopenic patients with hematological cancer. High platelet aggregation ruled out bleeding tendency in thrombocytopenic patients. Background Methods for testing platelet aggregation in thrombocytopenia are lacking. Objective To establish a flow-cytometric test of in vitro platelet aggregation independently of the patient's platelet count, and examine the association of aggregation with a bleeding history in thrombocytopenic patients. Patients/methods We established a flow-cytometric assay of platelet aggregation, and measured samples from healthy individuals preincubated with antiplatelet drugs, and samples from two patients with inherited platelet disorders. Then, we included 19 healthy individuals and 20 patients with platelet counts of ≤ 50 × 10(9) L(-1) , diagnosed with acute myeloid leukemia or myelodysplastic syndrome. We measured platelet aggregation and platelet activation by platelet surface expression of activated glycoprotein IIb-IIIa, P-selectin and CD63 after addition of agonists: collagen-related peptide, thrombin receptor-activating peptide (TRAP), and ADP. Results The platelet aggregation assay showed a low intraserial coefficient of variation of ≤ 3%. Similar results were obtained for platelet-rich plasma and isolated platelets at platelet counts of > 10 × 10(9) L(-1) ; otherwise, platelet isolation was required. The platelet aggregation percentage decreased with increasing antiplatelet drug concentration. Platelet aggregation in patients was reduced as compared with healthy individuals: 42% (interquartile range [IQR] 27-58) versus 66% (IQR 60-67) for TRAP; 41% (IQR 25-48) versus 70% (IQR 69-72) for collagen-related peptide; and 44% (IQR 30-53) versus 65% (IQR 46-72) for ADP. Platelet activation after

  1. Improved Flow Cytometric Assessment Reveals Distinct Microvesicle (Cell-Derived Microparticle) Signatures in Joint Diseases

    PubMed Central

    György, Bence; Szabó, Tamás G.; Turiák, Lilla; Wright, Matthew; Herczeg, Petra; Lédeczi, Zsigmond; Kittel, Ágnes; Polgár, Anna; Tóth, Kálmán; Dérfalvi, Beáta; Zelenák, Gergő; Böröcz, István; Carr, Bob; Nagy, György; Vékey, Károly; Gay, Steffen; Falus, András; Buzás, Edit I.

    2012-01-01

    Introduction Microvesicles (MVs), earlier referred to as microparticles, represent a major type of extracellular vesicles currently considered as novel biomarkers in various clinical settings such as autoimmune disorders. However, the analysis of MVs in body fluids has not been fully standardized yet, and there are numerous pitfalls that hinder the correct assessment of these structures. Methods In this study, we analyzed synovial fluid (SF) samples of patients with osteoarthritis (OA), rheumatoid arthritis (RA) and juvenile idiopathic arthritis (JIA). To assess factors that may confound MV detection in joint diseases, we used electron microscopy (EM), Nanoparticle Tracking Analysis (NTA) and mass spectrometry (MS). For flow cytometry, a method commonly used for phenotyping and enumeration of MVs, we combined recent advances in the field, and used a novel approach of differential detergent lysis for the exclusion of MV-mimicking non-vesicular signals. Results EM and NTA showed that substantial amounts of particles other than MVs were present in SF samples. Beyond known MV-associated proteins, MS analysis also revealed abundant plasma- and immune complex-related proteins in MV preparations. Applying improved flow cytometric analysis, we demonstrate for the first time that CD3+ and CD8+ T-cell derived SF MVs are highly elevated in patients with RA compared to OA patients (p = 0.027 and p = 0.009, respectively, after Bonferroni corrections). In JIA, we identified reduced numbers of B cell-derived MVs (p = 0.009, after Bonferroni correction). Conclusions Our results suggest that improved flow cytometric assessment of MVs facilitates the detection of previously unrecognized disease-associated vesicular signatures. PMID:23185418

  2. Flow cytometric method for measuring chromatin fragmentation in fixed sperm from yellow perch (Perca flavescens).

    PubMed

    Jenkins, J A; Draugelis-Dale, R O; Pinkney, A E; Iwanowicz, L R; Blazer, V S

    2015-03-15

    Declining harvests of yellow perch, Perca flavescens, in urbanized watersheds of Chesapeake Bay have prompted investigations of their reproductive fitness. The purpose of this study was to establish a flow cytometric technique for DNA analysis of fixed samples sent from the field to provide reliable gamete quality measurements. Similar to the sperm chromatin structure assay, measures were made on the susceptibility of nuclear DNA to acid-induced denaturation, but used fixed rather than live or thawed cells. Nuclei were best exposed to the acid treatment for 1 minute at 37 °C followed by the addition of cold (4 °C) propidium iodide staining solution before flow cytometry. The rationale for protocol development is presented graphically through cytograms. Field results collected in 2008 and 2009 revealed DNA fragmentation up to 14.5%. In 2008, DNA fragmentation from the more urbanized watersheds was significantly greater than from reference sites (P = 0.026) and in 2009, higher percentages of haploid testicular cells were noted from the less urbanized watersheds (P = 0.032) indicating better reproductive condition at sites with less urbanization. For both years, total and progressive live sperm motilities by computer-assisted sperm motion analysis ranged from 19.1% to 76.5%, being significantly higher at the less urbanized sites (P < 0.05). This flow cytometric method takes advantage of the propensity of fragmented DNA to be denatured under standard conditions, or 1 minute at 37 °C with 10% buffered formalin-fixed cells. The study of fixed sperm makes possible the restrospective investigation of germplasm fragmentation, spermatogenic ploidy patterns, and chromatin compaction levels from samples translocated over distance and time. The protocol provides an approach that can be modified for other species across taxa.

  3. Flow cytometric method for measuring chromatin fragmentation in fixed sperm from yellow perch (Perca flavescens)

    USGS Publications Warehouse

    Jenkins, Jill A.; Draugelis-Dale, Rassa O.; Pinkney, Alfred E.; Iwanowicz, Luke R.; Blazer, Vicki

    2015-01-01

    Declining harvests of yellow perch, Perca flavescens, in urbanized watersheds of Chesapeake Bay have prompted investigations of their reproductive fitness. The purpose of this study was to establish a flow cytometric technique for DNA analysis of fixed samples sent from the field to provide reliable gamete quality measurements. Similar to the sperm chromatin structure assay, measures were made on the susceptibility of nuclear DNA to acid-induced denaturation, but used fixed rather than live or thawed cells. Nuclei were best exposed to the acid treatment for 1 minute at 37 °C followed by the addition of cold (4 °C) propidium iodide staining solution before flow cytometry. The rationale for protocol development is presented graphically through cytograms. Field results collected in 2008 and 2009 revealed DNA fragmentation up to 14.5%. In 2008, DNA fragmentation from the more urbanized watersheds was significantly greater than from reference sites (P = 0.026) and in 2009, higher percentages of haploid testicular cells were noted from the less urbanized watersheds (P = 0.032) indicating better reproductive condition at sites with less urbanization. For both years, total and progressive live sperm motilities by computer-assisted sperm motion analysis ranged from 19.1% to 76.5%, being significantly higher at the less urbanized sites (P < 0.05). This flow cytometric method takes advantage of the propensity of fragmented DNA to be denatured under standard conditions, or 1 minute at 37 °C with 10% buffered formalin–fixed cells. The study of fixed sperm makes possible the restrospective investigation of germplasm fragmentation, spermatogenic ploidy patterns, and chromatin compaction levels from samples translocated over distance and time. The protocol provides an approach that can be modified for other species across taxa.

  4. A Flow Cytometric and Computational Approaches to Carbapenems Affinity to the Different Types of Carbapenemases

    PubMed Central

    Pina-Vaz, Cidália; Silva, Ana P.; Faria-Ramos, Isabel; Teixeira-Santos, Rita; Moura, Daniel; Vieira, Tatiana F.; Sousa, Sérgio F.; Costa-de-Oliveira, Sofia; Cantón, Rafael; Rodrigues, Acácio G.

    2016-01-01

    The synergy of carbapenem combinations regarding Enterobacteriaceae producing different types of carbapenemases was study through different approaches: flow cytometry and computational analysis. Ten well characterized Enterobacteriaceae (KPC, verona integron-encoded metallo-β-lactamases –VIM and OXA-48-like enzymes) were selected for the study. The cells were incubated with a combination of ertapenem with imipenem, meropenem, or doripenem and killing kinetic curves performed with and without reinforcements of the drugs. A cephalosporin was also used in combination with ertapenem. A flow cytometric assay with DiBAC4-(3), a membrane potential dye, was developed in order to evaluate the cellular lesion after 2 h incubation. A chemical computational study was performed to understand the affinity of the different drugs to the different types of enzymes. Flow cytometric analysis and time-kill assays showed a synergic effect against KPC and OXA-48 producing-bacteria with all combinations; only ertapenem with imipenem was synergic against VIM producing-bacteria. A bactericidal effect was observed in OXA-48-like enzymes. Ceftazidime plus ertapenem was synergic against ESBL-negative KPC producing-bacteria. Ertapenem had the highest affinity for those enzymes according to chemical computational study. The synergic effect between ertapenem and others carbapenems against different carbapenemase-producing bacteria, representing a therapeutic choice, was described for the first time. Easier and faster laboratorial methods for carbapenemase characterization are urgently needed. The design of an ertapenem derivative with similar affinity to carbapenemases but exhibiting more stable bonds was demonstrated as highly desirable. PMID:27555844

  5. A continuous-flow, high-throughput, high-pressure parahydrogen converter for hyperpolarization in a clinical setting.

    PubMed

    Hövener, Jan-Bernd; Bär, Sébastien; Leupold, Jochen; Jenne, Klaus; Leibfritz, Dieter; Hennig, Jürgen; Duckett, Simon B; von Elverfeldt, Dominik

    2013-02-01

    Pure parahydrogen (pH(2) ) is the prerequisite for optimal pH(2) -based hyperpolarization experiments, promising approaches to access the hidden orders of magnitude of MR signals. pH(2) production on-site in medical research centers is vital for the proliferation of these technologies in the life sciences. However, previously suggested designs do not meet our requirements for safety or production performance (flow rate, pressure or enrichment). In this article, we present the safety concept, design and installation of a pH(2) converter, operated in a clinical setting. The apparatus produces a continuous flow of four standard liters per minute of ≈98% enriched pH(2) at a pressure maximum of 50 bar. The entire production cycle, including cleaning and cooling to 25 K, takes less than 5 h, only ≈45 min of which are required for actual pH(2) conversion. A fast and simple quantification procedure is described. The lifetimes of pH(2) in a glass vial and aluminum storage cylinder are measured to be T(1C) (glass vial) =822 ± 29 min and T(1C) (Al cylinder) =129 ± 36 days, thus providing sufficiently long storage intervals and allowing the application of pH(2) on demand. A dependence of line width on pH(2) enrichment is observed. As examples, (1) H hyperpolarization of pyridine and (13) C hyperpolarization of hydroxyethylpropionate are presented.

  6. High throughput flow cytometry screening reveals a novel role for JAM-A as a cancer stem cell maintenance factor

    PubMed Central

    Lathia, Justin D.; Li, Meizhang; Sinyuk, Maksim; Alvarado, Alvaro G.; Flavahan, William A.; Stoltz, Kevin; Rosager, Ann Mari; Hale, James; Hitomi, Masahiro; Gallagher, Joseph; Wu, Qiulian; Martin, Jody; Vidal, Jason G.; Nakano, Ichiro; Dahlrot, Rikke H.; Hansen, Steinbjørn; McLendon, Roger E.; Sloan, Andrew E.; Bao, Shideng; Hjelmeland, Anita B.; Carson, Christian T.; Naik, Ulhas P.; Kristensen, Bjarne; Rich, Jeremy N.

    2014-01-01

    Summary Stem cells reside in niches that regulate the balance between self-renewal and differentiation. The identity of a stem cell is linked with the ability to interact with its niche through adhesion mechanisms. To identify targets that disrupt cancer stem cell (CSC) adhesion, we performed a flow cytometry screen on patient derived glioblastoma (GBM) cells and identified junctional adhesion molecule-A (JAM-A) as a CSC adhesion mechanism essential for self-renewal and tumor growth. JAM-A was dispensable for normal neural stem/progenitor cell (NPC) function and JAM-A expression was reduced in normal brain versus GBM. Targeting JAM-A compromises the self-renewal of CSCs. JAM-A expression negatively correlated to GBM patient prognosis. Our results demonstrate that novel GBM targeting strategies can be identified through screening adhesion receptors and JAM-A represents a novel mechanism for niche driven CSC maintenance. PMID:24373972

  7. Fabrication of continuous flow microfluidics device with 3D electrode structures for high throughput DEP applications using mechanical machining.

    PubMed

    Zeinali, Soheila; Çetin, Barbaros; Oliaei, Samad Nadimi Bavil; Karpat, Yiğit

    2015-07-01

    Microfluidics is the combination of micro/nano fabrication techniques with fluid flow at microscale to pursue powerful techniques in controlling and manipulating chemical and biological processes. Sorting and separation of bio-particles are highly considered in diagnostics and biological analyses. Dielectrophoresis (DEP) has offered unique advantages for microfluidic devices. In DEP devices, asymmetric pair of planar electrodes could be employed to generate non-uniform electric fields. In DEP applications, facing 3D sidewall electrodes is considered to be one of the key solutions to increase device throughput due to the generated homogeneous electric fields along the height of microchannels. Despite the advantages, fabrication of 3D vertical electrodes requires a considerable challenge. In this study, two alternative fabrication techniques have been proposed for the fabrication of a microfluidic device with 3D sidewall electrodes. In the first method, both the mold and the electrodes are fabricated using high precision machining. In the second method, the mold with tilted sidewalls is fabricated using high precision machining and the electrodes are deposited on the sidewall using sputtering together with a shadow mask fabricated by electric discharge machining. Both fabrication processes are assessed as highly repeatable and robust. Moreover, the two methods are found to be complementary with respect to the channel height. Only the manipulation of particles with negative-DEP is demonstrated in the experiments, and the throughput values up to 105 particles / min is reached in a continuous flow. The experimental results are compared with the simulation results and the limitations on the fabrication techniques are also discussed.

  8. Capillary flow layer-by-layer: a microfluidic platform for the high-throughput assembly and screening of nanolayered film libraries.

    PubMed

    Castleberry, Steven A; Li, Wei; Deng, Di; Mayner, Sarah; Hammond, Paula T

    2014-07-22

    Layer-by-layer (LbL) assembly is a powerful tool with increasing real world applications in energy, biomaterials, active surfaces, and membranes; however, the current state of the art requires individual sample construction using large quantities of material. Here we describe a technique using capillary flow within a microfluidic device to drive high-throughput assembly of LbL film libraries. This capillary flow layer-by-layer (CF-LbL) method significantly reduces material waste, improves quality control, and expands the potential applications of LbL into new research spaces. The method can be operated as a simple lab benchtop apparatus or combined with liquid-handling robotics to extend the library size. Here we describe and demonstrate the technique and establish its ability to recreate and expand on the known literature for film growth and morphology. We use the same platform to assay biological properties such as cell adhesion and proliferation and ultimately provide an example of the use of this approach to identify LbL films for surface-based DNA transfection of commonly used cell types.

  9. Automated flow-based anion-exchange method for high-throughput isolation and real-time monitoring of RuBisCO in plant extracts.

    PubMed

    Suárez, Ruth; Miró, Manuel; Cerdà, Víctor; Perdomo, Juan Alejandro; Galmés, Jeroni

    2011-06-15

    In this work, a miniaturized, completely enclosed multisyringe-flow system is proposed for high-throughput purification of RuBisCO from Triticum aestivum extracts. The automated method capitalizes on the uptake of the target protein at 4°C onto Q-Sepharose Fast Flow strong anion-exchanger packed in a cylindrical microcolumn (105 × 4 mm) followed by a stepwise ionic-strength gradient elution (0-0.8 mol/L NaCl) to eliminate concomitant extract components and retrieve highly purified RuBisCO. The manifold is furnished downstream with a flow-through diode-array UV/vis spectrophotometer for real-time monitoring of the column effluent at the protein-specific wavelength of 280 nm to detect the elution of RuBisCO. Quantitation of RuBisCO and total soluble proteins in the eluate fractions were undertaken using polyacrylamide gel electrophoresis (PAGE) and the spectrophotometric Bradford assay, respectively. A comprehensive investigation of the effect of distinct concentration gradients on the isolation of RuBisCO and experimental conditions (namely, type of resin, column dimensions and mobile-phase flow rate) upon column capacity and analyte breakthrough was effected. The assembled set-up was aimed to critically ascertain the efficiency of preliminary batchwise pre-treatments of crude plant extracts (viz., polyethylenglycol (PEG) precipitation, ammonium sulphate precipitation and sucrose gradient centrifugation) in terms of RuBisCO purification and absolute recovery prior to automated anion-exchange column separation. Under the optimum physical and chemical conditions, the flow-through column system is able to admit crude plant extracts and gives rise to RuBisCO purification yields better than 75%, which might be increased up to 96 ± 9% with a prior PEG fractionation followed by sucrose gradient step.

  10. Engineering an Acinetobacter regulon for biosensing and high-throughput enzyme screening in E. coli via flow cytometry

    PubMed Central

    Jha, Ramesh K.; Kern, Theresa L.; Fox, David T.; M. Strauss, Charlie E.

    2014-01-01

    We created a single cell sorting system to screen for enzyme activity in Escherichia coli producing 3,4 dihydroxy benzoate (34DHB). To do so, we engineered a transcription factor regulon controlling the expression of green fluorescent protein (GFP) for induction by 34DHB. An autoregulated transcription factor, pcaU, was borrowed from Acinetobacter sp ADP1 to E. coli and its promoter region adapted for activity in E. Coli. The engineered pcaU regulon was inducible at >5 μM exogenous 34DHB, making it a sensitive biosensor for this industrially significant nylon precursor. Addition of a second plasmid provided IPTG inducible expression of dehydroshikimate dehydratase enzyme (AsbF), which converts endogenous dehydroshikimate to 34DHB. This system produced GFP fluorescence in an IPTG dose-dependent manner, and was easily detected in single cell on flow cytometer despite a moderate catalytic efficiency of AsbF. Using fluorescence-activated cell sorting (FACS), individual cells carrying the active AsbF could be isolated even when diluted into a decoy population of cells carrying a mutant (inactivated) AsbF variant at one part in a million. The same biosensor was also effective for further optimization of itself. FACS on E. coli carrying randomized loci in the promoter showed several variants with enhanced response to 34DHB. PMID:24861620

  11. Flow Cytometric Analysis of Presynaptic Nerve Terminals Isolated from Rats Subjected to Hypergravity

    NASA Astrophysics Data System (ADS)

    Borisova, Tatiana

    2008-06-01

    Flow cytometric studies revealed an insignificant decrease in cell size heterogeneity and cytoplasmic granularity of rat brain nerve terminals (synaptosomes) isolated from animals subjected to centrifuge-induced hypergravity as compared to control ones. The analysis of plasma membrane potential using the potentiometric optical dye rhodamine 6G showed a decrease in fluorescence intensity by 10 % at steady state level in hypergravity synaptosomes. To monitor synaptic vesicle acidification we used pH-sensitive fluorescent dye acridine orange and demonstrated a lower fluorescence intensity level at steady state (10%) after hypergravity as compared to controls. Thus, exposure to hypergravity resulted in depolarization of the synaptosomal plasma membrane and diminution in synaptic vesicle acidification that may be a cause leading to altered synaptic neurotransmission.

  12. Flow cytometric analysis of DNA content differences in blood samples obtained by leucoconcentration.

    PubMed

    Pierrez, J; Guerci, A; Guerci, O

    1988-07-01

    The leucoconcentration technique allows rapid obtainment of cellular suspensions from total blood or bone marrow for flow cytometric analysis. The technique is based on picric acid in ethyl alcohol fixation and saponin red cell lysis, followed by mithramycin staining for DNA. It gives a good resolution of DNA distributions that allow detection of slight variations in DNA content. These results were obtained with cellular suspensions differing only in one X or Y chromosome (male, female, Klinefelter and Turner syndromes). In these studies the ratio of the DNA content of X and Y chromosomes agrees with the chromosomal mass ratio already reported by other authors, but the "absolute values" are 10-fold more compared to these same works. Our conclusion is that leucoconcentration technique followed by DNA staining with mithramycin increases the difference in the dye's penetration and binding between X and Y chromosomes.

  13. Standardizing flow cytometric assays in long-term population-based studies

    NASA Astrophysics Data System (ADS)

    Melzer, Susanne; Bocsi, Jozsef; Tárnok, Attila

    2015-03-01

    Quantification of leukocyte subpopulations and characterization of antigen-expression pattern on the cellular surface can play an important role in diagnostics. The state of cellular immunology on the single-cell level was analyzed by polychromatic flow cytometry in a recent comparative study within the average Leipzig population (LIFE - Leipzig Research Centre for Civilization Diseases). Data of 1699 subjects were recorded over a long-time period of three years (in a total of 1126 days). To ensure compatibility of such huge data sets, quality-controls on many levels (stability of instrumentation, low intra-laboratory variance and reader independent data analysis) are essential. The LIFE study aims to analyze various cytometric pattern to reveal the relationship between the life-style, the environmental effects and the individual health. We therefore present here a multi-step quality control procedure for long-term comparative studies.

  14. Flow cytometric determination of radiation-induced chromosome damage and its correlation with cell survival

    SciTech Connect

    Welleweerd, J.; Wilder, M.E.; Carpenter, S.G.; Raju, M.R.

    1984-07-01

    Chinese hamster M3-1 cells were irradiated with several doses of x rays or ..cap alpha.. particles from /sup 238/Pu. Propidium iodide-stained chromosome suspensions were prepared at different times after irradiation; cells were also assayed for survival. The DNA histograms of these chromosomes showed increased background counts with increased doses of radiation. This increase in background was cell-cycle dependent and was correlated with cell survival. The correlation between radiation-induced chromosome damage and cell survival was the same for X rays and ..cap alpha.. particles. Data are presented which indicate that flow cytometric analysis of chromosomes of irradiated cell populations can be a useful adjunct to classical cytogenic analysis of irradiation-induced chromosomal damage by virtue of its ability to express and measure chromosomal damage not seen by classical cytogenic methods.

  15. Detecting individual extracellular vesicles using a multicolor in situ proximity ligation assay with flow cytometric readout

    PubMed Central

    Löf, Liza; Ebai, Tonge; Dubois, Louise; Wik, Lotta; Ronquist, K. Göran; Nolander, Olivia; Lundin, Emma; Söderberg, Ola; Landegren, Ulf; Kamali-Moghaddam, Masood

    2016-01-01

    Flow cytometry is a powerful method for quantitative and qualitative analysis of individual cells. However, flow cytometric analysis of extracellular vesicles (EVs), and the proteins present on their surfaces has been hampered by the small size of the EVs – in particular for the smallest EVs, which can be as little as 40 nm in diameter, the limited number of antigens present, and their low refractive index. We addressed these limitations for detection and characterization of EV by flow cytometry through the use of multiplex and multicolor in situ proximity ligation assays (in situ PLA), allowing each detected EV to be easily recorded over background noise using a conventional flow cytometer. By targeting sets of proteins on the surface that are specific for distinct classes of EVs, the method allows for selective recognition of populations of EVs in samples containing more than one type of EVs. The method presented herein opens up for analyses of EVs using flow cytometry for their characterization and quantification. PMID:27681459

  16. Microfluidic structures for flow cytometric analysis of hydrodynamically focussed blood cells fabricated by ultraprecision micromachining.

    PubMed

    Kummrow, A; Theisen, J; Frankowski, M; Tuchscheerer, A; Yildirim, H; Brattke, K; Schmidt, M; Neukammer, J

    2009-04-07

    We present three-dimensional microfluidic structures with integrated optical fibers, mirrors and electrodes for flow cytometric analysis of blood cells. Ultraprecision milling technique was used to fabricate different flow cells featuring single-stage and two-stage cascaded hydrodynamic focusing of particles by a sheath flow. Two dimensional focussing of the sample fluid was proven by fluorescence imaging in horizontal and vertical directions and found to agree satisfactorily with finite element calculations. Focussing of the sample stream down to 5 microm at a particle velocity of 3 m s(-1) is accessible while maintaining stable operation for sample flow rates of up to 20 microL min(-1). In addition to fluorescence imaging, the micro-flow cells were characterised by measurements of pulse shapes and pulse height distributions of monodisperse microspheres. We demonstrated practical use of the microstructures for cell differentiation employing light scatter to distinguish platelets and red blood cells. Furthermore, T-helper lymphocytes labelled by monoclonal antibodies were identified by measuring side scatter and fluorescence.

  17. Optimized automated data analysis for the cytokinesis‐block micronucleus assay using imaging flow cytometry for high throughput radiation biodosimetry

    PubMed Central

    Rodrigues, M. A.; Probst, C. E.; Beaton‐Green, L. A.

    2016-01-01

    Abstract The cytokinesis‐block micronucleus (CBMN) assay is a well‐established technique that can be employed in triage radiation biodosimetry to estimate whole body doses of radiation to potentially exposed individuals through quantitation of the frequency of micronuclei (MN) in binucleated lymphocyte cells (BNCs). The assay has been partially automated using traditional microscope‐based methods and most recently has been modified for application on the ImageStreamX (ISX) imaging flow cytometer. This modification has allowed for a similar number of BNCs to be automatically scored as compared to traditional microscopy in a much shorter time period. However, the MN frequency measured was much lower than both manual and automated slide‐based methods of performing the assay. This work describes the optimized analysis template which implements newly developed functions in the IDEAS® data analysis software for the ISX that enhances specificity for BNCs and increases the frequency of scored MN. A new dose response calibration curve is presented in which the average rate of MN per BNC is of similar magnitude to those presented in the literature using automated CBMN slide scoring methods. In addition, dose estimates were generated for nine irradiated, blinded samples and were found to be within ±0.5 Gy of the delivered dose. Results demonstrate that the improved identification accuracy for MN and BNCs in the ISX‐based version of the CBMN assay will translate to increased accuracy when estimating unknown radiation doses received by exposed individuals following large‐scale radiological or nuclear emergencies. © 2016 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of ISAC PMID:27272602

  18. Flow cytometric DNA analysis of ducks accumulating 137Cs on a reactor reservoir

    SciTech Connect

    George, L.S.; Dallas, C.E.; Brisbin, I.L. Jr.; Evans, D.L. )

    1991-06-01

    The objective of this study was to detect red blood cell (rbc) DNA abnormalities in male, game-farm mallard ducks as they ranged freely and accumulated 137Cs (radiocesium) from an abandoned nuclear reactor cooling reservoir. Prior to release, the ducks were tamed to enable recapture at will. Flow cytometric measurements conducted at intervals during the first year of exposure yielded cell cycle percentages of DNA (G0/G1, S, G2 + M phases) of rbc, as well as coefficients of variation (CV) in the G0/G1 phase. DNA histograms of exposed ducks were compared with two sets of controls which were maintained 30 and 150 miles from the study site. 137Cs live wholebody burdens were also measured in these animals in a parallel kinetics study, and an approximate steady-state equilibrium was attained after about 8 months. DNA histograms from 2 of the 14 contaminated ducks revealed DNA aneuploid-like patterns after 9 months exposure. These two ducks were removed from the experiment at this time, and when sampled again 1 month later, one continued to exhibit DNA aneuploidy. None of the control DNA histograms demonstrated DNA aneuploid-like patterns. There were no significant differences in cell cycle percentages at any time point between control and exposed animals. A significant increase in CV was observed at 9 months exposure, but after removal of the two ducks with DNA aneuploidy, no significant difference was detected in the group monitored after 12 months exposure. An increased variation in the DNA and DNA aneuploidy could, therefore, be detected in duck rbc using flow cytometric analysis, with the onset of these effects being related to the attainment of maximal levels of 137Cs body burdens in the exposed animals.

  19. Flow cytometric maturity score as a novel prognostic parameter in patients with acute myeloid leukemia.

    PubMed

    Schneider, Tanja; Flörcken, Anne; Singh, Anju; Türkmen, Seval; Burmeister, Thomas; Anagnostopoulos, Ioannis; Pezzutto, Antonio; Dörken, Bernd; Westermann, Jörg

    2015-08-01

    The European LeukemiaNet (ELN) classification is widely accepted for risk stratification of patients with acute myeloid leukemia (AML). In order to establish immunophenotypic features that predict prognosis, the expression of single AML blast cell antigens has been evaluated with partly conflicting results; however, the influence of immunophenotypic blast maturity is largely unknown. In our study, 300 AML patients diagnosed at our institution between January 2003 and April 2012 were analyzed. A flow cytometric maturity score was developed in order to distinguish "mature" AML (AML-ma) from "immature" AML (AML-im) by quantitative expression levels of early progenitor cell antigens (CD34, CD117, and TdT). AML-ma showed significantly longer relapse-free survival (RFS) and overall survival (OS) than AML-im (p < 0.001). Interestingly, statistically significant differences in RFS and OS were maintained within the "intermediate-risk" group according to ELN (RFS, 7.0 years (AML-ma) vs. 3.3 years (AML-im); p = 0.002; OS, 5.1 years (AML-ma) vs. 3.0 years (AML-im); p = 0.022). Our novel flow cytometric score easily determines AML blast maturity and can predict clinical outcome. It remains to be clarified whether these results simply reflect an accumulation of favorable molecular phenotypes in the AML-ma subgroup or whether they rely on biological differences such as a higher proportion of leukemia stem cells and/or a higher degree of genetic instability within the AML-im subgroup.

  20. Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

    PubMed Central

    Baker, Gregory J.; Castro, Maria G.; Lowenstein, Pedro R.

    2016-01-01

    Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the β-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4–6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists’ discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the

  1. A new one-platform flow cytometric method for residual cell counting in platelet concentrates.

    PubMed

    Schmidt, Michael; Spengler, Hans-Peter; Lambrecht, Bernd; Hourfar, Michael K; Seifried, Erhard; Tonn, Torsten

    2009-12-01

    According to German regulations and guidelines, residual red blood cells (rRBCs) and residual white blood cells (rWBCs) must number fewer than 3 x 10(9) cells/unit and 1 x 10(6) cells/unit in platelet concentrates (PCs), respectively. Due to low levels of residual cells in final products, there is still a need for fast, reliable, and sensitive methods of automated detection of these cell types. In Part A, 21 PCs were spiked with predetermined numbers of red blood cells (RBCs) and white blood cells (WBCs). The linearity, precision, and accuracy of the BD Thrombo Count assay (BD Biosciences Europe) were tested and validated according to international guidelines. Finally in Part B, 100 PCs prepared from pooled buffy coats were tested by the BD Thrombo Count assay and compared with other methods, including Nageotte (rWBCs) and Neubauer (rRBCs) counting chambers and the flow cytometric BD LeucoCOUNT (Becton Dickinson) assay (rWBCs). The unspecific background of blank PC samples was fewer than 0.02 cells/microL for WBCs and fewer than 34 cells/microL for RBCs (mean, 21). Linear regression and precision analyses of spiked PC samples were determined for both WBCs (r(2) = 0.992; range, 0.6-6.0 WBCs/microL) and RBCs (r(2) = 0.999; 800-8000 RBCs/microL). No carryover of cells or drift in results was detected in the automated sample acquisition mode. Analysis according to statistical methods of Bland and Altman demonstrated a high correlation between BD Thrombo Count and the Neubauer manual counting chamber. This novel flow cytometric test is a quick and reliable single-tube assay that has been demonstrated as a potential alternative for the existing manual microscopic counting procedures that are both time-consuming and laborious.

  2. High-throughput proteomics

    NASA Astrophysics Data System (ADS)

    Lesley, Scott A.; Nasoff, Marc; Kreusch, Andreas; Spraggon, Glen

    2001-04-01

    Proteomics has become a major focus as researchers attempt to understand the vast amount of genomic information. Protein complexity makes identifying and understanding gene function inherently difficult. The challenge of studying proteins in a global way is driving the development of new technologies for systematic and comprehensive analysis of protein structure and function. We are addressing this challenge through instrumentation and approaches to rapidly express, purify, crystallize, and mutate large numbers of human gene products. Our approach applies the principles of HTS technologies commonly used in pharmaceutical development. Genes are cloned, expressed, and purified in parallel to achieve a throughput potential of hundreds per day. Our instrumentation allows us to produce tens of milligrams of protein from 96 separate clones simultaneously. Purified protein is used for several applications including a high-throughput crystallographic screening approach for structure determination using automated image analysis. To further understand protein function, we are integrating a mutagenesis and screening approach. By combining these key technologies, we hope to provide a fundamental basis for understanding gene function at the protein level.

  3. High throughput optical scanner

    SciTech Connect

    Basiji, David A.; van den Engh, Gerrit J.

    2001-01-01

    A scanning apparatus is provided to obtain automated, rapid and sensitive scanning of substrate fluorescence, optical density or phosphorescence. The scanner uses a constant path length optical train, which enables the combination of a moving beam for high speed scanning with phase-sensitive detection for noise reduction, comprising a light source, a scanning mirror to receive light from the light source and sweep it across a steering mirror, a steering mirror to receive light from the scanning mirror and reflect it to the substrate, whereby it is swept across the substrate along a scan arc, and a photodetector to receive emitted or scattered light from the substrate, wherein the optical path length from the light source to the photodetector is substantially constant throughout the sweep across the substrate. The optical train can further include a waveguide or mirror to collect emitted or scattered light from the substrate and direct it to the photodetector. For phase-sensitive detection the light source is intensity modulated and the detector is connected to phase-sensitive detection electronics. A scanner using a substrate translator is also provided. For two dimensional imaging the substrate is translated in one dimension while the scanning mirror scans the beam in a second dimension. For a high throughput scanner, stacks of substrates are loaded onto a conveyor belt from a tray feeder.

  4. Flow Cytometric Analysis: Four-Year Experience in a Tertiary Care Centre of Pakistan

    PubMed Central

    Ahmad, Imran N; Rahman, Muhammad; Ghazanfar, Haider

    2016-01-01

    Purpose:  This study summarizes a four-year experience from the analysis of hematolymphoid malignancies in Pakistani population using a database of six-colored flow cytometry. Methods: A cross-sectional survey of 323 specimens of hematolymphoid malignancies using six-colored flow cytometry (FC) was carried out in Shifa International Hospital, Islamabad, Pakistan from June 2012 to June 2016. The criterion for specimen adequacy was that the cases have abnormal populations by FC, and the specimen age (time from biopsy to being examined by the six-color FC tube) of three days or less was to be included in the study. Clinical follow-up of greater than six months was required for a negative flow cytometric study without a subsequent biopsy. Data analysis was done using  Statistical Package for the Social Sciences (SPSS) version 21. One-way analysis of variance (ANOVA) was used to compare diagnosis with some antibodies used. Results:  The number of specimen within certain age groups included were: 0-15 years; 111 (34.3%), 16-30 years; 65 (20.12%), 31-45 years; 47 (14.5%), 46-60 years; 46 (14.2%) and ≥ 60 years; 54 (16.7%). Hematological malignancies were documented in descending order of sequence with B-cell acute lymphoblastic leukemia (27.9%), acute myeloid leukemia (26.3%), chronic lymphocytic leukemia (13.3%), T cell acute lymphoblastic leukemia (7.7%), non-Hodgkin's lymphomas (5%), hairy cell leukemia (1.9%), chronic myeloid leukemia (0.3%), paroxysmal nocturnal hemoglobinuria (0.6%) and plasma cell dyscrasias (0.6%). The mean number of antibodies used were 12.68 ± 2.97. One-way ANOVA was used to compare diagnosis with some antibodies used. Statistical significance was found between diagnosis and number of antibodies used (F= 5.23 p<0.001). Conclusion:  B cell acute lymphoblastic leukemia is most commonly diagnosed at tertiary care units in Pakistan using six-colored flow cytometry. Adoption of these complicated techniques has reinforced the need for

  5. High-throughput pyrosequencing analysis of bacteria relevant to cometabolic and metabolic degradation of ibuprofen in horizontal subsurface flow constructed wetlands.

    PubMed

    Li, Yifei; Wu, Bing; Zhu, Guibing; Liu, Yu; Ng, Wun Jern; Appan, Adhityan; Tan, Soon Keat

    2016-08-15

    The potential toxicity of pharmaceutical residues including ibuprofen on the aquatic vertebrates and invertebrates has attracted growing attention to the pharmaceutical pollution control using constructed wetlands, but there lacks of an insight into the relevant microbial degradation mechanisms. This study investigated the bacteria associated with the cometabolic and metabolic degradation of ibuprofen in a horizontal subsurface flow constructed wetland system by high-throughput pyrosequencing analysis. The ibuprofen degradation dynamics, bacterial diversity and evenness, and bacterial community structure in a planted bed with Typha angustifolia and an unplanted bed (control) were compared. The results showed that the plants promoted the microbial degradation of ibuprofen, especially at the downstream zones of wetland. However, at the upstream one-third zone of wetland, the presence of plants did not significantly enhance ibuprofen degradation, probably due to the much greater contribution of cometabolic behaviors of certain non-ibuprofen-degrading microorganisms than that of the plants. By analyzing bacterial characteristics, we found that: (1) The aerobic species of family Flavobacteriaceae, family Methylococcaceae and genus Methylocystis, and the anaerobic species of family Spirochaetaceae and genus Clostridium_sensu_stricto were the most possible bacteria relevant to the cometabolic degradation of ibuprofen; (2) The family Rhodocyclaceae and the genus Ignavibacterium closely related to the plants appeared to be associated with the metabolic degradation of ibuprofen.

  6. High-Throughput Synthesis of Lignin Particles (∼30 nm to ∼2 μm) via Aerosol Flow Reactor: Size Fractionation and Utilization in Pickering Emulsions.

    PubMed

    Ago, Mariko; Huan, Siqi; Borghei, Maryam; Raula, Janne; Kauppinen, Esko I; Rojas, Orlando J

    2016-09-07

    An aerosol flow reactor was used for the first time for high-throughput, high yield synthesis of spherical lignin particles with given inherent hydrophilicity, depending on the precursor biomolecule. In situ fractionation via Berner type impactor afforded populations with characteristic sizes ranging from ∼30 nm to 2 μm. The as-produced, dry lignin particles displayed excellent mechanical integrity, even after redispersion under high shear in either mineral oil or water. They were effective in the stabilization of oil-in-water (O/W) Pickering emulsions with tunable droplet size, depending on the dimension of the lignin particles used for emulsification. The emulsion stability correlated with particle concentration as well as the respective lignin type. For the O/W emulsions stabilized with the more hydrophilic lignin particles, negligible changes in phase separation via Ostwald ripening and coalescence were observed over a period of time of more than two months. Together with the fact that the lignin particle concentrations used in emulsification were as low as 0.1%, our results reveal a remarkable ability to endow emulsified systems with high colloidal stability. Overall, we offer a new, high-yield, scalable nanomanufacturing approach to producing dry spherical lignin particles with size control and high production capacity. A number of emerging applications for these organic particles can be envisioned and, as a proof-of-concept, we illustrate here surfactant-free emulsification.

  7. High-throughput continuous hydrothermal flow synthesis of Zn-Ce oxides: unprecedented solubility of Zn in the nanoparticle fluorite lattice.

    PubMed

    Kellici, Suela; Gong, Kenan; Lin, Tian; Brown, Sonal; Clark, Robin J H; Vickers, Martin; Cockcroft, Jeremy K; Middelkoop, Vesna; Barnes, Paul; Perkins, James M; Tighe, Christopher J; Darr, Jawwad A

    2010-09-28

    High-throughput continuous hydrothermal flow synthesis has been used as a rapid and efficient synthetic route to produce a range of crystalline nanopowders in the Ce-Zn oxide binary system. High-resolution powder X-ray diffraction data were obtained for both as-prepared and heat-treated (850 degrees C for 10 h in air) samples using the new robotic beamline I11, located at Diamond Light Source. The influence of the sample composition on the crystal structure and on the optical and physical properties was studied. All the nanomaterials were characterized using Raman spectroscopy, UV-visible spectrophotometry, Brunauer-Emmett-Teller surface area and elemental analysis (via energy-dispersive X-ray spectroscopy). Initially, for 'as-prepared' Ce(1-x)Zn(x)O(y), a phase-pure cerium oxide (fluorite) structure was obtained for nominal values of x=0.1 and 0.2. Biphasic mixtures were obtained for nominal values of x in the range of 0.3-0.9 (inclusive). High-resolution transmission electron microscopy images revealed that the phase-pure nano-CeO(2) (x=0) consisted of ca 3.7 nm well-defined nanoparticles. The nanomaterials produced herein generally had high surface areas (greater than 150 m(2) g(-1)) and possessed combinations of particle properties (e.g. bandgap, crystallinity, size, etc.) that were unobtainable or difficult to achieve by other more conventional synthetic methods.

  8. A flow cytometric approach to the study of crustacean cellular immunity

    USGS Publications Warehouse

    Cardenas, W.; Jenkins, J.A.; Dankert, J.R.

    2000-01-01

    Responses of hemocytes from the crayfish Procambarus zonangulus to stimulation by fungal cell walls (Zymosan A) were measured by flow cytometry. Changes in hemocyte physical characteristics were assessed flow cytometrically using forward- and sidescatter light parameters, and viability was measured by two-color fluorescent staining with calcein-AM and ethidium homodimer 1. The main effects of zymosan A on crayfish hemocytes were reduction in cell size and viability compared to control mixtures (hemocytes in buffer only). Adding diethyldithiocarbamic acid, an inhibitor of phenoloxidase, to hemocyte to zymosan mixtures delayed the time course of cell size reduction and cell death compared to zymosan-positive controls. The inclusion of trypsin inhibitor in reaction mixtures further delayed the reduction in hemocyte size and cell death, thereby indicating that a proteolytic cascade, along with prophenoloxidase activation, played a key role in generating signal molecules which mediate these cellular responses. In addition to traditional methods such as microscopy and protein chemistry, flow cytometry can provide a simple, reproducible, and sensitve method for evaluating invertebrate hemocyte responses to immunological stimuli.

  9. Flow cytometric evaluation of disseminated intravascular coagulation in a canine endotoxemia model.

    PubMed

    Yu, Dohyeon; Noh, Dongho; Park, Jinho

    2015-01-01

    Sepsis is associated with substantial morbidity and mortality in dogs. Alterations in hemostasis by systemic inflammation play an important role in the pathophysiology of sepsis. To evaluate the functional hemostatic changes in sepsis, we evaluated coagulation profiles and flow cytometric measurement of P-selectin (CD62P) expression on platelets, as well as platelet-leukocyte aggregation from a lipopolysaccharide (LPS)-induced endotoxemia model in dogs (n = 7). A sublethal dose of LPS [1 mg/kg body weight (BW)] induced thrombocytopenia and increased activated partial thromboplastin time (aPTT), prothrombin time (PT), and D-dimer concentrations. Flow cytometry analysis showed a significant increase in P-selectin expression on platelets between 1 and 24 h of a total 48 h of the experiment. In addition, platelet-leukocyte aggregation was significantly increased in the early stage of endotoxemia (at 1 and < 6 h for platelet-monocyte aggregation and at 3 h for platelet-neutrophil aggregation). Our results suggest that CD62P expression on platelets and platelet-leukocyte aggregation, as measured by flow cytometry, can be useful biomarkers of disseminated intravascular coagulation (DIC) in canine sepsis. These functional changes contribute to our understanding of the pathophysiology of hemostasis in endotoxemia.

  10. Quality control procedures for flow cytometric applications in the hematology laboratory.

    PubMed

    D'hautcourt, J L

    1996-12-01

    Clinical diagnosis is one of the areas in which flow cytometry (FCM) has gained wide popularity and FCM now plays a crucial role in several aspects of medical hematology. It has progressively replaced many traditional laboratory tests due to its greater accuracy, sensitivity and rapidity. Unfortunately, among the very large number of its potential applications, only a minority of flow cytometric protocols have been standardized. Numerous factors are responsible for variation in analytical conditions and may affect results obtained by FCM. All these variables can be schematically divided into three major groups: factors related to the biological samples, immunological and accessory reagent factors and factors associated with the use of instruments. The quality control program must monitor and evaluate all aspects of the procedure. This includes the following main aspects: 1) performance of the flow cytometer, 2) specimen collection, transportation and maintenance of its integrity, 3) reagents, particularly monoclonal antibodies and 4) sample measurements, data acquisition and their interpretation. Procedures described here are designed to assess all the settings which affect the reliability, reproducibility and sensitivity of the cytometer in order to ensure identical conditions on a daily basis.

  11. Flow cytometric analysis of activated sludge with rRNA-targeted probes.

    PubMed Central

    Wallner, G; Erhart, R; Amann, R

    1995-01-01

    Samples from a wastewater treatment plant were hybridized with fluorescein-labeled oligonucleotide probes specific for members of the domains Bacteria and Eucarya; the alpha, beta, and gamma subclasses of the class Proteobacteria; or the genus Acinetobacter. Subsequently, they were counterstained with the DNA-specific dye Hoechst 33342 and analyzed by flow cytometry. By quantifying forward angle light scatter and Hoechst- and probe-conferred fluorescence as measures for cell size, DNA content, and rRNA content, respectively, not only relative abundances but also assessments of general metabolic activity for each of these groups were obtained. Hybridizations with a positive control probe binding to all bacteria showed that in the activated-sludge samples examined, 70 to 80% of the Hoechst-stained cells could unambiguously be identified by this method. The majority of the detected cells (approximately 40%) were beta-subclass Proteobacteria. Flow cytometric and microscopic counts were in general agreement. Discrepancies were found in particular for those populations that occurred predominantly in flocs (alpha subclass of the Proteobacteria) or chains (Acinetobacter spp.). Although the dispersal of aggregates needs to be improved, flow cytometry combined with rRNA-based in situ probing appears to be a powerful tool for the rapid and highly automated analysis of the microbial communities in activated sludge. PMID:7646023

  12. Flow Cytometric Chromosomal Sex Sorting of Stallion Spermatozoa Induces Oxidative Stress on Mitochondria and Genomic DNA.

    PubMed

    Balao da Silva, C M; Ortega-Ferrusola, C; Morrell, J M; Rodriguez Martínez, H; Peña, F J

    2016-02-01

    To date, the only repeatable method to select spermatozoa for chromosomal sex is the Beltsville sorting technology using flow cytometry. Improvement of this technology in the equine species requires increasing awareness of the modifications that the sorting procedure induces on sperm intactness. Oxidative stress is regarded as the major damaging phenomenon, and increasing evidence regards handling of spermatozoa - including sex sorting - as basic ground for oxidative damage. The aim of this study was to disclose whether the flow cytometric sorting procedure increases the production of reactive oxygen species (ROS), and to identify if ROS production relates to DNA damage in sorted spermatozoa using specific flow cytometry-based assays. After sorting, oxidative stress increased from 26% to 33% in pre- and post-incubation controls, to 46% after sex sorting (p < 0.05). Proportions of DNA fragmentation index post-sorting were approximately 10% higher (31.3%); an effect apparently conduced via oxidative DNA damage as revealed by the oxyDNA assay. The probable origin of this increased oxidative stress owes the removal of enough seminal plasma due to the unphysiological sperm extension, alongside a deleterious effect of high pressure on mitochondria during the sorting procedure. © 2015 Blackwell Verlag GmbH.

  13. Influences of fixatives on flow cytometric measurements of platelet P-selectin expression and fibrinogen binding.

    PubMed

    Hu, H; Daleskog, M; Li, N

    2000-11-01

    Sample fixation is an important issue in flow cytometric platelet assays. However, previous reports were less than consistent regarding the influence of sample fixation on the assays. We evaluated the effects of formaldehyde and paraformaldehyde fixation on platelet P-selectin expression and fibrinogen binding using whole-blood flow cytometry and a Coulter EPICS XL-MCL cytometer. Fluorescent-labeled whole-blood samples were diluted with HEPES-buffered saline or fixed with formaldehyde (0.2, 0.5, and 1. 0%) or paraformaldehyde (0.5, 1.0, and 2.0%). Platelet P-selectin expression was 1.1+/-0.3% and 39.6+/-13.7% in unfixed resting and 10(-5) M ADP stimulated samples, respectively. Resting P-selectin expression was not significantly altered by 0.2 or 0.5% formaldehyde fixation, but was slightly decreased by 1.0% formaldehyde fixation or PFA fixation. Formaldehyde fixation caused small increases of P-selectin expression in ADP-stimulated samples. Compared to platelet fibrinogen binding of unfixed resting (4.5+/-2.1%) and ADP-stimulated (56.7+/-22.6%) samples, formaldehyde or paraformaldehyde fixation had no significant influence on resting samples, but mildly increased fibrinogen binding in stimulated samples. Unfixed samples were stable for 2 h. Fixed samples were generally stable for at least 6 h, but not thereafter. Thus, formaldehyde and paraformaldehyde have mild but complex influences on platelet P-selectin expression and fibrinogen binding measurements. To evaluate the stabilities of unfixed and fixed samples, samples were analyzed after different durations (0, 1, 2, 4, 6, 12, and 24 h) of storage at 4 degrees C in the dark. The results suggest that sample manipulation without fixation may be used when the samples are analyzed within 2 h, and that fixation with 0.5-1.0% formaldehyde or paraformaldehyde seems to be preferable when sample analysis is delayed. Effects of fixation should be carefully evaluated when establishing flow cytometric platelet assays in

  14. Flow cytometric DNA hypertetraploidy is associated with unfavourable prognostic features in breast cancer.

    PubMed Central

    Pinto, A E; André, S; Nogueira, M; Mendonça, E; Soares, J

    1997-01-01

    AIM: Breast tumours with a DNA content higher than 4N (hypertetraploidy) are not well characterised. The aim of this study was to evaluate the clinical and biological characteristics of 51 hypertetraploid breast carcinomas selected from a series of 860 consecutive cases analysed by flow cytometry. METHODS: The clinicopathological characteristics of the hypertetraploid group were compared with those of a control group of 138 non-hypertetraploid breast carcinomas. Breast tumours from patients submitted to surgery as primary therapeutic approach (15 hypertetraploid and the 138 non-hypertetraploid) were TNM staged and classified according to the histological type and grade. The remaining 36 patients had advanced neoplastic disease at presentation and were classified by cytological criteria only. DNA flow cytometric analysis was performed on fresh-frozen samples stained with propidium iodide. Hormone receptors were analysed by immunocytochemistry. RESULTS: The incidence of hypertetraploid breast tumours was 5.9% (51 of 860). All the patients were women and the mean age at diagnosis was 65 years. There was a family history of breast cancer in 21.6% of cases. In the group of operated patients, 33.3% had pT3 tumours and 53.3% had axillary lymph node metastases. All but one tumour were invasive ductal carcinomas; the remaining was an invasive papillary carcinoma. Ten (66.7%) tumours were classified as poorly differentiated carcinomas. Oestrogen and progesterone receptors were negative in 33 (64.7%) and 38 (74.5%) tumours, respectively. At last follow up, 35 (72.9%) patients were alive, while 13 (27.1%) died of disease within three years of diagnosis. Statistical comparison of the clinicopathological features of hypertetraploid v non-hypertetraploid breast carcinomas yielded a significant difference in tumour size (p < 0.001), histological grade (p < 0.001), hormone receptor status (p < 0.001), and overall survival (p < 0.001) between the two groups. CONCLUSION: Flow

  15. Flow cytometric determination of bacterial populations in bottled natural mineral waters

    NASA Astrophysics Data System (ADS)

    Beisker, Wolfgang; Meier, H.

    1998-04-01

    In order to enhance the quality and safety of bottled natural mineral waters, new methodologies besides classical bacteriology have been evaluated. Multi laser flow cytometry has been used to identify bacterial populations based on their DNA content, physiological activity and phylogeny from in situ hybridization with rRNA targeted DNA probes. Due to the low content of organic material in these waters, the bacterial population are under conditions (low ribosome content, low activity, etc.) which makes it hard to detect them flow cytometrically. The numbers of bacteria are in the range between 1000 and 100,000 per ml (for uncarbonated waters). Filtration techniques to enrich the bacterial population have been developed in combination with specific staining and hybridization protocols. First results on some selected brands show, that most bacteria belong to the beta subclass of proteobacteria. If the DNA containing cells (DAPI staining) are counted as 100%, 84% could be stained with a eubacteria probe. From these 84% 68% belong to the beta subclass, 8.2% to the alpha and 0.3% to the gamma subclass of roteobacteria. 8.5% could be identified as cytophaga flexibacter. By optimizing DNA staining with cyanine dyes and enhancing the sensitivity of light scatter detection, the detection limit could be considerably lowered.

  16. Troglitazone-induced intracellular oxidative stress in rat hepatoma cells: a flow cytometric assessment.

    PubMed

    Narayanan, Padma Kumar; Hart, Timothy; Elcock, Fiona; Zhang, Cindy; Hahn, Laura; McFarland, David; Schwartz, Lester; Morgan, D Gwyn; Bugelski, Peter

    2003-03-01

    Troglitazone (TRO), a thiazolidinedione (TZD) peroxisome proliferator-activated receptor gamma agonist, was recently withdrawn from the market because of rare but serious hepatotoxicity. Previous studies investigating the cytotoxicity of TRO in cultured rat hepatocytes have conjectured about the role of oxidative stress in TRO-induced hepatotoxicity. Therefore, we investigated whether TRO induces oxidative stress and, if so, the portion of the TRO molecule responsible for the induction of oxidative stress. Novikoff rat hepatoma (N1S1) cells were incubated with TRO, troglitazone quinone (TQ), thiazolidinedione-phenoxyacetic acid (TD-PAA) or rosiglitazone (RSG). Membrane peroxidation, intracellular glutathione (GSH) content, and cellular viability were monitored simultaneously by multiparameter flow cytometry. TRO and TQ increased membrane peroxidation, decreased intracellular GSH, and decreased cell viability in a concentration-dependent manner. In contrast, TD-PAA and RSG neither increased membrane peroxidation nor induced loss of cell viability. In addition, TRO caused a concentration-dependent increase in intracellular superoxide generation accompanied by a collapse in mitochondrial membrane potential. Multiparameter flow cytometric evaluation of N1S1 cells indicated that the chromane ring of TRO, rather than the TZD moiety, may be responsible for oxidative stress and suggested that a direct effect on mitochondrial physiology may play a role in TRO-mediated hepatotoxicity. Copyright 2003 Wiley-Liss, Inc.

  17. Flow cytometric evaluation of the intracellular bacterium, Wolbachia pipientis, in mosquito cells

    PubMed Central

    Fallon, Ann M

    2014-01-01

    Wolbachia is an obligate intracellular bacterium (Anaplasmataceae, Rickettisales) that occurs in arthropods and filarial worms, and spreads by vertical transmission in the oocyte cytoplasm. In insects, reproductive distortions associated with Wolbachia, such as cytoplasmic incompatibility in mosquitoes, have potential value for controlling pests, including species that transmit human, animal and plant diseases. Wolbachia strains that propagate as a persistent infection in insect cell lines provide an important resource for developing the genetic tools that will facilitate these applications. Here I describe conditions for flow cytometric evaluation of Wolbachia growth in persistently infected mosquito cells. Cytometry parameters were established using uninfected mosquito cells and Escherichia coli as a surrogate for Wolbachia, and quantitation was correlated with cell counts determined with a Coulter electronic cell counter and bacterial counts based on optical density. The protocol was validated by showing depletion of Wolbachia in medium containing tetracycline and rifampicin, and sensitivity of Wolbachia to treatment of host cells with paraquat, an oxidizing agent, and lumiflavin, an inhibitor of riboflavin uptake. The Wolbachia peak on the flow cytometry histogram was shown to contain Wolbachia by DNA analysis using the polymerase chain reaction, and by infection of naive recipient cells. This approach will streamline investigation of Wolbachia growth in insect cell lines and facilitate identification of culture conditions that select for Wolbachia-infected cells. PMID:25300665

  18. Comparison of multidimensional flow cytometric data by a novel data mining technique

    NASA Astrophysics Data System (ADS)

    Leary, James F.; Smith, Jacob; Szaniszlo, Peter; Reece, Lisa M.

    2007-02-01

    Most flow/image cytometric data analysis methods look for clusters in the data corresponding to specific cell subpopulations. Comparisons between different cytometry datafiles often use human pattern recognition visualization of all the different combinations of variables ("parameters") two at a time in so-called bivariate scattergrams. Not only is this tedious, but it can miss potential clusters due to projection of higher dimensional dataspaces down onto two dimensional planes making them indiscernible as separate clusters. Novel data mining algorithms, implemented in software allow for the comparison of two or more higher dimensional datafiles without the requirement for reduction of dimensionality for human visualization. Of equal importance is the comparison of higher dimensional clusters which may move around slightly in space, yet still be "similar" according to algorithms which provide measures of similarity. This software, written in C/C++ and currently implemented in software with a Windows graphical user interface, allows for direct reading of FCS2.0 format flow cytometry datafiles of any number of parameters. In a few minutes or less, complex multiparameter data of two or more files can be compared on a personal computer or workstation. The software operates in either supervised or unsupervised mode, depending on whether the user wishes to include prior user knowledge or in a data mining discovery mode. Differences between these files can be exported as sub-datafiles which can be further analyzed using any other software that can read FCS2.0 data format.

  19. Flow cytometric analysis using SYBR Green I for genome size estimation in coffee.

    PubMed

    Ronildo Clarindo, Wellington; Roberto Carvalho, Carlos

    2011-02-01

    Plant genome size has been measured by flow cytometry using propidium iodide as a dye for nuclear DNA staining. However, some authors have reported the occurrence of genome size estimation errors, especially in plants rich in secondary metabolites, such as the coffee tree. In this context, we tested an alternative cytometric protocol using the SYBR Green I as a fluorochrome for stoichiometrically staining nuclear double-stranded DNA in Coffea canephora (2x) and Coffea arabica (4x). The results showed that the respective mean genome size measured from nuclei stained with SYBR Green I and propidium iodide was statistically identical. However, the G(0)/G(1) peaks of nuclei stained with SYBR Green I exhibited lower coefficient variations (1.57-2.85%) compared to those stained with propidium iodide (2.75-4.80%). Coefficient variation statistical data suggest that SYBR Green I is adequate for stoichiometric nuclei staining using this methodology. Our results provide evidence that SYBR Green I can be used in flow cytometry measurements of plants, with the advantages of minimizing errors in nuclear DNA content quantification, staining relatively quicker, with high affinity, and being less mutagenic than propidium iodide.

  20. Flow cytometric quantification and characterization of intracellular protein aggregates in yeast.

    PubMed

    Shiber, Ayala; Breuer, William; Ravid, Tommer

    2014-01-01

    The sequestration of misfolded proteins into aggregates is an integral pathway of the protein quality control network that becomes particularly prominent during proteotoxic stress and in various pathologies. Methods for systematic analysis of cellular aggregate content are still largely limited to fluorescence microscopy and to separation by biochemical techniques. Here, we describe an alternative approach, using flow cytometric analysis, applied to protein aggregates released from their intracellular milieu by mild lysis of yeast cells. Protein aggregates were induced in yeast by heat shock or by chaperone deprivation and labeled using GFP- or mCherry-tagged quality control substrate proteins and chaperones. The fluorescence-labeled aggregate particles were distinguishable from cell debris by flow cytometry. The assay was used to quantify the number of fluorescent aggregates per μg of cell lysate protein and for monitoring changes in the cellular content and properties of aggregates, induced by stress. The results were normalized to the frequencies of fluorescent reporter expression in the cell population, allowing quantitative comparison. The assay also provided a quantitative measure of co-localization of aggregate components, such as chaperones and quality control substrates, within the same aggregate particle. This approach may be extended by fluorescence-activated sorting and isolation of various protein aggregates, including those harboring proteins associated with conformation disorders.

  1. Early events in macrophage killing of Aspergillus fumigatus conidia: new flow cytometric viability assay.

    PubMed

    Marr, K A; Koudadoust, M; Black, M; Balajee, S A

    2001-11-01

    Detailed investigations of macrophage phagocytosis and killing of Aspergillus fumigatus conidia have been limited by technical difficulties in quantifying fungal uptake and viability. In order to study early events in cell pathogen ingestion and killing, we developed a new flow cytometry assay that utilizes the fungus-specific viability dye FUN-1. Metabolically active A. fumigatus conidia accumulate orange fluorescence in vacuoles, while dormant or dead conidia stain green. After incubation within THP-1 cells, recovered conidia are costained with propidium iodide (PI) to discriminate between dormant and dead cells. Flow cytometric measurements of FUN-1 metabolism and PI uptake provide indicators of conidial viability, dormancy, and death. Conidial phagocytosis and killing are also assessed by measurement of green and orange FUN-1 fluorescence within the THP-1 cell population. Compared to previously described methods, this assay has less error introduced by membrane permeability changes and serial dilution of filamentous fungal forms. Results suggest that the THP-1 cells kill conidia rapidly (within 6 h) after exposure. Conidia that are preexposed to human serum are ingested and killed more quickly than are nonopsonized conidia.

  2. Flow cytometric quantification of T cell proliferation and division kinetics in woodchuck model of hepatitis B.

    PubMed

    Gujar, Shashi A; Michalak, Tomasz I

    2005-01-01

    Woodchucks infected with woodchuck hepatitis virus (WHV) represent the closest natural animal model to study the immunopathogenesis of liver injury caused by essentially noncytopathic, highly human specific hepatitis B virus (HBV). The importance of antiviral T cell response in induction of hepatitis and in control of HBV replication has been demonstrated. However, the understanding of how these responses contribute to the development of different immunomorphological forms of liver disease and their outcomes remain elusive. In this study, we established and standardized a flow cytometry assay using peripheral blood mononuclear cells labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) to assess WHV-specific and mitogen-driven T lymphocyte proliferative responses in woodchucks. The assay is of significantly greater sensitivity than the adenine incorporation assay currently used when applied to measure either WHV-specific T cell responses in acute (P < 0.001) and chronic (P < 0.03) viral hepatitis or those induced by mitogens in both healthy and WHV-infected animals. It also provides a new type of information, not previously available, characterizing the strength of woodchuck T cell proliferative reactivity by measuring cell division rates. The study shows that woodchuck PBMC labeled with CFSE exhibit light scatter and fluorescence profiles compatible to those of human PBMC, allowing quantitation and deconvolution of the flow cytometric data by applying the existing analytical softwares. The availability of this novel assay should facilitate a more precise and comprehensive evaluation of hepadnavirus-specific and generalized T cell responses in experimental WHV hepatitis.

  3. Use of xantham gum to suspend large particles during flow cytometric analysis and sorting.

    PubMed

    Freyer, J P; Fillak, D; Jett, J H

    1989-11-01

    In this report we describe the use of xantham gum as a biologically inert material for increasing the viscosity of a suspension of cells or particles during flow cytometric analysis and sorting. A 0.1% concentration of xantham gum in culture medium or saline will increase the viscosity approximately 9-fold. For suspensions of multicellular spheroids 100-400 microns in diameter the measured sedimentation velocity was approximately 9 times slower than that in medium alone. Thus, spheroids of 100 microns diameter remain in suspension in 0.1% xantham gum for 66 min, compared to 7.5 min in culture medium. This allows extended periods of sorting without stirring or agitating the sample suspension. The xantham gum solution is noncytotoxic for periods up to 8 h as measured by clonogenicity assay. Xantham gum has the added advantage that the viscosity is significantly reduced when the solution is subjected to shear stress, such as during flow. This technique should be applicable to extended sorting of suspensions of spheroids, plant cells, and other large particles, as well as for analyzing and sorting single cells for extended periods.

  4. Flow cytometric analysis of DNA binding and cleavage by cell surface-displayed homing endonucleases.

    PubMed

    Volná, Petra; Jarjour, Jordan; Baxter, Sarah; Roffler, Steve R; Monnat, Raymond J; Stoddard, Barry L; Scharenberg, Andrew M

    2007-01-01

    LAGLIDADG homing endonucleases (LHEs) cleave 18-24 bp DNA sequences and are promising enzymes for applications requiring sequence-specific DNA cleavage amongst genome-sized DNA backgrounds. Here, we report a method for cell surface display of LHEs, which facilitates analysis of their DNA binding and cleavage properties by flow cytometry. Cells expressing surface LHEs can be stained with fluorescently conjugated double-stranded oligonucleotides (dsOligos) containing their respective target sequences. The signal is absolutely sequence specific and undetectable with dsOligos carrying single base-pair substitutions. LHE-dsOligo interactions facilitate rapid enrichment and viable recovery of rare LHE expressing cells by both fluorescence-activated cell sorting (FACS) and magnetic cell sorting (MACS). Additionally, dsOligos conjugated with unique fluorophores at opposite termini can be tethered to the cell surface and used to detect DNA cleavage. Recapitulation of DNA binding and cleavage by surface-displayed LHEs provides a high-throughput approach to library screening that should facilitate rapid identification and analysis of enzymes with novel sequence specificities.

  5. Noninvasive high-throughput single-cell analysis of the intracellular pH of Saccharomyces cerevisiae by ratiometric flow cytometry.

    PubMed

    Valkonen, Mari; Mojzita, Dominik; Penttilä, Merja; Bencina, Mojca

    2013-12-01

    The ability of cells to maintain pH homeostasis in response to environmental changes has elicited interest in basic and applied research and has prompted the development of methods for intracellular pH measurements. Many traditional methods provide information at population level and thus the average values of the studied cell physiological phenomena, excluding the fact that cell cultures are very heterogeneous. Single-cell analysis, on the other hand, offers more detailed insight into population variability, thereby facilitating a considerably deeper understanding of cell physiology. Although microscopy methods can address this issue, they suffer from limitations in terms of the small number of individual cells that can be studied and complicated image processing. We developed a noninvasive high-throughput method that employs flow cytometry to analyze large populations of cells that express pHluorin, a genetically encoded ratiometric fluorescent probe that is sensitive to pH. The method described here enables measurement of the intracellular pH of single cells with high sensitivity and speed, which is a clear improvement compared to previously published methods that either require pretreatment of the cells, measure cell populations, or require complex data analysis. The ratios of fluorescence intensities, which correlate to the intracellular pH, are independent of the expression levels of the pH probe, making the use of transiently or extrachromosomally expressed probes possible. We conducted an experiment on the kinetics of the pH homeostasis of Saccharomyces cerevisiae cultures grown to a stationary phase after ethanol or glucose addition and after exposure to weak acid stress and glucose pulse. Minor populations with pH homeostasis behaving differently upon treatments were identified.

  6. High-throughput quantitation of nefazodone and its metabolites in human plasma by high flow direct-injection LC-MS/MS.

    PubMed

    Mao, Yan; Huang, Mike-Qingtao; Xia, Yuan-Qing; Jemal, Mohammed

    2007-04-11

    A rapid, selective and sensitive high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) method coupled with high flow direct-injection on-line extraction has been developed and validated for the simultaneous quantitation of nefazodone and its three active metabolites, hydroxynefazodone, triazole-dione (BMS-180492) and m-chlorophyenylpiperazine (mCPP) in human plasma. The method utilized d7-nefazodone, d7-hydroxynefazodone, d4-BMS-180492 and d4-mCPP as internal standards (IS). The plasma samples were injected into the LC-MS/MS system after simply adding the internal standard solution and centrifuging. The required extraction and chromatographic separation of the analytes were achieved on an Oasis HLB column (on-line extraction column, 1 mm x 50 mm, 30 microm) and a conventional Luna C8 column (analytical column, 4.6 mm x 50 mm, 5 microm). Detection was by positive ion electrospray tandem mass spectrometry. The total analysis run time for each sample was 2 min, which included the time needed for on-line extraction, chromatographic separation and LC-MS/MS analysis. The assay was validated for each analyte and the concentrations ranged from 2.0 to 500 ng/ml for nefazodone, hydroxynefazodone and mCPP and from 4.0 to 1000 ng/ml for BMS-180492, respectively. The assay was used for the high-throughput sample analysis of thousands of pharmacokinetic study samples and was proven to be rapid, accurate, precise, sensitive, specific and rugged.

  7. Early changes in flow cytometric DNA profiles induced by californium-252 neutron brachytherapy in squamocellular carcinomas of the uterine cervix.

    PubMed

    Tacev, T; Zaloudík, J; Janáková, L; Vagunda, V

    1998-01-01

    Ninety-five squamocellular carcinomas of the uterine cervix, clinical Stages II and III, were treated by either four schedules combining 252-californium neutron-gamma-radiotherapy with different proportions of a neutron component (9, 6 and 3 Gy) or gamma-irradiation alone. Flow cytometric DNA profiles were obtainable in 72 cases before treatment and 56 cases were monitored for DNA content by flow cytometry (FCM) in weekly intervals by analysis of sequential microbiopsies for one month during and after radiotherapy. DNA aneuploidy was reduced from 40% (25/63) to 19% (9/47) one week within therapy in neutron-treated groups, but not after initial gamma-radiotherapy alone. Extinction of DNA aneuploid subpopulations occurred after neutron therapy in all remaining aneuploid tumors (9/9) during further monitoring, but only in 40% (2/5) of tumors after sole gamma-irradiation. In contrast, proliferation index by more than 50% was more often achieved in groups with a higher gamma-radiation component than after neutrons only. When all therapy-induced DNA flow cytometric events are taken together for evaluation of the effects of various radiotherapy schedules, it appears that the regimen with the maximal neutron dose may not be optimal for all tumors. It is hypothesized that the differences in the early flow cytometric DNA profiles may select the DNA aneuploid squamous cell uterine cervical carcinomas as candidates for combined neutron-brachytherapy, while highly proliferating DNA near-diploid tumors may profit more from treatment with a higher gamma-radiotherapy component. However, these early DNA flow cytometric findings need to be correlated with clinical course of the disease to validate this hypothesis, a process which will be completed at the end of the expected five-year clinical outcome in 2000.

  8. A Flow Cytometric Clonogenic Assay Reveals the Single-Cell Potency of Doxorubicin

    PubMed Central

    Maass, Katie F.; Kulkarni, Chethana; Quadir, Mohiuddin A.; Hammond, Paula T.; Betts, Alison M.; Wittrup, K. Dane

    2015-01-01

    Standard cell proliferation assays use bulk media drug concentration to ascertain the potency of chemotherapeutic drugs; however, the relevant quantity is clearly the amount of drug actually taken up by the cell. To address this discrepancy, we have developed a flow cytometric clonogenic assay to correlate the amount of drug in a single cell with the cell’s ability to proliferate using a cell tracing dye and doxorubicin, a naturally fluorescent chemotherapeutic drug. By varying doxorubicin concentration in the media, length of treatment time, and treatment with verapamil, an efflux pump inhibitor, we introduced 105 – 1010 doxorubicin molecules per cell; then used a dye-dilution assay to simultaneously assess the number of cell divisions. We find that a cell’s ability to proliferate is a surprisingly conserved function of the number of intracellular doxorubicin molecules, resulting in single-cell IC50 values of 4 – 12 million intracellular doxorubicin molecules. The developed assay is a straightforward method for understanding a drug’s single-cell potency and can be used for any fluorescent or fluorescently-labeled drug, including nanoparticles or antibody-drug conjugates. PMID:26344409

  9. A Flow Cytometric Clonogenic Assay Reveals the Single-Cell Potency of Doxorubicin.

    PubMed

    Maass, Katie F; Kulkarni, Chethana; Quadir, Mohiuddin A; Hammond, Paula T; Betts, Alison M; Wittrup, Karl Dane

    2015-12-01

    Standard cell proliferation assays use bulk media drug concentration to ascertain the potency of chemotherapeutic drugs; however, the relevant quantity is clearly the amount of drug actually taken up by the cell. To address this discrepancy, we have developed a flow cytometric clonogenic assay to correlate the amount of drug in a single cell with the cell's ability to proliferate using a cell tracing dye and doxorubicin, a naturally fluorescent chemotherapeutic drug. By varying doxorubicin concentration in the media, length of treatment time, and treatment with verapamil, an efflux pump inhibitor, we introduced 10(5) -10(10) doxorubicin molecules per cell; then used a dye-dilution assay to simultaneously assess the number of cell divisions. We find that a cell's ability to proliferate is a surprisingly conserved function of the number of intracellular doxorubicin molecules, resulting in single-cell IC50 values of 4-12 million intracellular doxorubicin molecules. The developed assay is a straightforward method for understanding a drug's single-cell potency and can be used for any fluorescent or fluorescently labeled drug, including nanoparticles or antibody-drug conjugates.

  10. Multi-parameter flow cytometric analysis of uterine immune cell fluctuations over the murine estrous cycle.

    PubMed

    Diener, Kerrilyn R; Robertson, Sarah A; Hayball, John D; Lousberg, Erin L

    2016-02-01

    Investigating immune cell populations within various reproductive tissues commonly utilises flow cytometric methods. With advances in fluorophore technology and equipment capabilities, multiple cell types from a single tissue sample can be identified by using different combinations of cell surface markers to distinguish specific cell populations. Here a protocol optimized for mouse uterine tissue was used to show the proportional changes in dendritic cells, monocyte/macrophages, T and B cells, NK and NK T cells, and the granulocytes, neutrophils and eosinophils at each of the four stages of the estrous cycle. Importantly, we demonstrate that use of anti-SiglecF or assessment of FSC/SSC plots could be used to differentiate monocyte/macrophage and eosinophil populations that otherwise cannot be distinguished by use of the common combination of antibodies against F4/80 and CD11b. Our results clearly indicate that within the uterus a dynamic population of immune cells resides, with many cell types reaching peak abundance at estrus and metestrus phases of the cycle, consistent with their importance in the response to paternal antigens and/or pathogens encountered after insemination.

  11. Cytokinetic investigations in human breast cancer by flow cytometrically recorded DNA/protein distributions.

    PubMed

    Weiss, H; Görlich, M; Frege, J; Granetzny, A; Streller, B; Nitschke, U; Weiher, U

    1996-01-01

    This prospective study characterizes T1-T2 breast carcinomas (N = 114) and fibroadenomas (N = 16) by cell kinetic parameters derived from flow cytometrically recorded DNA/protein histograms. Ploidy level, cell cycle distribution and the number of cell subpopulations (SP) characterized by correlating DNA and protein values were assessed. The subpopulations were derived from the three-dimensional plot. The estrogen receptor (ER) status was determined biochemically (N = 61). Within the G1/0 cell peak 1-6 SP were evident in principle. Depending on the number of SP, two subsets were established: subset 1 with < or = 2 SP, subset 2 with > or = 3 SP. They differed significantly in proliferative activity expressed in the percentage of cells in the G2M phase. Subset 2 showed the higher activity. Analysis of subset distributions revealed that subset 1 prevails in favourable prognostic cases as ER positive cases (P < 0.03), lobular carcinomas (P < 0.01) and LN- cases (P < 0.03), whereas subset 2 prevails in the unfavourable counterparts. Analysis of variance showed that the main effect on proliferative activity indicated by G2M% is due to subpopulation composition rather than histologic type, nodal status or ER status (P < 0.01, P < 0.002, P < 0.05), not even due to ploidy level (P < 0.0001). The rationale for subset stratification may be cytogenetic variability connected with protein content heterogeneity accounting for kinetic SP.

  12. Flow cytometric analysis of lymphocyte proliferative responses to food allergens in dogs with food allergy.

    PubMed

    Fujimura, Masato; Masuda, Kenichi; Hayashiya, Makio; Okayama, Taro

    2011-10-01

    Two different allergy tests, antigen-specific immunoglobulin E quantification (IgE test) and flow cytometric analysis of antigen-specific proliferation of peripheral lymphocytes (lymphocyte proliferation test), were performed to examine differences in allergic reactions to food allergens in dogs with food allergy (FA). Thirteen dogs were diagnosed as FA based on clinical findings and elimination diet trials. Seven dogs clinically diagnosed with canine atopic dermatitis (CAD) were used as a disease control group, and 5 healthy dogs were used as a negative control group. In the FA group, 19 and 33 allergen reactions were identified using the serum IgE test and the lymphocyte proliferation test, respectively. Likewise, in the CAD group, 12 and 6 allergen reactions and in the healthy dogs 3 and 0 allergen reactions were identified by each test, respectively. A significant difference was found between FA and healthy dogs in terms of positive allergen detection by the lymphocyte proliferation test, suggesting that the test can be useful to differentiate FA from healthy dogs but not from CAD. Both tests were repeated in 6 of the dogs with FA after a 1.5- to 5-month elimination diet trial. The IgE concentrations in 9 of 11 of the positive reactions decreased by 20-80%, whereas all the positive reactions in the lymphocyte proliferation test decreased to nearly zero (P<0.05), suggesting that lymphocytes against food allergens may be involved in the pathogenesis of canine FA.

  13. Flow cytometric evaluation of hypoxic cells in solid experimental tumours using fluorescence immunodetection.

    PubMed Central

    Hodgkiss, R. J.; Jones, G.; Long, A.; Parrick, J.; Smith, K. A.; Stratford, M. R.; Wilson, G. D.

    1991-01-01

    Numerous methods have been proposed for the detection of hypoxic cells using nitroimidazoles labelled with both radioactive and stable isotopes where the isotopic label becomes bound as a result of reductive metabolism of the nitro group. A new probe for hypoxia, 7-(4'-(2-nitroimidazol-l-yl)-butyl)-theophylline, is described where an immunologically recognisable hapten (theophylline) is covalently linked to a 2-nitroimidazole. Bioreduction of the nitroimidazole leads to binding of bioreductive metabolites, and hence the theophylline side-chain, to intracellular molecules. Immunochemical procedures are then used to stain cells containing the bound theophylline using an FITC-conjugated anti-serum. Flow cytometric analysis of stained cells is facilitated by co-staining cellular DNA, which allows discrimination of single cells in the sample and rejection of cell clumps and debris. The alternative use of an immunoperoxidase-conjugated anti-serum has been used to demonstrate the localisation of hypoxic cells in frozen tumour sections. Images Figure 8 PMID:1989649

  14. A flow cytometric study of chromosomes from rat kangaroo and Chinese hamster cells.

    PubMed

    Stöhr, M; Hutter, K J; Frank, M; Futterman, G; Goerttler, K

    1980-01-01

    Chromosomes from rat kangaroo (PTK) and chinese hamster (CHV 79) cells have been prepared for quantitative flow-cytometric analysis. The preparation time was otimized down to 30 (PTK) and 40 min (CHV 79). DAPI was used as a AT-sensitive fluorescent dye to stain for monoparameter DNA measurements. Simultaneous two-parameter DNA-protein analysis was carried out with DAPI and SR 101 (as a general protein fluorochrome) in combination. The karyotype of the PTK cells with 13 (14) chromosomes was separated into 10DNA peaks. The X-chromosome bearing the nucleolus organizer region generates a distinct peak. The karyotype of the CHV 79 cells with 22 chromosomes was separated inot 15 peaks. The DNA profile obtained indicates a geometric grading of the chromosomal amount of AT components in teh karyotype of this particular cell line. The simultaneous DNA-protein analysis performed show enough sensitivity of the instrument utilizing hihg power UV excitation illumination to discriminate the two color emission consisting of blue (DAPI) and red (SR 101) fluorescence. Color overlapping could be completely avoided. Additionally, the quality (number, location, and resolution of peaks) of the DNA distribution was not influences by the simultaneous application of a second fluorescent stain. Fluorescence activated electronic sorting applied on chromosomal fluorescence distributions providing purified fractions of chromosomes for subsequent biochemical and biological determinations is discussed.

  15. Differentiation of A31T6 proadipocytes to adipocytes: A flow cytometric analysis

    SciTech Connect

    Smyth, M.J.; Wharton, W. )

    1992-03-01

    A flow cytometric assay has been developed which provides precise, quantitative information on the accumulation of cytoplasmic triglycerides in individual A31T6 proadipocytes as they differentiate into adipocytes. The opportunity to measure multiple optical parameter on a cell-by-cell basis has enabled us to monitor phenotypic aspects of differentiation with a greater level of sensitivity than was previously possible. Using the fluorescent hydrophobic probe, Nile red, they have found that as a cell proceeds along the differentiation pathway, the gold fluorescence signal from the cell increases, reflecting the accumulation of cytoplasmic lipid droplets. They have determined (1) the presence of an undifferentiated population of cells whose existence is not detected by conventional phase microscopy, (2) that insulin is no required to drive differentiation in this system, (3) that exposure to a combination of insulin and dexamethasone results in a lower accumulation of lipid in a cell than does exposure to either agent alone, and (4) that A31T6 cells show the same response to differentiation-promoting agents whether applied at the time of plating or at confluence.

  16. Combined flow cytometric analysis of surface and intracellular antigens reveals surface molecule markers of human neuropoiesis.

    PubMed

    Turaç, Gizem; Hindley, Christopher J; Thomas, Ria; Davis, Jason A; Deleidi, Michela; Gasser, Thomas; Karaöz, Erdal; Pruszak, Jan

    2013-01-01

    Surface molecule profiles undergo dynamic changes in physiology and pathology, serve as markers of cellular state and phenotype and can be exploited for cell selection strategies and diagnostics. The isolation of well-defined cell subsets is needed for in vivo and in vitro applications in stem cell biology. In this technical report, we present an approach for defining a subset of interest in a mixed cell population by flow cytometric detection of intracellular antigens. We have developed a fully validated protocol that enables the co-detection of cluster of differentiation (CD) surface antigens on fixed, permeabilized neural cell populations defined by intracellular staining. Determining the degree of co-expression of surface marker candidates with intracellular target population markers (nestin, MAP2, doublecortin, TUJ1) on neuroblastoma cell lines (SH-SY5Y, BE(2)-M17) yielded a combinatorial CD49f(-)/CD200(high) surface marker panel. Its application in fluorescence-activated cell sorting (FACS) generated enriched neuronal cultures from differentiated cell suspensions derived from human induced pluripotent stem cells. Our data underlines the feasibility of using the described co-labeling protocol and co-expression analysis for quantitative assays in mammalian neurobiology and for screening approaches to identify much needed surface markers in stem cell biology.

  17. Evaluation of fluorescein diacetate for flow cytometric determination of cell viability in orthopaedic research.

    PubMed

    Bell, R S; Bourret, L A; Bell, D F; Gebhardt, M C; Rosenberg, A; Berrey, H B; Treadwell, B V; Tomford, W W; Mankin, H J

    1988-01-01

    Accurate estimation of cellular viability is important both in research and in aspects of orthopaedic clinical practice. We have been interested in the potential for flow cytometric application of fluorescein diacetate (FDA) in evaluating chondrocyte survival following cryopreservation of osteochondral allografts as well as in the assessment of sarcoma necrosis following preoperative chemotherapy. In order to evaluate the suitability of this method for cell viability assays, this study compared FDA with more traditional methodology (trypan blue, clonigenic assay, metabolic activity analysis, measurement of DNA synthesis, and histological assessment of necrosis). Both chondrocytes and sarcoma cells were exposed to various experimental injuries prior to viability analysis. Although it is evident from these experiments that FDA accurately reflects cell survival after physical injury, it underestimates the effect of chemotherapy on cell reproductive potential in vitro. However, FDA is highly correlated with histological assessment of tumor viability after chemotherapy in vivo. It is apparent that the methodology chosen for determination of viability should be appropriate for the type of experimental injury and should analyze the cell function (i.e., metabolic activity or reproductive capacity) that is appropriate for the experimental model.

  18. Dissociation of skeletal muscle for flow cytometric characterization of immune cells in macaques.

    PubMed

    Liang, Frank; Ploquin, Aurélie; Hernández, José DelaO; Fausther-Bovendo, Hugues; Lindgren, Gustaf; Stanley, Daphne; Martinez, Aiala Salvador; Brenchley, Jason M; Koup, Richard A; Loré, Karin; Sullivan, Nancy J

    2015-10-01

    The majority of vaccines and several treatments are administered by intramuscular injection. The aim is to engage and activate immune cells, although they are rare in normal skeletal muscle. The phenotype and function of resident as well as infiltrating immune cells in the muscle after injection are largely unknown. While methods for obtaining and characterizing murine muscle cell suspensions have been reported, protocols for nonhuman primates (NHPs) have not been well defined. NHPs comprise important in vivo models for studies of immune cell function due to their high degree of resemblance with humans. In this study, we developed and systematically compared methods to collect vaccine-injected muscle tissue to be processed into single cell suspensions for flow cytometric characterization of immune cells. We found that muscle tissue processed by mechanical disruption alone resulted in significantly lower immune cell yields compared to enzymatic digestion using Liberase. Dendritic cell subsets, monocytes, macrophages, neutrophils, B cells, T cells and NK cells were readily detected in the muscle by the classic human markers. The methods for obtaining skeletal muscle cell suspension established here offer opportunities to increase the understanding of immune responses in the muscle, and provide a basis for defining immediate post-injection vaccine responses in primates. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Fine needle aspirate flow cytometric phenotyping characterizes immunosuppressive nature of the mesothelioma microenvironment

    PubMed Central

    Lizotte, Patrick H.; Jones, Robert E.; Keogh, Lauren; Ivanova, Elena; Liu, Hongye; Awad, Mark M.; Hammerman, Peter S.; Gill, Ritu R.; Richards, William G.; Barbie, David A.; Bass, Adam J.; Bueno, Raphael; English, Jessie M.; Bittinger, Mark; Wong, Kwok-Kin

    2016-01-01

    With the emergence of checkpoint blockade and other immunotherapeutic drugs, and the growing adoption of smaller, more flexible adaptive clinical trial designs, there is an unmet need to develop diagnostics that can rapidly immunophenotype patient tumors. The ability to longitudinally profile the tumor immune infiltrate in response to immunotherapy also presents a window of opportunity to illuminate mechanisms of resistance. We have developed a fine needle aspirate biopsy (FNA) platform to perform immune profiling on thoracic malignancies. Matching peripheral blood, bulk resected tumor, and FNA were analyzed from 13 mesothelioma patients. FNA samples yielded greater numbers of viable cells when compared to core needle biopsies. Cell numbers were adequate to perform flow cytometric analyses on T cell lineage, T cell activation and inhibitory receptor expression, and myeloid immunosuppressive checkpoint markers. FNA samples were representative of the tumor as a whole as assessed by head-to-head comparison to single cell suspensions of dissociated whole tumor. Parallel analysis of matched patient blood enabled us to establish quality assurance criteria to determine the accuracy of FNA procedures to sample tumor tissue. FNA biopsies provide a diagnostic to rapidly phenotype the tumor immune microenvironment that may be of great relevance to clinical trials. PMID:27539742

  20. Flow cytometric assay for quantifying opsonophagocytosis and killing of Staphylococcus aureus by peripheral blood leukocytes.

    PubMed Central

    Martin, E; Bhakdi, S

    1992-01-01

    We describe a novel flow cytometric method for quantifying opsonophagocytosis and killing of Staphylococcus aureus in cell-rich plasma obtained after dextran sedimentation of erythrocytes. To analyze opsonophagocytosis, phagocytes were labeled with a phycoerythrin-conjugated monoclonal antibody and were incubated with viable staphylococci containing carboxyfluorescein as a vital fluorescent dye. Phagocytosing cells assumed a dual, orange-green fluorescence. The relative numbers of bacteria associating with phagocytes could be determined by quantifying the decrease of free green fluorescent particles. A parallel incubation of fluorescent bacteria with unlabeled cell-rich plasma was performed to assess phagocytic killing. Blood cells were lysed with 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate. This detergent spared viable bacteria, and residual green fluorescent particles were counted. The decrease in the number of these particles relative to the controls yielded the degree of killing. At bacteria-to-phagocyte ratios of 1:1 and 10:1, approximately 36 and 75% of the phagocytes participated in opsonophagocytosis, respectively. Over 90% of the staphylococci were phagocyte associated after 30 to 60 min. Killing rates were on the order of 66% +/- 12% and 80% +/- 7% after 1 and 2 h of incubation, respectively. These numbers, which were confirmed by colony countings, were significantly lower than those reported in the majority of past reports. PMID:1400987

  1. A flow-cytometric gram-staining technique for milk-associated bacteria.

    PubMed

    Holm, Claus; Jespersen, Lene

    2003-05-01

    A Gram-staining technique combining staining with two fluorescent stains, Oregon Green-conjugated wheat germ agglutinin (WGA) and hexidium iodide (HI) followed by flow-cytometric detection is described. WGA stains gram-positive bacteria while HI binds to the DNA of all bacteria after permeabilization by EDTA and incubation at 50 degrees C for 15 min. For WGA to bind to gram-positive bacteria, a 3 M potassium chloride solution was found to give the highest fluorescence intensity. A total of 12 strains representing some of the predominant bacterial species in bulk tank milk and mixtures of these were stained and analyzed by flow cytometry. Overall, the staining method showed a clear differentiation between gram-positive and gram-negative bacterial populations. For stationary-stage cultures of seven gram-positive bacteria and five gram-negative bacteria, an average of 99% of the cells were correctly interpreted. The method was only slightly influenced by the growth phase of the bacteria or conditions such as freezing at -18 degrees C for 24 h. For any of these conditions, an average of at least 95% of the cells were correctly interpreted. When stationary-stage cultures were stored at 5 degrees C for 14 days, an average of 86% of the cells were correctly interpreted. The Gram-staining technique was applied to the flow cytometry analysis of bulk tank milk inoculated with Staphylococcus aureus and Escherichia coli. These results demonstrate that the technique is suitable for analyzing milk samples without precultivation.

  2. Expanding the potential of standard flow cytometry by extracting fluorescence lifetimes from cytometric pulse shifts

    PubMed Central

    Cao, Ruofan; Naivar, Mark A; Wilder, Mark; Houston, Jessica P

    2014-01-01

    Fluorescence lifetime measurements provide information about the fluorescence relaxation, or intensity decay, of organic fluorophores, fluorescent proteins, and other inorganic molecules that fluoresce. The fluorescence lifetime is emerging in flow cytometry and is helpful in a variety of multiparametric, single cell measurements because it is not impacted by nonlinearity that can occur with fluorescence intensity measurements. Yet time-resolved cytometry systems rely on major hardware modifications making the methodology difficult to reproduce. The motivation of this work is, by taking advantage of the dynamic nature of flow cytometry sample detection and applying digital signal processing methods, to measure fluorescence lifetimes using an unmodified flow cytometer. We collect a new lifetime-dependent parameter, referred to herein as the fluorescence-pulse-delay (FPD), and prove it is a valid representation of the average fluorescence lifetime. To verify we generated cytometric pulses in simulation, with light emitting diode (LED) pulsation, and with true fluorescence measurements of cells and microspheres. Each pulse is digitized and used in algorithms to extract an average fluorescence lifetime inherent in the signal. A range of fluorescence lifetimes is measurable with this approach including standard organic fluorophore lifetimes (∼1 to 22 ns) as well as small, simulated shifts (0.1 ns) under standard conditions (reported herein). This contribution demonstrates how digital data acquisition and signal processing can reveal time-dependent information foreshadowing the exploitation of full waveform analysis for quantification of similar photo-physical events within single cells. © 2014 The Authors. Published by Wiley Periodicals, Inc. PMID:25274073

  3. Flow cytometric viability assessment of lactic acid bacteria starter cultures produced by fluidized bed drying.

    PubMed

    Bensch, Gerald; Rüger, Marc; Wassermann, Magdalena; Weinholz, Susann; Reichl, Udo; Cordes, Christiana

    2014-06-01

    For starter culture production, fluidized bed drying is an efficient and cost-effective alternative to the most frequently used freeze drying method. However, fluidized bed drying also poses damaging or lethal stress to bacteria. Therefore, investigation of impact of process variables and conditions on viability of starter cultures produced by fluidized bed drying is of major interest. Viability of bacteria is most frequently assessed by plate counting. While reproductive growth of cells can be characterized by the number of colony-forming units, it cannot provide the number of viable-but-nonculturable cells. However, in starter cultures, these cells still contribute to the fermentation during food production. In this study, flow cytometry was applied to assess viability of Lactobacillus plantarum starter cultures by membrane integrity analysis using SYBR®Green I and propidium iodide staining. The enumeration method established allowed for rapid, precise and sensitive determination of viable cell concentration, and was used to investigate effects of fluidized bed drying and storage on viability of L. plantarum. Drying caused substantial membrane damage on cells, most likely due to dehydration and oxidative stress. Nevertheless, high bacterial survival rates were obtained, and granulates contained in the average 2.7 × 10(9) viable cells/g. Furthermore, increased temperatures reduced viability of bacteria during storage. Differences in results of flow cytometry and plate counting suggested an occurrence of viable-but-nonculturable cells during storage. Overall, flow cytometric viability assessment is highly feasible for rapid routine in-process control in production of L. plantarum starter cultures, produced by fluidized bed drying.

  4. A Flow-Cytometric Gram-Staining Technique for Milk-Associated Bacteria

    PubMed Central

    Holm, Claus; Jespersen, Lene

    2003-01-01

    A Gram-staining technique combining staining with two fluorescent stains, Oregon Green-conjugated wheat germ agglutinin (WGA) and hexidium iodide (HI) followed by flow-cytometric detection is described. WGA stains gram-positive bacteria while HI binds to the DNA of all bacteria after permeabilization by EDTA and incubation at 50°C for 15 min. For WGA to bind to gram-positive bacteria, a 3 M potassium chloride solution was found to give the highest fluorescence intensity. A total of 12 strains representing some of the predominant bacterial species in bulk tank milk and mixtures of these were stained and analyzed by flow cytometry. Overall, the staining method showed a clear differentiation between gram-positive and gram-negative bacterial populations. For stationary-stage cultures of seven gram-positive bacteria and five gram-negative bacteria, an average of 99% of the cells were correctly interpreted. The method was only slightly influenced by the growth phase of the bacteria or conditions such as freezing at −18°C for 24 h. For any of these conditions, an average of at least 95% of the cells were correctly interpreted. When stationary-stage cultures were stored at 5°C for 14 days, an average of 86% of the cells were correctly interpreted. The Gram-staining technique was applied to the flow cytometry analysis of bulk tank milk inoculated with Staphylococcus aureus and Escherichia coli. These results demonstrate that the technique is suitable for analyzing milk samples without precultivation. PMID:12732558

  5. Flow cytometric evaluation of red blood cell chimerism after bone marrow transplantation in Iranian patients: a preliminary study.

    PubMed

    Shaiegan, Mojgan; Hadjati, Esmerdis; Aghaiipour, Mahnaz; Iravani, Masoud; David, Gaelle; Bernard, Daniel

    2006-10-01

    The aim of this study was to evaluate mixed red cells population and red blood cell chimerism after hematopoietic stem cell transplantation. Red blood cell chimerism after hematopoietic stem cell transplantation was analyzed using a series of fluorescein isothiocyanate-conjugated monoclonal antibodies (BioAtlantic, France) directed against ABH, Rh (D, C, E, c, e), Kell, Duffy, Kidd, and Ss antigens on blood samples of 14 patients with hematologic disorders undergoing hematopoietic stem cell transplantation, by flow cytometric method on days 15, 30, and 60 after transplantation. All patients showed expression of donor red cell antigens within days 15 - 30 after hematopoietic stem cell transplantation. Graft versus host disease and ABO incompatibility did not affect the expression of chimerism. Flow cytometric analysis is a simple, accurate, and valuable test which is of significant help in monitoring chimerism in allogeneic hematopoietic stem cell transplantation.

  6. Surface free energy activated high-throughput cell sorting.

    PubMed

    Zhang, Xinru; Zhang, Qian; Yan, Tao; Jiang, Zeyi; Zhang, Xinxin; Zuo, Yi Y

    2014-09-16

    Cell sorting is an important screening process in microbiology, biotechnology, and clinical research. Existing methods are mainly based on single-cell analysis as in flow cytometric and microfluidic cell sorters. Here we report a label-free bulk method for sorting cells by differentiating their characteristic surface free energies (SFEs). We demonstrated the feasibility of this method by sorting model binary cell mixtures of various bacterial species, including Pseudomonas putida KT2440, Enterococcus faecalis ATCC 29212, Salmonella Typhimurium ATCC 14028, and Escherichia coli DH5α. This method can effectively separate 10(10) bacterial cells within 30 min. Individual bacterial species can be sorted with up to 96% efficiency, and the cell viability ratio can be as high as 99%. In addition to its capacity of sorting evenly mixed bacterial cells, we demonstrated the feasibility of this method in selecting and enriching cells of minor populations in the mixture (presenting at only 1% in quantity) to a purity as high as 99%. This SFE-activated method may be used as a stand-alone method for quickly sorting a large quantity of bacterial cells or as a prescreening tool for microbial discrimination. Given its advantages of label-free, high-throughput, low cost, and simplicity, this SFE-activated cell sorting method has potential in various applications of sorting cells and abiotic particles.

  7. Prospects and limits of the flow cytometric seed screen – insights from Potentilla sensu lato (Potentilleae, Rosaceae)

    PubMed Central

    Dobeš, Christoph; Lückl, Andrea; Hülber, Karl; Paule, Juraj

    2013-01-01

    The flow cytometric seed screen allows for identification of reproductive modes of seed formation and inference of the ploidy of contributing gametes. However, the lack of a mathematical formalization to infer male/female genomic contributions, and the prerequisite of a binucleate female contribution to the endosperm limits its applicability. We evaluated this assumption combining a DNA-based progeny survey with a comparison of the cytology of reproductive pathways co-occurring within single individuals representing 14 Potentilleae species from six phylogenetic lineages. A numerical framework valid for sexual and pseudogamous taxa was developed, enabling quantification of female and male genomes contributing to embryo and endosperm independent of gametophyte origins, numbers of sperm involved and ploidy of parents. The inference strongly depended on accurate peak index estimation. The endosperm of Potentilleae species received a binucleate female contribution in five evolutionary lineages whereas endosperm formation remained uncertain in the Tormentillae. A modified flow cytometric seed screen protocol was developed to cope with low endosperm contents. Evolutionary conservation of a binucleate female contribution to the endosperm suggested wide applicability of flow cytometric seed screen – at least in the Potentilleae. However, alternative progeny surveys and precise embryo/endosperm ploidy estimates are required for a comprehensive understanding of the cytology of seed formation. PMID:23425259

  8. Multiplex ready flow cytometric immunoassay for total insulin like growth factor 1 in serum of cattle.

    PubMed

    Bremer, Maria Gabriëlle Eleonore Gerarda; Smits, Nathalie Gabriëlle Esther; Haasnoot, Willem; Nielen, Michel Wilhelmus Franciscus

    2010-05-01

    The European Union has banned the use of recombinant bovine somatotropins (rbST, growth hormones) to increase milk yield in dairy cattle. As direct detection of rbST in serum is problematic, methods based on the detection of changes in multiple rbST-dependent biomarkers have high potential for monitoring rbST abuse. In this study immunoassays were developed for total insulin-like growth factor 1 (IGF-1) in cow sera. Ultimately aiming at combination with other rbST-dependent biomarker assays two multiplex formats were studied and compared critically, a multi-channel surface plasmon resonance (SPR)-based biosensor and flow cytometry combined with color encoded microbeads. Moreover, a new dedicated sample pretreatment was developed for the dissociation of complexed IGF-1 in serum, while keeping other biomarkers in solution. Compared to the SPR biosensor immunoassay, the flow cytometric immunoassay (FCIA) was more sensitive, less antibody-consuming and less vulnerable to necessary but interfering reagents from the sample treatment. In an initial in-house validation study the developed FCIA showed to be fast, specific, robust, and a high repeatability and reliability, and generated realistic IGF-1 values for bovine serum, without compromising the potential for simultaneous detection of other biomarkers. Due to the xMAP technology, in which 100 different bead sets can be measured simultaneously, the total IGF-1 assay can easily be extended with other immunoassays for candidate biomarkers. Preliminary results about a FCIA for IGF-1 multiplexing with insulin-like growth factor binding protein 2 (IGFBP2) are presented which strongly supported both the FCIA multiplex format as well as the generic nature of the developed sample pretreatment.

  9. Flow cytometric method for detecting thiazole orange-positive (reticulated) platelets in thrombocytopenic horses.

    PubMed

    Russell, K E; Perkins, P C; Grindem, C B; Walker, K M; Sellon, D C

    1997-10-01

    To evaluate a method for detecting thiazole orange-positive (TO+, reticulated) platelets in equine blood, using flow cytometry. 16 healthy, equine infectious anemia virus (EIAV)-negative horses and ponies; 9 thrombocytopenic, EIAV-positive horses and ponies; and 2 thrombocytopenic, EIAV-negative horses. Blood from healthy and thrombocytopenic horses was collected by jugular venipuncture. Appropriate sample requirement and incubation time for the assay were evaluated, using blood anticoagulated with EDTA or sodium citrate, or platelet-rich plasma in sodium citrate. The sample of blood or platelet-rich plasma was incubated with thiazole orange, and flow cytometric analysis was performed. Percentage of circulating TO+ platelets was determined from fluorescence (FL-1) logarithmic histograms. Healthy ponies (n = 9) had 1.28 to 2.83% (mean +/- SD, 2.03 +/- 0.50%) and horses (n = 7) had 0.9 to 3.44% (2.12 +/- 1.14%) TO+ platelets in circulation. Thrombocytopenic ponies (n = 7) had 11.14 to 48.41% (26.51 +/- 11.99%) and thrombocytopenic horses (n = 4) had 2.33 to 8.52% (6.19 +/- 2.68%) TO+ platelets in circulation. Mean platelet counts for the thrombocytopenic ponies and horses were 24,400 +/- 20,500 and 39,300 +/- 13,500 platelets/microliters, respectively (reference range, 94,000 to 232,000 platelets/ microliters). Thiazole orange-positive platelets can be detected in equine blood and percentages of TO+ platelets are increased in thrombocytopenic horses. Enumeration of TO+ platelets may prove to be a helpful noninvasive clinical measurement of bone marrow platelet production and aid in the assessment of platelet kinetics in thrombocytopenic horses.

  10. Formaldehyde-fixation of platelets for flow cytometric measurement of phosphatidylserine exposure is feasible.

    PubMed

    Rochat, Sophie; Alberio, Lorenzo

    2015-01-01

    Strong platelet activation results in a redistribution of negatively charged phospholipids from the cytosolic to the outer leaflet of the cellular membrane. Annexin V has a high affinity to negatively charged phospholipids and can be used to identify procoagulant platelets. Formaldehyde fixation can cause factitious Annexin V binding. Our aim was to evaluate a method for fixing platelets avoiding additional Annexin V binding. We induced expression of negatively charged phospholipids on the surface of a fraction of platelets by combined activation with convulxin and thrombin in the presence of Annexin V-fluorescein isothiocyanate and calcium. Aliquots of resting and activated platelets were fixed with a low concentration, calcium-free formaldehyde solution. Both native platelets and fixed platelets were analyzed by flow cytometry immediately and after a 24-h storage at 4°C. We observed that the percentage of Annexin V positive resting platelets ranged from 1.5 to 9.3% for the native samples and from 0.4 to 12.8% for the fixed samples (P=0.706, paired t-test). The amount of Annexin V positive convulxin/thrombin activated platelets varied from 12.9 to 35.4% without fixation and from 15.3 to 36.3% after formalin fixation (P=0.450). After a 24-h storage at 4°C, Annexin V positive platelets significantly increased both in the resting and in the convulxin/thrombin activated samples of native platelets (both P<0.001), while results for formalin fixed platelets did not differ from baseline values (P=0.318 for resting fixed platelets; P=0.673 for activated fixed platelets). We conclude that platelet fixation with a low concentration, calcium-free formaldehyde solution does not alter the proportion of Annexin V positive platelets. This method can be used to investigate properties of procoagulant platelets by multicolor flow-cytometric analysis requiring fixation steps.

  11. Flow cytometric analysis of lectin binding to in vitro-cultured Perkinsus marinus surface carbohydrates

    USGS Publications Warehouse

    Gauthier, J.D.; Jenkins, J.A.; La Peyre, Jerome F.

    2004-01-01

    Parasite surface glycoconjugates are frequently involved in cellular recognition and colonization of the host. This study reports on the identification of Perkinsus marinus surface carbohydrates by flow cytometric analyses of fluorescein isothiocyanate-conjugated lectin binding. Lectin-binding specificity was confirmed by sugar inhibition and Kolmogorov-Smirnov statistics. Clear, measurable fluorescence peaks were discriminated, and no parasite autofluorescence was observed. Parasites (GTLA-5 and Perkinsus-1 strains) harvested during log and stationary phases of growth in a protein-free medium reacted strongly with concanavalin A and wheat germ agglutinin, which bind to glucose-mannose and N-acetyl-D-glucosamine (GlcNAc) moieties, respectively. Both P. marinus strains bound with lower intensity to Maclura pomifera agglutinin, Bauhinia purpurea agglutinin, soybean agglutinin (N-acetyl-D-galactosamine-specific lectins), peanut agglutinin (PNA) (terminal galactose specific), and Griffonia simplicifolia II (GlcNAc specific). Only background fluorescence levels were detected with Ulex europaeus agglutinin I (L-fucose specific) and Limulus polyphemus agglutinin (sialic acid specific). The lectin-binding profiles were similar for the 2 strains except for a greater relative binding intensity of PNA for Perkinsus-1 and an overall greater lectin-binding capacity of Perkinsus-1 compared with GTLA-5. Growth stage comparisons revealed increased lectin-binding intensities during stationary phase compared with log phase of growth. This is the first report of the identification of surface glycoconjugates on a Perkinsus spp. by flow cytometry and the first to demonstrate that differential surface sugar expression is growth phase and strain dependent. ?? American Society of Parasitologists 2004.

  12. Flow cytometric assay for analysis of cytotoxic effects of potential drugs on human peripheral blood leukocytes

    NASA Astrophysics Data System (ADS)

    Nieschke, Kathleen; Mittag, Anja; Golab, Karolina; Bocsi, Jozsef; Pierzchalski, Arkadiusz; Kamysz, Wojciech; Tarnok, Attila

    2014-03-01

    Toxicity test of new chemicals belongs to the first steps in the drug screening, using different cultured cell lines. However, primary human cells represent the human organism better than cultured tumor derived cell lines. We developed a very gentle toxicity assay for isolation and incubation of human peripheral blood leukocytes (PBL) and tested it using different bioactive oligopeptides (OP). Effects of different PBL isolation methods (red blood cell lysis; Histopaque isolation among others), different incubation tubes (e.g. FACS tubes), anticoagulants and blood sources on PBL viability were tested using propidium iodide-exclusion as viability measure (incubation time: 60 min, 36°C) and flow cytometry. Toxicity concentration and time-depended effects (10-60 min, 36 °C, 0-100 μg /ml of OP) on human PBL were analyzed. Erythrocyte lysis by hypotonic shock (dH2O) was the fastest PBL isolation method with highest viability (>85%) compared to NH4Cl-Lysis (49%). Density gradient centrifugation led to neutrophil granulocyte cell loss. Heparin anticoagulation resulted in higher viability than EDTA. Conical 1.5 mL and 2 mL micro-reaction tubes (both polypropylene (PP)) had the highest viability (99% and 97%) compared to other tubes, i.e. three types of 5.0 mL round-bottom tubes PP (opaque-60%), PP (blue-62%), Polystyrene (PS-64%). Viability of PBL did not differ between venous and capillary blood. A gentle reproducible preparation and analytical toxicity-assay for human PBL was developed and evaluated. Using our assay toxicity, time-course, dose-dependence and aggregate formation by OP could be clearly differentiated and quantified. This novel assay enables for rapid and cost effective multiparametric toxicological screening and pharmacological testing on primary human PBL and can be adapted to high-throughput-screening.°z

  13. Flow cytometric bacterial cell counts challenge conventional heterotrophic plate counts for routine microbiological drinking water monitoring.

    PubMed

    Van Nevel, S; Koetzsch, S; Proctor, C R; Besmer, M D; Prest, E I; Vrouwenvelder, J S; Knezev, A; Boon, N; Hammes, F

    2017-04-15

    Drinking water utilities and researchers continue to rely on the century-old heterotrophic plate counts (HPC) method for routine assessment of general microbiological water quality. Bacterial cell counting with flow cytometry (FCM) is one of a number of alternative methods that challenge this status quo and provide an opportunity for improved water quality monitoring. After more than a decade of application in drinking water research, FCM methodology is optimised and established for routine application, supported by a considerable amount of data from multiple full-scale studies. Bacterial cell concentrations obtained by FCM enable quantification of the entire bacterial community instead of the minute fraction of cultivable bacteria detected with HPC (typically < 1% of all bacteria). FCM measurements are reproducible with relative standard deviations below 3% and can be available within 15 min of samples arriving in the laboratory. High throughput sample processing and complete automation are feasible and FCM analysis is arguably less expensive than HPC when measuring more than 15 water samples per day, depending on the laboratory and selected staining procedure(s). Moreover, many studies have shown FCM total (TCC) and intact (ICC) cell concentrations to be reliable and robust process variables, responsive to changes in the bacterial abundance and relevant for characterising and monitoring drinking water treatment and distribution systems. The purpose of this critical review is to initiate a constructive discussion on whether FCM could replace HPC in routine water quality monitoring. We argue that FCM provides a faster, more descriptive and more representative quantification of bacterial abundance in drinking water. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. FlowFP: A Bioconductor Package for Fingerprinting Flow Cytometric Data

    PubMed Central

    Rogers, Wade T.; Holyst, Herbert A.

    2009-01-01

    A new software package called flowFP for the analysis of flow cytometry data is introduced. The package, which is tightly integrated with other Bioconductor software for analysis of flow cytometry, provides tools to transform raw flow cytometry data into a form suitable for direct input into conventional statistical analysis and empirical modeling software tools. The approach of flowFP is to generate a description of the multivariate probability distribution function of flow cytometry data in the form of a “fingerprint.” As such, it is independent of a presumptive functional form for the distribution, in contrast with model-based methods such as Gaussian Mixture Modeling. FlowFP is computationally efficient and able to handle extremely large flow cytometry data sets of arbitrary dimensionality. Algorithms and software implementation of the package are described. Use of the software is exemplified with applications to data quality control and to the automated classification of Acute Myeloid Leukemia. PMID:19956416

  15. Flow cytometric DNA analysis as a diagnostic aid for cervical condyloma and cancer.

    PubMed

    Tsou, K C; Hong, D H; Varello, M; Giuntoli, R; Wheeler, J E; Atkinson, B F; Mangan, C; Mikuta, J

    1984-11-01

    Flow cytometric DNA analysis data (FCDA) were obtained from 324 samples provided through the Gynecology-Oncology Clinic. These samples consisted of 294 combined endoectocervical and vaginal smears and 30 peritoneal washings. Using a conventional scheme for G0/G1, S + G2/M and the coefficient of variation with computer correction for the cell-cycle kinetics, it was possible to assign a diagnostic Class I, II, III or V similar to that used by the Cytology Laboratory. These data were then compared with the histopathologic and colposcopic diagnoses. The correlation between FCDA and cytologic results were essentially similar to the previous data obtained from only endocervical sampling. The most interesting finding in this study was the recognition of an FCDA pattern showing a higher DNA content in the G0/G1 and the early S regions in 70 of 94 (74.5%) of samples from patients with condyloma acuminata. All condyloma samples were diagnosed either by cytologic, histopathologic, or colposcopic examination, or a combination of two or three. All biopsy specimens were then reviewed by one pathologist to verify any discrepancies. The relationship of this pattern to the viral etiology of this disease is discussed with the three methods of diagnosis and electron microscopic observations. It is suggested that, based on this study. FCDA analysis of pap smears may also be useful in determining the presence of condyloma in a gynecology clinic. The potential value of FCDA analysis from peritoneal washings for the diagnosis of gynecologic cancer can not be ascertained in this preliminary investigation because of insufficient samples.

  16. Evaluation of the S phase distribution of flow cytometric DNA histograms by autoradiography and computer algorithms.

    PubMed

    Sheck, L E; Muirhead, K A; Horan, P K

    1980-09-01

    Cell sorting and tritiated thymidine autoradiography were used to define the distribution of S phase cells in flow cytometric DNA histograms obtained from exponential mouse lymphoma cells (L5178Y). The numbers of labeled S phase cells, autoradiographically determined from cells sorted at 2-channel intervals in the G1/early S and late S/G2M regions of the histogram, were compared with the numbers of computed S phase cells in comparable 2-channel intervals as predicted by several computer algorithms used to extract cell cycle phase distributions from DNA histograms. Polynomial and multirectangle algorithms gave computed estimates of total %S in close agreement with the tritiated thymidine labeling index for the cell population, while multi-Gaussian algorithms underestimated %S. Interval autoradiographic and algorithm studies confirmed these results in that no significant differences were found between the autoradiographic S phase distribution and S phase distributions calculated by the polynomial and multirectangle models. However, S phase cells were significantly underestimated in G1/early S by a constrained multi-Gaussian model and in both G1/early S and late S/G2 by an unconstrained multi-Gaussian model. For the particular cell line (L5178Y), staining protocol (mithramycin following ethanol fixation) and instrumentation (Coulter TPS-2 cell sorter) used in this study, close agreement between computed %S and tritiated thymidine labeling index was found to be a reliable indicator of an algorithm's success in resolving S phase cells in the G1/S and S/G2 transition regions of the DNA histograms.

  17. Flow cytometric detection of micronuclei by combined staining of DNA and membranes

    SciTech Connect

    Wessels, J.M.; Nuesse, M.

    1995-03-01

    A new staining method is presented for flow cytometric measurement of micronuclei (MN) in cell cultures and human lymphocytes using membrane-specific fluorescent dyes in addition to DNA staining. Several combinations of fluorescent membrane and DNA dyes were studied for a better discrimination of MN from debris in a suspension of nuclei and micronuclei. For staining of membranes, the lipophilic dyes 2-hydroxyethyl-7,12,17-tris(methoxyethyl)porphycene (HEPn) and 1,6-diphenyl-1,3,5-hexatriene (DPH) were used in combination with ethidium bromide (EB), proflavine (PF), and Hoechst 33258 (HO). Due to their spectral properties, HO or EB combined with HEPn were not as suitable for the discrimination of MN from debris as was HEPn in combination with PF. With HEPn in combination with PF, however, additional noise was found at low fluorescence intensities, probably due to free fluorescent dye molecules in the solution. The optimal simultaneous staining of membranes and DNA was obtained using a combination of DPH and EB. The induction of MN in Chinese hamster and mouse NIH-3T3 cells by UV-B illumination was studied with this new staining technique. UV-B illumination (280-360 nm) induced MN in both cell lines. Chinese hamster cells were found to be more sensitive to these wavelengths. Illumination with wavelengths above 360 nm did not induce MN in either cell line. The results obtained from human lymphocytes using the combination of EB or DPH were comparable to the results obtained with the combination of EB and HO. 23 refs., 7 figs.

  18. Flow Cytometric Assessment of Bacterial Abundance in Soils, Sediments and Sludge

    PubMed Central

    Frossard, Aline; Hammes, Frederik; Gessner, Mark O.

    2016-01-01

    Bacterial abundance is a fundamental measure in microbiology, but its assessment is often tedious, especially for soil, and sediment samples. To overcome this limitation, we adopted a time-efficient flow-cytometric (FCM) counting method involving cell detachment and separation from matrix particles by centrifugation in tubes receiving sample suspensions and Histodenz® solution. We used this approach to assess bacterial abundances in diverse soils (natural and agricultural), sediments (streams and lakes) and sludge from sand-filters in a drinking water treatment plant and compared the results to bacterial abundances determined by two established methods, epifluorescence microscopy (EM) and adenosine triphosphate (ATP) quantification. Cell abundances determined by FCM and EM correlated fairly well, although absolute cell abundances were generally lower when determined by FCM. FCM also showed significant relations with cell counts converted from ATP concentrations, although estimates derived from ATP determinations were typically higher, indicating the presence of ATP sources other than bacteria. Soil and sediment organic matter (OM) content influenced the goodness of fit between counts obtained with EM and FCM. In particular, bacterial abundance determined by FCM in samples containing less than 10% OM, such as stream sediment, was particularly well correlated with the cell counts assessed by EM. Overall, these results suggest that FCM following cell detachment and purification is a useful approach to increase sample throughput for determining bacterial abundances in soils, sediments and sludge. However, notable scatter and only partial concordance among the FCM and reference methods suggests that protocols require further improvement for assessments requiring high precision, especially when OM contents in samples are high. PMID:27379043

  19. Flow-cytometric screening of aggregation-inhibitors using a fluorescence-assisted intracellular method.

    PubMed

    Lindberg, Hanna; Sandersjöö, Lisa; Meister, Sebastian W; Uhlén, Mathias; Löfblom, John; Ståhl, Stefan

    2017-01-01

    Aggregation of misfolded peptides and proteins is a key event in several neurodegenerative diseases. Suggested treatments of such disorders aim to inhibit the initial aggregation process. Here, we have developed an intracellular, function-based screening method, intended for isolation of aggregation-inhibitors from combinatorial protein libraries by flow-cytometric cell sorting. The method is based on fusion of aggregation-prone peptides to a fluorescent protein, functioning as a solubility reporter. Co-expression of a protein-based aggregation-inhibitor should prevent aggregation and thus increase the whole-cell fluorescence. We evaluated the method using the aggregation-prone Alzheimer's-related amyloid-β (Aβ) peptide in fusion to green-fluorescent protein (GFP), and an Aβ aggregation-inhibiting Affibody molecule. To adapt the method for library applications, the inhibitor was linked to an mCherry reporter for normalization of protein expression levels. We found that aggregation propensity correlates with fluorescence intensity, as co-expression of the Affibody-inhibitor increased the whole-cell fluorescence relative to a non-inhibitor. Employing improved cultivation parameters, we furthermore demonstrated efficient rescue from aggregation of an α-synuclein-derived protein using a different type of aggregation-inhibitor. Importantly, we also showed that the Aβ aggregation-inhibiting Affibody molecule could be isolated from a 1:10,000 background of non-inhibitors, with around 3,500-fold enrichment, in one cycle of fluorescence-based cell sorting. In conclusion, our new method represents a promising approach for generation of novel protein-based aggregation-inhibitors. Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Flow Cytometric Assessment of Membrane Integrity of Ethanol-Stressed Oenococcus oeni Cells

    PubMed Central

    Graça da Silveira, M.; Vitória San Romão, M.; Loureiro-Dias, Maria C.; Rombouts, Frans M.; Abee, Tjakko

    2002-01-01

    The practical application of commercial malolactic starter cultures of Oenococcus oeni surviving direct inoculation in wine requires insight into the mechanisms involved in ethanol toxicity and tolerance in this organism. Exposure to ethanol resulted in an increase in the permeability of the cytoplasmic membrane, enhancing passive proton influx and concomitant loss of intracellular material (absorbing at 260 nm). Cells grown in the presence of 8% (vol/vol) ethanol revealed adaptation to ethanol stress, since these cells showed higher retention of compounds absorbing at 260 nm. Moreover, for concentrations higher than 10% (vol/vol), lower rates of passive proton influx were observed in these ethanol-adapted cells, especially at pH 3.5. The effect of ethanol on O. oeni cells was studied as the ability to efficiently retain carboxyfluorescein (cF) as an indicator of membrane integrity and enzyme activity and the uptake of propidium iodide (PI) to assess membrane damage. Flow cytometric analysis of both ethanol-adapted and nonadapted cells with a mixture of the two fluorescent dyes, cF and PI, revealed three main subpopulations of cells: cF-stained intact cells; cF- and PI-stained permeable cells, and PI-stained damaged cells. The subpopulation of O. oeni cells that maintained their membrane integrity, i.e., cells stained only with cF, was three times larger in the population grown in the presence of ethanol, reflecting the protective effect of ethanol adaptation. This information is of major importance in studies of microbial fermentations in order to assign bulk activities measured by classical methods to the very active cells that are effectively responsible for the observations. PMID:12450832

  1. Ligand receptor dynamics at streptavidin-coated particle surfaces: A flow cytometric and spectrofluorimetric study

    SciTech Connect

    Buranda, T. |; Jones, G.M.; Nolan, J.P.; Keij, J.; Lopez, G.P.; Sklar, L.A. |

    1999-04-29

    The authors have studied the binding of 5-((N-(5-(N-(6-(biotinoyl)amino)hexanoyl)amino)pentyl)thioureidyl)fluorescein (fluorescein biotin) to 6.2 {micro}m diameter, streptavidin-coated polystyrene beads using a combination of fluorimetric and flow cytometric methods. They have determined the average number of binding sites per bead, the extent of fluorescein quenching upon binding to the bead, and the association and dissociation kinetics. The authors estimate the site number to be {approx}1 million per bead. The binding of the fluorescein biotin ligand occurs in steps where the insertion of the biotin moiety into one receptor pocket is followed immediately by the capture of the fluorescein moiety by a neighboring binding pocket; fluorescence quenching is a consequence of this secondary binding. At high surface coverage, the dominant mechanism of quenching appears to be via the formation of nonfluorescent nearest-neighbor aggregates. At early times, the binding process is characterized by biphasic association and dissociation kinetics which are remarkably dependent on the initial concentration of the ligand. The rate constant for binding to the first receptor pocket of a streptavidin molecule is {approx}(1.3 {+-} 0.3) {times} 10{sup 7} 1{sup {minus}1} S{sup {minus}1}. The rate of binding of a second biotin may be reduced due to steric interference. The early time dissociative behavior is in sharp contrast to the typical stability associated with this system. The early time dissociative behavior is in sharp contrast to the typical stability associated with this system. The dissociation rate constant is as high as 0.05 s{sup {minus}1} shortly after binding, but decreases by 3 orders of magnitude after 3 h of binding. Potential sources for the time dependence of the dissociation rate constant are discussed.

  2. High Throughput Transcriptomics @ USEPA (Toxicology ...

    EPA Pesticide Factsheets

    The ideal chemical testing approach will provide complete coverage of all relevant toxicological responses. It should be sensitive and specific It should identify the mechanism/mode-of-action (with dose-dependence). It should identify responses relevant to the species of interest. Responses should ideally be translated into tissue-, organ-, and organism-level effects. It must be economical and scalable. Using a High Throughput Transcriptomics platform within US EPA provides broader coverage of biological activity space and toxicological MOAs and helps fill the toxicological data gap. Slide presentation at the 2016 ToxForum on using High Throughput Transcriptomics at US EPA for broader coverage biological activity space and toxicological MOAs.

  3. 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 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. 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.

  4. Cytometric fingerprints: evaluation of new tools for analyzing microbial community dynamics.

    PubMed

    Koch, Christin; Harnisch, Falk; Schröder, Uwe; Müller, Susann

    2014-01-01

    Optical characteristics of individual bacterial cells of natural communities can be measured with flow cytometry (FCM) in high throughput. The resulting data are visualized in cytometric histograms. These histograms represent individual cytometric fingerprints of microbial communities, e.g., at certain time points or microenvironmental conditions. Up to now four tools for analyzing the variation in these cytometric fingerprints are available but have not yet been systematically compared regarding application: Dalmatian Plot, Cytometric Histogram Image Comparison (CHIC), Cytometric Barcoding (CyBar), and FlowFP. In this article these tools were evaluated concerning (i) the required experience of the operator in handling cytometric data sets, (ii) the detection level of changes, (iii) time demand for analysis, and (iv) software requirements. As an illustrative example, FCM was used to characterize the microbial community structure of electroactive microbial biofilms. Their cytometric fingerprints were determined, analyzed with all four tools, and correlated to experimental and functional parameters. The source of inoculum (four different types of wastewater samples) showed the strongest influence on the microbial community structure and biofilm performance while the choice of substrate (acetate or lactate) had no significant effect in the present study. All four evaluation tools were found suitable to monitor structural changes of natural microbial communities. The Dalmatian Plot was shown to be most sensitive to operator impact but nevertheless provided an overview on community shifts. CHIC, CyBar, and FlowFP showed less operator dependence and gave highly resolved information on community structure variation on different detection levels. In conclusion, experimental and productivity parameters correlated with the biofilm structures and practical process integration details were available from cytometric fingerprint analysis.

  5. A robust, high-throughput assay to determine the phagocytic activity of clinical antibody samples

    PubMed Central

    Ackerman, Margaret E.; Moldt, Brian; Wyatt, Richard T; Dugast, Anne-Sophie; McAndrew, Elizabeth; Tsoukas, Stephen; Jost, Stephanie; Berger, Christoph T.; Sciaranghella, Gaia; Liu, Qingquan; Irvine, Darrell J; Burton, Dennis R.; Alter, Galit

    2011-01-01

    Phagocytosis can be induced via the engagement of Fcγ receptors by antibody-opsonized material. Furthermore, the efficiency of antibody-induced effector functions has been shown to be dramatically modulated by changes in antibody glycosylation. Because infection can modulate antibody glycans, which in turn modulate antibody functions, assays capable of determining the induction of effector functions rather than neutralization or titer provide a valuable opportunity to more fully characterize the quality of the adaptive immune response. Here we describe a robust and high-throughput flow cytometric assay to define the phagocytic activity of antigen-specific antibodies from clinical samples. This assay employs a monocytic cell line that expresses numerous Fc receptors: including inhibitory and activating, and high and low affinity receptors—allowing complex phenotypes to be studied. We demonstrate the adaptability of this high-throughput, flow-based assay to measure antigen-specific antibody-mediated phagocytosis against an array of viruses, including influenza, HIV, and dengue. The phagocytosis assay format further allows for simultaneous analysis of cytokine release, as well as determination of the role of specific Fcγ-receptor subtypes, making it a highly useful system for parsing differences in the ability of clinical and vaccine induced antibody samples to recruit this critical effector function. PMID:21192942

  6. A robust, high-throughput assay to determine the phagocytic activity of clinical antibody samples.

    PubMed

    Ackerman, Margaret E; Moldt, Brian; Wyatt, Richard T; Dugast, Anne-Sophie; McAndrew, Elizabeth; Tsoukas, Stephen; Jost, Stephanie; Berger, Christoph T; Sciaranghella, Gaia; Liu, Qingquan; Irvine, Darrell J; Burton, Dennis R; Alter, Galit

    2011-03-07

    Phagocytosis can be induced via the engagement of Fcγ receptors by antibody-opsonized material. Furthermore, the efficiency of antibody-induced effector functions has been shown to be dramatically modulated by changes in antibody glycosylation. Because infection can modulate antibody glycans, which in turn modulate antibody functions, assays capable of determining the induction of effector functions rather than neutralization or titer provide a valuable opportunity to more fully characterize the quality of the adaptive immune response. Here we describe a robust and high-throughput flow cytometric assay to define the phagocytic activity of antigen-specific antibodies from clinical samples. This assay employs a monocytic cell line that expresses numerous Fc receptors: including inhibitory and activating, and high and low affinity receptors--allowing complex phenotypes to be studied. We demonstrate the adaptability of this high-throughput, flow-based assay to measure antigen-specific antibody-mediated phagocytosis against an array of viruses, including influenza, HIV, and dengue. The phagocytosis assay format further allows for simultaneous analysis of cytokine release, as well as determination of the role of specific Fcγ-receptor subtypes, making it a highly useful system for parsing differences in the ability of clinical and vaccine induced antibody samples to recruit this critical effector function. Copyright © 2010 Elsevier B.V. All rights reserved.

  7. Lysis matters: red cell lysis with FACS Lyse affects the flow cytometric enumeration of circulating leukemic blasts.

    PubMed

    Einwallner, Elisa; Subasic, Almira; Strasser, Andrea; Augustin, Dorothea; Thalhammer, Renate; Steiner, Irene; Schwarzinger, Ilse

    2013-04-30

    The whole blood lysis method has become a standard procedure to remove red cells prior to immunophenotypic analysis of leukocytes. In the present study we investigated the influence of four different lysis protocols on the flow cytometric recovery of leukemic blasts. 32 blast cells containing blood samples were stained with anti-CD45 and anti-CD34 monoclonal antibody combinations. Red cell lysis was performed with FACS Lysing Solution and BD PharmLyse™ (Becton Dickinson and Company BD Biosciences, San Jose, CA; n=32) as well as Optilyse C and IOTest 3 (Immunotech SAS, Marseille; n=15 out of 32). Flow cytometric enumeration of blasts was performed on a FACS-Canto flow cytometer. The percentage of blasts after treatment with FACS Lyse was significantly smaller than after PharmLyse™ (p<0.0001), Optilyse C (p<0.0001), or IOTest 3 (p<0.0001), respectively. The difference between PharmLyse™ and Optilyse C (p=0.93), PharmLyse™ and IOTest 3 (p=0.31), and Optilyse C and IOTest 3 (p=0.34) was not significant. These results emphasize the importance of harmonization of red cell lysis protocols for the application of flow cytometry in hematological neoplasms. Copyright © 2013 Elsevier B.V. All rights reserved.

  8. Effect of intra-cellular trafficking on flow cytometric measurement of neutrophil's oxidative status in iron deficient pregnant females.

    PubMed

    Youssef, Soha R; Hendawy, Sherif F; Boshnak, Noha H; Sedhom, Mariana S

    2017-03-27

    Iron deficiency and iron deficiency anemia are prevalent among pregnant women particularly in developing countries. This study aimed to evaluate the iron status among Egyptian pregnant women and its impact on their neutrophil's count and antimicrobial functions. Ninety pregnant females underwent complete blood count, iron profile, flow cytometric studies for neutrophil myeloperoxidase expression & oxidative burst using dihydrorhodamine 123 (DHR) after phorbol-12-myristate-13-acetate (PMA) stimulation as well as neutrophil phagocytic and lytic indices. According to percent saturation 54/90 women (60%) were iron deficient (<15% saturation) (cases) and 36/90 (40%) were iron sufficient (controls). A higher proportion of iron deficient pregnant women were in their third trimester compared to controls. No significant difference was found between the iron deficient & sufficient groups as regards anemia despite a positive correlation between haemoglobin level and percent saturation (P=.02). Both the phagocytic and lytic indices were significantly lower among the cases compared to controls (P=.014 & .002 respectively). Cases and controls were comparable as regards flow cytometric studies of neutrophils' myeloperoxidase and oxidative burst (P>.05). No significant correlation was found between any of the iron profile parameters and the oxidative burst by flow cytometry. Functional microphage assay (phagocytic and lytic indices) may be more relevant and cost effective than flow cytometry assays of myeloperoxidase and oxidative burst in reflecting either iron status or cellular immunity in pregnancy. © 2017 Wiley Periodicals, Inc.

  9. Flow cytometric identification and enumeration of photosynthetic sulfur bacteria and potential for ecophysiological studies at the single-cell level.

    PubMed

    Casamayor, Emilio O; Ferrera, Isabel; Cristina, Xavier; Borrego, Carles M; Gasol, Josep M

    2007-08-01

    We show the potential of flow cytometry as a fast tool for population identification and enumeration of photosynthetic sulfur bacteria. Purple (PSB) and green sulfur bacteria (GSB) oxidize hydrogen sulfide to elemental sulfur that can act as storage compound to be further oxidized to sulfate generating the reducing power required for growth. Both groups have different elemental sulfur allocation strategies: whereas PSB store elemental sulfur as intracellular inclusions, GSB allocate sulfur globules externally. We used well-characterized laboratory strains and complex natural photosynthetic populations developing in a sharply stratified meromictic lake to show that PSB and GSB could be detected, differentiated and enumerated in unstained samples using a blue laser-based flow cytometer. Variations in cell-specific pigment content and the dynamics of sulfur accumulation, both intra- and extracellularly, were also detected in flow cytometric plots as sulfur accumulation changed the light scatter characteristics of the cells. These data were used to show the potential for studies on the metabolic status and the rate of activity at the single-cell level. Flow cytometric identification and enumeration resulted in faster and more precise analyses than previous approaches, and may open the door to more complex ecophysiological experiments with photosynthetic sulfur bacteria in mixed cultures and natural environments.

  10. Flow Cytometric Investigation of Filamentation, Membrane Patency, and Membrane Potential in Escherichia coli following Ciprofloxacin Exposure

    PubMed Central

    Wickens, H. J.; Pinney, R. J.; Mason, D. J.; Gant, V. A.

    2000-01-01

    Ninety-eight percent of the cells in a population of Escherichia coli in log-phase growth lost colony-forming ability after being exposed for 3 h to the quinolone antibiotic ciprofloxacin at four times the MIC in nutrient broth, a concentration easily reached in vivo. Flow cytometric analysis, however, demonstrated that only 68% of this bacterial population had lost membrane potential, as judged by the membrane potential-sensitive dye bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)], and only 30% could no longer exclude the nucleic acid-binding dye propidium iodide (PI), reflecting lost membrane integrity, efflux mechanisms, or both. Subsequent removal of ciprofloxacin and resuspension in nutrient broth resulted in renewed cell division after 2 h, with a calculated postantibiotic effect (PAE) time of 57 min. The proportion of DiBAC- and PI-fluorescent cells in this recovering population remained stable for more than 4 h after antibiotic removal. Eighty percent of cells present at drug removal were filamentous. Their number subsequently decreased with time, and the increase in particle count seen at the end of the PAE resulted from the division of short cells. Exposure to ciprofloxacin in the presence of the protein synthesis inhibitor chloramphenicol increased colony-forming ability to 60% of starting population numbers. In contrast to ciprofloxacin alone, this antibiotic combination resulted in insignificant filamentation and no dye uptake. Subsequent drug removal and resuspension in nutrient broth resulted in the appearance of filaments within 1 h, with 69% of the population forming filaments at 3 h. Dye uptake was also seen, with 20% of the population fluorescing with either dye after 4 h. We were unable to relate dye uptake to the viable count. Cell division resumed 240 min after removal of both drugs, yielding a PAE calculated at 186 min. Inhibition of protein synthesis with chloramphenicol prevented ciprofloxacin-induced changes in bacterial

  11. Single tube, six-color flow cytometric analysis is a sensitive and cost-effective technique for assaying clonal plasma cells.

    PubMed

    Marsee, Derek K; Li, Betty; Dorfman, David M

    2010-05-01

    Bone marrow flow cytometric analysis is a powerful and rapid tool for evaluating plasma cell myeloma. By using a noncontrolled patient population in various stages of diagnosis and treatment, we compared 6-color (single-tube) and 4-color (multiple-tube) flow cytometric immunophenotyping protocols. Prospective comparison in 52 cases demonstrated improved ability to detect clonal plasma cells or identical diagnoses in 100% of the cases using 6-color, single-tube analysis. In cases in which 6-color flow cytometric analysis improved detection of a clonal population, concurrent biopsy showed less than 5% involvement by plasma cell myeloma, suggesting that 6-color flow cytometry has an advantage in patients with a low disease burden. In addition, the simplification of the procedure resulted in substantial savings in technologist time and reagent costs. Taken together, this study demonstrates that 6-color flow cytometry is an excellent, cost-effective means to assay for clonal plasma cells in a noncontrolled patient population.

  12. A low-cost flow cytometric assay for the detection and quantification of apoptosis using an anionic halogenated fluorescein dye.

    PubMed

    Meyer, Mervin; Essack, Magbubah; Kanyanda, Stonard; Rees, Jasper

    2008-09-01

    We describe here a technical improvement of an established colorimetric method used to detect and measure the occurrence of apoptosis in mammalian cells during in vitro cell culture. This assay uses an anionic halogenated fluorescein dye that is taken up by apoptotic cells at the stage of phosphatidylserine externalization. We demonstrate that apoptotic cells stained with this dye can be detected by flow cytometric analysis. Furthermore, we show that the modified method compares well with the standard annexin-V-based apoptosis assay and that it is significantly more cost-effective than the annexin-V assay.

  13. Flow cytometric analysis of expression of interleukin-2 receptor beta chain (p70-75) on various leukemic cells

    SciTech Connect

    Hoshino, S.; Oshimi, K.; Tsudo, M.; Miyasaka, M.; Teramura, M.; Masuda, M.; Motoji, T.; Mizoguchi, H. )

    1990-08-15

    We analyzed the expression of the interleukin-2 receptor (IL-2R) beta chain (p70-75) on various leukemic cells from 44 patients by flow cytometric analysis using the IL-2R beta chain-specific monoclonal antibody, designated Mik-beta 1. Flow cytometric analysis demonstrated the expression of the IL-2R beta chain on granular lymphocytes (GLs) from all eight patients with granular lymphocyte proliferative disorders (GLPDs), on adult T-cell leukemia (ATL) cells from all three patients with ATL, and on T-cell acute lymphoblastic leukemia (T-ALL) cells from one of three patients with T-ALL. Although GLs from all the GLPD patients expressed the IL-2R beta chain alone and not the IL-2R alpha chain (Tac-antigen: p55), ATL and T-ALL cells expressing the beta chain coexpressed the alpha chain. In two of seven patients with common ALL (cALL) and in both patients with B-cell chronic lymphocytic leukemia, the leukemic cells expressed the alpha chain alone. Neither the alpha chain nor the beta chain was expressed on leukemic cells from the remaining 28 patients, including all 18 patients with acute nonlymphocytic leukemia, five of seven patients with cALL, all three patients with multiple myeloma, and two of three patients with T-ALL. These results indicate that three different forms of IL-2R chain expression exist on leukemic cells: the alpha chain alone; the beta chain alone; and both the alpha and beta chains. To examine whether the results obtained by flow cytometric analysis actually reflect functional aspects of the expressed IL-2Rs, we studied the specific binding of 125I-labeled IL-2 (125I-IL-2) to leukemic cells in 18 of the 44 patients. In addition, we performed 125I-IL-2 crosslinking studies in seven patients. The results of IL-2R expression of both 125I-IL-2 binding assay and crosslinking studies were in agreement with those obtained by flow cytometric analysis.

  14. Flow cytometric and radioisotopic determinations of platelet survival time in normal cats and feline leukemia virus-infected cats

    SciTech Connect

    Jacobs, R.M.; Boyce, J.T.; Kociba, G.J.

    1986-01-01

    This study demonstrates the potential usefulness of a flow cytometric technique to measure platelet survival time in cats utilizing autologous platelets labeled in vitro with fluorescein isothiocyanate (FITC). When compared with a 51Cr method, no significant differences in estimated survival times were found. Both the 51Cr and FITC-labeling procedures induced similar changes in platelet shape and collagen-induced aggregation. Platelets labeled with FITC had significantly greater volumes compared with those of glutaraldehyde-fixed platelets. These changes were primarily related to the platelet centrifugation and washing procedures rather than the labels themselves. This novel technique potentially has wide applicability to cell circulation time studies as flow cytometry equipment becomes more readily available. Problems with the technique are discussed. In a preliminary study of the platelet survival time in feline leukemia virus (FeLV)-infected cats, two of three cats had significantly reduced survival times using both flow cytometric and radioisotopic methods. These data suggest increased platelet turnover in FeLV-infected cats.

  15. Postlarval muscle growth in fish: a DNA flow cytometric and morphometric analysis.

    PubMed

    Alfei, L; Maggi, F; Parvopassu, F; Bertoncello, G; De Vita, R

    1989-01-01

    The mechanism of postlarval fish myotomal growth was investigated in trout (Salmo gairdneri) by means of morphometric and cytofluorometric analysis. The mechanism by which new fibres are added during postlarval growth (hyperplasia) is not fully understood. In histological cross sections these new fibres have a small diameter which give the muscle a "mosaic" appearance. One hypothesis suggested that they could be derived from the proliferative activity of satellite cells. DNA cytofluorometric analysis of nuclei suspensions obtained from trout white myotomal muscle during different developmental stages (eleutherembyronic; alevin; yearling and adult) showed a consistently low S-cytometric phase during all stage in which myofibres of small diameters were present. The percentage of such small fibres, determined by morphometric analysis, suggested that satellite cells are the proliferative population. In fact, their percentages, as determined by morphometric analysis in histological section, bear a linear relationship with the S-cytometric phase percent nuclei (R = 0.927). Only in adults (67 cm in size) there was a significant decrease in the S-cytometric phase. At this stage, in histological sections, the myotomal muscle no longer had a "mosaic" appearance because of the disappearance of the small fibres. It may, therefore, be supposed that in the cm 67 adult specimens, the proliferative population is entering the G0 phase. It is known, in fact, that muscle growth proceeds only by fibre hypertrophy in trout longer than 70 cm in length (Stickland, 1983).

  16. Rapid Detection and Enumeration of Giardia lamblia Cysts in Water Samples by Immunomagnetic Separation and Flow Cytometric Analysis ▿ †

    PubMed Central

    Keserue, Hans-Anton; Füchslin, Hans Peter; Egli, Thomas

    2011-01-01

    Giardia lamblia is an important waterborne pathogen and is among the most common intestinal parasites of humans worldwide. Its fecal-oral transmission leads to the presence of cysts of this pathogen in the environment, and so far, quantitative rapid screening methods are not available for various matrices, such as surface waters, wastewater, or food. Thus, it is necessary to establish methods that enable reliable rapid detection of a single cyst in 10 to 100 liters of drinking water. Conventional detection relies on cyst concentration, isolation, and confirmation by immunofluorescence microscopy (IFM), resulting in low recoveries and high detection limits. Many different immunomagnetic separation (IMS) procedures have been developed for separation and cyst purification, so far with variable but high losses of cysts. A method was developed that requires less than 100 min and consists of filtration, resuspension, IMS, and flow cytometric (FCM) detection. MACS MicroBeads were used for IMS, and a reliable flow cytometric detection approach was established employing 3 different parameters for discrimination from background signals, i.e., green and red fluorescence (resulting from the distinct pattern emitted by the fluorescein dye) and sideward scatter for size discrimination. With spiked samples, recoveries exceeding 90% were obtained, and false-positive results were never encountered for negative samples. Additionally, the method was applicable to naturally occurring cysts in wastewater and has the potential to be automated. PMID:21685159

  17. Nucleic acid specificity of an acridine derivative permits its use for flow cytometric analysis of the cell cycle.

    PubMed

    Jayat-Vignoles, C; Ratinaud, M H

    1997-02-01

    3-amino-6-methoxy-9-(2-hydroxyethylamine) acridine (AMHA) is an acridine derivative, which is easily excited in near ultraviolet and which emits a bright green fluorescence. The dye was preferentially incorporated into nucleic structures as attested by microscopic and cytometric analyses after RNase and DNase treatments. The affinity for RNA seemed low and similar to that observed for propidium iodide. AMHA was quickly accumulated in fixed cells, and in appropriate concentrations (10-50 microM) was a DNA- and RNA-specific dye. AMHA probably exhibits an adenine-thymine specificity, as suggested by its quenching after bromodeoxyuridine uptake: the fluorescence quenching was similar to that obtained for Hoechst 33258. After cell treatment by RNase and in the presence of MgCl2, AMHA staining allowed flow cytometric analysis of the cell-cycle distribution. The resulting histograms were similar to those obtained with propidium iodide (CV near 3.5%, and similar cell cycle distribution). Thus, AMHA is a suitable fluorescent dye for efficient analysis of the cell cycle by flow cytometry.

  18. A simplified flow cytometric assay identifies children with acute lymphoblastic leukemia who have a superior clinical outcome

    PubMed Central

    Coustan-Smith, Elaine; Ribeiro, Raul C.; Stow, Patricia; Zhou, Yinmei; Pui, Ching-Hon; Rivera, Gaston K.; Pedrosa, Francisco; Campana, Dario

    2006-01-01

    Bone marrow normal lymphoid progenitors (CD19+, CD10+, and/or CD34+) are exquisitely sensitive to corticosteroids and other antileukemic drugs. We hypothesized that, in patients with B-lineage acute lymphoblastic leukemia (ALL), cells with this phenotype detected early in treatment should be leukemic rather than normal. We therefore developed a simple and inexpensive flow cytometric assay for such cells and prospectively applied it to bone marrow samples collected on day 19 from 380 children with B-lineage ALL. In 211 patients (55.5%), these cells represented 0.01% or more of the mononuclear cells; results correlated remarkably well with those of more complex flow cytometric and molecular minimal residual disease (MRD) evaluations. Among 84 uniformly treated children, the 10-year incidence of relapse or remission failure was 28.8% ± 7.1% (SE) for the 42 patients with 0.01% or more leukemic cells on day 19 detected by the simplified assay versus 4.8% ± 3.3% for the 42 patients with lower levels (P = .003). These assay results were the strongest predictor of outcome, even after adjustment for competing clinicobiologic variables. Thus, this new assay would enable most treatment centers to identify a high proportion of children with ALL who have an excellent early treatment response and a high likelihood of cure. (Blood. 2006;108:97-102) PMID:16537802

  19. Rapid detection and enumeration of Giardia lamblia cysts in water samples by immunomagnetic separation and flow cytometric analysis.

    PubMed

    Keserue, Hans-Anton; Füchslin, Hans Peter; Egli, Thomas

    2011-08-01

    Giardia lamblia is an important waterborne pathogen and is among the most common intestinal parasites of humans worldwide. Its fecal-oral transmission leads to the presence of cysts of this pathogen in the environment, and so far, quantitative rapid screening methods are not available for various matrices, such as surface waters, wastewater, or food. Thus, it is necessary to establish methods that enable reliable rapid detection of a single cyst in 10 to 100 liters of drinking water. Conventional detection relies on cyst concentration, isolation, and confirmation by immunofluorescence microscopy (IFM), resulting in low recoveries and high detection limits. Many different immunomagnetic separation (IMS) procedures have been developed for separation and cyst purification, so far with variable but high losses of cysts. A method was developed that requires less than 100 min and consists of filtration, resuspension, IMS, and flow cytometric (FCM) detection. MACS MicroBeads were used for IMS, and a reliable flow cytometric detection approach was established employing 3 different parameters for discrimination from background signals, i.e., green and red fluorescence (resulting from the distinct pattern emitted by the fluorescein dye) and sideward scatter for size discrimination. With spiked samples, recoveries exceeding 90% were obtained, and false-positive results were never encountered for negative samples. Additionally, the method was applicable to naturally occurring cysts in wastewater and has the potential to be automated.

  20. Confounding effects of platelets on flow cytometric analysis and cell-sorting experiments using blood-derived cells.

    PubMed

    McFarland, David C; Zhang, Cindy; Thomas, Heath C; T L, Ratliff

    2006-02-01

    Flow cytometric analysis and cell-sorting of peripheral blood leukocytes is commonplace; however, platelet contamination is typically ignored during immunophenotypic analysis and sorting of blood-derived cells. Red blood cells, platelets, T & B lymphocytes, monocytes, and granulocytes were sorted from rat blood preparations. Presort enrichment was performed by differential centrifugation for all cell types. Additionally, leukocyte samples were prepared by ammonium chloride lysis of red blood cells. Unless proper precautions were taken, significant numbers of platelets were sorted along with (nonplatelet) cells of interest. The amount of platelet contamination varied greatly from experiment to experiment with the highest level of leukocyte-platelet association observed in the neutrophil/granulocyte population in samples prepared using ammonium chloride-based red blood cell-lysing solution. Addition of an immunophenotypic marker for platelet identification is a simple, yet prudent, measure to help evaluate the impact of platelets on immunophenotypic staining when performing flow cytometric analysis or sorting of blood-derived cells and should become a routine practice. Platelet presence in postsort fractions can be due to free platelets as well as target cell-associated platelets and both sources of contamination must be addressed. (c) 2005 Wiley-Liss, Inc.

  1. Comparison of drug release from liquid crystalline monoolein dispersions and solid lipid nanoparticles using a flow cytometric technique

    PubMed Central

    Dawoud, Mohamed Z.; Nasr, Mohamed

    2016-01-01

    Colloidal lipid particles such as solid lipid nanoparticles and liquid crystalline nanoparticles have great opportunities as drug carriers especially for lipophilic drugs intended for intravenous administration. In order to evaluate drug release from these nanoparticles and determine their behavior after administration, emulsion droplets were used as a lipophilic compartment to which the transfer of a model drug was measured. The detection of the model drug transferred from monoolein cubic particles and trimyristin solid lipid nanoparticles into emulsion droplets was performed using a flow cytometric technique. A higher rate and amount of porphyrin transfer from the solid lipid nanoparticles compared to the monoolein cubic particles was observed. This difference might be attributed to the formation of a highly ordered particle which leads to the expulsion of drug to the surface of the crystalline particle. Furthermore, the sponge-like structure of the monoolein cubic particles decreases the rate and amount of drug transferred. In conclusion, the flow cytometric technique is a suitable technique to study drug transfer from these carriers to large lipophilic acceptors. Monoolein cubic particles with their unique structure can be used successfully as a drug carrier with slow drug release compared with trimyristin nanoparticles. PMID:27006901

  2. A cluster analysis method for identification of subpopulations of cells in flow cytometric list-mode arrays

    NASA Technical Reports Server (NTRS)

    Li, Z. K.

    1985-01-01

    A specialized program was developed for flow cytometric list-mode data using an heirarchical tree method for identifying and enumerating individual subpopulations, the method of principal components for a two-dimensional display of 6-parameter data array, and a standard sorting algorithm for characterizing subpopulations. The program was tested against a published data set subjected to cluster analysis and experimental data sets from controlled flow cytometry experiments using a Coulter Electronics EPICS V Cell Sorter. A version of the program in compiled BASIC is usable on a 16-bit microcomputer with the MS-DOS operating system. It is specialized for 6 parameters and up to 20,000 cells. Its two-dimensional display of Euclidean distances reveals clusters clearly, as does its 1-dimensional display. The identified subpopulations can, in suitable experiments, be related to functional subpopulations of cells.

  3. Flow cytometric kinetic assay of calcium mobilization in whole blood platelets using Fluo-3 and CD41.

    PubMed

    do Céu Monteiro, M; Sansonetty, F; Gonçalves, M J; O'Connor, J E

    1999-04-01

    Platelet activation plays a major role in the physiology and pathology of hemostasis. Flow cytometry is a promising approach for the structural and functional analysis of platelets. However, the choice of adequate biological parameters and most technical issues are still under discussion. A rise in cytosolic free Ca2+ is a key early event that follows platelet stimulation and precedes several activation responses, including shape change, aggregation, secretion, and expression of procoagulant activity. Our objective was to set up a fast and sensitive flow cytometric method to determine the kinetics of intracellular Ca2+ mobilization in platelets, which could be performed with the least artifactual perturbation of platelet function. Anticoagulated blood was diluted in Tyrode's buffer and incubated with Fluo-3-acetoxymethyl ester prior to staining with phycoerytrin-conjugated antiplatelet GPIIb/IIIa complex monoclonal antibody. Platelets were identified by a gate including only CD41+ events. After the determination of baseline Fluo-3 green fluorescence on a flow cytometer (EPICS XL-MCL, Coulter Electronics, Hialeah, FL), adequate agonists were added and time-dependent changes in Fluo-3 fluorescence were recorded on-line for up to 3 min. In these conditions, a very fast and transient increase of cytosolic-free Ca2+ was observed following the addition of thrombin, a strong platelet agonist. Stimulation with adenosine diphosphate (ADP), a weak agonist, also resulted in evident increase of Ca2+ levels. Our results show that this flow cytometric kinetic method provides a simple and sensitive tool to assess in vitro the time course and intensity of signal transduction responses to different platelet agonists under near physiological conditions. In this way, it may be useful to evaluate the degree of platelet reactivity and thus to monitor antiplatelet therapy.

  4. High-Throughput Sequencing Technologies

    PubMed Central

    Reuter, Jason A.; Spacek, Damek; Snyder, Michael P.

    2015-01-01

    Summary The human genome sequence has profoundly altered our understanding of biology, human diversity and disease. The path from the first draft sequence to our nascent era of personal genomes and genomic medicine has been made possible only because of the extraordinary advancements in DNA sequencing technologies over the past ten years. Here, we discuss commonly used high-throughput sequencing platforms, the growing array of sequencing assays developed around them as well as the challenges facing current sequencing platforms and their clinical application. PMID:26000844

  5. High throughput protein production screening

    DOEpatents

    Beernink, Peter T.; Coleman, Matthew A.; Segelke, Brent W.

    2009-09-08

    Methods, compositions, and kits for the cell-free production and analysis of proteins are provided. The invention allows for the production of proteins from prokaryotic sequences or eukaryotic sequences, including human cDNAs using PCR and IVT methods and detecting the proteins through fluorescence or immunoblot techniques. This invention can be used to identify optimized PCR and WT conditions, codon usages and mutations. The methods are readily automated and can be used for high throughput analysis of protein expression levels, interactions, and functional states.

  6. Ultra-Fast and Optimized Method for the Preparation of Rodent Testicular Cells for Flow Cytometric Analysis

    PubMed Central

    2009-01-01

    Homogeneity of cell populations is a prerequisite for the analysis of biochemical and molecular events during male gamete differentiation. Given the complex organization of the mammalian testicular tissue, various methods have been used to obtain enriched or purified cell populations, including flow cell sorting. Current protocols are usually time-consuming and may imply loss of short-lived RNAs, which is undesirable for expression profiling. We describe an optimized method to speed up the preparation of suitable testicular cell suspensions for cytometric analysis of different spermatogenic stages from rodents. The procedure takes only 15 min including testis dissection, tissue cutting, and processing through the Medimachine System (Becton Dickinson). This method could be a substitute for the more tedious and time-consuming cell preparation techniques currently in use. PMID:19495915

  7. Flow cytometric analyses of the viability, surface marker expression and function of lymphocytes from children following cryopreservation.

    PubMed

    Chen, Xi; Zhang, Hui; Mou, Wenjun; Qi, Zhan; Ren, Xiaoya; Wang, Guoliang; Jiao, Hong; Kong, Xiaohui; Gui, Jingang

    2016-11-01

    Flow cytometric analysis is important for the investigation and clinical preparation of lymphocytes from children. However, the strict requirement of cell freshness and inter‑assay variability limits the application of this methodology for pediatric investigations. Therefore, it is necessary to identify a reliable cryopreservative method capable of maintaining high cell viability and proper cell function in lymphocytes from children. In the present study, eight commonly‑used cell cyropreservative methods were used, and their effects on cell viability, surface marker expression and cell function were examined. In addition, how these methods affect the distribution of T‑cell receptor Vβ subfamilies were also determined. The results of the present study provided valuable experimental evidence, based on which the optimal method for the cryopreservation of lymphocytes from children in pediatric investigations and clinical applications can be selected.

  8. Effects of Hoechst33342 staining on the viability and flow cytometric sex-sorting of frozen-thawed ram sperm.

    PubMed

    Quan, Guo Bo; Ma, Yuan; Li, Jian; Wu, Guo Quan; Li, Dong Jiang; Ni, Yi Na; Lv, Chun Rong; Zhu, Lan; Hong, Qiong Hua

    2015-02-01

    Cytometric sorting of frozen-thawed sperm can overcome difficulties caused by the unavailability of sorting facilities on farms where semen is collected from male livestock. In order to optimize the cytometric sex-sorting procedure, effects of Hoechst33342 staining on the viability and cytometric sorting efficiency of frozen-thawed ram sperm were evaluated. The frozen-thawed sperm were stained with Hoechst33342 at various dye concentrations (80 μM, 120 μM, 160 μM, 200 μM, 240 μM, or 320 μM) for 45 min to evaluate effects of dye dose. The frozen-thawed sperm were stained with 160 μM Hoechst33342 for various durations (0 min, 15 min, 30 min, 45 min, 60 min, 75 min, or 90 min) to evaluate effects of staining duration. Sperm motility and moving velocity were analyzed using a computer-assisted sperm analysis system (CASAS). Acrosome status, membrane integrity, and distribution of phosphatidylserine (PS) in Hoechst33342-stained sperm were analyzed using flow cytometry after staining with fluorescein isothiocyanate-labeled lectin from pisum sativum (FITC-PSA), Annexin V, or propidium iodide (PI). The fertility of Hoechst33342-stained sperm was analyzed by in vitro fertilization (IVF). A high-speed cell sorter was used to evaluate effects of Hoechst33342 staining on cytometric sex-sorting of frozen-thawed sperm. The motility, moving velocity, membrane integrity, and PS distribution of Hoechst33342-stained sperm were significantly different from that of immediately thawed sperm (P<0.05). However, there is no significant difference existing among the Hoechst33342-stained groups with respect to the above evaluated parameters. Additionally, along with the staining durations, the adverse effects of the staining procedure on sperm showed a steady increase. However, Hoechst33342 staining did not damage acrosome and in vitro fertilizing capability of frozen-thawed ram sperm. Results of cytometric sorting indicated that frozen-thawed sperm can be efficiently sorted into two

  9. Flow Cytometric Measurement of Blood Cells with BCR-ABL1 Fusion Protein in Chronic Myeloid Leukemia.

    PubMed

    Löf, Liza; Arngården, Linda; Olsson-Strömberg, Ulla; Siart, Benjamin; Jansson, Mattias; Dahlin, Joakim S; Thörn, Ingrid; Christiansson, Lisa; Hermansson, Monica; Larsson, Anders; Ahlstrand, Erik; Wålinder, Göran; Söderberg, Ola; Rosenquist, Richard; Landegren, Ulf; Kamali-Moghaddam, Masood

    2017-04-04

    Chronic myeloid leukemia (CML) is characterized in the majority of cases by a t(9;22)(q34;q11) translocation, also called the Philadelphia chromosome, giving rise to the BCR-ABL1 fusion protein. Current treatment with tyrosine kinase inhibitors is directed against the constitutively active ABL1 domain of the fusion protein, and minimal residual disease (MRD) after therapy is monitored by real-time quantitative PCR (RQ-PCR) of the fusion transcript. Here, we describe a novel approach to detect and enumerate cells positive for the BCR-ABL1 fusion protein by combining the in situ proximity ligation assay with flow cytometry as readout (PLA-flow). By targeting of the BCR and ABL1 parts of the fusion protein with one antibody each, and creating strong fluorescent signals through rolling circle amplification, PLA-flow allowed sensitive detection of cells positive for the BCR-ABL1 fusion at frequencies as low as one in 10,000. Importantly, the flow cytometric results correlated strongly to those of RQ-PCR, both in diagnostic testing and for MRD measurements over time. In summary, we believe this flow cytometry-based method can serve as an attractive approach for routine measurement of cells harboring BCR-ABL1 fusions, also allowing simultaneously assessment of other cell surface markers as well as sensitive longitudinal follow-up.

  10. Flow cytometric detection of endothelial microparticles (EMP): effects of centrifugation and storage alter with the phenotype studied.

    PubMed

    van Ierssel, Sabrina H; Van Craenenbroeck, Emeline M; Conraads, Viviane M; Van Tendeloo, Viggo F; Vrints, Christiaan J; Jorens, Philippe G; Hoymans, Vicky Y

    2010-04-01

    Endothelial microparticles (EMP) are released into the circulation in case of endothelial disturbance, and are therefore increasingly investigated as a biomarker reflecting disease activity. Numerous pre-analytic methods have been proposed for their flow cytometric enumeration, but standardization is still lacking. In this study we evaluated the influence of centrifugation and storage conditions on EMP quantification. Platelet-poor plasma (PPP) from 10 healthy volunteers was prepared by centrifugation at 1,550 g for 20 minutes twice. A first aliquot of PPP was analyzed immediately, a second after storage at 4 degrees C for 7 hours. A third and fourth aliquot were snap-frozen and stored at -80 degrees C for 7 and 28 days. A final aliquot was further centrifuged at 10,000g for 10 minutes and analyzed immediately. EMP were defined as CD31+CD42b-, CD62E+, CD144+ or CD144+CD105+ particles, smaller than 1.0 microm. High speed centrifugation led to a significant loss of CD31+CD42b- EMP (p=0.004). A good correlation between PPP and high speed centrifuged PPP was only found for CD144+ EMP (Kendall tau b=0.611, p=0.025). Storage at 4 degrees C did not affect EMP quantification. However, freezing at -80 degrees C increased CD31+CD42b- and CD62E+ EMP counts, and lowered CD144+ EMP (p<0.05). Nevertheless, the agreement among the different storage conditions was relatively good (Kendall coefficient of concordance >0.487; p<0.05). The flow cytometric detection of EMP varies with the centrifugation protocol and the storage method used, and these changes also depend on the phenotype studied. The results of this study caution against comparing study results gathered with different EMP laboratory protocols. (c) 2010 Elsevier Ltd. All rights reserved.

  11. Flow cytometric evaluation of physico-chemical impact on Gram-positive and Gram-negative bacteria

    PubMed Central

    Fröhling, Antje; Schlüter, Oliver

    2015-01-01

    Since heat sensitivity of fruits and vegetables limits the application of thermal inactivation processes, new emerging inactivation technologies have to be established to fulfill the requirements of food safety without affecting the produce quality. The efficiency of inactivation treatments has to be ensured and monitored. Monitoring of inactivation effects is commonly performed using traditional cultivation methods which have the disadvantage of the time span needed to obtain results. The aim of this study was to compare the inactivation effects of peracetic acid (PAA), ozonated water (O3), and cold atmospheric pressure plasma (CAPP) on Gram-positive and Gram-negative bacteria using flow cytometric methods. E. coli cells were completely depolarized after treatment (15 s) with 0.25% PAA at 10°C, and after treatment (10 s) with 3.8 mg l−1 O3 at 12°C. The membrane potential of CAPP treated cells remained almost constant at an operating power of 20 W over a time period of 3 min, and subsequently decreased within 30 s of further treatment. Complete membrane permeabilization was observed after 10 s O3 treatment, but treatment with PAA and CAPP did not completely permeabilize the cells within 2 and 4 min, respectively. Similar results were obtained for esterase activity. O3 inactivates cellular esterase but esterase activity was detected after 4 min CAPP treatment and 2 min PAA treatment. L. innocua cells and P. carotovorum cells were also permeabilized instantaneously by O3 treatment at concentrations of 3.8 ± 1 mg l−1. However, higher membrane permeabilization of L. innocua and P. carotovorum than of E. coli was observed at CAPP treatment of 20 W. The degree of bacterial damage due to the inactivation processes is highly dependent on treatment parameters as well as on treated bacteria. Important information regarding the inactivation mechanisms can be obtained by flow cytometric measurements and this enables the definition of critical process parameters. PMID

  12. Flow cytometric measurement of calcium influx in murine T cell hybrids using Fluo-3 and an organic-anion transport inhibitor.

    PubMed

    Baus, E; Urbain, J; Leo, O; Andris, F

    1994-07-12

    A method is described to facilitate flow cytometric analysis of calcium mobilization upon stimulation of murine T cell hybrids. In these transformed cell lines, the accuracy of cytometric measurement of free cytoplasmic calcium with Fluo-3 is compromised by the rapid loss of the intracellular dye. We have found that the addition of sulfinpyrazone, a known organic-anion transporter inhibitor in epithelial cells and in macrophages, severely impairs the leakage of the Fluo-3 probe from the cytoplasmic matrix. Under appropriate conditions, sulfinpyrazone has little effect on the cell physiology and permits the detection of calcium influx in a variety of murine T cell hybrids.

  13. High-throughput discovery metabolomics.

    PubMed

    Fuhrer, Tobias; Zamboni, Nicola

    2015-02-01

    Non-targeted metabolomics by mass spectrometry has established as the method of choice for investigating metabolic phenotypes in basic and applied research. Compared to other omics, metabolomics provides broad scope and yet direct information on the integrated cellular response with low demand in material and sample preparation. These features render non-targeted metabolomics ideally suited for large scale screens and discovery. Here we review the achievements and potential in high-throughput, non-targeted metabolomics. We found that routine and precise analysis of thousands of small molecular features in thousands of complex samples per day and instrument is already reality, and ongoing developments in microfluidics and integrated interfaces will likely further boost throughput in the next few years. Copyright © 2014 Elsevier Ltd. All rights reserved.

  14. Clinical flow cytometric screening of SAP and XIAP expression accurately identifies patients with SH2D1A and XIAP/BIRC4 mutations.

    PubMed

    Gifford, Carrie E; Weingartner, Elizabeth; Villanueva, Joyce; Johnson, Judith; Zhang, Kejian; Filipovich, Alexandra H; Bleesing, Jack J; Marsh, Rebecca A

    2014-07-01

    X-linked lymphoproliferative disease is caused by mutations in two genes, SH2D1A and XIAP/BIRC4. Flow cytometric methods have been developed to detect the gene products, SAP and XIAP. However, there is no literature describing the accuracy of flow cytometric screening performed in a clinical lab setting. We reviewed the clinical flow cytometric testing results for 656 SAP and 586 XIAP samples tested during a 3-year period. Genetic testing was clinically performed as directed by the managing physician in 137 SAP (21%) and 115 XIAP (20%) samples. We included these samples for analyses of flow cytometric test accuracy. SH2D1A mutations were detected in 15/137 samples. SAP expression was low in 13/15 (sensitivity 87%, CI 61-97%). Of the 122 samples with normal sequencing, SAP was normal in 109 (specificity 89%, CI 82-94%). The positive predictive values (PPVs) and the negative predictive values (NPVs) were 50% and 98%, respectively. XIAP/BIRC4 mutations were detected in 19/115 samples. XIAP expression was low in 18/19 (sensitivity 95%, CI 73-100%). Of the 96 samples with normal sequencing, 59 had normal XIAP expression (specificity 61%, CI 51-71%). The PPVs and NPVs were 33% and 98%, respectively. Receiver-operating characteristic analysis was able to improve the specificity to 75%. Clinical flow cytometric screening tests for SAP and XIAP deficiencies offer good sensitivity and specificity for detecting genetic mutations, and are characterized by high NPVs. We recommend these tests for patients suspected of having X-linked lymphoproliferative disease type 1 (XLP1) or XLP2. © 2014 Clinical Cytometry Society.

  15. Class II–Associated Invariant Chain Peptide Expression Represents a Novel Parameter for Flow Cytometric Detection of Acute Promyelocytic Leukemia

    PubMed Central

    van Luijn, Marvin M.; Westers, Theresia M.; Chamuleau, Martine E.D.; van Ham, S. Marieke; Ossenkoppele, Gert J.; van de Loosdrecht, Arjan A.

    2011-01-01

    Because of severe bleeding complications, patients with acute promyelocytic leukemia (APL) have to be treated with all-trans retinoic acid immediately following diagnosis. In addition to morphology, flow cytometry contributes to a rapid detection of APL according to phenotypic characteristics of leukemic cells. In some patients, these analyses are inconclusive or even contradictory to diagnosis. Previously, we showed the clinical and functional impact of class II–associated invariant chain peptide (CLIP) in acute myeloid leukemia (AML). This study focuses on the analysis of CLIP expression on leukemic cells to characterize HLA-DR–negative AML, including APL. We demonstrate exclusive and significant CLIP expression in all cases of typical and variant APL, as compared to other HLA-DR–negative non–APL-type AML. CLIP appears to be a highly sensitive and specific flow cytometric marker, resolving discrepant identification of both genetic subgroups. Our findings show the additive value of CLIP analysis for a fast and unequivocal recognition of APL by flow cytometry in conjunction with morphology. PMID:21907692

  16. Flow cytometric enumeration of bacteria using TO-PRO®-3 iodide as a single-stain viability dye.

    PubMed

    Kerstens, Monique; Boulet, Gaëlle; Tritsmans, Christian; Horemans, Tessa; Hellings, Mario; Delputte, Peter; Maes, Louis; Cos, Paul

    2014-12-01

    Quantification of bacteria using conventional viable plate counting (VPC) is labor-intensive and time-consuming. Flow cytometry (FCM) can be proposed as a faster alternative. This study aimed to develop a flow cytometric, single-stain approach using TO-PRO®-3 iodide (TP3) for the quantification of Staphylococcus aureus, Escherichia coli, and Bacillus subtilis cells. Live or dead bacterial suspensions were stained with TP3 and analyzed using a FACSCalibur flow cytometer. After optimization of staining parameters and instrument settings, an excellent separation of viable and dead cells was achieved for all species. The quantitative performance of the technique was assessed by analyzing serial dilutions of bacterial suspensions using FCM and VPC. A highly linear correlation (r2 > 0.99) was observed between the colony forming units (CFU)/mL as determined by FCM and by VPC over a concentration range of about 104 to 108 CFU/mL. As such, FCM quantification of viable bacteria using TP3 can be considered as an accurate and reliable alternative for VPC. The monostain procedure is easy to apply and cost-effective, and it allows bacterial enumeration in a broad variety of samples.

  17. A new four-color flow cytometric assay to detect apoptosis in lymphocyte subsets of cultured peripheral blood cells.

    PubMed

    Hasper, H J; Weghorst, R M; Richel, D J; Meerwaldt, J H; Olthuis, F M; Schenkeveld, C E

    2000-06-01

    Human peripheral blood lymphocytes kept in culture after isolation die by an apoptotic process. Detection of apoptosis with labeled Annexin V to demonstrate loss of plasma membrane asymmetry is sensitive, specific, and easy using flow cytometry. This is true in lymphoblastic cell lines when combining Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI). However, measurement of apoptosis by flow cytometry in isolated human lymphocytes using Annexin V-FITC/PI is disturbed by the presence of a variable percentage of erythrocytes in the isolated lymphocyte population. To overcome this problem, we have developed and tested a new four-color flow cytometric assay to detect apoptosis in lymphocyte subsets of cultured peripheral blood cells. Peripheral blood lymphocytes are isolated by density gradient centrifugation. Nucleus-containing cells are selected using CD45-phycoerythrin (PE). The lymphocyte subset of interest is selected using CD4, CD8, or CD19 energy-coupled dye (ECD) labeling. Apoptosis is detected using Annexin V-FITC with 7-amino-Actinomycin-D (7-AAD) to distinguish early apoptotic from late apoptotic lymphocytes. We have developed a new technique to detect apoptosis in isolated human peripheral blood lymphocyte subsets with good reproducibility, coefficient of variation < 17%. We now have a validated tool to study apoptosis in subsets of isolated human lymphocytes to increase our knowledge of pathogenesis and therapies in lymphoreticular malignancies. Copyright 2000 Wiley-Liss, Inc.

  18. Metaproteomic and metagenomic analyses of defined oceanic microbial populations using microwave cell fixation and flow cytometric sorting.

    PubMed

    Mary, Isabelle; Oliver, Anna; Skipp, Paul; Holland, Ross; Topping, Juliette; Tarran, Glen; Scanlan, David J; O'Connor, C David; Whiteley, Andrew S; Burkill, Peter H; Zubkov, Mikhail V

    2010-10-01

    A major obstacle in the molecular investigation of natural, especially oceanic, microbial cells is their adequate preservation for further land-based molecular analyses. Here, we examined the use of microwaves for cell fixation before high-speed flow cytometric sorting to define the metaproteomes and metagenomes of key microbial populations. The microwave fixation procedure was established using cultures of Synechococcus cyanobacteria, the photosynthetic eukaryote Micromonas pusilla and the gammaproteobacterium Halomonas variabilis. Shotgun proteomic analyses showed that the profile of microwave-fixed and -unfixed Synechococcus sp. WH8102 cells was the same, and hence proteome identification of microwave-fixed sorted cells by nanoLC-MS/MS is possible. Microwave-fixed flow-sorted Synechococcus cells can also be successfully used for whole-genome amplification and fosmid library construction. We then carried out successful metaproteomic and metagenomic analyses of microwave-fixed Synechococcus cells flow sorted from concentrates of microbial cells, collected in the North Atlantic Ocean. Thus, the microwave fixation procedure developed appears to be useful for molecular studies of microbial populations in aquatic ecosystems.

  19. DNA flow cytometric evaluation of cell cycle distribution in ulcerative colitis: a proposed method for assessing severity of disease.

    PubMed Central

    Bortoluzzi, F; Valentini, M; Cernigoi, C; Toffoli, G; Boiocchi, M; Poletti, M; Sozzi, M; Fornasarig, M; Cannizzaro, R; Bertolissi, E

    1995-01-01

    The assessment of disease severity in ulcerative colitis depends mainly on subjective variables, and an objective method of assessing mucosal inflammation is needed. Determination of the synthetic phase of the cell cycle is an accurate expression of inflammatory activity in the colonic mucosa. The aim of the study was to find out if the proliferative index or the synthetic phase (S phase) of the colonic mucosa of patients with ulcerative colitis, as evaluated by DNA flow cytometry, is a reliable and reproducible marker of disease activity. Sixty consecutive patients with ulcerative colitis of different degrees of activity were entered into the study and submitted to colonoscopy plus multiple rectal biopsies. Disease severity was defined for each patient by means of a clinical, endoscopic, and histological score. Flow cytometry was used to calculate the proliferative index and the S phase of the cell cycle. A statistically significant correlation (p < 0.001) was found between all indices of severity. It is suggested that flow cytometric evaluation of the cell cycle in the rectal mucosa may be an efficient method of assessing severity of disease and efficacy of medical treatment in ulcerative colitis. PMID:7890236

  20. Overview of very small embryonic-like stem cells (VSELs) and methodology of their identification and isolation by flow cytometric methods.

    PubMed

    Zuba-Surma, Ewa K; Ratajczak, Mariusz Z

    2010-01-01

    The protocols presented here describe the procedures employed to identify and isolate very small embryonic-like stem cells (VSELs) using flow cytometric technologies including fluorescence-activated cell sorting (FACS). We describe the recommended steps in detail for their successful identification and isolation from adult tissues. These protocols were initially established to isolate such cells from murine bone marrow (BM) and human cord blood (CB) and may also be employed to isolate these primitive cells from other adult organs and embryonic tissues. Here, we focus on some critical parameters/key points required for the successful identification and purification of these rare cells by employing classical flow cytometry. In the last part of this unit, we also discuss a novel flow cytometric tool, ImageStream, an imaging flow cytometer, which allows better identification and morphological analysis of sorted cells.

  1. Flow cytometric method for the assessment of the minimal inhibitory concentrations of antibacterial agents to Mycoplasma agalactiae.

    PubMed

    Assunção, Patrícia; Antunes, Nuno T; Rosales, Ruben S; de la Fe, Christian; Poveda, Carlos; Poveda, José B; Davey, Hazel M

    2006-10-01

    In this study, flow cytometry was evaluated for the determination of the minimal inhibitory concentrations (MIC) of seven antibacterial agents (enrofloxacin, ciprofloxacin, gentamicin, streptomycin, chloramphenicol, oxytetracycline, and tylosin) on Mycoplasma (M.) agalactiae. Flow cytometry was able to detect M. agalactiae inhibition from 6 h postincubation, although it seems that definitive MIC values determined by flow cytometry were only possible at 12-h postincubation. However, the results obtained by the traditional method were only obtained at 24 h, when a visible change in the medium had occurred. At 24 h, both methods gave the same result for six antibacterial agents (enrofloxacin, ciprofloxacin, gentamicin, streptomycin, chloramphenicol, and oxytetracycline); whereas flow cytometry gave slightly higher MIC for tylosin. This was attributed to the fact that the M. agalactiae growth that had occurred in the tubes containing tylosin was not enough to visibly change the color of the medium. Futhermore, flow cytometry detected that inhibitory concentrations of oxytetracycline, chloramphenicol, and tylosin as judged at 24 h were not able to inhibit the M. agalactiae growth after 48 h. MIC values of enrofloxacin and ciprofloxacin were sufficient only to maintain the total counts per milliliter throughout the time matched samples, whereas higher concentrations of theses antibacterial agents reduced the total counts per milliliter over the course of the experiment. The main advantage of the flow cytometric method is that MIC results for M. agalactiae can be obtained in a shorter time than is possible with the traditional method. The method presented makes identification of resistant populations of M. agalactiae possible and, unlike the traditional method, allows the effect of each antibacterial agent to be determined in real-time at the single-cell level.

  2. High Throughput Plasma Water Treatment

    NASA Astrophysics Data System (ADS)

    Mujovic, Selman; Foster, John

    2016-10-01

    The troublesome emergence of new classes of micro-pollutants, such as pharmaceuticals and endocrine disruptors, poses challenges for conventional water treatment systems. In an effort to address these contaminants and to support water reuse in drought stricken regions, new technologies must be introduced. The interaction of water with plasma rapidly mineralizes organics by inducing advanced oxidation in addition to other chemical, physical and radiative processes. The primary barrier to the implementation of plasma-based water treatment is process volume scale up. In this work, we investigate a potentially scalable, high throughput plasma water reactor that utilizes a packed bed dielectric barrier-like geometry to maximize the plasma-water interface. Here, the water serves as the dielectric medium. High-speed imaging and emission spectroscopy are used to characterize the reactor discharges. Changes in methylene blue concentration and basic water parameters are mapped as a function of plasma treatment time. Experimental results are compared to electrostatic and plasma chemistry computations, which will provide insight into the reactor's operation so that efficiency can be assessed. Supported by NSF (CBET 1336375).

  3. [Flow cytometric analysys of Enterococcus faecalis pAD1(-) strains response to cAD1 pheromone].

    PubMed

    Jarzembowski, Tomasz; Bryl, Ewa; Galiński, Janusz; Witkowski, Jacek M

    2005-01-01

    Conjugative plasmids transfer in Enterococcus faecalis is inducted by sex pheromones. The pheromone is excreted by recipient cells and induces expression of aggregation protein AS in donor cells. This protein is involved in formation of matting aggregates. Use of flow cytometry and anti-As monoclonal antibodies allowed collect of interesting data pheromone response. However, according to our knowledge, no study focused on unspecific influence on particular pheromone for plasmid-free recipient strains. Six pAD1 (-) and tree pAD1 (+) Enterococcus faecalis stains were cultivated for 18h in BHI, with and without cAD1 pheromone (Sigma, Germany), respectively. The bacteria were washed, stained with carboksyfluorescein (FCDA, and analyzed by flow cytometry in FACS BD scan cytometr. Relative fluorescence and size of aggregation was used to compare influence on particular strains. Surprisingly, the results shows divergence in fluorescence, size of aggregates and degree of correlation between fluorescence of aggregates and their sizes among pAD1(-) strains, allowing for distinguish of two groups. Three of studied strains have higher fluorescence than pAD (+) stains. Correlation between fluorescence and size of aggregates, significant higher than in pAD1(+) stains, decrease from r = 0.88 to r=0.74 in reaction to cAD1. The strains if other group fluorize with lower intensity than pAD1 (+). Furthermore, 30.4% pAD1 (-) of second group have no detectable fluorescence. In contrast to pAD1 (-) ) strains of the first group and pA1 (+) strains, low (r=0.55) correlation between fluorescence and size of aggregates of group II increase up to r=0.74 after incubation with cAD1 pheromone. Previous study of these pAD1 (-) strains, currently assigned to group II, shown their low frequency of collecting aph2" gene encoded on other conjugative plasmid, pMG. According to these results, such flow cytometric analysis may be used to predict ability of strain to collect unrelated conjugative

  4. A new simple tubular flow cell for use with variable angle spectroscopic ellipsometry: A high throughput in situ protein adsorption study

    NASA Astrophysics Data System (ADS)

    Byrne, T. M.; Trussler, S.; McArthur, M. A.; Lohstreter, L. B.; Bai, Zhijun; Filiaggi, M. J.; Dahn, J. R.

    2009-09-01

    A simple and novel flow cell design is presented here for use with variable angle spectroscopic ellipsometry (VASE) to study the adsorption of liquid-borne species on reflective surfaces. The flow cell allows a sample as large as 6 mm × 75 mm to be probed point by point and at any common ellipsometric angle of incidence, unlike other designs. Using our flow cell system with VASE, combinatorial films of Al 1-xNb x, Al 1-xTa x, and Al 1-xTi x (0 ⩽ x ⩽ 1) were tested in situ for fibrinogen affinity along their 75 mm long compositional gradients. Fibrinogen adsorption on the films was found to be closely correlated to the various surface oxide fractions, with high alumina content at the surface leading to low amounts of adsorbed fibrinogen for each binary library. Adsorbed amounts measured in situ were in agreement with previously obtained values found using ex situ techniques.

  5. CD33 monoclonal antibody conjugated Au cluster nano-bioprobe for targeted flow-cytometric detection of acute myeloid leukaemia

    NASA Astrophysics Data System (ADS)

    Retnakumari, Archana; Jayasimhan, Jasusri; Chandran, Parwathy; Menon, Deepthy; Nair, Shantikumar; Mony, Ullas; Koyakutty, Manzoor

    2011-07-01

    Protein stabilized gold nanoclusters (Au-NCs) are biocompatible, near-infrared (NIR) emitting nanosystems having a wide range of biomedical applications. Here, we report the development of a Au-NC based targeted fluorescent nano-bioprobe for the flow-cytometric detection of acute myeloid leukaemia (AML) cells. Au-NCs with ~ 25-28 atoms showing bright red-NIR fluorescence (600-750 nm) and average size of ~ 0.8 nm were prepared by bovine serum albumin assisted reduction-cum-stabilization in aqueous phase. The protein protected clusters were conjugated with monoclonal antibody against CD33 myeloid antigen, which is overexpressed in ~ 99.2% of the primitive population of AML cells, as confirmed by immunophenotyping using flow cytometry. Au-NC-CD33 conjugates having average size of ~ 12 nm retained bright fluorescence over an extended duration of ~ a year, as the albumin protein protects Au-NCs against degradation. Nanotoxicity studies revealed excellent biocompatibility of Au-NC conjugates, as they showed no adverse effect on the cell viability and inflammatory response. Target specificity of the conjugates for detecting CD33 expressing AML cells (KG1a) in flow cytometry showed specific staining of ~ 95.4% of leukaemia cells within 1-2 h compared to a non-specific uptake of ~ 8.2% in human peripheral blood cells (PBMCs) which are CD33low. The confocal imaging also demonstrated the targeted uptake of CD33 conjugated Au-NCs by leukaemia cells, thus confirming the flow cytometry results. This study demonstrates that novel nano-bioprobes can be developed using protein protected fluorescent nanoclusters of Au for the molecular receptor targeted flow cytometry based detection and imaging of cancer cells.

  6. Wide-field imaging and flow cytometric analysis of cancer cells in blood by fluorescent nanodiamond labeling and time gating

    PubMed Central

    Hui, Yuen Yung; Su, Long-Jyun; Chen, Oliver Yenjyh; Chen, Yit-Tsong; Liu, Tzu-Ming; Chang, Huan-Cheng

    2014-01-01

    Nanodiamonds containing high density ensembles of negatively charged nitrogen-vacancy (NV−) centers are promising fluorescent biomarkers due to their excellent photostability and biocompatibility. The NV− centers in the particles have a fluorescence lifetime of up to 20 ns, which distinctly differs from those (<10 ns) of cell and tissue autofluorescence, making it possible to achieve background-free detection in vivo by time gating. Here, we demonstrate the feasibility of using fluorescent nanodiamonds (FNDs) as optical labels for wide-field time-gated fluorescence imaging and flow cytometric analysis of cancer cells with a nanosecond intensified charge-coupled device (ICCD) as the detector. The combined technique has allowed us to acquire fluorescence images of FND-labeled HeLa cells in whole blood covered with a chicken breast of ~0.1-mm thickness at the single cell level, and to detect individual FND-labeled HeLa cells in blood flowing through a microfluidic device at a frame rate of 23 Hz, as well as to locate and trace FND-labeled lung cancer cells in the blood vessels of a mouse ear. It opens a new window for real-time imaging and tracking of transplanted cells (such as stem cells) in vivo. PMID:24994610

  7. Implementation of a simplified flow cytometric assays for minimal residual disease monitoring in childhood acute lymphoblastic leukemia.

    PubMed

    Vinhas, Ester; Lucena-Silva, Norma; Pedrosa, Francisco

    2016-06-25

    Monitoring minimal residual disease (MRD) in B-lineage acute lymphoblastic leukemia (ALL) to assess treatment response is crucial for risk assignment. Flow cytometry can be used to monitor MRD in ALL but the implementation of this approach requires extensive expertise. If resources are limited, the costs of full flow cytometric MRD testing might be prohibitive. We evaluated the applicability of a previously reported simplified MRD assay, designed to distinguish leukemic from normal lymphoblastic during remission induction therapy. Fifty-nine samples from children with ALL, enrolled in the RE-LLA study at a pediatric oncology center in Recife (Brazil), were evaluated for MRD on day 19 and on day 26 of remission induction therapy. We compared results obtained by a trainee in Recife and an expert. The method was implemented successfully and the concordance between results obtained by the trainee and the expert was practically absolute at the end of the study. It is possible to implement reliable measurements of MRD during remission induction therapy in childhood ALL despite limited resources. The simplicity of the MRD method used in this study does not require extensive prior training in leukemia immunophenotyping. © 2016 International Clinical Cytometry Society. © 2016 International Clinical Cytometry Society.

  8. HLA-targeted flow cytometric sorting of blood cells allows separation of pure and viable microchimeric cell populations.

    PubMed

    Drabbels, Jos J M; van de Keur, Carin; Kemps, Berit M; Mulder, Arend; Scherjon, Sicco A; Claas, Frans H J; Eikmans, Michael

    2011-11-10

    Microchimerism is defined by the presence of low levels of nonhost cells in a person. We developed a reliable method for separating viable microchimeric cells from the host environment. For flow cytometric cell sorting, HLA antigens were targeted with human monoclonal HLA antibodies (mAbs). Optimal separation of microchimeric cells (present at a proportion as low as 0.01% in artificial mixtures) was obtained with 2 different HLA mAbs, one targeting the chimeric cells and the other the background cells. To verify purity of separated cell populations, flow-sorted fractions of 1000 cells were processed for DNA analysis by HLA-allele-specific and Y-chromosome-directed real-time quantitative PCR assays. After sorting, PCR signals of chimeric DNA markers in the positive fractions were significantly enhanced compared with those in the presort samples, and they were similar to those in 100% chimeric control samples. Next, we demonstrate applicability of HLA-targeted FACS sorting after pregnancy by separating chimeric maternal cells from child umbilical cord mononuclear cells. Targeting allelic differences with anti-HLA mAbs with FACS sorting allows maximal enrichment of viable microchimeric cells from a background cell population. The current methodology enables reliable microchimeric cell detection and separation in clinical specimens.

  9. A Simple Flow Cytometric Method to Measure Glucose Uptake and Glucose Transporter Expression for Monocyte Subpopulations in Whole Blood.

    PubMed

    Palmer, Clovis S; Anzinger, Joshua J; Butterfield, Tiffany R; McCune, Joseph M; Crowe, Suzanne M

    2016-08-12

    Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of monocytes induces effector functions and a concomitant shift from oxidative to glycolytic metabolism that is accompanied by increased glucose transporter expression. This increased glycolytic metabolism is also observed for trained immunity of monocytes, a form of innate immunological memory. Although in vitro protocols examining glucose transporter expression and glucose uptake by monocytes have been described, none have been examined by multi-parametric flow cytometry in whole blood. We describe a multi-parametric flow cytometric protocol for the measurement of fluorescent glucose analog 2-NBDG uptake in whole blood by total monocytes and the classical (CD14(++)CD16(-)), intermediate (CD14(++)CD16(+)) and non-classical (CD14(+)CD16(++)) monocyte subpopulations. This method can be used to examine glucose transporter expression and glucose uptake for total monocytes and monocyte subpopulations during homeostasis and inflammatory disease, and can be easily modified to examine glucose uptake for other leukocytes and leukocyte subpopulations within blood.

  10. Wide-field imaging and flow cytometric analysis of cancer cells in blood by fluorescent nanodiamond labeling and time gating

    NASA Astrophysics Data System (ADS)

    Hui, Yuen Yung; Su, Long-Jyun; Chen, Oliver Yenjyh; Chen, Yit-Tsong; Liu, Tzu-Ming; Chang, Huan-Cheng

    2014-07-01

    Nanodiamonds containing high density ensembles of negatively charged nitrogen-vacancy (NV-) centers are promising fluorescent biomarkers due to their excellent photostability and biocompatibility. The NV- centers in the particles have a fluorescence lifetime of up to 20 ns, which distinctly differs from those (<10 ns) of cell and tissue autofluorescence, making it possible to achieve background-free detection in vivo by time gating. Here, we demonstrate the feasibility of using fluorescent nanodiamonds (FNDs) as optical labels for wide-field time-gated fluorescence imaging and flow cytometric analysis of cancer cells with a nanosecond intensified charge-coupled device (ICCD) as the detector. The combined technique has allowed us to acquire fluorescence images of FND-labeled HeLa cells in whole blood covered with a chicken breast of ~0.1-mm thickness at the single cell level, and to detect individual FND-labeled HeLa cells in blood flowing through a microfluidic device at a frame rate of 23 Hz, as well as to locate and trace FND-labeled lung cancer cells in the blood vessels of a mouse ear. It opens a new window for real-time imaging and tracking of transplanted cells (such as stem cells) in vivo.

  11. Evaluating the Safety of Somatic Periosteal Cells by Flow-Cytometric Analysis Monitoring the History of DNA Damage.

    PubMed

    Kawase, Tomoyuki; Hayama, Kazuhide; Tsuchimochi, Makoto; Nagata, Masaki; Okuda, Kazuhiro; Yoshie, Hiromasa; Burns, Douglas M; Nakata, Koh

    2016-04-01

    In preparing cell-based products for regenerative therapy, cell quality should be strictly controlled. Methodologies for evaluating cell viability, identity, and purity are established and used routinely, whereas current methodologies for evaluating cell safety, particularly genetic integrity or tumorigenicity, are time-consuming and relatively insensitive. As part of developing a more practical screening system, the authors previously demonstrated that γ-H2AX and p53 were useful markers for evaluating the history of DNA damage. To validate these markers further and develop a more quantitative methodology, single cell-based expression of these markers and two additional candidates have now been examined using flow cytometry (FCM). FCM analysis and immunofluorescent staining demonstrated that γ-ray-irradiation suppressed proliferation, enlarged cells, and cell nuclei, and immediately upregulated γ-H2AX and p21(waf1) in large numbers of cells for up to 12 days. Gamma-H2AX foci were formed in the nuclei of many affected cells. An initial sharp increase in p53 expression declined slowly over 12 days, while Rb expression increased linearly. The present findings suggest that this high-throughput, cell-based, combinational evaluation of protein markers and cell size enables a small number of cells with a history of DNA damage to be detected quickly and routinely from within a very large cell population. Using this screening methodology will improve the ability to verify the quality of cell-based products used in regenerative therapy.

  12. Flow cytometric assessment of susceptibilities of Streptococcus pyogenes to erythromycin and rokitamycin.

    PubMed

    Braga, Pier Carlo; Bovio, Cinzia; Culici, Maria; Dal Sasso, Monica

    2003-01-01

    The effects of erythromycin (a 14-membered ring macrolide) and rokitamycin (a 16-membered ring macrolide) on the viability of the Streptococcus pyogenes M phenotype were studied by means of flow cytometry and fluorescence microscopy by using a combination of two fluorochromes (syto 9 and propidium iodide) that stains live bacteria green and dead bacteria red. In order to apply the flow cytometry, a bacterial sonication procedure was expressly set up to separate single cells from the long, intralaced S. pyogenes chains of up to 30 to 40 cells that have previously prevented the application of flow cytometry to this type of bacteria. The association of flow cytometry using an appropriate sonication procedure, together with a combination of fluorescent probes, offered the possibility of very quickly investigating the different microbiological effects of rokitamycin at 2 microg/ml, which was active on the S. pyogenes M phenotype, and of erythromycin at doses of up to 32 microg/ml, which was not.

  13. Flow Cytometric Single-Cell Identification of Populations in Synthetic Bacterial Communities.

    PubMed

    Rubbens, Peter; Props, Ruben; Boon, Nico; Waegeman, Willem

    2017-01-01

    Bacterial cells can be characterized in terms of their cell properties using flow cytometry. Flow cytometry is able to deliver multiparametric measurements of up to 50,000 cells per second. However, there has not yet been a thorough survey concerning the identification of the population to which bacterial single cells belong based on flow cytometry data. This paper not only aims to assess the quality of flow cytometry data when measuring bacterial populations, but also suggests an alternative approach for analyzing synthetic microbial communities. We created so-called in silico communities, which allow us to explore the possibilities of bacterial flow cytometry data using supervised machine learning techniques. We can identify single cells with an accuracy >90% for more than half of the communities consisting out of two bacterial populations. In order to assess to what extent an in silico community is representative for its synthetic counterpart, we created so-called abundance gradients, a combination of synthetic (i.e., in vitro) communities containing two bacterial populations in varying abundances. By showing that we are able to retrieve an abundance gradient using a combination of in silico communities and supervised machine learning techniques, we argue that in silico communities form a viable representation for synthetic bacterial communities, opening up new opportunities for the analysis of synthetic communities and bacterial flow cytometry data in general.

  14. Flow Cytometric Single-Cell Identification of Populations in Synthetic Bacterial Communities

    PubMed Central

    Boon, Nico; Waegeman, Willem

    2017-01-01

    Bacterial cells can be characterized in terms of their cell properties using flow cytometry. Flow cytometry is able to deliver multiparametric measurements of up to 50,000 cells per second. However, there has not yet been a thorough survey concerning the identification of the population to which bacterial single cells belong based on flow cytometry data. This paper not only aims to assess the quality of flow cytometry data when measuring bacterial populations, but also suggests an alternative approach for analyzing synthetic microbial communities. We created so-called in silico communities, which allow us to explore the possibilities of bacterial flow cytometry data using supervised machine learning techniques. We can identify single cells with an accuracy >90% for more than half of the communities consisting out of two bacterial populations. In order to assess to what extent an in silico community is representative for its synthetic counterpart, we created so-called abundance gradients, a combination of synthetic (i.e., in vitro) communities containing two bacterial populations in varying abundances. By showing that we are able to retrieve an abundance gradient using a combination of in silico communities and supervised machine learning techniques, we argue that in silico communities form a viable representation for synthetic bacterial communities, opening up new opportunities for the analysis of synthetic communities and bacterial flow cytometry data in general. PMID:28122063

  15. A high-throughput in vivo micronucleus assay for genome instability screening in mice

    PubMed Central

    Balmus, Gabriel; Karp, Natasha A; Ng, Bee Ling; Jackson, Stephen P; Adams, David J; McIntyre, Rebecca E

    2016-01-01

    We describe a sensitive, robust, high-throughput method for quantifying the formation of micronuclei, markers of genome instability, in mouse erythrocytes. Micronuclei are whole chromosomes or chromosome segments that have been separated from the nucleus. Other methods of detection rely on labour-intensive, microscopy-based techniques. Here, we describe a 2-d, 96-well plate-based flow cytometric method of micronucleus scoring that is simple enough for a research technician experienced in flow cytometry to perform. The assay detects low levels of genome instability that cannot be readily identified by classic phenotyping, using 25 μl of blood. By using this assay, we have screened >10,000 blood samples and discovered novel genes that contribute to vertebrate genome maintenance, as well as novel disease models and mechanisms of genome instability disorders. We discuss experimental design considerations, including statistical power calculation, we provide troubleshooting tips, and we discuss factors that contribute to a false-positive increase in the number of micronucleated red blood cells and to experimental variability. PMID:25551665

  16. Laser-based flow cytometric analysis of genotoxicity of humans exposed to ionizing radiation during the Chernobyl accident

    NASA Astrophysics Data System (ADS)

    Jensen, Ronald H.; Bigbee, William L.; Langlois, Richard G.; Grant, Stephen G.; Pleshanov, Pavel G.; Chirkov, Andre A.; Pilinskaya, Maria A.

    1991-05-01

    An analytical technique has been developed that allows laser-based flow cytometric measurement of the frequency of red blood cells that have lost allele-specific expression of a cell surface antigen due to genetic toxicity in bone marrow precursor cells. Previous studies demonstrated a correlation of such effects with the exposure of each individual to mutagenic phenomena, such as ionizing radiation, and the effects can persist for the lifetime of each individual. During the emergency response to the nuclear power plant accidert at Chemobyl, Ukraine, USSR, a number of people were exposed to whole body doses of ioniing radiation. Some of these individuals were tested with this laser-based assay and found to express a dose-dependent increase in the frequency of variant red blood cells that appears to be a persistent biological effect. This effect is similar to that which was previously observed in individuals who were exposed to ionizing radiation at Hiroshima in 1945 because of the A-bomb explosion. All data indicate that this assay might well be used as a biodosimeter to estimate radiation dose and also as an element to be used for estimating the risk of each individual to develop cancer due to radiation exposure.

  17. Estimation of the Whitefly Bemisia tabaci Genome Size Based on k-mer and Flow Cytometric Analyses.

    PubMed

    Chen, Wenbo; Hasegawa, Daniel K; Arumuganathan, Kathiravetpillai; Simmons, Alvin M; Wintermantel, William M; Fei, Zhangjun; Ling, Kai-Shu

    2015-07-28

    Whiteflies of the Bemisia tabaci (Hemiptera: Aleyrodidae) cryptic species complex are among the most important agricultural insect pests in the world. These phloem-feeding insects can colonize over 1000 species of plants worldwide and inflict severe economic losses to crops, mainly through the transmission of pathogenic viruses. Surprisingly, there is very little genomic information about whiteflies. As a starting point to genome sequencing, we report a new estimation of the genome size of the B. tabaci B biotype or Middle East-Asia Minor 1 (MEAM1) population. Using an isogenic whitefly colony with over 6500 haploid male individuals for genomic DNA, three paired-end genomic libraries with insert sizes of ~300 bp, 500 bp and 1 Kb were constructed and sequenced on an Illumina HiSeq 2500 system. A total of ~50 billion base pairs of sequences were obtained from each library. K-mer analysis using these sequences revealed that the genome size of the whitefly was ~682.3 Mb. In addition, the flow cytometric analysis estimated the haploid genome size of the whitefly to be ~690 Mb. Considering the congruency between both estimation methods, we predict the haploid genome size of B. tabaci MEAM1 to be ~680-690 Mb. Our data provide a baseline for ongoing efforts to assemble and annotate the B. tabaci genome.

  18. A whole blood flow cytometric determination of platelet activation by unfractionated and low molecular weight heparin in vitro.

    PubMed

    Klein, Bernd; Faridi, Andreé; von Tempelhoff, G F; Heilmann, Lothar; Mittermayer, Christian; Rath, Werner

    2002-12-15

    The influence of unfractionated (Heparin-Natrium) and low-molecular heparin (Fragmin(R)) on platelet activation in whole blood was investigated by FACS analysis in vitro using antibodies against glycoprotein (gp) IIb/IIIa (CD 41), GMP 140 (CD 62P), gp 53 (CD 63) and fibrinogen. Samples were also labeled with anti-gp Ib (CD 42b). Neither unfractionated heparin (UFH) nor low molecular weight heparin (LMWH) led to significant (i.e., p<0.05) changes in fluorescence intensities of platelets labeled with anti-gp IIb/IIIa or anti-gp 53. Significant platelet activation due to unfractionated heparin could be observed by labeling with anti-GMP 140 (UFH: p=0.009; LMWH: p=0.16). The proportion of platelets with surface-bound fibrinogen was significantly increased (UFH: p=0.00006; LMWH: p=0.008). After incubation with heparins, activation ability of platelets by adenosine diphosphate (ADP) was significantly increased. The potentiating action of unfractionated heparin was larger. Therefore, flow cytometric results of platelet activation in patients receiving heparin should be interpreted carefully.

  19. Laser-based flow cytometric analysis of genotoxicity of humans exposed to ionizing radiation during the Chernobyl accident

    SciTech Connect

    Jensen, R.H.; Bigbee, W.L.; Langlois, R.G.; Grant, S.G. ); Pleshanov, P.G. ); Chirkov, A.A. ); Pilinskaya, M.A. )

    1990-09-12

    An analytical technique has been developed that allows laser-based flow cytometric measurement of the frequency of red blood cells that have lost allele-specific expression of a cell surface antigen due to genetic toxicity in bone marrow precursor cells. Previous studies demonstrated a correlation of such effects with the exposure of each individual to mutagenic phenomena, such as ionizing radiation, and the effects can persist for the lifetime of each individual. During the emergency response to the nuclear power plant accident at Chernobyl, Ukraine, USSR, a number of people were exposed to whole body doses of ionizing radiation. Some of these individuals were tested with this laser-based assay and found to express a dose-dependent increase in the frequency of variant red blood cells that appears to be a persistent biological effect. All data indicate that this assay might well be used as a biodosimeter to estimate radiation dose and also as an element to be used for estimating the risk of each individual to develop cancer due to radiation exposure. 17 refs., 5 figs.

  20. Estimation of the Whitefly Bemisia tabaci Genome Size Based on k-mer and Flow Cytometric Analyses

    PubMed Central

    Chen, Wenbo; Hasegawa, Daniel K.; Arumuganathan, Kathiravetpillai; Simmons, Alvin M.; Wintermantel, William M.; Fei, Zhangjun; Ling, Kai-Shu

    2015-01-01

    Whiteflies of the Bemisia tabaci (Hemiptera: Aleyrodidae) cryptic species complex are among the most important agricultural insect pests in the world. These phloem-feeding insects can colonize over 1000 species of plants worldwide and inflict severe economic losses to crops, mainly through the transmission of pathogenic viruses. Surprisingly, there is very little genomic information about whiteflies. As a starting point to genome sequencing, we report a new estimation of the genome size of the B. tabaci B biotype or Middle East-Asia Minor 1 (MEAM1) population. Using an isogenic whitefly colony with over 6500 haploid male individuals for genomic DNA, three paired-end genomic libraries with insert sizes of ~300 bp, 500 bp and 1 Kb were constructed and sequenced on an Illumina HiSeq 2500 system. A total of ~50 billion base pairs of sequences were obtained from each library. K-mer analysis using these sequences revealed that the genome size of the whitefly was ~682.3 Mb. In addition, the flow cytometric analysis estimated the haploid genome size of the whitefly to be ~690 Mb. Considering the congruency between both estimation methods, we predict the haploid genome size of B. tabaci MEAM1 to be ~680–690 Mb. Our data provide a baseline for ongoing efforts to assemble and annotate the B. tabaci genome. PMID:26463411

  1. Flow cytometric analysis of immunoglobulin heavy chain expression in B-cell lymphoma and reactive lymphoid hyperplasia

    PubMed Central

    Grier, David D; Al-Quran, Samer Z; Cardona, Diana M; Li, Ying; Braylan, Raul C

    2012-01-01

    The diagnosis of B-cell lymphoma (BCL) is often dependent on the detection of clonal immunoglobulin (Ig) light chain expression. In some BCLs, the determination of clonality based on Ig light chain restriction may be difficult. The aim of our study was to assess the utility of flow cytometric analysis of surface Ig heavy chain (HC) expression in lymphoid tissues in distinguishing lymphoid hyperplasias from BCLs, and also differentiating various BCL subtypes. HC expression on B-cells varied among different types of hyperplasias. In follicular hyperplasia, IgM and IgD expression was high in mantle cells while germinal center cells showed poor HC expression. In other hyperplasias, B cell compartments were blurred but generally showed high IgD and IgM expression. Compared to hyperplasias, BCLs varied in IgM expression. Small lymphocytic lymphomas had lower IgM expression than mantle cell lymphomas. Of importance, IgD expression was significantly lower in BCLs than in hyperplasias, a finding that can be useful in differentiating lymphoma from reactive processes. PMID:22400070

  2. Genetic alterations within the retinoblastoma locus in colorectal carcinomas. Relation to DNA ploidy pattern studied by flow cytometric analysis.

    PubMed Central

    Meling, G. I.; Lothe, R. A.; Børresen, A. L.; Hauge, S.; Graue, C.; Clausen, O. P.; Rognum, T. O.

    1991-01-01

    Alterations within the retinoblastoma (Rb) gene, as detected by the VNTR probe p68RS2.0, and flow cytometric DNA pattern have been analysed in 255 colorectal carcinomas. A total of 35.3% of the tumours had alterations within the Rb gene. Amplification of one allele was demonstrated in 29.5% of the tumours, and loss of heterozygosity was found in 11.5%. No association was found between amplification within the Rb gene and clinicopathological characteristics of the patients. The high frequency of alterations demonstrated within the Rb gene, suggests that this gene is involved in colorectal carcinogenesis with amplification as by far the most abundant genetic alteration. This may imply that the Rb gene has an oncogene-like function in colorectal carcinomas, rather than acting as a tumour suppressor gene. Sixty-three per cent of the carcinomas were DNA aneuploid, and a significant association was demonstrated between amplification within the Rb gene and DNA aneuploidy (P less than 0.01). Two other chromosome loci were analysed, on chromosome 1p (probe pYNZ2) and on chromosome 2p (probe pYNH24), respectively. On chromosome 1p, heterozygous loss was found in 22.2% of the tumours, indicating an involvement of this chromosome in a subset of colorectal carcinomas. Images Figure 1 PMID:1911187

  3. Flow cytometric analysis of Pig-a gene mutation and chromosomal damage induced by procarbazine hydrochloride in CD-1 mice.

    PubMed

    Phonethepswath, Souk; Avlasevich, Svetlana L; Torous, Dorothea K; Mereness, Jared; Bemis, Jeffrey C; Macgregor, James T; Dertinger, Stephen D

    2013-05-01

    Procarbazine is a genotoxic carcinogen whose DNA-damaging activities are not reliably detected in vitro. We evaluated the in vivo genotoxic effects of procarbazine on hematopoietic cells of male CD-1 mice using a multi-endpoint study design that scored micronucleated reticulocyte (MN-RET) frequency and gene mutation at the Pig-a locus. CD-1 mice were treated for 3 days with procarbazine, up to 150 mg/kg/day. Blood samples collected on Day 3 exhibited robust induction of MN-RETs, with the high dose group exhibiting a mean 29-fold increase. Blood collected 15 and 30 days after treatment began was analyzed for Pig-a mutation with a dual labeling method that facilitated mutant cell frequency measurements in both total erythrocytes and the reticulocyte subpopulation. Procarbazine significantly increased mutant reticulocyte frequencies by Day 15. Mutant erythrocyte responses were also apparent, with a peak incidence observed for the high dose group on Day 30. These results demonstrate that the complex metabolism and resulting genotoxicity of procarbazine is best evaluated in intact animal models, and show that the flow cytometric methods employed offer a means to efficiently monitor both in vivo chromosomal damage and mutation. Copyright © 2013 Wiley Periodicals, Inc.

  4. Fragment filtration: a method for the accurate determination of flow cytometric kinetic data from bone marrow aspirates

    SciTech Connect

    Zbroja, R.A.; Wass, J.; Vincent, P.C.; Young, G.A.

    1986-02-01

    The extent to which bone marrow obtained by conventional aspiration is contaminated by peripheral blood has been confirmed and quantitated. In marrow aspirates from normal subjects the median percentage of nucleated cells that had originated from the peripheral blood was 32% (range 2.5%-64%), in patients with acute leukemia 23% (range 0.5%-96.5%), in patients with chronic leukemia 59% (range 17%-76%), and in patients with lymphoma 31% (range 0.5%-74%). Flow cytometric (FCM) DNA analysis of conventional marrow aspirates from a range of subjects significantly underestimated the proportions of S-phase cells present, when compared with results from trephines obtained at the same time. Having shown, using 51Cr-labeled red cells in mice, that circulating red cells do not reenter the marrow parenchyma, a mathematical correction for contaminating blood similar to that described by Holdrinet et al. was devised. This correction improved the S-phase cell estimate from aspirated marrows, and the corrected values were not significantly different from values from paired trephine samples. A previously described technique for collecting fragments by filtration of aspirated marrow has been adapted for FCM analysis as a more direct way of overcoming problems due to blood contamination. This method was shown to yield estimates of S-phase cells not significantly different from those in paired marrow trephines and offers an alternative to routine trephine biopsies for FCM analysis of marrow cell kinetics.

  5. Flow cytometric enumeration of parasitemia in cultures of Plasmodium falciparum stained with SYBR Green I and CD235A.

    PubMed

    Jang, Jin Woo; Kim, Ju Yeon; Yoon, Jung; Yoon, Soo Young; Cho, Chi Hyun; Han, Eun Taek; An, Seong Soo A; Lim, Chae Seung

    2014-01-01

    A flow cytometric (FACS) detection method for Plasmodium falciparum cultures (P. falciparum) was developed using SYBR Green I and CD235A and compared against the Giemsa stained microscopic examination. The cultured P. falciparum were spiked into red blood cells (RBCs) to yield parasitemia, ranging from 0.01% to 22.0%. FACS analysis demonstrated a clear separation between P. falciparum infected and uninfected RBCs. The measured percentage of parasitemia by FACS revealed higher precision (CV of 2.2-37.2%) with the sensitivity of 0.01% parasitemia than Giemsa stained microscopic examination (CV of 7.2-66.0%). High correlation of measured parasitaemia (r=0.98, P<0.05) was observed between FACS and Giemsa stained microscopic analyses. The higher levels of parasitaemia detection were observed in all ranges by FACS in comparison to Giemsa stained microscopic analysis. The currently reported FACS method using SYBR Green I and CD235A is potentially useful for measuring parasitemia in treating patients.

  6. A flow cytometric assay to quantify invasion of red blood cells by rodent Plasmodium parasites in vivo.

    PubMed

    Lelliott, Patrick M; Lampkin, Shelley; McMorran, Brendan J; Foote, Simon J; Burgio, Gaetan

    2014-03-17

    Malaria treatments are becoming less effective due to the rapid spread of drug resistant parasites. Increased understanding of the host/parasite interaction is crucial in order to develop treatments that will be less prone to resistance. Parasite invasion of the red blood cell (RBC) is a critical aspect of the parasite life cycle and is, therefore, a promising target for the development of malaria treatments. Assays for analysing parasite invasion in vitro have been developed, but no equivalent assays exist for in vivo studies. This article describes a novel flow cytometric in vivo parasite invasion assay. Experiments were conducted with mice infected with erythrocytic stages of Plasmodium chabaudi adami strain DS. Exogenously labelled blood cells were transfused into infected mice at schizogony, and collected blood samples stained and analysed using flow cytometry to specifically detect and measure proportions of labelled RBC containing newly invaded parasites. A combination of antibodies (CD45 and CD71) and fluorescent dyes, Hoechst (DNA) and JC-1 (mitochondrial membrane potential), were used to differentiate parasitized RBCs from uninfected cells, RBCs containing Howell-Jolly bodies, leukocytes and RBC progenitors. Blood cells were treated ex vivo with proteases to examine the effects on in vivo parasite invasion. The staining and flow cytometry analysis method was accurate in determining the parasitaemia down to 0.013% with the limit of detection at 0.007%. Transfused labelled blood supported normal rates of parasite invasion. Protease-treated red cells resulted in 35% decrease in the rate of parasite invasion within 30 minutes of introduction into the bloodstream of infected mice. The invasion assay presented here is a versatile method for the study of in vivo red cell invasion efficiency of Plasmodium parasites in mice, and allows direct comparison of invasion in red cells derived from two different populations. The method also serves as an accurate

  7. Bivalent response to long-term storage in liquid-preserved boar semen: a flow cytometric analysis.

    PubMed

    Henning, Heiko; Petrunkina, Anna M; Harrison, Robin A P; Waberski, Dagmar

    2012-07-01

    The fertility of liquid-preserved boar semen declines during storage at 17°C, insemination trials even indicating early losses in fertilizing ability within the first 24-48 h of storage. Standard semen parameters barely reflect these changes in semen quality, and new approaches for assessment of functional changes in stored spermatozoa are needed. Capacitation, the essential prefertilization step for spermatozoa in the female genital tract, is specifically induced in vitro by bicarbonate. Therefore, we have investigated changes in responsiveness of boar spermatozoa to bicarbonate during storage. Ejaculates of 14 boars were diluted in Beltsville thawing solution, cooled to 17°C and stored for 12, 24, 72, 120, and 168 h before investigation. At each time, basic semen quality was characterized by sperm motility and viability. Subsequently, washed subsamples were incubated in variants of an in vitro fertilization (IVF) medium and assessed for kinetic changes of viability (plasma membrane integrity) and intracellular calcium concentration using flow cytometry in combination with propidium iodide and Fluo-3. By this means, it was possible to determine specific effects of bicarbonate and calcium on sperm subpopulations over incubation time. During storage, standard semen parameters remained on a high level. However, flow cytometric analysis of sperm responses to capacitating and control media revealed two opposing effects of storage. There was a loss of response to bicarbonate in part of the live sperm population but an increasing degree of instability in the rest. Assessment of response to capacitating media by flow cytometry appears a markedly more sensitive way of monitoring sperm functionality during storage than the standard semen parameters of motility and viability.

  8. HPC enumeration with the Sysmex XE-2100 can guide further flow cytometric CD34(+) measurements and timing of leukaphereses.

    PubMed

    Oelschlaegel, U; Bornhaeuser, M; Thiede, C; Ehninger, G; Hoelig, K

    2003-01-01

    The aim of this study was to evaluate whether HPC counts measured with the hematology analyzer can predict CD34+ levels in peripheral blood and in the apheresis product, as detected by standard flow cytometry. The main focus was the evaluation of HPC counts in poor mobilizers. Progenitor cell quantification was performed measuring HPC counts provided by the Sysmex XE-2100 hematology analyzer and CD34+ counts obtained in parallel by flow cytometry. Peripheral blood of patients who had received chemotherapy and G-CSF (142 measurements) and healthy donors mobilized with G-CSF alone (106 measurements) was investigated HPC counts in peripheral blood were also correlated with apheresis yield. HPC counts were significantly higher than CD34+ counts (3.5 fold inpatients and 1.7 fold in healthy donors, p= 0.0015). Our data indicate that HPC counts < or = 10/microL in pretreated patients predict a low probability of adequate CD34+ counts in peripheral blood and yields < 2 x 10(6)/kg in subsequent aphereses. Furthermore, repetitive low HPC enumerations in an individual were followed by insufficientCD34+ counts in peripheral blood or aphereses in 81% of investigations. In healthy donors low HPC counts (< or = 10/microL; 12/106 measurements) did not exclusively predict low CD34+ counts (median 23/microL). HPC counts can be used to schedule the start of CD34+ measurements(threshold > 10 HPC/microL) in patients mobilized after chemotherapy for autologous donation. Thus, expensive and time-consuming CD34+ enumerations can perhaps be minimized HPC measurements cannot completely replace flow cytometric CD34+ enumeration. In particular healthy stem-cell donors should be monitored with both methods to exclude false negative HPC measurements.

  9. Flow Cytometric Microsphere-Based Immunoassay: Analysis of Secreted Cytokines in Whole-Blood Samples from Asthmatics

    PubMed Central

    Camilla, Christophe; Mély, Laurent; Magnan, Antoine; Casano, Brice; Prato, Sabine; Debono, Stephane; Montero, Felix; Defoort, Jean-Philippe; Martin, Marie; Fert, Vincent

    2001-01-01

    The ability of flow cytometry to resolve multiple parameters was used in a microsphere-based flow cytometric assay for the simultaneous determination of several cytokines in a sample. The flow cytometer microsphere-based assay (FMBA) for cytokines consists of reagents and dedicated software, specifically designed for the quantitative determination of cytokines. We have made several improvements in the multiplex assay: (i) dedicated software specific for the quantitative multiplex assay that processes data automatically, (ii) a stored master calibration curve with a two-point recalibration to adjust the stored curve periodically, and (iii) an internal standard to normalize the detection step in each sample. Overall analytical performance, including sensitivity, reproducibility, and dynamic range, was investigated for interleukin-4 (IL-4), IL-6, IL-10, IL-12, gamma interferon (IFN-γ), and tumor necrosis factor alpha. These assays were found to be reproducible and accurate, with a sensitivity in the picograms-per-milliliter range. Results obtained with FMBA correlate well with commercial enzyme-linked immunosorbent assay data (r > 0.98) for all cytokines assayed. This multiplex assay was applied to the determination of cytokine profiles in whole blood from atopic and nonatopic patients. Our results show that atopic subjects' blood produces more IL-4 (P = 0.003) and less IFN-γ (P = 0.04) than the blood of nonatopic subjects. However, atopic asthmatic subjects' blood produces significantly more IFN-γ than that of atopic nonasthmatic subjects (P = 0.03). The results obtained indicate that the FMBA technology constitutes a powerful system for the quantitative, simultaneous determination of secreted cytokines in immune diseases. PMID:11427426

  10. Belgian consensus recommendations for flow cytometric immunophenotyping. The Belgian Association for Cytometry/Belgische Vereniging voor Cytometrie/Association Belge de Cytométrie.

    PubMed

    Van Bockstaele, D R; Deneys, V; Philippé, J; Bernier, M; Kestens, L; Chatelain, B; De Waele, M; Demanet, C

    1999-04-01

    This paper summarises the guidelines and recommendations that were generated during a number of discussion forums attended by the majority of Belgian cytometry laboratory professionals. These forums focused on the rational and optimal use of flow cytometric evaluations in the clinical laboratory setting. The aim was to improve the coherence of the testing panels and the quality of the results and--as such--the clinical diagnostic information. It was also the aim to provide the Belgian prescribing physician and interested laymen with an updated overview of the flow cytometric possibilities. Emphasis is placed on immunophenotyping of haematological malignancies, hematopoietic progenitor cell counting and follow-up of the viral infection caused by the human immunodeficiency virus.

  11. Flow cytometric-membrane potential detection of sodium channel active marine toxins: application to ciguatoxins in fish muscle and feasibility of automating saxitoxin detection.

    PubMed

    Manger, Ronald; Woodle, Doug; Berger, Andrew; Dickey, Robert W; Jester, Edward; Yasumoto, Takeshi; Lewis, Richard; Hawryluk, Timothy; Hungerford, James

    2014-01-01

    Ciguatoxins are potent neurotoxins with a significant public health impact. Cytotoxicity assays have allowed the most sensitive means of detection of ciguatoxin-like activity without reliance on mouse bioassays and have been invaluable in studying outbreaks. An improvement of these cell-based assays is presented here in which rapid flow cytometric detection of ciguatoxins and saxitoxins is demonstrated using fluorescent voltage sensitive dyes. A depolarization response can be detected directly due to ciguatoxin alone; however, an approximate 1000-fold increase in sensitivity is observed in the presence of veratridine. These results demonstrate that flow cytometric assessment of ciguatoxins is possible at levels approaching the trace detection limits of our earlier cytotoxicity assays, however, with a significant reduction in analysis time. Preliminary results are also presented for detection of brevetoxins and for automation and throughput improvements to a previously described method for detecting saxitoxins in shellfish extracts.

  12. Flow cytometric determination of endocytosis of viable labelled Legionella pneumophila by Acanthamoeba palestinensis.

    PubMed

    Harf, C; Goffinet, S; Meunier, O; Monteil, H; Colin, D A

    1997-03-01

    Endocytosis of fluorescently labelled cells of Legionella pneumophila (L. pneumophila) by free-living Acanthamoeba palestinensis (A. palestinensis) has been studied using flow cytometry. L. pneumophila cells were labelled with CM-DiI, a lipophilic fluorescent probe under conditions that did not modify viability. Coculturing the bacteria with amoebae was accompanied by rapid endocytosis; after 5 min, 90% of the amoebae had internalized bacteria. This percentage remained unchanged during further coculture, but the number of bacteria ingested per amoeba increased. Moreover, the number of ingested bacteria was found to be dependent on the size of the amoeba. The validity of the internalization analyzed by flow cytometry was confirmed by observation using epifluorescence and phase contrast microscopy. CM-DiI labelling associated with flow cytometry provides a very valuable technique for the determination of bacteria endocytosis by free-living amoeba.

  13. Flow Cytometric Analysis of Myeloid Cells in Human Blood, Bronchoalveolar Lavage, and Lung Tissues

    PubMed Central

    Yu, Yen-Rei A.; Hotten, Danielle F.; Malakhau, Yuryi; Volker, Ellen; Ghio, Andrew J.; Noble, Paul W.; Kraft, Monica; Hollingsworth, John W.; Gunn, Michael D.

    2016-01-01

    Clear identification of specific cell populations by flow cytometry is important to understand functional roles. A well-defined flow cytometry panel for myeloid cells in human bronchoalveolar lavage (BAL) and lung tissue is currently lacking. The objective of this study was to develop a flow cytometry–based panel for human BAL and lung tissue. We obtained and performed flow cytometry/sorting on human BAL cells and lung tissue. Confocal images were obtained from lung tissue using antibodies for cluster of differentiation (CD)206, CD169, and E cadherin. We defined a multicolor flow panel for human BAL and lung tissue that identifies major leukocyte populations. These include macrophage (CD206+) subsets and other CD206− leukocytes. The CD206− cells include: (1) three monocyte (CD14+) subsets, (2) CD11c+ dendritic cells (CD14−, CD11c+, HLA-DR+), (3) plasmacytoid dendritic cells (CD14−, CD11c−, HLA-DR+, CD123+), and (4) other granulocytes (neutrophils, mast cells, eosinophils, and basophils). Using this panel on human lung tissue, we defined two populations of pulmonary macrophages: CD169+ and CD169− macrophages. In lung tissue, CD169− macrophages were a prominent cell type. Using confocal microscopy, CD169+ macrophages were located in the alveolar space/airway, defining them as alveolar macrophages. In contrast, CD169− macrophages were associated with airway/alveolar epithelium, consistent with interstitial-associated macrophages. We defined a flow cytometry panel in human BAL and lung tissue that allows identification of multiple immune cell types and delineates alveolar from interstitial-associated macrophages. This study has important implications for defining myeloid cells in human lung samples. PMID:26267148

  14. High throughput detection of miRNAs and gene-specific mRNA at the single-cell level by flow cytometry

    PubMed Central

    Porichis, Filippos; Hart, Meghan G.; Griesbeck, Morgane; Everett, Holly L.; Hassan, Muska; Baxter, Amy E.; Lindqvist, Madelene; Miller, Sara M.; Soghoian, Damien Z.; Kavanagh, Daniel G.; Reynolds, Susan; Norris, Brett; Mordecai, Scott K.; Nguyen, Quan; Lai, Chunfai; Kaufmann, Daniel E.

    2014-01-01

    Fluorescent in situ hybridization (FISH) is a method that uses fluorescent probes to detect specific nucleic acid sequences at the single cell level. Here we describe optimized protocols that exploit a highly sensitive FISH method based on branched DNA technology to detect mRNA and miRNA in human leukocytes. This technique can be multiplexed and combined with fluorescent antibody protein staining to addressa variety of questions in heterogeneous cell populations. We demonstrate antigen-specific upregulation of IFNγ and IL-2 mRNAs in HIV- and CMV-specific T cells. We show simultaneous detection of cytokine mRNA and corresponding protein in single cells. We apply this method to detect mRNAs for which flow antibodies against the corresponding proteins are poor or are not available. We use this technique to show modulation of a microRNA critical for T cell function, miR-155. We adapt this assay for simultaneous detection of mRNA and proteins by Image Stream technology. PMID:25472703

  15. Role of autofluorescence in flow cytometric analysis of Escherichia coli treated with bactericidal antibiotics.

    PubMed

    Renggli, Sabine; Keck, Wolfgang; Jenal, Urs; Ritz, Daniel

    2013-09-01

    Bactericidal antibiotics kill by different mechanisms as a result of a specific interaction with their cellular targets. Over the past few years, alternative explanations for cidality have been proposed based on a postulated common pathway, depending on the intracellular production of reactive oxygen species. Detection of hydroxyl radicals relies on staining with specific fluorescent dyes that can penetrate the cell and are detected using flow cytometry. Flow cytometry has become an important tool in microbiology to study characteristics of individual cells within large heterogeneous cellular populations. We show here that Escherichia coli treated with different bactericidal antibiotics exhibits increased autofluorescence when analyzed by flow cytometry. We present evidence suggesting that this change in autofluorescence is caused by altered cell morphology upon antibiotic treatment. Consistent with this view, mutant cells that fail to elongate upon norfloxacin treatment show no increased auto-fluorescence response. Finally, we present data demonstrating that changes in autofluorescence can impact the results with fluorescent probes when using flow cytometry and confound the findings obtained with specific dyes. In summary, recent findings that correlate the exposure to cidal antibiotics with the production of reactive oxygen species need to be reconsidered in light of such changes in autofluorescence. Conclusive evidence for an increase of hydroxyl radicals after treatment with such drugs is still missing.

  16. Analysis of synthetic and biological microparticles on several flow cytometric platforms

    EPA Science Inventory

    Microparticles (MPs) are membrane vesicles (0.1 to 1 urn) released from cells upon activation. The limit of detection ofmost standard flow cytometers is just below 1 urn. Recent advances enable detection of particles lower than 0.5 urn, Synthetic. beads are used to define size ra...

  17. Analysis of synthetic and biological microparticles on several flow cytometric platforms

    EPA Science Inventory

    Microparticles (MPs) are membrane vesicles (0.1 to 1 urn) released from cells upon activation. The limit of detection ofmost standard flow cytometers is just below 1 urn. Recent advances enable detection of particles lower than 0.5 urn, Synthetic. beads are used to define size ra...

  18. Role of Autofluorescence in Flow Cytometric Analysis of Escherichia coli Treated with Bactericidal Antibiotics

    PubMed Central

    Renggli, Sabine; Keck, Wolfgang; Jenal, Urs

    2013-01-01

    Bactericidal antibiotics kill by different mechanisms as a result of a specific interaction with their cellular targets. Over the past few years, alternative explanations for cidality have been proposed based on a postulated common pathway, depending on the intracellular production of reactive oxygen species. Detection of hydroxyl radicals relies on staining with specific fluorescent dyes that can penetrate the cell and are detected using flow cytometry. Flow cytometry has become an important tool in microbiology to study characteristics of individual cells within large heterogeneous cellular populations. We show here that Escherichia coli treated with different bactericidal antibiotics exhibits increased autofluorescence when analyzed by flow cytometry. We present evidence suggesting that this change in autofluorescence is caused by altered cell morphology upon antibiotic treatment. Consistent with this view, mutant cells that fail to elongate upon norfloxacin treatment show no increased auto-fluorescence response. Finally, we present data demonstrating that changes in autofluorescence can impact the results with fluorescent probes when using flow cytometry and confound the findings obtained with specific dyes. In summary, recent findings that correlate the exposure to cidal antibiotics with the production of reactive oxygen species need to be reconsidered in light of such changes in autofluorescence. Conclusive evidence for an increase of hydroxyl radicals after treatment with such drugs is still missing. PMID:23836867

  19. Flow cytometric enumeration of bacterial in the coral surface mucus layer.

    PubMed

    Bettarel, Yvan; Thanh, Mai Chi; Patrice, Got; Antoinette, Adingra; Nadège, Kouadio-Ngbesso; Bui, Van Ngoc; Thierry, Bouvier

    2016-09-01

    The direct counts of bacteria inhabiting coral mucus were performed by flow cytometry testing four fluorescent dyes (SYBR®Green I, HCS, TOPRO®3, SYTO®62) with three different scleractinian species. Results obtained with SYTO62 were the most reliable based on the comparison with standardized epifluorescence counts and the resolution of cytograms.

  20. Flow cytometric gating for spleen monocyte and DC subsets: differences in autoimmune NOD mice and with acute inflammation.

    PubMed

    Dong, Matthew B; Rahman, M Jubayer; Tarbell, Kristin V

    2016-05-01

    The role of antigen presenting cells (APCs) in the pathogenesis of autoimmune and other inflammatory diseases is now better understood due to advances in multicolor flow cytometry, gene expression analysis of APC populations, and functional correlation of mouse to human APC populations. A simple but informative nomenclature of conventional and plasmacytoid dendritic cell subsets (cDC1, cDC2, pDC) and monocyte-derived populations incorporates these advances, but accurate subset identification is critical. Ambiguous gating schemes and alterations of cell surface markers in inflammatory condition can make comparing results between studies difficult. Both acute inflammation, such as TLR-ligand stimulation, and chronic inflammation as found in mouse models of autoimmunity can alter DC subset gating. Here, we address these issues using in vivo CpG stimulation as an example of acute inflammation and the non-obese diabetic (NOD) mouse as a model of chronic inflammation.We provide a flow cytometric antibody panel and gating scheme that differentiate 2 monocytic and 3DC subsets in the spleen both at steady state and after CpG stimulation. Using this method, we observed differences in the composition of NOD DCs that have been previously reported, and newly identified increases in the number of NOD monocyte-derived DCs. Finally, we established a protocol for DC phosphoflow to measure the phosphorylation state of intracellular proteins, and use it to confirm functional differences in the identified subsets. Therefore, we present optimized methods for distinguishing monocytic and DC populations with and without inflammation and/or autoimmunity associated with NOD mice.

  1. Flow Cytometric and 16S Sequencing Methodologies for Monitoring the Physiological Status of the Microbiome in Powdered Infant Formula Production

    PubMed Central

    Anvarian, Amir H. P.; Cao, Yu; Srikumar, Shabarinath; Fanning, Séamus; Jordan, Kieran

    2016-01-01

    The aim of this study was to develop appropriate protocols for flow cytometric (FCM) and 16S rDNA sequencing investigation of the microbiome in a powdered infant formula (PIF) production facility. Twenty swabs were collected from each of the three care zones of a PIF production facility and used for preparing composite samples. For FCM studies, the swabs were washed in 200 mL phosphate buffer saline (PBS). The cells were harvested by three-step centrifugation followed by a single stage filtration. Cells were dispersed in fresh PBS and analyzed with a flow cytometer for membrane integrity, metabolic activity, respiratory activity and Gram characteristics of the microbiome using various fluorophores. The samples were also plated on agar plates to determine the number of culturable cells. For 16S rDNA sequencing studies, the cells were harvested by centrifugation only. Genomic DNA was extracted using a chloroform-based method and used for 16S rDNA sequencing studies. Compared to the dry low and high care zones, the wet medium care zone contained a greater number of viable, culturable, and metabolically active cells. Viable but non-culturable cells were also detected in dry-care zones. In total, 243 genera were detected in the facility of which 42 were found in all three care zones. The greatest diversity in the microbiome was observed in low care. The genera present in low, medium and high care were mostly associated with soil, water, and humans, respectively. The most prevalent genera in low, medium and high care were Pseudomonas, Acinetobacter, and Streptococcus, respectively. The integration of FCM and metagenomic data provided further information on the density of different species in the facility. PMID:27446009

  2. Flow Cytometric and 16S Sequencing Methodologies for Monitoring the Physiological Status of the Microbiome in Powdered Infant Formula Production.

    PubMed

    Anvarian, Amir H P; Cao, Yu; Srikumar, Shabarinath; Fanning, Séamus; Jordan, Kieran

    2016-01-01

    The aim of this study was to develop appropriate protocols for flow cytometric (FCM) and 16S rDNA sequencing investigation of the microbiome in a powdered infant formula (PIF) production facility. Twenty swabs were collected from each of the three care zones of a PIF production facility and used for preparing composite samples. For FCM studies, the swabs were washed in 200 mL phosphate buffer saline (PBS). The cells were harvested by three-step centrifugation followed by a single stage filtration. Cells were dispersed in fresh PBS and analyzed with a flow cytometer for membrane integrity, metabolic activity, respiratory activity and Gram characteristics of the microbiome using various fluorophores. The samples were also plated on agar plates to determine the number of culturable cells. For 16S rDNA sequencing studies, the cells were harvested by centrifugation only. Genomic DNA was extracted using a chloroform-based method and used for 16S rDNA sequencing studies. Compared to the dry low and high care zones, the wet medium care zone contained a greater number of viable, culturable, and metabolically active cells. Viable but non-culturable cells were also detected in dry-care zones. In total, 243 genera were detected in the facility of which 42 were found in all three care zones. The greatest diversity in the microbiome was observed in low care. The genera present in low, medium and high care were mostly associated with soil, water, and humans, respectively. The most prevalent genera in low, medium and high care were Pseudomonas, Acinetobacter, and Streptococcus, respectively. The integration of FCM and metagenomic data provided further information on the density of different species in the facility.

  3. Single-laboratory validation of a multiplex flow cytometric immunoassay for the simultaneous detection of coccidiostats in eggs and feed.

    PubMed

    Bienenmann-Ploum, Monique E; Vincent, Ursula; Campbell, Katrina; Huet, Anne-Catherine; Haasnoot, Willem; Delahaut, Philippe; Stolker, Linda A A M; Elliott, Christopher T; Nielen, Michel W F

    2013-11-01

    Coccidiostats are authorized in the European Union (EU) to be used as poultry feed additives. Maximum (residue) levels (M(R)Ls) have been set within the EU for consumer and animal protection against unintended carry-over, and monitoring is compulsory. This paper describes the single-laboratory validation of a previously developed multiplex flow cytometric immunoassay (FCIA) as screening method for coccidiostats in eggs and feed and provides and compares different approaches for the calculation of the cut-off levels which are not described in detail within Commission Decision 2002/657/EC. Comparable results were obtained between the statistical (reference) approach and the rapid approaches. With the most rapid approach, the cut-off levels for narasin/salinomycin, lasalocid, diclazuril, nicarbazin (DNC) and monensin in egg, calculated as percentages of inhibition (%B/B0), were 60, 32, 76, 80 and 84, respectively. In feed, the cut-off levels for narasin/salinomycin, lasalocid, nicarbazin (DNC) and monensin were 70, 64, 72 and 78, respectively, and could not be determined for diclazuril. For all analytes, except for diclazuril in feed, the rate of false positives (false non-compliant) in blank samples was lower than 1 %, and the rate of false negatives (false compliant) at the M(R)Ls was below 5 %. Additionally, very good correlations (r ranging from 0.994 to 0.9994) were observed between two different analysers, a sophisticated flow cytometer (FlexMAP 3D(®)) and a more cost-efficient and transportable planar imaging detector (MAGPIX(®)), hence demonstrating adequate transferability.

  4. Flow cytometric characterization of peripheral blood leukocyte populations of 3 neotropical snake species: Boa constrictor, Bothrops jararaca, and Crotalus durissus.

    PubMed

    Carvalho, Marcelo P N; Queiroz-Hazarbassanov, Nicolle G T; Massoco, Cristina O; Rossi, Silmara; Sant'Anna, Sávio S; Catão-Dias, José L; Grego, Kathleen F

    2016-06-01

    The reptilian immune system is represented by innate, humoral, and cell-mediated mechanisms, involving different types of blood leukocytes. The development of optimized methods for the advanced study of origin and function of reptilian blood leukocytes is needed. The purpose of the study was to optimize leukocyte density gradient isolation protocols from snake peripheral blood samples, and characterize recovered cells by flow cytometry based on size and internal complexity for a qualitative and semi-quantitative assessment of leukocyte populations in one boa (Boa constrictor), and 2 viper species (Bothrops jararaca, Crotalus durissus). Blood samples from 30 snakes (10 from each species, 5 males and 5 females) were collected in tubes with sodium heparin. Fresh blood was centrifuged with either ficoll-paque PLUS or percoll density gradients for leukocyte isolation. Flow cytometric leukocyte gates were defined based on size (forward scatter [FSC]) and internal complexity (side scatter [SSC]). Relative leukocyte differential counts after sorting the cells in these gates in one snake for each species were compared to conventional light microscopic differential counts on unsorted isolated leukocytes. There was no statistical difference in the relative leukocyte populations, including heterophils, azurophils, and small and large lymphocytes between samples isolated by ficoll or percoll. Four leukocyte gates were identified based on their location in FSC/SSC cytograms. The relative leukocyte differential counts after sorting in single animals showed some agreement with the light microscopy differential count on unsorted cells. Based on FSC and SSC, 4 distinct leukocyte populations were found in ficoll or percoll density gradient isolated leukocytes from peripheral blood from boa and viper species. Further optimization of the technique should allow the performance of functional assays. © 2016 American Society for Veterinary Clinical Pathology.

  5. Current International Flow Cytometric Practices for the Detection and Monitoring of Paroxysmal Nocturnal Haemoglobinuria (PNH) clones: A UK NEQAS Survey.

    PubMed

    Fletcher, Matthew; Whitby, Liam; Whitby, Alison; Barnett, David

    2016-03-02

    Background Paroxysmal Nocturnal Haemoglobinuria (PNH) is a rare acquired genetic disorder, with an incidence of approximately 1.3 new cases per million population per year. Evidence from the UK National External Quality Assessment Service for Leucocyte Immunophenotyping (UK NEQAS LI) programme suggested major discrepancies on how PNH testing is undertaken. To investigate this we surveyed laboratories in the UK NEQAS LI PNH programme and report here the findings. Method A questionnaire was distributed to all centres registered in UK NEQAS LI flow cytometry programmes (n=1587). Comprising several subsections, it covered the majority of clinical flow cytometric practices. Participants completed a general section and then the subsections relevant to their laboratory repertoire. One subsection contained 34 questions regarding practices in PNH clone detection. Results A total of 105 laboratories returned results for the PNH section; the results demonstrated lack of consensus in all areas of PNH testing. Variation was seen in gating and testing strategies, sensitivity levels and final reporting of test results. Several incorrect practices were highlighted such as inappropriate antibody selection and failure to wash the red blood cells (RBCs) prior to analysis. Conclusion Despite the availability of consensus guidelines there appears to be no agreement in the detection and monitoring of PNH. We found only fourteen centres using methods compatible with the International Clinical Cytometry Society guidelines. Of specific note we found that no two laboratories used the same method. This technical variation could lead to incorrect diagnoses, highlighting the need for better adoption and understanding of consensus practices. This article is protected by copyright. All rights reserved.

  6. Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

    PubMed Central

    Süring, Katrin; Bach, Sabine; Höflich, Conny; Straff, Wolfgang

    2016-01-01

    Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 µm in diameter in inhalable particulate matter (PM10, particle size ≤10 µm in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions. So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles >0.5 µm. PMID:27911374

  7. Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10.

    PubMed

    Süring, Katrin; Bach, Sabine; Höflich, Conny; Straff, Wolfgang

    2016-11-19

    Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 µm in diameter in inhalable particulate matter (PM10, particle size ≤10 µm in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions. So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles >0.5 µm.

  8. Flow cytometric analysis of the response of Eimeria tenella (Coccidia) sporozoites to coccidiocidal effects of ionophores.

    PubMed

    Fuller, A L; Golden, J; McDougald, L R

    1995-12-01

    Fluorescein diacetate (FDA) and propidium iodide (PI) were used as indicators of membrane integrity after Eimeria tenella sporozoites were treated with polyether ionophores. Flow cytometry was used to quantitate the structural and functional effects based on red or green fluorescence and shape index of the sporozoites. Two field isolates (FS119 and FS139) were essentially resistant to polyether ionophores administered under practical conditions, whereas a laboratory strain was considered sensitive. The shape of sporozoites changed after treatment with ionophores, and this could be detected by flow cytometry. Green-fluorescing cells declined in number as the membranes were compromised by ionophore treatment. Red-fluorescing cells increased as the compromised membranes allowed entry of PI to bind with the nucleic acids. These effects were generally slower to develop in ionophore-tolerant field isolates compared with the sensitive laboratory strain. The effect of lasalocid on FDA and PI uptake, change in shape of the sporozoites, and lysis of sporozoites was more rapid than that of monensin or salinomycin. The 2 field isolates responded at different rates to salinomycin and monensin. Flow cytometry was a sensitive and accurate instrument for analysis of the effects of ionophores on sensitive and resistant lines of coccidia.

  9. Simple flow cytometric detection of haemozoin containing leukocytes and erythrocytes for research on diagnosis, immunology and drug sensitivity testing.

    PubMed

    Frita, Rosangela; Rebelo, Maria; Pamplona, Ana; Vigario, Ana M; Mota, Maria M; Grobusch, Martin P; Hänscheid, Thomas

    2011-03-31

    Malaria pigment (haemozoin, Hz) has been the focus of diverse research efforts. However, identification of Hz-containing leukocytes or parasitized erythrocytes is usually based on microscopy, with inherent limitations. Flow cytometric detection of depolarized Side-Scatter is more accurate and its adaptation to common bench top flow cytometers might allow several applications. These can range from the ex-vivo and in-vitro detection and functional analysis of Hz-containing leukocytes to the detection of parasitized Red-Blood-Cells (pRBCs) to assess antimalarial activity. A standard benchtop flow cytometer was adapted to detect depolarized Side-Scatter. Synthetic and Plasmodium falciparum Hz were incubated with whole blood and PBMCs to detect Hz-containing leukocytes and CD16 expression on monocytes. C5BL/6 mice were infected with Plasmodium berghei ANKA or P. berghei NK65 and Hz-containing leukocytes were analysed using CD11b and Gr1 expression. Parasitized RBC from infected mice were identified using anti-Ter119 and SYBR green I and were analysed for depolarized Side Scatter. A highly depolarizing RBC population was monitored in an in-vitro culture incubated with chloroquine or quinine. A flow cytometer can be easily adapted to detect depolarized Side-Scatter and thus, intracellular Hz. The detection and counting of Hz containing leukocytes in fresh human or mouse blood, as well as in leukocytes from in-vitro experiments was rapid and easy. Analysis of CD14/CD16 and CD11b/Gr1 monocyte expression in human or mouse blood, in a mixed populations of Hz-containing and non-containing monocytes, appears to show distinct patterns in both types of cells. Hz-containing pRBC and different maturation stages could be detected in blood from infected mice. The analysis of a highly depolarizing population that contained mature pRBC allowed to assess the effect of chloroquine and quinine after only 2 and 4 hours, respectively. A simple modification of a flow cytometer allows for

  10. Simple flow cytometric detection of haemozoin containing leukocytes and erythrocytes for research on diagnosis, immunology and drug sensitivity testing

    PubMed Central

    2011-01-01

    Background Malaria pigment (haemozoin, Hz) has been the focus of diverse research efforts. However, identification of Hz-containing leukocytes or parasitized erythrocytes is usually based on microscopy, with inherent limitations. Flow cytometric detection of depolarized Side-Scatter is more accurate and its adaptation to common bench top flow cytometers might allow several applications. These can range from the ex-vivo and in-vitro detection and functional analysis of Hz-containing leukocytes to the detection of parasitized Red-Blood-Cells (pRBCs) to assess antimalarial activity. Methods A standard benchtop flow cytometer was adapted to detect depolarized Side-Scatter. Synthetic and Plasmodium falciparum Hz were incubated with whole blood and PBMCs to detect Hz-containing leukocytes and CD16 expression on monocytes. C5BL/6 mice were infected with Plasmodium berghei ANKA or P. berghei NK65 and Hz-containing leukocytes were analysed using CD11b and Gr1 expression. Parasitized RBC from infected mice were identified using anti-Ter119 and SYBR green I and were analysed for depolarized Side Scatter. A highly depolarizing RBC population was monitored in an in-vitro culture incubated with chloroquine or quinine. Results A flow cytometer can be easily adapted to detect depolarized Side-Scatter and thus, intracellular Hz. The detection and counting of Hz containing leukocytes in fresh human or mouse blood, as well as in leukocytes from in-vitro experiments was rapid and easy. Analysis of CD14/CD16 and CD11b/Gr1 monocyte expression in human or mouse blood, in a mixed populations of Hz-containing and non-containing monocytes, appears to show distinct patterns in both types of cells. Hz-containing pRBC and different maturation stages could be detected in blood from infected mice. The analysis of a highly depolarizing population that contained mature pRBC allowed to assess the effect of chloroquine and quinine after only 2 and 4 hours, respectively. Conclusions A simple

  11. Flow cytometric assessment of Streptococcus mutans viability after exposure to blue light-activated curcumin.

    PubMed

    Manoil, Daniel; Filieri, Anna; Gameiro, Cécile; Lange, Norbert; Schrenzel, Jacques; Wataha, John C; Bouillaguet, Serge

    2014-09-01

    Streptococcus mutans biofilms are considered as primary causative agents of dental caries. Photodynamic antimicrobial chemotherapy (PACT) has been recently proposed as a strategy for inactivating dental biofilms. This study aimed to investigate the effect of blue light-activated curcumin on S. mutans viability and to explore its potential as a new anti-caries therapeutic agent. The effect of different concentrations and incubation times of photo-activated curcumin on the survival of S. mutans in planktonic and biofilm models of growth was assessed by flow cytometry. Streptococcus mutans in planktonic suspensions or biofilms formed on hydroxyapatite disks were incubated for 5 or 10min with curcumin prior to blue light activation. Bacteria were labeled with SYTO 9 and propidium iodide before viability was assessed by flow cytometry. Results were statistically analyzed using one-way ANOVA and Tukey multiple comparison intervals (α=0.05). For planktonic cultures, 0.2μM of light-activated curcumin significantly reduced S. mutans viability (p<0.05). For biofilm cultures, light-activated curcumin at concentration of 40-60μM only suppressed viability by 50% (p<0.05). Independently of the mode of growth, incubation time has no significant effect on PACT efficiency. This study indicates that blue light-activated curcumin can efficiently inactivate planktonic cultures of S. mutans whereas biofilms were more resistant to treatment. Flow cytometry allowed the detection of bacteria with damaged membranes that were unable to replicate and grow after cell sorting. Further studies seem warranted to optimize the efficacy of light-activated curcumin against S. mutans biofilms. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. Flow cytometric analysis of apoptosis in cryoconserved chicken primordial germ cells.

    PubMed

    Sawicka, Dorota; Chojnacka-Puchta, Luiza; Zielinski, Marcin; Plucienniczak, Grazyna; Plucienniczak, Andrzej; Bednarczyk, Marek

    2015-03-01

    Our research aimed to compare the effects of four cryoprotectants and four slow freezing programs on the viability and apoptosis of primordial germ cells (PGCs) in vitro. PGCs were collected from chicken embryonic blood at Hamburger and Hamilton (HH) stages 14-16 and purified by Percoll density gradient centrifugation and then subjected to cryopreservation. We applied microscopy to determine the survival of PGCs after trypan blue staining and flow cytometry to examine apoptosis and viability after annexin V kit staining. We also examined the functionality of cryopreserved PGCs in vivo. Significant differences in viability of PGCs determined via microscopy and flow cytometry were observed. The most unfavorable combination for slow freezing PGCs was program 3 and MIX H (10% DMSO and 5% glycerol in Hank's solution supplemented with 10% FBS) as the cryoprotectant (48.43 and 15.37% live and early apoptotic PGCs, respectively). The highest average percentage of live PGCs (93.1%) and the lowest percentage of early apoptotic PGCs (6.5%) were achieved by slow freezing PGCs in the presence of DMSO F (10% DMSO in FBS) via program 1. Therefore, this method was chosen for the in vivo test. Cryopreserved (group 1) and freshly isolated (group 2) PGCs were transfectedwith a pEGFP-N1 plasmid, cultured under antibiotic selection, and then injected into 3-day-old embryos. After 5 days of incubation, we identified the EGFP marker gene in the gonads of 40 and 45% of recipients in groups 1 and 2, respectively. This is the first study to apply flow cytometry to examine the apoptosis and viability of cryopreserved PGCs. The in vitro and in vivo findings showed that the developed PGC cryoconservation method, depending on slow freezing at the rate of 2°C/min (program 1) in the presence of 10% DMSO F, is an improvement over previous cryoconservation methods and may be a useful tool for the ex situ strategy of poultry biodiversity preservation.

  13. Detection of tumor cells in body cavity fluids by flow cytometric and immunocytochemical analysis.

    PubMed

    Krishan, Awtar; Ganjei-Azar, Parvin; Jorda, Merce; Hamelik, Ronald M; Reis, Isildinha M; Nadji, Mehrdad

    2006-08-01

    Measurement of electronic volume versus DNA content of nuclei can be used to discriminate between normal and malignant cells. Epithelial membrane antigen immunocytochemistry (EMA-ICC), a helpful ancillary test in body cavity fluids, is not universally accurate for detecting malignancy in effusions. The current study was undertaken to determine if multiparametric flow cytometry (based on simultaneous analysis of light scatter, nuclear volume, DNA, and nuclear protein content) in combination with (EMA-ICC) could be used for the detection of malignant cells in peritoneal and pleural fluids. We studied 130 body cavity fluids (68 peritoneal and 62 pleural fluids) by conventional cytology and multiparametric laser flow cytometry. EMA-ICC was performed using EMA antibodies and L-SAB detection system (DakoCytomation, Carpinteria, CA). EMA-ICC had significantly higher sensitivity than conventional cytology (79% versus 59%, P = 0.016) and ploidy (79% versus 38%, P = 0.001). Cytology had significantly higher specificity than ploidy (97% versus 82%, P = 0.012). The differences in specificity between EMA-ICC and ploidy (87% versus 82%, P= 0.607) or EMA-ICC and cytology (87% versus 97%, P = 0.109) were not statistically significant. However, assuming serial testing, sensitivity increased significantly for the combinations of cytology and EMA-ICC (79.4%, P = 0.016) and cytology and ploidy (73.5%, P = 0.004) as compared to cytology alone (58.8%). Also, the combination of cytology and ploidy had a higher sensitivity than ploidy alone (73% versus 38%, P < 0.0001). However, the sensitivity associated with the three tests used in serial (85.3%) was not significantly different from the sensitivities corresponding to the combination of cytology and EMA-ICC (79%) or cytology and ploidy (73%). Multiparametric flow cytometry utilizing high resolution DNA, nuclear volume, protein measurement, and ICC, in combination with cytomorphology, may be a valuable tool for rapid identification of

  14. Endoreduplicative standards for calibration of flow cytometric C-Value measurements.

    PubMed

    Galbraith, David W

    2014-04-01

    It has been estimated that there are, globally, as many as 400,000 species of the angiosperms (the flowering plants). Of these, a minimal proportion has been characterized at the cytological level. Urgency is required in initiating a systematic and comprehensive census, due to species extinction as a consequence of anthropogenic activities. Fundamental to eukaryotes is the 2C-value, the amount of DNA contained within the nucleus of the unreduced gametes. Flow cytometry provides an ideal method for determining C-values, but the values archived in the Kew Plant C-value Database represent <2% of these species. Complicating the issue is a proliferation of different, and inconsistent standards for C-value measurements utilizing flow cytometry, and variability associated with different instrument platforms and using different staining procedures. In previous work, the use of flow cytometry for analysis of plant nuclear DNA contents for species spanning much of the range of genome sizes found in the angiosperms was described. For this work, an endoreduplicative species (Arabidopsis thaliana L.) was particularly helpful as an internal standard for genome size calibration. Such a standard is compromised if it overlaps in DNA content than that of the species whose genome size is sought. This report describes the use of a second species displaying endoreduplication, Capsicum annuum L., for similar standardization. The results (a) indicate accurate reporting of nuclear DNA contents across a range 0.32-423.68 pg, (b) confirm that endoreduplication increases nuclear DNA contents by complete replication of the genome, and (c) provide a means for quality control of linearity in instrumentation over defined dynamic ranges. © 2014 International Society for Advancement of Cytometry.

  15. Optimal cellular preservation for high dimensional flow cytometric analysis of multicentre trials.

    PubMed

    Ng, Amanda A P; Lee, Bernett T K; Teo, Timothy S Y; Poidinger, Michael; Connolly, John E

    2012-11-30

    High dimensional flow cytometry is best served by centralized facilities. However, the difficulties around sample processing, storage and shipment make large scale international studies impractical. We therefore sought to identify optimized fixation procedures which fully leverage the analytical capability of high dimensional flow cytometry without the need for complex cell processing or a sustained cold chain. Whole blood staining procedure was employed to investigate the applicability of fixatives including Cyto-Chex® Blood Collection tube (Streck), Transfix® (Cytomark), 1% and 4% paraformaldehyde to centralized analysis of field trial samples. Samples were subjected to environmental conditions which mimic field studies, without refrigerated shipment and analyzed across 10 days, based on cell count and marker expression. This study showed that Cyto-Chex® demonstrated the least variability in absolute cell count relative to samples analyzed directly from donors in the absence of fixation. Transfix® was better at preserving the marker expression among all fixatives. However, Transfix® caused marked increased cell membrane permeabilization and was detrimental to intracellular marker identification. Paraformaldehyde fixation, at either 1% or 4% concentrations, was unfavorable for cell preservation under the conditions tested and thus not recommended. Using these data, we have created an online interactive tool which enables researchers to evaluate the impact of different fixatives on their panel of interest. In this study, we have identified Cyto-Chex® as the optimal cellular preservative for high dimensional flow cytometry in large scale studies for shipped whole blood samples, even in the absence of a sustained cold chain. Copyright © 2012 Elsevier B.V. All rights reserved.

  16. Flow cytometric analysis of T lymphocyte proliferation in vivo by EdU incorporation.

    PubMed

    Sun, Xiaojing; Zhang, Chunpan; Jin, Hua; Sun, Guangyong; Tian, Yue; Shi, Wen; Zhang, Dong

    2016-12-01

    Monitoring T lymphocyte proliferation, especially in vivo, is essential for the evaluation of adaptive immune reactions. Flow cytometry-based proliferation assays have advantages in measuring cell division of different T lymphocyte subsets at the same time by multicolor labelling. In this study, we aimed to establish the use of 5-Ethynyl-2'-deoxyuridine (EdU) incorporation in vivo to monitor T lymphocyte proliferation by flow cytometry with an adoptive transfer model. We found that fixation followed by permeabilization preserved T cell surface antigens and had no obvious effects on the fluorescence intensity of APC, PE, PE-Cy7, FITC and PerCP-Cy5.5 when the concentration of the permeabilization reagents was optimized. However, the click reaction resulted in a significant decrease in the fluorescence intensity of PE and PE-Cy7, and surface staining after the click reaction improved the fluorescence intensity. Thus, an extra step of blocking with PBS with 3% FBS between the click reaction and cell surface staining is needed. Furthermore, the percentage of EdU-positive cells increased in a dose-dependent manner, and the saturated dose of EdU was 20mg/kg. Intraperitoneal and intravenous injection had no differences in lymphocyte proliferation detection with EdU in vivo. In addition, T cell proliferation measured by EdU incorporation was comparable to BrdU but was lower than CFSE labelling. In conclusion, we optimized the protocols for EdU administration in vivo and staining in vitro, providing a feasible method for the measurement of T lymphocyte proliferation with EdU incorporation by flow cytometry in vivo.

  17. Flow cytometric determination of genome size in European sunbleak Leucaspius delineatus (Heckel, 1843).

    PubMed

    Filipiak, Marta; Tylko, Grzegorz; Kilarski, Wincenty

    2012-04-01

    The aim of this study was to compare DNA content in hepatocyte and erythrocyte nuclei of the European sunbleak, Leucaspius delineatus, in relation to nuclear and cell size by means of flow cytometry and fluorescence microscopy. The DNA standards, chicken and rainbow trout erythrocytes, were prepared in parallel with both cell types, with initial separation of liver cells in pepsin solution followed by cell filtering. Standards and investigated cells were stained with a mixture of propidium iodide, citric acid, and Nonidet P40 in the presence of RNAse, and fluorescence of at least 50,000 nuclei was analyzed by flow cytometry. Average cell size was determined by flow cytometry, using fresh cell suspension in relation to latex beads of known diameter. The size of nuclei was examined on the basis of digital micrographs obtained by fluorescence microscopy after nuclei staining with DAPI. The sunbleak's erythrocyte nuclei contain 2.25 ± 0.06 pg of DNA, whereas the hepatocyte nuclei contain 2.46 ± 0.06 pg of DNA. This difference in DNA content was determined spectroscopically using isolated DNA from the two cell types. The modal diameters of the erythrocytes and hepatocytes were estimated to be 5.1 ± 0.2 and 22.3 ± 5.0 μm, respectively, and the corresponding modal dimensions of their nuclei (measured as surface area) were 15.2 and 21.4 μm(2), respectively. The nucleoplasmic index, as calculated from diameters estimated from surface area of nuclear profiles, was 2.51 for the erythrocytes compared with 0.08 for hepatocytes.

  18. Quantitative assessment of BAX transcript and flow cytometric expression in acute myeloid leukemia: a prospective study.

    PubMed

    Sharawat, Surender Kumar; Raina, Vinod; Kumar, Lalit; Sharma, Atul; Bakhshi, Radhika; Vishnubhatla, Sreenivas; Gupta, Ritu; Bakhshi, Sameer

    2014-10-01

    Quantitative assessment of BAX transcripts and protein in acute myeloid leukemia (AML). We quantitatively evaluated BAX gene transcripts by real-time polymerase chain reaction (TaqMan probe chemistry) and protein expression by flow cytometry. Consecutive 112 AML patients with a median age of 16 (1-59) years were recruited in the study. By flow cytometry, the percentage expression was in linear correlation with relative median fluorescent intensity (RMFI; R = 0.4425; P < 0.001). However, there was no linear relationship between the transcript copies of the BAX with its RMFI (R = -0.0559; P = 0.586). The expression of the BAX at both protein and transcript level was significantly higher in AML patients as compared with normal control. RMFI of the BAX were higher in the cohort with lower white blood cell count (P = 0.029). None of the other baseline characteristics correlated with either the BAX transcript or the RMFI. BAX expression did not correlate with complete remission rate, event free, disease free, and overall survival. BAX gene expression in AML was evaluated first time with two different methods but did not correlate with the survival outcome.

  19. Flow cytometric measurement of the cellular propagation of TDP-43 aggregation.

    PubMed

    Zeineddine, Rafaa; Whiten, Daniel R; Farrawell, Natalie E; McAlary, Luke; Hanspal, Maya A; Kumita, Janet R; Wilson, Mark R; Yerbury, Justin J

    2017-05-04

    Amyotrophic lateral sclerosis is a devastating neuromuscular degenerative disease characterized by a focal onset of motor neuron loss, followed by contiguous outward spreading of pathology including TAR DNA-binding protein of 43 kDa (TDP-43) aggregates. Previous work suggests that TDP-43 can move between cells. Here we used a novel flow cytometry technique (FloIT) to analyze TDP-43 inclusions and propagation. When cells were transfected to express either mutant G294A TDP-43 fused to GFP or wild type TDP-43fused to tomato red and then co-cultured, flow cytometry detected intact cells containing both fusion proteins and using FloIT detected an increase in the numbers of inclusions in lysates from cells expressing wild type TDP-43-tomato. Furthermore, in this same model, FloIT analyses detected inclusions containing both fusion proteins. These results imply the transfer of TDP-43 fusion proteins between cells and that this process can increase aggregation of wild-type TDP-43 by a mechanism involving co-aggregation with G294A TDP-43.

  20. Flow cytometric analysis of regulatory T cells during hyposensitization of acquired allergic contact dermatitis.

    PubMed

    Fraser, Kathleen; Abbas, Mariam; Hull, Peter R

    2014-01-01

    We previously demonstrated that repeated intradermal steroid injections administered at weekly intervals into positive patch-test sites induce hyposensitization and desensitization. To examine changes in CD4CD25CD127lo/ regulatory T cells during the attenuation of the patch-test response. Ten patients with known allergic contact dermatitis were patch tested weekly for 10 weeks. The patch-test site was injected intradermally with 2 mg triamcinolone. At weeks 1 and 7, a biopsy was performed on the patch-test site in 6 patients, and flow cytometry was performed assessing CD4CD25CD127lo/ regulatory T cells. Secondary outcomes were clinical score, reaction size, erythema, and temperature. Statistical analysis included regression, correlation, and repeated-measures analysis of variance. The percentage of CD4CD25CD127lo/ regulatory T cells, measured by flow cytometry, increased from week 1 to week 7 by an average of 19.2%. The average grade of patch-test reaction decreased from +++ (vesicular reaction) to ++ (palpable erythema). The mean drop in temperature following treatment was 0.28°C per week. The mean area decreased 8.6 mm/wk over 10 weeks. Intradermal steroid injections of weekly patch-test reactions resulted in hyposensitization of the allergic contact dermatitis reaction. CD4CD25CD127lo/ regulatory T cells showed a tendency to increase; however, further studies are needed to determine if this is significant.

  1. Flow cytometric analysis to evaluate physiological alterations in herbicide-exposed Chlamydomonas moewusii cells.

    PubMed

    Prado, Raquel; Rioboo, Carmen; Herrero, Concepción; Suárez-Bregua, Paula; Cid, Angeles

    2012-03-01

    Investigation of herbicide toxicology in non-target aquatic primary producers such as microalgae is of great importance from an ecological point of view. In order to study the toxicity of the widely used herbicide paraquat on freshwater green microalga Chlamydomonas moewusii, physiological changes associated with 96 h-exposures to this pollutant were monitored using flow cytometry (FCM) technique. Intracellular reactive oxygen species concentration, cytoplasmic membrane potential, metabolic activity and cell protein content were monitored to evaluate the toxicological impact of paraquat on algal physiology. Results showed that herbicide paraquat induced oxidative stress in C. moewusii cells, as it indicated the increase of both superoxide anion and hydrogen peroxide levels observed in non-chlorotic cells of cultures exposed to increasing herbicide concentrations. Furthermore, a progressive increase in the percentage of depolarised cells and a decrease in the metabolic activity level were observed in response to paraquat when non-chlorotic cells were analysed. Chlorotic cells were probably non-viable cells, based on the cytoplasmic membrane depolarisation, its metabolically non-active state and its drastically reduced protein content. In view of the obtained results, we have concluded that a range of significant physiological alterations, detected by flow cytometry, occur when C. moewusii, an ubiquitous microalga in freshwater environments, is challenged with environmentally relevant concentrations of the herbicide paraquat.

  2. Flow cytometric monitoring of hormone receptor expression in human solid tumors

    NASA Astrophysics Data System (ADS)

    Krishan, Awtar

    2002-05-01

    Hormone receptor expression in human breast and prostate tumors is of diagnostic and therapeutic importance. With the availability of anti-estrogen, androgen and progesterone antibodies, immunohistochemistry has become a standard tool for determination of receptor expression in human tumor biopsies. However, this method is dependent on examination of a small number of cells under a microscope and the data obtained in most cases is not quantitative. As most of the commercially used anti-hormone antibodies have nuclear specificity, we have developed methods for isolation and antigen unmasking of nuclei from formalin fixed/paraffin embedded archival human tumors. After immunostaining with the antibodies and propidium iodide (for DNA content and cell cycle analysis), nuclei are analyzed by multiparametric laser flow cytometry for hormone receptor expression, DNA content, aneuploidy and cell cycle determination. These multiparametric methods are especially important for retrospective studies seeking to correlate hormone receptor expression with clinical response to anti-hormonal therapy of human breast and prostate tumors.

  3. Determination of micro-litre volumes with high accuracy for flow cytometric blood cell counting

    NASA Astrophysics Data System (ADS)

    Reitz, S.; Kummrow, A.; Kammel, M.; Neukammer, J.

    2010-07-01

    We have gravimetrically calibrated the volumes dispensed by 1 mL syringes in the range between 1 µL and 100 µL using ultra-pure water. Protocols are based on series of consecutive difference measurements of masses in order to precisely compensate for evaporation, being the most important disturbing quantity. We determined expanded uncertainties of volume measurements for glass syringes of typically 0.2% (expansion factor 2) when dispensing volumes of 10 µL. For polypropylene syringes, selected with respect to the manufacturer, expanded uncertainties of 0.25% (expansion factor 2) were observed. Calibrated syringes were applied for measuring concentrations of blood cells in a flow cytometer demonstrating the capability to determine reference measurement values. Since the direct interaction of blood cells and syringe walls may lead to cell adhesion, glass syringes as well as (disposable) polypropylene syringes were calibrated.

  4. Flow cytometric enumeration of absolute lymphocyte number in peripheral blood using two parameters of light scatter.

    PubMed

    Smart, Y C; Cox, J; Murphy, B; Enno, A; Burton, R C

    1985-03-01

    A method was developed to measure the absolute lymphocyte count (ALC) of whole blood using the Spectrum III automated flow cytometer. Ninety-nine samples of human peripheral blood were analysed on the Spectrum and the Coulter Counter S Plus II, to allow for comparison of the two machines. Regression analysis was used to test the extent of agreement between the sets of measurements on the two machines. The results demonstrated that the slope of the regression line was not significantly different from one, indicating a high level of correlation between Spectrum and Coulter ALC's. However, the mean difference between Coulter and Spectrum ALC's was not equal to zero, with the Spectrum giving counts approximately 10% lower than those of the Coulter machine. This is attributed to the different ways by which the two machines define a lymphocyte, the Spectrum III by two parameters of light scatter and the Coulter S Plus II by the single parameter of cell volume.

  5. AIEOP-BFM consensus guidelines 2016 for flow cytometric immunophenotyping of Pediatric acute lymphoblastic leukemia.

    PubMed

    Dworzak, Michael N; Buldini, Barbara; Gaipa, Giuseppe; Ratei, Richard; Hrusak, Ondrej; Luria, Drorit; Rosenthal, Eti; Bourquin, Jean-Pierre; Sartor, Mary; Schumich, Angela; Karawajew, Leonid; Mejstrikova, Ester; Maglia, Oscar; Mann, Georg; Ludwig, Wolf-Dieter; Biondi, Andrea; Schrappe, Martin; Basso, Giuseppe

    2017-02-10

    Immunophenotyping by flow cytometry (FCM) is a worldwide mainstay in leukemia diagnostics. For concordant multicentric application, however, a gap exists between available classification systems, technologic standardization, and clinical needs. The AIEOP-BFM consortium induced an extensive standardization and validation effort between its nine national reference laboratories collaborating in immunophenotyping of pediatric acute lymphoblastic leukemia (ALL). We elaborated common guidelines which take advantage of the possibilities of multi-color FCM: marker panel requirements, immunological blast gating, in-sample controls, tri-partite antigen expression rating (negative vs. weak or strong positive) with capturing of blast cell heterogeneities and subclone formation, refined ALL subclassification, and a dominant lineage assignment algorithm able to distinguish "simple" from bilineal/"complex" mixed phenotype acute leukemia (MPAL) cases, which is essential for choice of treatment. These guidelines are a first step toward necessary inter-laboratory standardization of pediatric leukemia immunophenotyping for a concordant multicentric application. © 2017 International Clinical Cytometry Society.

  6. Side scatter versus CD45 flow cytometric plot can distinguish acute leukaemia subtypes.

    PubMed

    Saksena, Annapurna; Gautam, Parul; Desai, Parth; Gupta, Naresh; Dubey, A P; Singh, Tejinder

    2016-05-01

    Flow cytometry is an important tool to diagnose acute leukaemia. Attempts are being made to find the minimal number of antibodies for correctly diagnosing acute leukaemia subtypes. The present study was designed to evaluate the analysis of side scatter (SSC) versus CD45 flow dot plot to distinguish acute myeloid leukaemia (AML) from acute lymphoblastic leukaemia (ALL), with minimal immunological markers. One hundred consecutive cases of acute leukaemia were evaluated for blast cluster on SSC versus CD45 plots. The parameters studied included visual shape, CD45 and side scatter expression, continuity with residual granulocytes/lymphocytes/monocytes and ratio of maximum width to maximum height (w/h). The final diagnosis of ALL and AML and their subtypes was made by morphology, cytochemistry and immunophenotyping. Two sample Wilcoxon rank-sum (Mann Whitney) test and Kruskal-Wallis equality-of-populations rank tests were applied to elucidate the significance of the above ratios of blast cluster for diagnosis of ALL, AML and their subtypes. Receiver operating characteristic (ROC) curves were generated and the optimal cut-offs of the w/h ratio to distinguish between ALL and AML determined. Of the 100 cases, 57 of ALL and 43 cases of AML were diagnosed. The median w/h ratio of blast population was 3.8 for ALL and 1 for AML (P<0.001). ROC had area under curve of 0.9772.The optimal cut-off of the w/h ratio for distinction of ALL from AML was found to be 1.6. Our findings suggest that if w/h ratio on SSC versus CD45 plot is less than 1.6, AML may be considered, and if it is more than 1.6, ALL may be diagnosed. Using morphometric analysis of the blast cluster on SSC versus CD45, it was possible to distinguish between ALL and AML, and their subtypes.

  7. Neurocytometry: Flow Cytometric Sorting of Specific Neuronal Populations from Human and Rodent Brain.

    PubMed

    Martin, David; Xu, Jiaxi; Porretta, Connie; Nichols, Charles D

    2017-02-15

    Flow cytometry has the potential to facilitate understanding of the heterogeneous responses of diverse brain cell populations to a variety of stimuli. However, existing methods of applying flow cytometry to brain tissues are each limited in certain ways. They either require genetically labeled cells to achieve separation of specific populations, are not applicable to previously fixed tissue, or are not compatible with downstream mRNA analysis. Here, we describe a group of related methods that overcome many previous limitations and allow robust sorting and downstream molecular analysis of highly enriched populations of specific neuronal and non-neuronal cells from any mammalian brain. We illustrate these techniques, which are compatible with antibodies for both nuclear and non-nuclear epitopes and do not require transgenic animals, with three examples. First, we describe the separation and downstream mRNA analysis of four types of cortical interneurons (somatostatin, parvalbumin, calretinin, and calbindin) from paraformaldehyde-fixed rat brain sections. Second, we demonstrate separation of neurons and non-neurons from zinc-fixed mouse brain cortical sections followed by analysis of enzymatic activity (ACE2 activity) and mRNA expression. Third, we show that routinely fixed post-mortem human brain can be analyzed by isolating parvalbumin-containing neurons from cortical samples that were fixed for periods of up to 8 weeks in formalin. In each case, sorted cell identity was confirmed with mRNA analysis. The neurocytometry methodology described here has the potential to significantly expand studies to analyze the effects of drugs, environmental manipulations, and disease states on the nucleic acid and protein content of specific brain cell populations.

  8. Two-dimensional polyacrylamide gel electrophoresis of membrane proteins from flow cytometrically sorted ram sperm.

    PubMed

    Leahy, T; Marti, J I; Crossett, B; Evan, G; Maxwell, W M C

    2011-03-15

    Membrane proteins orchestrate key events required for participation of sperm in fertilisation. These proteins may be removed or altered due to the mechanical and dilution stressors associated with sex-sorting of sperm. Ram sperm were incubated with Hoechst 33342 and flow-sorted. Sex-selected (viable, orientated) and waste (separated into non-viable or non-orientated) sperm populations were collected, or sperm were not sorted. Sperm membrane proteins were extracted and characterised by one- and two-dimensional PAGE. Densiometric analysis of protein bands separated by one-dimensional PAGE showed proteins of 30 and 28 kDa as doublet bands on non-sorted sperm, and single bands on sex-sorted sperm, and the proportion of a 14 kDa protein was 3-fold higher in non-sorted compared to sorted sperm. Proteins in the 14 kDa band were identified by mass spectroscopy as a bovine Fibronectin type-2 protein (Fn-2), cytochrome oxidase 5a (Cox5a) and a sperm membrane associated protein (SLLP1). The abundance of these proteins in the two-dimensional gels was lowest in the sorted sperm population identified as viable during sorting (orientated and non-orientated sperm) and highest in the non-viable sperm population (P < 0.001). We concluded that the membrane protein profile was different for sex-sorted compared with non-sorted sperm, due to the selection of plasma membrane-intact cells in the flow-sorted population. This provided further evidence that sex-sorting selected a homogenous population of sperm with superior function to non-sorted sperm. Furthermore, this was apparently the first time sperm membrane acrosome associated protein was reported in ram sperm, and it was demonstrated that seminal plasma proteins remained on the sperm membrane after sex-sorting. Copyright © 2011 Elsevier Inc. All rights reserved.

  9. Practical High-Throughput Experimentation for Chemists

    PubMed Central

    2017-01-01

    Large arrays of hypothesis-driven, rationally designed experiments are powerful tools for solving complex chemical problems. Conceptual and practical aspects of chemical high-throughput experimentation are discussed. A case study in the application of high-throughput experimentation to a key synthetic step in a drug discovery program and subsequent optimization for the first large scale synthesis of a drug candidate is exemplified. PMID:28626518

  10. High-throughput optimization of surfaces for antibody immobilization using metal complexes.

    PubMed

    Muir, Ben W; Barden, Michael C; Collett, Simon P; Gorse, Alain-Dominique; Monteiro, Raisa; Yang, Liqun; McDougall, Nicole A; Gould, Sharon; Maeji, N Joe

    2007-04-01

    Using a high-throughput surface discovery approach, we have generated a 1600-member library of metal-containing surfaces and screened them for antibody binding potential. The surface library assembly involved graft modification of argon plasma-treated polyvinylidenedifluoride (PVDF) membranes with alternating maleic anhydride-styrene copolymer followed by anhydride ring opening with a range of secondary amines and microarray contact printing of transition metal complexes. The microarrays of metal-containing surfaces were then tested for their antibody binding capacity by incubation with a biotinylated mouse antibody in a chemiluminescence assay. A total of 11 leads were identified from the first screen, constituting a "hit" rate of 0.7%. A smaller 135-member surface library was then synthesized and screened to optimize existing hits and generate additional leads. To demonstrate the applicability of these surfaces to other formats, high-binding surface leads were then transferred onto Luminex beads for use in a bead flow cytometric immunoassay. The novel one-step antibody coupling process increased assay sensitivity of a Luminex tumor necrosis factor immunoassay. These high-binding surfaces do not require prior incorporation of polyhistidine tags or posttreatments such as oxidation to achieve essentially irreversible binding of immunoglobulin G.

  11. Optimal Percoll concentration facilitates flow cytometric analysis for annexin V/propidium iodine-stained ischemic brain tissues.

    PubMed

    Juan, Wei-Sheng; Lin, Hsiao-Wen; Chen, Ying-Hsin; Chen, Hung-Yi; Hung, Yu-Chang; Tai, Shih-Huang; Huang, Sheng-Yang; Chen, Tsung-Ying; Lee, E-Jian

    2012-05-01

    We sought to determine the optimal Percoll concentration for ischemic rat brain prepared for flow cytometric (FC) measurements. Animals were subjected to the right middle cerebral artery (MCA) occlusion, and were euthanized at 3, 12, 24, and 72 h after reperfusion onset. The brains were processed by different concentrations (unisolated, 20, 25, 30, or 40%) of Percoll and stained with annexin V/propidium iodine (PI). Ischemic brain damage was evaluated by FC analysis and image analysis for histologic sections. The relative susceptibility of different phenotypes of cells to necrotic and apoptotic damage were evaluated by the FC analyses for the immunohistochemistry, PI, and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)-processed brain tissues. Our results showed that FC analysis effectively detected the extent and maturation of apoptotic/necrotic brain damage, and the results were consistent with those determined from histologic brain sections. Neuron was more vulnerable to apoptosis than glia, whereas both cellular phenotypes were compatible in susceptibility for necrotic cell death. Percoll at a low concentration (20%) could effectively remove tissue debris without affecting membranous integrity of the injured neurons. Conversely, high percentages of Percoll (30-40%) substantially increased membranous damage for the injured cells. These results supported the application of FC to determine the extent and progression in time, as well as relative phenotypes of apoptotic/necrotic cell deaths following ischemic damage. We highlighted the use of Percoll at low percentages to facilitate the removal of tissue debris and to improve membrane integrity preservation for the injured neurons. Copyright © 2012 International Society for Advancement of Cytometry.

  12. Development of a flow cytometric immunoassay for recombinant bovine somatotropin-induced antibodies in serum of dairy cows.

    PubMed

    Smits, Nathalie Gabriëlle Esther; Bremer, Maria Gabriëlle Eleonore Gerarda; Ludwig, Susann Katrina Julie; Nielen, Michel Wilhelmus Franciscus

    2012-05-01

    Administration of recombinant bovine somatotropin (rbST) to enhance milk production in dairy cows is banned within the European Union. Therefore, methods for pinpointing rbST abuse are required. Due to the problematic detection of rbST itself in serum, methods are also focused on detecting changes in rbST-related biomarkers. In this study, a fast and easy-to-perform microsphere-based flow cytometric immunoassay (FCIA) for detection of rbST-induced antibodies in serum was developed. Until now, detection of rbST-induced antibodies was also problematic due to non-specific binding of serum proteins resulting in a high rate of false positive results. Therefore, five different sample preparation methods, i.e. dilution, octanoic acid precipitation, filtration, protein G purification, and a previously described generic FCIA sample preparation were critically compared to overcome non-specific binding to the microspheres. Only the generic FCIA sample pretreatment was effective in reducing non-specific binding. As a result, an absolute decision level for detecting rbST antibodies in serum of dairy cows was determined and its applicability was demonstrated. In accordance with biological expectations from literature, rbST antibodies were induced in three out of four rbST-treated dairy cows. These rbST-induced antibodies were successfully detected for up to 4 weeks after the last rbST treatment, whereas no false positive results were obtained for 27 untreated dairy cows. This is the first method, able to overcome the interference of serum proteins and therefore, can be applied with high confidence for screening unknown herds of cattle for rbST antibodies, an important biomarker for pinpointing at rbST abuse in cattle. Copyright © 2011 John Wiley & Sons, Ltd.

  13. Discriminating Active Tuberculosis from Latent Tuberculosis Infection by flow cytometric measurement of CD161-expressing T cells

    PubMed Central

    Yang, Qianting; Xu, Qian; Chen, Qi; Li, Jin; Zhang, Mingxia; Cai, Yi; Liu, Haiying; Zhou, Yiping; Deng, Guofang; Deng, Qunyi; Zhou, Boping; Kornfeld, Hardy; Chen, Xinchun

    2015-01-01

    Interferon-gamma Release Assays (IGRAs) significantly increases the possibility for early diagnosis of tuberculosis, but IGRAs alone cannot discriminate active TB from LTBI. Therefore, fast and reliable discrimination of active tuberculosis, especially bacteriology negative tuberculosis, from LTBI is a great necessity. Here we established an assay based on flow cytometric multiparameter assay assessing expression of CD161 along with CD3, CD4, and CD8, whereby a set of indices formulated by the percentages of CD3+CD161+, CD3+CD4+CD161+ and CD3+CD8+CD161+ T cells multiplied with lymphocyte/monocyte ratio were established. Application of the CD3+CD8+CD161+ index to compare a cohort of active tuberculosis with a cohort of LTBI or health control yielded 0.7662 (95% confidence interval [CI] 0.6559–0.8552) or 0.7922 (95%  CI 0.6846–0.8763) for sensitivity and 0.9048 (95%  CI 0.8209–0.9580) or 0.8939 (95% CI 0.8392–0.9349) for specificity when the TB cohort was AFB+; the corresponding results were 0.7481 (95%  CI 0.6648–0.8198) or 0.7557 (95%  CI 0.6730–0.8265) for sensitivity and 0.8571 (95%  CI 0.7637–0.9239) or 0.8603 (95%  CI 0.8008–0.9075) for specificity when the TB cohort was AFB−. Our results reveal that in combination with IGRAs, CD161-based indices provide a novel, fast diagnostic solution addressing the limitation of current tuberculosis diagnostics. PMID:26643453

  14. Antibody affinity maturation through combining display of two-chain paired antibody and precision flow cytometric sorting.

    PubMed

    Sun, Shuang; Yang, Xiao; Wang, Haifeng; Zhao, Yun; Lin, Yan; Ye, Chen; Fang, Xiangdong; Hang, Haiying

    2016-07-01

    Recombination of antibody light and heavy chain libraries greatly increases the size of a two-chain paired antibody library, thus easing the construction of large antibody libraries. Here, light and heavy chain variable domains paired by a coiled coil were applied to a bacterial inner membrane display system. However, the probability of the correct pairing of light and heavy chains through random recombination after each round of flow cytometric sorting and cloning was very low in the presence of mostly unmatched light and heavy chain genes, resulting in inefficient enrichment; a target antibody clone in the ratio of 1:100,000 negative control spheroplasts was unable to be enriched by six rounds of sorting and cloning by a conventional sorting strategy (sorting the top 1 %). By just sorting the top 0.000025 % of spheroplasts, we succeeded in enriching the target antibody clone mixed with negative control spheroplasts in a ratio of 1:10(8) by just one round of sorting and cloning. Furthermore, using this gating strategy, we efficiently enriched for an antibody clone with an affinity slightly better than the parent antibody clone from mixed spheroplasts which were present in the ratio of 1 better affinity clone to 10 parent clones to 10(6) negative control clones after just two rounds of sorting and cloning, suggesting that this gating strategy is highly sensitive in distinguishing between clones with a small difference in affinity and also enriching for clones with a higher affinity. Taken together, the combination of the display of a two-chain paired antibody library and the use of stringent gating has significantly increased the efficiency of the antibody maturation system.

  15. Relevance of Flow Cytometric Auto-Crossmatch to the Post-transplant Course of Kidney Transplant Recipients.

    PubMed

    Demir, E; Yeğit, O; Erol, A; Akgül, S U; Çalışkan, B; Bayraktar, A; Çalışkan, Y; Türkmen, A; Savran, F O; Sever, M S

    2017-04-01

    The crossmatch test is essential prior to kidney transplantation (tx) to confirm compatibility between the donor and the recipient. However, its results can be misleading due to "undetectable antibodies" in the recipient's serum. To establish if undetectable autoantibodies are responsible for a positive result, an auto-crossmatch test can be performed. In this study, we aim to determine the long-term prognostic value of auto-flow cytometric auto-crossmatch (FCXM) test on kidney survival in kidney tx recipients. The primary outcome variable was reduced renal function. Secondary endpoints were incidence of biopsy-confirmed chronic antibody-mediated rejection (CAMR) and recurrent glomerulonephritis (GN). There were no differences regarding initial serum creatinine levels between the study and control groups (P = .441). Patients who had positive auto-B FCXM had a significantly reduced renal function compared with the control group (P = .016). Four patients developed biopsy-confirmed CAMR in the study group and 1 patient in the control group (P = .047). Five patients had biopsy-confirmed recurrent GN in the GN study group, and only 1 patient had recurrent GN in the GN control group (P = .026). Kidney transplant recipients with positive auto-FCXM test had significantly reduced renal function and a higher incidence of recurrent GN and CAMR compared with the control group. The findings of this study suggest a potential role of auto-antibody causing positive auto-FCXM test result, meanwhile increasing the risk of CAMR, recurrent GN, and new-onset diabetes after tx. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Performance of the flow cytometric E-screen assay in screening estrogenicity of pure compounds and environmental samples.

    PubMed

    Vanparys, Caroline; Depiereux, Sophie; Nadzialek, Stéphanie; Robbens, Johan; Blust, Ronny; Kestemont, Patrick; De Coen, Wim

    2010-09-15

    In vitro estrogenicity screens are believed to provide a first prioritization step in hazard characterization of endocrine disrupting chemicals. When applied to complex environmental matrices or mixture samples, they have been indicated valuable in estimating the overall estrogen-mimicking load. In this study, the performance of an adapted format of the classical E-screen or MCF-7 cell proliferation assay was profoundly evaluated to rank pure compounds as well as influents and effluents of sewage treatment plants (STPs) according to estrogenic activity. In this adapted format, flow cytometric cell cycle analysis was used to allow evaluation of the MCF-7 cell proliferative effects after only 24 h of exposure. With an average EC(50) value of 2 pM and CV of 22%, this assay appears as a sensitive and reproducible system for evaluation of estrogenic activity. Moreover, estrogenic responses of 17 pure compounds corresponded well, qualitatively and quantitatively, with other in vitro and in vivo estrogenicity screens, such as the classical E-screen (R(2)=0.98), the estrogen receptor (ER) binding (R(2)=0.84) and the ER transcription activation assay (R(2)=0.87). To evaluate the applicability of this assay for complex samples, influents and effluents of 10 STPs covering different treatment processes, were compared and ranked according to estrogenic removal efficiencies. Activated sludge treatment with phosphorus and nitrogen removal appeared most effective in eliminating estrogenic activity, followed by activated sludge, lagoon and filter bed. This is well in agreement with previous findings based on chemical analysis or biological activity screens. Moreover, ER blocking experiments indicated that cell proliferative responses were mainly ER mediated, illustrating that the complexity of the end point, cell proliferation, compared to other ER screens, does not hamper the interpretation of the results. Therefore, this study, among other E-screen studies, supports the use of

  17. Comparison of quantitative flow cytometric data provided by panels with lower and increased color number

    NASA Astrophysics Data System (ADS)

    Bocsi, József; Mittag, Anja; Pierzchalski, Arkadiusz; Baumgartner, Adolf; Dähnert, Ingo; Tárnok, Attila

    2012-03-01

    To date the flow cytometry (FCM) industry is booming with new generations of commercial clinical instruments. Long-term clinical studies have the dilemma that moving to new instruments being capable of more complex cell-analysis makes it difficult to compare new data with those obtained on older instruments with less complex analysis panels. Since 15 years we conduct follow-up studies on children with congenital heart diseases. In this period we moved from 2- to 3- and now to 10-color FCM immunophenotyping panels. Questions arise how to compare and transfer data from lower to higher level of complexity. Two comparable antibody panels for leukocyte immunophenotyping (12-tube 2-colors, and 9-tube 4-colors) were measured on a BD FACScalibur FCM (calibration: Spherotech beads) in 19 blood samples from children with congenital heart disease. This increase of colors was accompanied by moving antibodies that were in the 2-color panel either FITC or PE labeled to red dyes such as PerCP or APC. Algorithms were developed for bridging data for quantitative characterization of antigen expression (mean fluorescence intensity) and frequency of different cell subpopulations in combination with rainbow bead standard data. This approach worked for the most relevant antibodies (CD3, CD4, CD8 etc.) well, but rendered substantial uncertainty for activation markers (CD69 etc.). Our techniques are particularly well suited to the analysis in long-term studies and have the potential to compare older and recent results in a standardized way.

  18. Flow cytometric and autoradiographic studies of human kidney carcinomas surgically removed after preirradiation.

    PubMed Central

    Linden, W. A.; Köllermann, M.; König, K.

    1980-01-01

    In a randomized study of human renal adenocarcinomas the tumours were either preirradiated with 25 Gy of 42 MeV X-rays or removed without pretreatment. Nephrectomy was performed 3 1/2 weeks after the end of irradiation or in the untreated series immediately after diagnosis. The impairment of cell proliferation after irradiation was determined by flow cytometry (FCM) and 3H-autoradiography after in vitro incubation. From FCM DNA distributions the fractions of cells in the phases of cell cycle were computed. The results show good agreement between the S-phase fractions determined by FCM and autoradiography. Preliminary data from 14 patients, 5 preirradiated and 9 unirradiated show that the fraction of cells in S-phase was reduced from 0.06 +/- 0.03 in the unirradiated patients to 0.01 +/- 0.01 in the irradiated ones. 12 of the 14 adenocarcinomas had diploid DNA content (2C) and 2 were hyperdiploid. The results demonstrate that FCM can supplement the techniques available for the assessment of radiation response of human tumours. PMID:7000121

  19. A flow cytometric method for estimating S-phase duration in plants

    PubMed Central

    Mickelson-Young, Leigh; Wear, Emily; Mulvaney, Patrick; Lee, Tae-Jin; Szymanski, Eric S.; Allen, George; Hanley-Bowdoin, Linda; Thompson, William

    2016-01-01

    The duration of the DNA synthesis stage (S phase) of the cell cycle is fundamental in our understanding of cell cycle kinetics, cell proliferation, and DNA replication timing programs. Most S-phase duration estimates that exist for plants are based on indirect measurements. We present a method for directly estimating S-phase duration by pulse-labeling root tips or actively dividing suspension cells with the halogenated thymidine analog 5-ethynl-2'-deoxyuridine (EdU) and analyzing the time course of replication with bivariate flow cytometry. The transition between G1 and G2 DNA contents can be followed by measuring the mean DNA content of EdU-labeled S-phase nuclei as a function of time after the labeling pulse. We applied this technique to intact root tips of maize (Zea mays L.), rice (Oryza sativa L.), barley (Hordeum vulgare L.), and wheat (Triticum aestivum L.), and to actively dividing cell cultures of Arabidopsis (Arabidopsis thaliana (L.) Heynh.) and rice. Estimates of S-phase duration in root tips were remarkably consistent, varying only by ~3-fold, although the genome sizes of the species analyzed varied >40-fold. PMID:27697785

  20. Usefulness of Flow Cytometric Analysis for Detecting Leptomeningeal Diseases in Non-Hodgkin Lymphoma

    PubMed Central

    Shin, Sang-Yong; Kim, Hee-Jin; Oh, Young Lyun; Kim, Seok Jin; Kim, Won Seog

    2016-01-01

    Background The clinical usefulness of flow cytometry (FCM) for the diagnosis of leptomeningeal diseases (LMD) in non-Hodgkin lymphomas has been suggested in previous studies but needs to be further validated. With this regards, we evaluated the use of FCM for LMD in a series of Korean patients with non-Hodgkin lymphoma. Methods FCM and cytomorphology were conducted using samples obtained from clinically suspected LMD patients, follow-up LMD patients, and those with high risk of developing tumorigenic diseases. We then compared results of FCM and cytomorphology. In total, 55 and 47 CSF samples were analyzed by FCM and cytomorphology, respectively. Results Of the samples analyzed, 25.5% (14/55) and 12.8% (6/47) were positive by FCM and cytomorphology, respectively. No samples were determined as negative by FCM but positive by cytomorphology. Seven patients were positive only by FCM and negative by cytomorphology, and six among them were clinically confirmed to have LMD either by follow-up cytomorphology or imaging study. Conclusions We observed a high detection rate of tumor cells by FCM compared with cytomorphology. FCM study can be useful in early sensitive detection of LMD. PMID:26915608

  1. Establishing the flow cytometric assessment of myeloid cells in kidney ischemia/reperfusion injury.

    PubMed

    Williams, Timothy M; Wise, Andrea F; Alikhan, Maliha A; Layton, Daniel S; Ricardo, Sharon D

    2014-03-01

    Polychromatic flow cytometry is a powerful tool for assessing populations of cells in the kidney through times of homeostasis, disease and tissue remodeling. In particular, macrophages have been identified as having central roles in these three settings. However, because of the plasticity of myeloid cells it has been difficult to define a specific immunophenotype for these cells in the kidney. This study developed a gating strategy for identifying and assessing monocyte and macrophage subpopulations, along with neutrophils and epithelial cells in the healthy kidney and following ischemia/reperfusion (IR) injury in mice, using antibodies against CD45, CD11b, CD11c, Ly6C, Ly6G, F4/80, CSF-1R (CD115), MHC class II, mannose receptor (MR or CD206), an alternatively activated macrophage marker, and the epithelial cell adhesion marker (EpCAM or CD326). Backgating analysis and assessment of autofluorescence was used to extend the knowledge of various cell types and the changes that occur in the kidney at various time-points post-IR injury. In addition, the impact of enzymatic digestion of kidneys on cell surface markers and cell viability was assessed. Comparisons of kidney myeloid populations were also made with those in the spleen. These results provide a useful reference for future analyses of therapies aimed at modulating inflammation and enhancing endogenous remodeling following kidney injury.

  2. Development of a novel flow cytometric approach to evaluate fish sperm chromatin using fixed samples

    USGS Publications Warehouse

    Jenkins, Jill A.

    2013-01-01

    The integrity of the paternal DNA is essential for the accurate transmission of genetic information, yet fertilization is not inhibited by chromatin breakage. Some methods are available for the sensitive detection of DNA damage and can be applied in studies of environmental toxicology, carcinogenesis, aging, and assisted reproduction techniques in both clinical and experimental settings. Because semen samples obtained from remote locations undergo chromatin damage prior to laboratory assessment, the present study was undertaken to evaluate treatments for effective chromatin staining in the development of a DNA fragmentation assay using fixed milt from yellow perch (Perca flavescens). Similar to the sperm chromatin structure assay (SCSA), susceptibility of nuclear DNA to acid-induced denaturation was measured by flow cytometry (FCM). Use of 10% buffered formalin for milt fixation allowed easier peak discrimination than 4% paraformaldehyde. The effects of time and temperature of incubation in 0.08 N HCl were evaluated in order to determine the ideal conditions for promoting DNA decondensation and making strand breaks more available for staining and detection by FCM. The best results were obtained with incubation at 37°C for 1 minute, followed by cold propidium iodide staining for 30 minutes.

  3. Flow Cytometric Analysis of Mononuclear Phagocytes in Nondiseased Human Lung and Lung-Draining Lymph Nodes

    PubMed Central

    Desch, A. Nicole; Gibbings, Sophie L.; Goyal, Rajni; Kolde, Raivo; Bednarek, Joe; Bruno, Tullia; Slansky, Jill E.; Jacobelli, Jordan; Mason, Robert; Ito, Yoko; Messier, Elise; Randolph, Gwendalyn J.; Prabagar, Miglena; Atif, Shaikh M.; Segura, Elodie; Xavier, Ramnik J.; Bratton, Donna L.; Janssen, William J.; Henson, Peter M.

    2016-01-01

    Rationale: The pulmonary mononuclear phagocyte system is a critical host defense mechanism composed of macrophages, monocytes, monocyte-derived cells, and dendritic cells. However, our current characterization of these cells is limited because it is derived largely from animal studies and analysis of human mononuclear phagocytes from blood and small tissue resections around tumors. Objectives: Phenotypic and morphologic characterization of mononuclear phagocytes that potentially access inhaled antigens in human lungs. Methods: We acquired and analyzed pulmonary mononuclear phagocytes from fully intact nondiseased human lungs (including the major blood vessels and draining lymph nodes) obtained en bloc from 72 individual donors. Differential labeling of hematopoietic cells via intrabronchial and intravenous administration of antibodies within the same lobe was used to identify extravascular tissue-resident mononuclear phagocytes and exclude cells within the vascular lumen. Multiparameter flow cytometry was used to identify mononuclear phagocyte populations among cells labeled by each route of antibody delivery. Measurements and Main Results: We performed a phenotypic analysis of pulmonary mononuclear phagocytes isolated from whole nondiseased human lungs and lung-draining lymph nodes. Five pulmonary mononuclear phagocytes were observed, including macrophages, monocyte-derived cells, and dendritic cells that were phenotypically distinct from cell populations found in blood. Conclusions: Different mononuclear phagocytes, particularly dendritic cells, were labeled by intravascular and intrabronchial antibody delivery, countering the notion that tissue and blood mononuclear phagocytes are equivalent systems. Phenotypic descriptions of the mononuclear phagocytes in nondiseased lungs provide a precedent for comparative studies in diseased lungs and potential targets for therapeutics. PMID:26551758

  4. Multiplex flow cytometric immunoassay for serum biomarker profiling of recombinant bovine somatotropin.

    PubMed

    Smits, Nathalie Gabriëlle Esther; Ludwig, Susann Katrina Julie; Van der Veer, Grisha; Bremer, Maria Gabriëlle Eleonore Gerarda; Nielen, Michel Wilhelmus Franciscus

    2013-01-07

    Recombinant bovine somatotropin (rbST) is licensed for enhancing milk production in dairy cows in some countries, for instance the United States, but is banned in Europe. Serum biomarker profiling can be an adequate approach to discriminate between treated and untreated groups. In this study a multiplex screening tool of a small set of biomarkers for pinpointing recombinant bovine somatotropin (rbST) (ab)use was developed and evaluated: insulin-like growth factor 1 (IGF-1), IGF binding protein 2 (IGFBP2) and rbST-induced antibodies were selected as rbST dependent markers and combined in one parallel assay format. For this, the color-encoded microspheres were used in a suspension array, with a dedicated flow cytometer. Serum samples obtained from an animal experiment with rbST-treated and untreated dairy cows were measured with the developed triplex immunoassay and biomarker responses on rbST treatment were evaluated. This resulted in characteristic treatment-dependent responses for all three individual biomarkers. Combining these results with the statistical prediction model k-nearest neighbours (kNN), resulted in good discrimination of treated and untreated animals: an overall sensitivity (true positive rate) of 89.1% and an overall specificity (true negative rate) of 97.7% were reached. Therefore, this is the first multiplex method which can be applied with high confidence for screening of unknown herds of cattle pinpointing at rbST (ab)use.

  5. Flow cytometric measurement of ABO antibodies in ABO-incompatible living donor kidney transplantation.

    PubMed

    Yung, Gisella Puga; Valli, Piero V; Starke, Astrid; Mueller, Regula J; Fehr, Thomas; Cesar-Ozpamir, Marija; Schanz, Urs; Weber, Markus; Wüthrich, Rudolf P; Seebach, Jörg D; Stussi, Georg

    2007-12-27

    Due to different detection methods, a comparison of anti-A/B antibody (Ab) levels among transplantation centers after living donor ABO-incompatible kidney transplantation is problematic. In the present study, anti-A/B Ab levels were determined prior to, and after, blood group A-to-O kidney transplantation using a recently established semiquantitative flow cytometry-based method, ABO fluorescence-activated cell sorting (ABO-FACS), and compared with standard agglutination titers and indirect antiglobulin testing. Pretransplant agglutination titers were reduced from 1:64 to 1:4, by a total of 14 Glycosorb A column immunoadsorptions (IADSs). Compared with the agglutination titers, antidonor immunoglobulin (Ig) M ABO-FACS mean fluorescence intensity ratios (MFIRs) decreased faster and remained low. No difference was observed using donor type or third-party A red blood cells (RBCs) for the ABO-FACS. Glycosorb A columns were not specific, also reducing anti-B and antiporcine IgM levels, which was confirmed by detecting anti-A/B and antiporcine Abs in the column eluates. In conclusion, analysis of pre- and posttransplant Abs from ABO-incompatible kidney transplant recipients by ABO-FACS allows a better understanding of Ab kinetics, which may improve the design of future IADS protocols.

  6. Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

    PubMed Central

    Boltze, Johannes; Wagner, Daniel-Christoph; Weise, Gesa

    2016-01-01

    Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed. Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair. PMID:26967380

  7. Flow cytometric analysis of red-eared slider turtles (Trachemys scripta) from Tar Creek Superfund Site.

    PubMed

    Hays, Kimberly A; McBee, Karen

    2007-05-01

    Tar Creek Superfund Site (TCSFS) was heavily mined from the 1890s to 1970 and currently is contaminated with lead, zinc, and cadmium. Flow cytometry (FCM) was used to measure variation in nuclear DNA content of red blood cells collected from Trachemys scripta living within TCSFS and reference sites, Lake Carl Blackwell (LCB) and Sequoyah National Wildlife Refuge (SNWR). We also used atomic absorption spectrometry to measure Pb in blood and carapace and Cd in blood samples of turtles from TCSFS and SNWR. Mean coefficients of variation around the G(1) peak ranged from 5.33 to 5.48 and showed no significant difference between contaminated and reference populations; however, there was a significantly higher frequency of aneuploidy at TCSFS when compared with both reference populations. Blood Pb levels were not significantly different between TCSFS and SNWR populations. Pb levels in carapace samples did not differ significantly between sites; however, Pb levels were higher in carapace than blood for both populations. Blood Cd was significantly higher in animals at TCSFS than SNWR.

  8. Dormancy in Stationary-Phase Cultures of Micrococcus luteus: Flow Cytometric Analysis of Starvation and Resuscitation

    PubMed Central

    Kaprelyants, Arseny S.; Kell, Douglas B.

    1993-01-01

    Cultures of the copiotrophic bacterium Micrococcus luteus were stored in spent growth medium for an extended period of time following batch culture. After an initial decrease, the total cell counts remained constant at approximately 60 to 70% of the counts at the beginning of storage. The level of viability, as judged by plate counts, decreased to less than 0.05%, while respiration and the ability to accumulate the lipophilic cation rhodamine 123 decreased to undetectable levels. However, using penicillin pretreatment (to remove viable cells) and flow cytometry and by monitoring both the total and viable counts, we found that at least 50% of the cells in populations of 75-day-old cultures were not dead but were dormant. Resuscitation in liquid medium was accompanied by the appearance of a population of larger cells, which could accumulate rhodamine 123 and reduce the dye 5-cyano-2,3-ditolyl tetrazolium chloride to a fluorescent formazan, while a similar fraction of the population was converted to colony-forming, viable cells. We surmise that dormancy may be far more common than death in starving microbial cultures. PMID:16349059

  9. Flow cytometric leukocyte population patterns in brown bullhead from three Ohio rivers

    SciTech Connect

    Torsella, T.A.; Neiheisel, T.; Cormier, S.M.; Bercz, P.

    1994-12-31

    Brown bullhead (A. nebulosus) were collected from three Ohio rivers: Old Woman Creek (OWC), classified as clean, Cuyahoga (CR) and Black Rivers (BR), documented as polluted. Each river was sampled in April and September 1993. Patterns of leukocyte distribution (prepared via density gradient) by GSH content and oxidative burst capacity, using fluorescent probes were determined by flow cytometry. Sample processing was in blind coded batches. Three principal classes of leukocytes were identified. Type A; large, very granular with high GSH reserves and marked capacity for oxidative burst. Type B were smaller, less granular, contained low GSH and negligible oxidative burst capacity. Type C were small, less granular, possessing intermediate GSH and peroxidative activity. ANOVA of the subset distributions and mean fluorescent intensities by sex, site and season, disclosed: Type C were significantly (p < 0.001) elevated in the spring OWC males, whereas type A dominated in the spring CR and spring BR males. No differences in Type A/Type C patterns were seen in the spring females. In the fall sampling, significant dominance (p < 0.001) of Type A was seen in both sexes of the OWC fish, the CR and BR fish showed a predominance of Type C. These may be explained by chemical stressors, affecting immune competence. Sex differences in the spring were attributed to hormonal (spawning) influences.

  10. Improved sensitivity in flow cytometric intracellular ionized calcium measurement using fluo-3/Fura Red fluorescence ratios.

    PubMed

    Novak, E J; Rabinovitch, P S

    1994-10-01

    Measurement of changes in intracellular ionized calcium concentrations ([Ca2+]i) has proved to be of wide use in the study of cellular responses to activating stimuli. The fluorescent dye Indo-1 has successfully been used in flow cytometry for this purpose, and when used as a ratiometric indicator it provides optimum sensitivity and accuracy. Unfortunately, this dye requires ultraviolet (UV) excitation which is often not available. We show here that similar results can be obtained using a ratio of green to red fluorescence from the simultaneous loading of the dyes Fura Red and fluo-3. Both Fura Red and fluo-3 are excited using the commonly available blue 488 nm laser line. With appropriate concentrations of the two dyes, the magnitude of response with the fluo-3/Fura Red ratio is greater than that achieved with indo-1, while the intercellular variation in measurement is similar to that seen with indo-1. Analyses can be simultaneously combined with immunofluorescent detection of PE-labeled antibodies to enable [Ca2+]i measurement within cell subsets.

  11. Correlation of prognosis to nuclear roundness and to flow cytometric light scatter.

    PubMed

    Mohler, J L; Partin, A W; Coffey, D S

    1987-05-01

    The application of quantitative pathology to the study of malignant tumors is discussed, with emphasis on the correlation between nuclear roundness and prognosis in prostatic carcinoma. Stage A2 prostatic carcinoma patients with 4-year follow-ups and surgically treated stage B1 and B2 prostatic carcinoma patients with 15-year follow-ups were characterized by manual digitization of cancerous nuclei. A form factor that assessed nuclear shape produced an accurate separation of those patients who developed metastatic disease from those who did not. The digitized nuclear roundness correlated with the perpendicular light scatter measured by flow cytometry. When combined with forward light scatter, perpendicular light scatter separated four tumor cell lines of different metastatic potential in an animal model of prostatic cancer; a relative grading index based on these findings seemed capable of identifying human patients with more extensive disease in a preliminary study. The observation of freshly aspirated viable prostatic carcinoma cells led us to appreciate membrane ruffling, pseudopodal extension and translational movements that appear to distinguish these cells from their benign counterparts. Time-lapse cinematography, image digitization and Fourier analysis allow for the objective description of cell motion and should provide new and exciting tools for the pathologist.

  12. Effects of 50 Hz magnetic fields on mouse spermatogenesis monitored by flow cytometric analysis

    SciTech Connect

    Vita, R. de; Cavallo, D.; Raganella, L.; Eleuteri, P.; Grollino, M.G.; Calugi, A.

    1995-12-01

    Flow cytometry (FCM) was performed to monitor the cellular effects of extremely-low-frequency magnetic field on mouse spermatogenesis. Groups of five male hybrid F1 mice aged 8--10 weeks were exposed to 50 Hz magnetic field. The strength of the magnetic field was 1.7 mT. Exposure times of 2 and 4 h were chosen. FCM measurements were performed 7, 14, 21, 28, 35, and 42 days after treatment. For each experimental point, a sham-treated group was used as a control. The possible effects were studied by analyzing the DNA content distribution of the different cell types involved in spermatogenesis and using the elongated spermatids as the reference population. The relative frequencies of the various testicular cell types were calculated using specific software. In groups exposed for 2 h, no effects were observed. In groups exposed for 4 h, a statistically significant (P < 0.001) decrease in elongated spermatids was observed at 28 days after treatment. This change suggests a possible cytotoxic and/or cytostatic effect on differentiating spermatogonia. However, further studies are being carried out to investigate the effects of longer exposure times.

  13. Flow cytometric analysis of genome size variation in some Passiflora species.

    PubMed

    Souza, Margarete Magalhães; Palomino, Guadalupe; Pereira, Telma Nair Santana; Pereira, Messias Gonzaga; Viana, Alexandre Pio

    2004-01-01

    Nuclear genome size variation was studied in eight taxa of Passiflora. Nuclear DNA content was estimated by flow cytometry of nuclei stained by propidium iodide. 2C DNA content ranged from 3.16-5.36 pg for diploids and 1.83 pg for tetraploid. Differences in nuclear genome size were observed among Passiflora species (pg): P. suberosa 1.83, P. edulis f. edulis 3.16, P. edulis f. flavicarpa (Brazil) 3.19, P. edulis f. flavicarpa (Mexico) 3.21, P. mucronata 3.40, Passiflora edmundoi 3.43, P. laurifolia 3.88, P. giberti 3.92, P. quadrangularis 5.36, the largest value being up to 192% greater than the smallest. The means of 2C DNA content were compared by the Tukey test, and the differences in genome size permitted the recognition of five taxa groups. The result was the same for the means 2C genome size (Mbp) values. The genetic parameters were studied with their respective estimators, phenotypic variance (sigma2F), genotypic variability (PhiG), and the genotypic determination index (H2). The genotypic determination index presented high magnitude estimates (greater than 99%) emphasizing the reliability of the results and demonstrating the efficiency of determining the DNA content in the species using only one leaf per plant. Passiflora species show great phenotypic variability and have different geographic distribution that might implicate in genetic diversity.

  14. Monitoring microbiological changes in drinking water systems using a fast and reproducible flow cytometric method.

    PubMed

    Prest, E I; Hammes, F; Kötzsch, S; van Loosdrecht, M C M; Vrouwenvelder, J S

    2013-12-01

    Flow cytometry (FCM) is a rapid, cultivation-independent tool to assess and evaluate bacteriological quality and biological stability of water. Here we demonstrate that a stringent, reproducible staining protocol combined with fixed FCM operational and gating settings is essential for reliable quantification of bacteria and detection of changes in aquatic bacterial communities. Triplicate measurements of diverse water samples with this protocol typically showed relative standard deviation values and 95% confidence interval values below 2.5% on all the main FCM parameters. We propose a straightforward and instrument-independent method for the characterization of water samples based on the combination of bacterial cell concentration and fluorescence distribution. Analysis of the fluorescence distribution (or so-called fluorescence fingerprint) was accomplished firstly through a direct comparison of the raw FCM data and subsequently simplified by quantifying the percentage of large and brightly fluorescent high nucleic acid (HNA) content bacteria in each sample. Our approach enables fast differentiation of dissimilar bacterial communities (less than 15 min from sampling to final result), and allows accurate detection of even small changes in aquatic environments (detection above 3% change). Demonstrative studies on (a) indigenous bacterial growth in water, (b) contamination of drinking water with wastewater, (c) household drinking water stagnation and (d) mixing of two drinking water types, univocally showed that this FCM approach enables detection and quantification of relevant bacterial water quality changes with high sensitivity. This approach has the potential to be used as a new tool for application in the drinking water field, e.g. for rapid screening of the microbial water quality and stability during water treatment and distribution in networks and premise plumbing. Copyright © 2013 Elsevier Ltd. All rights reserved.

  15. Flow cytometric analysis of CD123 is useful for immunophenotyping classical Hodgkin lymphoma.

    PubMed

    Fromm, Jonathan R

    2011-03-01

    Although the diagnosis of classical Hodgkin lymphoma (CHL) is traditionally made morphologically in tissue sections, it is now possible to diagnose CHL by flow cytometry (FC). In an effort to identify additional antigens on Hodgkin and Reed-Sternberg (HRS) cells that might be useful for immunophenotyping this lymphoma by FC, we examined the expression of CD123 (α chain of the IL-3R) on HRS cells and compared this with the expression of CD123 in other lymph node samples (372 tissue specimens, including 98 reactive and 274 neoplastic cases), using a nine-color FC reagent combination including anti-CD123. CD123 was found to be expressed on the majority of HRS cell populations (59% of 59 CHL lymph nodes examined), rarely on B-cell non-Hodgkin lymphomas (NHLs) (3 of 3 hairy cell leukemia cases, 3 of 29 chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) cases, 1 of 55 large B-cell lymphoma cases, but not on 95 other B-cell NHL examined), and in none of the 16 T-cell NHL cases examined. In reactive lymph nodes, CD123 expression was largely limited to plasmacytoid dendritic cells and rare histiocyte populations that can readily be distinguished from HRS populations. As the expression of this antigen is shown to be relatively limited in B- and T-NHL and in reactive lymph node populations, assessment of CD123 expression is useful for supporting the FC diagnosis of CHL. Copyright © 2010 International Clinical Cytometry Society.

  16. Flow cytometric analysis of the cell cycle: mathematical modeling and biological interpretation.

    PubMed

    Pierrez, J; Ronot, X

    1992-09-01

    Estimation of the repartition of asynchronous cells in the cell cycle can be explained by two hypotheses: the cells are supposed to be distributed into three groups: cells with a 2c DNA content (G0/1 phase), cells with a 4c DNA content (G2 + M phase) and cells with a DNA content ranging from 2c to 4c (S phase); there is a linear relationship between the amount of fluorescence emitted by the fluorescent probe which reveals the DNA and the DNA content. According to these hypotheses, the cell cycle can be represented by the following equation: [formula: see text] All the solutions for this equation are approximations. Non parametric methods (or graphical methods: rectangle, peak reflect) only use one or two phase(s) of the cell cycle, the remaining phase(s) being estimated by exclusion. In parametric methods (Dean & Jett, Baisch II, Fried), the DNAT(x) distribution is supposed to be known and is composed of two gaussians (representative of G0/1 and G2 + M) and a P(x,y) function representative of S phase. Despite the generality, these models are not applicable to all sample types, particularly heterogeneous cell populations with various DNA content. In addition, the cell cycle is dependent on several regulation points (transition from quiescence to proliferation, DNA synthesis initiation, mitosis induction) and biological perturbations can also lead to cytokinesis perturbations. Before the emergence of flow cytometry, the current view of cell cycle resided in the assessment of cell proliferation (increase in cell number) or the kinetic of molecules incorporation (DNA precursors).(ABSTRACT TRUNCATED AT 250 WORDS)

  17. Flow cytometric analysis of somaclonal variation in lineages of Hosta sports detects polyploidy and aneuploidy chimeras.

    PubMed

    Zonneveld, B J M; Pollock, W I

    2012-11-01

    Somaclonal variation of some 124 specially selected cultivars of Hosta Tratt. (Hostaceae) was investigated. Nuclear DNA contents (2C-value) were measured by flow cytometry of leaves and roots of L1, L2 and L3 layers derived from apical meristems. These values were then converted to inferred ploidies by comparing the measured 2C-values and ploidy with those of the parent plant. During tissue-culture propagation, on occasion diploid (L1-L2-L3 = 2-2-2) hostas give rise to polyploids, such as fully tetraploids (4-4-4), and periclinal chimeras, such as partial tetraploids (4-2-2). Continual propagation can result in partial tetraploids becoming full tetraploids. Nuclear DNA of some diploids increased with incomplete chromosome sets resulting in fully aneuploids, such as hostas with a DNA ploidy of L1-L2-L3 = 2.5-2.5-2.5 and 3.7-3.7-3.7, and even in aneuploid periclinal chimeras, such as L1-L2-L3 = 2.5-2-2 and 3.8-2-2. The polyploidy of L1, irrespective of the ploidy of L2 and L3, is found to mainly determine the thickness of leaves. Also the higher the ploidy of L1, the wider and more intense in color is the leaf margin. The measurements of Hosta cultivars and their lineages of sports show that chromosome losses or gains are an important source of new cultivars. The complexity of chromosomal distribution in lineages of several Hosta cultivars is discussed. © 2012 German Botanical Society and The Royal Botanical Society of the Netherlands.

  18. Flow cytometric assessment of Cd genotoxicity in three plants with different metal accumulation and detoxification capacities.

    PubMed

    Monteiro, M S; Rodriguez, E; Loureiro, J; Mann, R M; Soares, A M V M; Santos, C

    2010-09-01

    Cadmium (Cd) is a widespread environmental contaminant, strongly mutagenic and known to cause DNA damage in plants. In this work, flow cytometry (FCM) was applied to determine if in vivo exposure to Cd would induce genotoxic effects at the genome level. The hyper-accumulator Thlaspi caerulescens (J. & C. Presl), the related non-accumulator Thlaspi arvense L. and the accumulator crop species Lactuca sativa L. were germinated in distilled water and grown in modified Hoagland's medium with increasing concentrations of Cd(NO3)2 (0, 1, 10 and 100 microM). After 28 days of exposure, shoot and root growth was recorded and the tissues were harvested for Cd and FCM analysis. In general, roots from treated plants contained higher content of Cd than leaves and growth inhibition was observed in the treated plants. Nuclear DNA content was estimated and the G0/G1 full peak coefficient of variation (FPCV), as an indicator of clastogenic damage, was recorded. In T. arvense and T. caerulescens no significant differences were detected between control and exposed plants. Leaves of L. sativa exposed to 10 microM Cd presented a statistically significant increase in FPCV values in comparison with the control group. Furthermore, roots exposed to 100 microM Cd presented a reduction in nuclear DNA content and an increase in FPCV when compared to the control. FCM data indicates that no major DNA damage was induced on both Cd-exposed Thlaspi species and L. sativa leaves. On the contrary, results obtained with L. sativa roots suggests clastogenic damage in these organs exposed to 100 microM of Cd. Copyright (c) 2010 Elsevier Inc. All rights reserved.

  19. Flow cytometric studies of bicarbonate-mediated Ca2+ influx in boar sperm populations.

    PubMed

    Harrison, R A; Mairet, B; Miller, N G

    1993-06-01

    Boar spermatozoa loaded with the Ca2+ probe fluo-3 were incubated in various Tyrode's-based media similar to those used for in vitro fertilization (IVF), and samples were then analysed by two-colour flow cytometry; propidium iodide was included in the media to detect membrane-damaged ("dead") cells. If media contained bicarbonate/CO2 (a component thought to promote capacitation), part of the live sperm population experienced a considerable influx of Ca2+ into both head and tail compartments. The percentage of responding cells reached a maximum after about 30 min, but both during and after this period there was also a steady increase in the number of dead cells. This bicarbonate-mediated increase in cell death took place in the absence of external Ca2+. Evidence was obtained that the entry of propidium iodide was preceded by a change in permeability of the plasma membrane, detectable by leakage of carboxydichlorofluorescein, and it was therefore deduced that the Ca2+ influx detected by fluo-3 was due to destabilization of the plasma membrane. A similar response could be produced by both caffeine and papaverine (best known as phosphodiesterase inhibitors), but neither cyclic AMP nor activators of adenylate cyclase had any effect. There was no influence of substrate on the process, but, in comparison to poly(vinyl alcohol), serum albumin enhanced it. The precise relevance of this destabilization to capacitation is not yet clear, but it seems significant that the process is mediated or enhanced by components often specifically included in IVF media, and that different individual cells respond after different times.

  20. Flow cytometric analysis of gut mucosal lymphocytes supports an impaired Th1 cytokine profile in spondyloarthropathy

    PubMed Central

    Van Damme, N; De Vos, M; Baeten, D; Demetter, P; Mielants, H; Verbruggen, G; Cuvelier, C; Veys, E; De Keyser, F

    2001-01-01

    OBJECTIVE—To quantify the fraction of gut mucosal lymphocytes expressing the T helper type 1 (Th1) cytokines, interferon γ (IFNγ) and interleukin (IL)2, and the Th2 cytokines, IL4 and IL10, at the single cell level in patients with spondyloarthropathy (SpA) in comparison with healthy controls.
METHODS—An improved extraction protocol was used for the enrichment of intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs) from colonic and ileal biopsy specimens obtained from patients with SpA (n=20) and healthy controls (n=13). After stimulation with phorbol ester/ionomycin, expression of the intracellular cytokines IFNγ, IL2, IL4, and IL10 was determined in CD3+, CD3+CD8+ and CD3+CD8− T cells by flow cytometry.
RESULTS—In colonic LPLs, a significant decrease in IFNγ-producing CD3+ cells was observed (p=0.02) in patients with SpA. In the CD3+CD8− subset, the proportion of cells producing IFNγ and IL2 was decreased in patients with SpA (p=0.021 and p=0.027 respectively). In ileal LPLs, the percentage of IL10-producing CD3+CD8− cells was significantly increased (p=0.046).
CONCLUSION—An impaired Th1 cytokine profile is observed in gut mucosal lymphocytes from patients with SpA. This adds to the existing evidence that the gut mucosal immune apparatus is involved in the pathogenesis of SpA.

 PMID:11302872

  1. Flow-cytometric study of vital cellular functions in Escherichia coli during solar disinfection (SODIS).

    PubMed

    Berney, Michael; Weilenmann, Hans-Ulrich; Egli, Thomas

    2006-06-01

    The effectiveness of solar disinfection (SODIS), a low-cost household water treatment method for developing countries, was investigated with flow cytometry and viability stains for the enteric bacterium Escherichia coli. A better understanding of the process of injury or death of E. coli during SODIS could be gained by investigating six different cellular functions, namely: efflux pump activity (Syto 9 plus ethidium bromide), membrane potential [bis-(1,3-dibutylbarbituric acid)trimethine oxonol; DiBAC4(3)], membrane integrity (LIVE/DEAD BacLight), glucose uptake activity (2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose; 2-NBDG), total ATP concentration (BacTiter-Glo) and culturability (pour-plate method). These variables were measured in E. coli K-12 MG1655 cells that were exposed to either sunlight or artificial UVA light. The inactivation pattern of cellular functions was very similar for both light sources. A UVA light dose (fluence) of <500 kJ m(-2) was enough to lower the proton motive force, such that efflux pump activity and ATP synthesis decreased significantly. The loss of membrane potential, glucose uptake activity and culturability of >80 % of the cells was observed at a fluence of approximately 1500 kJ m(-2), and the cytoplasmic membrane of bacterial cells became permeable at a fluence of >2500 kJ m(-2). Culturable counts of stressed bacteria after anaerobic incubation on sodium pyruvate-supplemented tryptic soy agar closely correlated with the loss of membrane potential. The results strongly suggest that cells exposed to >1500 kJ m(-2) solar UVA (corresponding to 530 W m(-2) global sunlight intensity for 6 h) were no longer able to repair the damage and recover. Our study confirms the lethal effect of SODIS with cultivation-independent methods and gives a detailed picture of the 'agony' of E. coli when it is stressed with sunlight.

  2. Flow cytometric functional analysis of multidrug resistance by Fluo-3: a comparison with rhodamine-123.

    PubMed

    Koizumi, S; Konishi, M; Ichihara, T; Wada, H; Matsukawa, H; Goi, K; Mizutani, S

    1995-09-01

    Using four cell lines including drug-sensitive K562/Parent cells, P-glycoprotein (Pgp)-mediated multidrug resistant (MDR) K562/VCR, K562/ADR and revertant K562/ADR-R cells, two fluorescent agents, Fluo-3 and rhodamine-123 (Rh-123), were compared as indicators in a functional assay of MDR. Cells were incubated with 4 microM Fluo-3 or 1 microM Rh-123 for 45 min and then the intracellular accumulation of the agent was measured using a flow cytometer. Verapamil (20 microM) or cepharanthine (biscoclaurine alkaloid, 10 microM) was added just before the fluorescent agents. Efflux patterns were also studied 60 min after incubation with or without verapamil and cepharanthine. Increased intracellular accumulation and a delayed efflux pattern of Fluo-3 by verapamil and cepharanthine were demonstrated in multidrug resistant K562/VCR and K562/ADR cells, indicating that Fluo-3 is another good indicator of MDR. However, a similar, but lower, increase in uptake and a delayed efflux pattern of Fluo-3 by verapamil and cepharanthine were also demonstrated even in Pgp-non-overexpressed K562/Parent cells. In contrast, accumulation of Rh-123 was not affected by verapamil and cepharanthine. To further study the Pgp dependency of Fluo-3, another cell line, K562/NC16 expressing minimum MDR1 mRNA, was cloned. Increased uptake and a delayed efflux pattern of Fluo-3, but not Rh-123, with verapamil or cepharanthine were again demonstrated in K562/NC16 cells, indicating that intracellular accumulation of Fluo-3 may be non-specifically influenced by verapamil and cepharanthine at very low levels of Pgp-related MDR, while the influx and efflux patterns of Rh-123 may be specifically affected by Pgp overexpression.

  3. Functional defects in phagocytic cells following thermal injury. Application of flow cytometric analysis.

    PubMed Central

    Duque, R. E.; Phan, S. H.; Hudson, J. L.; Till, G. O.; Ward, P. A.

    1985-01-01

    Defective phagocytic cell function may partially account for the morbidity and mortality associated with thermal injury. In experimental thermal injury in the rat, small circulating blood volumes increase the difficulty in obtaining significant data. Furthermore, purification and or elicitation procedures have the potential for altering the cell surface characteristics and/or the functional response of the cell in question. We have examined the circulating neutrophils and pulmonary alveolar macrophages of anesthetized rats following a 16-20% body surface area scald injury to the shaved back. The circulating neutrophils of thermally injured rats were examined by flow cytometry following stimulation with phorbol myristate acetate (PMA) (100 ng/ml) in terms of the change in fluorescence intensity of the potentiometric cyanine dye, dipentyloxocarboxyanine and the formation of the oxidized product of 2',7'-dichlorofluorescin diacetate-loaded cells. The alveolar macrophages were examined after stimulation with PMA (100 ng/ml) in terms of the change in fluorescence intensity of the potentiometric dye, dipropylthiodicarbocyanine and the generation of superoxide production, as assessed by the superoxide dismutase inhibitable reduction of cytochrome c. Both cells exhibited a profound inhibition of cell function 4 hours after the insult, with partial return toward control values at later time points. Furthermore, the plasma of thermally injured rats, 4 hours after the burn was inhibitory to normal rat neutrophils. Fluorescent compounds suggestive of in vivo lipid peroxidation were maximally detectable at this time point. Further research is needed to establish the role of these products in the induction of phagocytic cell dysfunction. PMID:2981471

  4. Flow cytometric sorting of fecal bacteria after in situ hybridization with polynucleotide probes.

    PubMed

    Bruder, Lena M; Dörkes, Marcel; Fuchs, Bernhard M; Ludwig, Wolfgang; Liebl, Wolfgang

    2016-10-01

    The gut microbiome represents a key contributor to human physiology, metabolism, immune function, and nutrition. Elucidating the composition and genetics of the gut microbiota under various conditions is essential to understand how microbes function individually and as a community. Metagenomic analyses are increasingly used to study intestinal microbiota. However, for certain scientific questions it is sufficient to examine taxon-specific submetagenomes, covering selected bacterial genera in a targeted manner. Here we established a new variant of fluorescence in situ hybridization (FISH) combined with fluorescence-activated cell sorting (FACS), providing access to the genomes of specific taxa belonging to the complex community of the intestinal microbiota. In contrast to standard oligonucleotide probes, the RNA polynucleotide probe used here, which targets domain III of the 23S rRNA gene, extends the resolution power in environmental samples by increasing signal intensity. Furthermore, cells hybridized with the polynucleotide probe are not subjected to harsh pretreatments, and their genetic information remains intact. The protocol described here was tested on genus-specifically labeled cells in various samples, including complex fecal samples from different laboratory mouse types that harbor diverse intestinal microbiota. Specifically, as an example for the protocol described here, RNA polynucleotide probes could be used to label Enterococcus cells for subsequent sorting by flow cytometry. To detect and quantify enterococci in fecal samples prior to enrichment, taxon-specific PCR and qPCR detection systems have been developed. The accessibility of the genomes from taxon-specifically sorted cells for subsequent molecular analyses was demonstrated by amplification of functional genes. Copyright © 2016 Elsevier GmbH. All rights reserved.

  5. Flow cytometric detection of alpha-1-acid glycoprotein on feline circulating leucocytes.

    PubMed

    Paltrinieri, S; Marchini, I; Gelain, M E

    2012-08-01

    To assess whether alpha-1-acid glycoprotein (AGP) can be detected on the membrane of feline circulating leucocytes. The presence of AGP on circulating leucocytes was investigated in both clinically healthy cats and cats with different diseases. A group of feline coronavirus (FCoV)-positive cats, comprising cats with feline infectious peritonitis (FIP) and cats not affected by FIP but seropositive for FCoV, were included in this study because the serum concentration of AGP increases during FCoV infection. Flow cytometry (using an anti-feline AGP antibody), serum protein electrophoresis, routine haematology and measurement of the serum AGP concentration were performed using blood samples from 32 healthy cats (19 FCoV-seropositive), 13 cats with FIP and 12 with other diseases (6 FCoV-seropositive). The proportion of cats with AGP-positive leucocytes in the different groups (e.g. controls vs sick; FIP vs other diseases, etc.) or in cats with different intensities of inflammatory response was compared using a Chi-square test. AGP-positive leucocytes were found in 23% of cats. Compared with controls, the proportion of patients with positive granulocytes and monocytes was higher among sick cats (especially cats with diseases other than FIP) and cats with high serum AGP concentration, but not in cats with leucocytosis or that were FCoV-seropositive. AGP-positive leucocytes can be found in feline blood, especially during inflammation. Conversely, no association between AGP-positive leucocytes and FIP was found. Further studies are needed to elucidate the mechanism responsible for this finding and its diagnostic role in cats with inflammation. © 2012 The Authors. Australian Veterinary Journal © 2012 Australian Veterinary Association.

  6. Flow-cytometric determination of genotoxic effects of exposure to petroleum in mink and sea otters

    USGS Publications Warehouse

    Bickham, J.W.; Mazet, J.A.; Blake, J.; Smolen, M.J.; Lou, Y.; Ballachey, B.E.

    1998-01-01

    Three experiments were conducted to investigate the genotoxic effects of crude oil on mink and sea otters, In the first experiment, the effects on mink of chronic exposure to weathered Prudhoe Bay crude oil were studied, Female mink were fed a diet that included weathered crude oil for a period of 3 weeks prior to mating, during pregnancy and until weaning. Kits were exposed through lactation and by diet after weaning until 4 months of age. Kidney and liver tissues of the kits were examined using flow cytometry (FCM) and it was found that the genome size was increased in kidney samples from the experimental group compared to the control group. This effect was probably due to some type of DNA amplification and it could have been inherited from the exposed mothers or have been a somatic response to oil exposure in the pups, No evidence of clastogenic effects, as measured by the coefficient of variation (CV) of the G(1) peak, was found in kidney or liver tissue. In the second experiment, yearling female mink were exposed either by diet or externally to crude oil or bunker C fuel oil. Evidence for clastogenic damage was found in spleen tissue for the exposure groups, but not in kidney tissue. No evidence of increased genome size was observed. In the third experiment, blood was obtained from wild-caught sea otters in Prince William Sound. The sea otters represented two populations: one from western Prince William Sound that was potentially exposed to oil from the Exxon Valdez oil spill and a reference population from eastern Prince William Sound that did not receive oil from the spill. The spill had occurred 1.5 years prior to obtaining the blood samples. Although the mean CVs did not differ between the populations, the exposed population had a significantly higher variance of CV measurements and five out of 15 animals from the exposed population had CVs higher than the 95% confidence limits of the reference population, It is concluded that FCM is a sensitive indicator

  7. Color encoded microbeads-based flow cytometric immunoassay for polycyclic aromatic hydrocarbons in food.

    PubMed

    Meimaridou, Anastasia; Haasnoot, Willem; Noteboom, Linda; Mintzas, Dimitrios; Pulkrabova, Jana; Hajslová, Jana; Nielen, Michel W F

    2010-07-05

    Food contamination caused by chemical hazards such as persistent organic pollutants (POPs) is a worldwide public health concern and requires continuous monitoring. The chromatography-based analysis methods for POPs are accurate and quite sensitive but they are time-consuming, laborious and expensive. Thus, there is a need for validated simplified screening tools, which are inexpensive, rapid, have automation potential and can detect multiple POPs simultaneously. In this study we developed a flow cytometry-based immunoassay (FCIA) using a color-encoded microbeads technology to detect benzo[a]pyrene (BaP) and other polycyclic aromatic hydrocarbons (PAHs) in buffer and food extracts as a starting point for the future development of rapid multiplex assays including other POPs in food, such as polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs). A highly sensitive assay for BaP was obtained with an IC(50) of 0.3 microg L(-1) using a monoclonal antibody (Mab22F12) against BaP, similar to the IC(50) of a previously described enzyme-linked immunosorbent assay (ELISA) using the same Mab. Moreover, the FCIA was 8 times more sensitive for BaP compared to a surface plasmon resonance (SPR)-based biosensor immunoassay (BIA) using the same reagents. The selectivity of the FCIAs was tested, with two Mabs against BaP for 25 other PAHs, including two hydroxyl PAH metabolites. Apart from BaP, the FCIAs can detect PAHs such as indenol[1,2,3-cd]pyrene (IP), benz[a]anthracene (BaA), and chrysene (CHR) which are also appointed by the European Food Safety Authority (EFSA) as suitable indicators of PAH contamination in food. The FCIAs results were in agreement with those obtained with gas chromatography-mass spectrometry (GC-MS) for the detection of PAHs in real food samples of smoked carp and wheat flour and has great potential for the future routine application of this assay in a simplex or multiplex format in combination with simplified extraction procedure which

  8. Image and flow cytometric analysis of gold nanoparticle uptake by macrophages

    NASA Astrophysics Data System (ADS)

    Fixler, Dror; Ankri, Rinat; Weiss, Ronald; Grahnert, Anja; Melzer, Susanne; Tárnok, Attila

    2016-03-01

    Background/Aim: In atherosclerosis stable and vulnerable atherosclerotic plaque types are distinguished that behave differently concerning rupture, thrombosis and clinical events. The stable are rich in M2 macrophages. The unstable are rich in inflammatory M1 macrophages and are highly susceptible to rupture, setting patients at risk for thrombotic events when they undergo invasive diagnosis such as coronary angiography. Therefore, novel approaches for non-invasive detection and classification of vulnerable plaques in vivo are needed. Whereas classical approaches fail to differentiate between both plaque types, a new biophotonic method (combination of the diffusion reflection (DR) method with flow cytometry (FCM) or image cytometry (IC)) to analyze gold nanoparticle (GNP) loading of plaques could overcome this limitation. Methods: Two types of GNP were used three variants of gold nanorods (GNRI with 40x18 nm, II 65x25 nm and III 52x13 nm in size) and gold nanospheres (GNS with an average diameter of 18.5 nm). The GNS had an absorption peak at 520 nm and the GNR at 630 nm. Monocytes were isolated from human buffy blood samples, differentiated into macrophages and their subtypes and labelled with GNR and GNS for 3 and 24 h. GNS and GNR loading were determined by FCM and/or IC. Macrophages within tissue-like phantoms were analyzed by the DR system. Results: After GNR labelling of macrophages the FCM light scatter values increased up to 3.7 fold and the DR slope changed from an average slope of 0.196 (macrophages only) to an average slope of 0.827 (macrophages labelled with GNR). But, GNRIII did not present much higher DR slopes than the control phantoms, indicating that macrophages take up GNRIII in a lower amount than GNRI or II. IC and microscopy showed that all particle variants were taken up by the cells in a heterogeneous fashion. Conclusion and outlook: The combination of FCM and DR measurements provides a potential novel, highly sensitive and non

  9. Flow cytometric immunophenotyping is of great value to diagnosis of natural killer cell neoplasms involving bone marrow and peripheral blood.

    PubMed

    Jiang, Neng-Gang; Jin, Yong-Mei; Niu, Qian; Zeng, Ting-Ting; Su, Jun; Zhu, Huan-Ling

    2013-01-01

    Natural killer (NK) cell neoplasms are unusual disorders. In this study we compared results of flow cytometric immunophenotype (FCI) with cytomorphology, histopathology and clinical findings in a series of patients with NK cell neoplasms with peripheral blood and/or bone marrow involvement, and the FCI of neoplastic and normal NK cells were compared. Retrospective data and specimens (bone marrow aspiration or peripheral blood) from 71 cases of NK cell neoplasms were obtained. All patients have been demonstrated laboratory and clinical features consistent with NK cell neoplasms, and the subtypes were determined by integrated clinical estimation. Routine 4-color flow cytometry (FCM) using a NK/T cell related antibody panels was performed. NK cell neoplasms were divided into two major subtypes by FCI, namely malignant NK cell lymphoma, including extranodal nasal type NK cell lymphoma (ENKL, 11 cases) and aggressive NK cell lymphoma/leukemia (ANKL, 43 cases), and relative indolent chronic lymphoproliferative disorder of NK cell (CLPD-NK, 17 cases). The former exhibited stronger CD56-expressing, larger forward scatter (FSC) and more usually CD7- and CD16-missing. FCI of CLPD-NK was similar to normal NK cells, but CD56-expressing was abnormal, which was negative in five cases and partially or dimly expressed in eight cases. Cytomorphologic abnormal cells were found on bone marrow slides of 4 cases of ENKL and 30 cases of ANKL. Eight cases of ENKL were positive in bone marrow biopsies, and other three cases were negative. In 32 cases of ANKL which bone marrow biopsies were applied, 21 cases were positive in the first biopsies. Lymphocytosis was found only in six cases of CLPD-NK by cytomorphology, and biopsy pathology was not much useful for diagnosing CLPD-NK. These results suggest that FCM analysis of bone marrow and peripheral blood was superior to cytomorphology, bone marrow biopsy, and immunohistochemistry in sensitivity and early diagnosis for ANKL, stage III

  10. Flow Cytometric and Cytokine ELISpot Approaches To Characterize the Cell-Mediated Immune Response in Ferrets following Influenza Virus Infection

    PubMed Central

    DiPiazza, Anthony; Richards, Katherine; Batarse, Frances; Lockard, Laura; Zeng, Hui; García-Sastre, Adolfo; Albrecht, Randy A.

    2016-01-01

    ABSTRACT Influenza virus infections represent a significant socioeconomic and public health burden worldwide. Although ferrets are considered by many to be ideal for modeling human responses to influenza infection and vaccination, efforts to understand the cellular immune response have been severely hampered by a paucity of standardized procedures and reagents. In this study, we developed flow cytometric and T cell enzyme-linked immunosorbent spot (ELISpot) approaches to characterize the leukocyte composition and antigen-specific T cell response within key lymphoid tissues following influenza virus infection in ferrets. Through a newly designed and implemented set of serological reagents, we used multiparameter flow cytometry to directly quantify the frequency of CD4+ and CD8+ T cells, Ig+ B cells, CD11b+ myeloid-derived cells, and major histocompatibility complex (MHC) class II-positive antigen-presenting cells (APCs) both prior to and after intranasal infection with A/California/04/09 (H1N1). We found that the leukocyte composition was altered at 10 days postinfection, with notable gains in the frequency of T cells and myeloid cells within the draining lymph node. Furthermore, these studies revealed that the antigen specificity of influenza virus-reactive CD4 and CD8 T cells was very broad, with recognition of the viral HA, NA, M1, NS1, and NP proteins, and that total reactivity to influenza virus postinfection represented approximately 0.1% of the circulating peripheral blood mononuclear cells (PBMC). Finally, we observed distinct patterns of reactivity between individual animals, suggesting heterogeneity at the MHC locus in ferrets within commercial populations, a finding of considerable interest in efforts to move the ferret model forward for influenza vaccine and challenge studies. IMPORTANCE Ferrets are an ideal animal model to study transmission, diseases, and vaccine efficacies of respiratory viruses because of their close anatomical and physiological

  11. Comparative genotoxicity of nanosilver in human liver HepG2 and colon Caco2 cells evaluated by a flow cytometric in vitro micronucleus assay.

    PubMed

    Sahu, Saura C; Njoroge, Joyce; Bryce, Steven M; Yourick, Jeffrey J; Sprando, Robert L

    2014-11-01

    Two widely used in vitro cell culture models, human liver HepG2 cells and human colon Caco2 cells, and flow cytometry techniques were evaluated as tools for rapid screening of potential genotoxicity of food-related nanosilver. Comparative genotoxic potential of 20 nm silver was evaluated in HepG2 and Caco2 cell cultures by a flow cytometric-based in vitro micronucleus assay. The nanosilver, characterized by the dynamic light scattering, transmission electron microscopy and inductively coupled plasma-mass spectrometry analysis, showed no agglomeration of the silver nanoparticles. The inductively coupled plasma-mass spectrometry and transmission electron microscopy analysis demonstrated the uptake of 20 nm silver by both cell types. The 20 nm silver exposure of HepG2 cells increased the concentration-dependent micronucleus formation sevenfold at 10 µg ml(-1) concentration in attached cell conditions and 1.3-fold in cell suspension conditions compared to the vehicle controls. However, compared to the vehicle controls, the 20 nm silver exposure of Caco2 cells increased the micronucleus formation 1.2-fold at a concentration of 10 µg ml(-1) both in the attached cell conditions as well as in the cell suspension conditions. Our results of flow cytometric in vitro micronucleus assay appear to suggest that the HepG2 cells are more susceptible to the nanosilver-induced micronucleus formation than the Caco2 cells compared to the vehicle controls. However, our results also suggest that the widely used in vitro models, HepG2 and Caco2 cells and the flow cytometric in vitro micronucleus assay are valuable tools for the rapid screening of genotoxic potential of nanosilver and deserve more careful evaluation.

  12. Flow Cytometric Quantification of Peripheral Blood Cell β-Adrenergic Receptor Density and Urinary Endothelial Cell-Derived Microparticles in Pulmonary Arterial Hypertension

    PubMed Central

    Rose, Jonathan A.; Wanner, Nicholas; Cheong, Hoi I.; Queisser, Kimberly; Barrett, Patrick; Park, Margaret; Hite, Corrine; Naga Prasad, Sathyamangla V.; Erzurum, Serpil; Asosingh, Kewal

    2016-01-01

    Pulmonary arterial hypertension (PAH) is a heterogeneous disease characterized by severe angiogenic remodeling of the pulmonary artery wall and right ventricular hypertrophy. Thus, there is an increasing need for novel biomarkers to dissect disease heterogeneity, and predict treatment response. Although β-adrenergic receptor (βAR) dysfunction is well documented in left heart disease while endothelial cell-derived microparticles (Ec-MPs) are established biomarkers of angiogenic remodeling, methods for easy large clinical cohort analysis of these biomarkers are currently absent. Here we describe flow cytometric methods for quantification of βAR density on circulating white blood cells (WBC) and Ec-MPs in urine samples that can be used as potential biomarkers of right heart failure in PAH. Biotinylated β-blocker alprenolol was synthesized and validated as a βAR specific probe that was combined with immunophenotyping to quantify βAR density in circulating WBC subsets. Ec-MPs obtained from urine samples were stained for annexin-V and CD144, and analyzed by a micro flow cytometer. Flow cytometric detection of alprenolol showed that βAR density was decreased in most WBC subsets in PAH samples compared to healthy controls. Ec-MPs in urine was increased in PAH compared to controls. Furthermore, there was a direct correlation between Ec-MPs and Tricuspid annular plane systolic excursion (TAPSE) in PAH patients. Therefore, flow cytometric quantification of peripheral blood cell βAR density and urinary Ec-MPs may be useful as potential biomarkers of right ventricular function in PAH. PMID:27270458

  13. The effects of orange juice clarification on the physiology of Escherichia coli; growth-based and flow cytometric analysis.

    PubMed

    Anvarian, Amir H P; Smith, Madeleine P; Overton, Tim W

    2016-02-16

    Orange juice (OJ) is a food product available in various forms which can be processed to a greater or lesser extent. Minimally-processed OJ has a high consumer perception but presents a potential microbiological risk due to acid-tolerant bacteria. Clarification of OJ (such as removal of cloud) is a common processing step in many OJ products. However, many of the antimicrobial components of OJ such as essential oils are present in the cloud fraction. Here, the effect of clarification by filtration on the viability and physiology of Escherichia coli K-12 was tested using total viable count (TVC) and flow cytometric (FCM) analysis. The latter technique was also used to monitor intracellular pH during incubation in OJ. Removal of the OJ cloud fraction was shown to have dramatic effects on bacterial viability and physiology during storage at a range of incubation temperatures. For instance, at 4 °C, a significantly lower number of healthy cells and a significantly higher number of injured cells were observed in 0.22 μm-filtered OJ at 24h post-inoculation, compared to filtered OJ samples containing particles between 0.22 μm and 11 μm in size. Similarly, there was a significant difference between the number of healthy bacteria in the 0.7 μm-filtered OJ and both 0.22 μm-filtered and 1.2 μm-filtered OJ after 24 hour incubation at 22.5 °C. This indicated that OJ cloud between 0.7 μm and 0.22 μm in size might have an adverse effect on the viability of E. coli K-12. Furthermore, FCM allowed the rapid analysis of bacterial physiology without the requirement for growth on agar plates, and revealed the extent of the viable but non-culturable (VBNC) population. For example, at 4 °C, while the FCM viable count did not substantially decrease until 48 h, decreases in TVC were observed between 0 and 48 hour incubation, due to a subset of injured bacteria entering the VBNC state, hence being unable to grow on agar plates. This study highlights the application of FCM in

  14. High-throughput theoretical design of lithium battery materials

    NASA Astrophysics Data System (ADS)

    Shi-Gang, Ling; Jian, Gao; Rui-Juan, Xiao; Li-Quan, Chen

    2016-01-01

    The rapid evolution of high-throughput theoretical design schemes to discover new lithium battery materials is reviewed, including high-capacity cathodes, low-strain cathodes, anodes, solid state electrolytes, and electrolyte additives. With the development of efficient theoretical methods and inexpensive computers, high-throughput theoretical calculations have played an increasingly important role in the discovery of new materials. With the help of automatic simulation flow, many types of materials can be screened, optimized and designed from a structural database according to specific search criteria. In advanced cell technology, new materials for next generation lithium batteries are of great significance to achieve performance, and some representative criteria are: higher energy density, better safety, and faster charge/discharge speed. Project supported by the National Natural Science Foundation of China (Grant Nos. 11234013 and 51172274) and the National High Technology Research and Development Program of China (Grant No. 2015AA034201).

  15. Flow cytometric analysis of platelet cyclooxygenase-1 and -2 and surface glycoproteins in patients with immune thrombocytopenia and healthy individuals.

    PubMed

    Rubak, Peter; Kristensen, Steen D; Hvas, Anne-Mette

    2017-06-01

    Immature platelets may contain more platelet enzymes such as cyclooxygenase (COX)-1 and COX-2 than mature platelets. Patients with immune thrombocytopenia (ITP) have a higher fraction of immature platelets and can therefore be utilized as a biological model for investigating COX-1 and COX-2 platelet expression. The aims were to develop flow cytometric assays for platelet COX-1 and COX-2 and to investigate the COX-1 and COX-2 platelet expression, platelet turnover, and platelet glycoproteins in ITP patients (n = 10) compared with healthy individuals (n = 30). Platelet count and platelet turnover parameters (mean platelet volume (MPV), immature platelet fraction (IPF), and immature platelet count (IPC)) were measured by flow cytometry (Sysmex XE-5000). Platelet COX-1, COX-2, and the glycoproteins (GP)IIb, IX, Ib, Ia, and IIIa were all analyzed by flow cytometry (Navios) and expressed as median fluorescence intensity. COX analyses were performed in both whole blood and platelet rich plasma (PRP), whereas platelet glycoproteins were analyzed in whole blood only. ITP patients had significantly lower platelet count (55 × 10(9)/L) than healthy individuals (240 × 10(9)/L, p < 0.01), but a higher MPV (p = 0.03) and IPF (p < 0.01). IPC was similar for the two groups (p = 0.74). PRP had significantly lower MPV (p < 0.01) and significantly higher platelet count and IPC (both p-values <0.03) when compared with whole blood. IPF was similar for PRP and whole blood (p = 0.18). COX-1 expression was 10 times higher and COX-2 expression was 50% higher in PRP than in whole blood (pCOX-1 < 0.01, pCOX-2 < 0.01). Platelet COX-1 expression was higher in ITP patients than healthy individuals using whole blood (pCOX-1 < 0.01) and PRP, though this was nonsignificant in PRP (pCOX-1 = 0.17). In ITP patients, positive correlations were found between platelet turnover and COX-1 expression (all p-values <0.01, rho = 0.80-0.94), whereas healthy individuals showed significant though weaker

  16. EuroFlow antibody panels for standardized n-dimensional flow cytometric immunophenotyping of normal, reactive and malignant leukocytes

    PubMed Central

    van Dongen, J J M; Lhermitte, L; Böttcher, S; Almeida, J; van der Velden, V H J; Flores-Montero, J; Rawstron, A; Asnafi, V; Lécrevisse, Q; Lucio, P; Mejstrikova, E; Szczepański, T; Kalina, T; de Tute, R; Brüggemann, M; Sedek, L; Cullen, M; Langerak, A W; Mendonça, A; Macintyre, E; Martin-Ayuso, M; Hrusak, O; Vidriales, M B; Orfao, A

    2012-01-01

    Most consensus leukemia & lymphoma antibody panels consist of lists of markers based on expert opinions, but they have not been validated. Here we present the validated EuroFlow 8-color antibody panels for immunophenotyping of hematological malignancies. The single-tube screening panels and multi-tube classification panels fit into the EuroFlow diagnostic algorithm with entries defined by clinical and laboratory parameters. The panels were constructed in 2–7 sequential design–evaluation–redesign rounds, using novel Infinicyt software tools for multivariate data analysis. Two groups of markers are combined in each 8-color tube: (i) backbone markers to identify distinct cell populations in a sample, and (ii) markers for characterization of specific cell populations. In multi-tube panels, the backbone markers were optimally placed at the same fluorochrome position in every tube, to provide identical multidimensional localization of the target cell population(s). The characterization markers were positioned according to the diagnostic utility of the combined markers. Each proposed antibody combination was tested against reference databases of normal and malignant cells from healthy subjects and WHO-based disease entities, respectively. The EuroFlow studies resulted in validated and flexible 8-color antibody panels for multidimensional identification and characterization of normal and aberrant cells, optimally suited for immunophenotypic screening and classification of hematological malignancies. PMID:22552007

  17. High Throughput Determination of Critical Human Dosing ...

    EPA Pesticide Factsheets

    High throughput toxicokinetics (HTTK) is a rapid approach that uses in vitro data to estimate TK for hundreds of environmental chemicals. Reverse dosimetry (i.e., reverse toxicokinetics or RTK) based on HTTK data converts high throughput in vitro toxicity screening (HTS) data into predicted human equivalent doses that can be linked with biologically relevant exposure scenarios. Thus, HTTK provides essential data for risk prioritization for thousands of chemicals that lack TK data. One critical HTTK parameter that can be measured in vitro is the unbound fraction of a chemical in plasma (Fub). However, for chemicals that bind strongly to plasma, Fub is below the limits of detection (LOD) for high throughput analytical chemistry, and therefore cannot be quantified. A novel method for quantifying Fub was implemented for 85 strategically selected chemicals: measurement of Fub was attempted at 10%, 30%, and 100% of physiological plasma concentrations using rapid equilibrium dialysis assays. Varying plasma concentrations instead of chemical concentrations makes high throughput analytical methodology more likely to be successful. Assays at 100% plasma concentration were unsuccessful for 34 chemicals. For 12 of these 34 chemicals, Fub could be quantified at 10% and/or 30% plasma concentrations; these results imply that the assay failure at 100% plasma concentration was caused by plasma protein binding for these chemicals. Assay failure for the remaining 22 chemicals may

  18. High-throughput computing in the sciences.

    PubMed

    Morgan, Mark; Grimshaw, Andrew

    2009-01-01

    While it is true that the modern computer is many orders of magnitude faster than that of yesteryear; this tremendous growth in CPU clock rates is now over. Unfortunately, however, the growth in demand for computational power has not abated; whereas researchers a decade ago could simply wait for computers to get faster, today the only solution to the growing need for more powerful computational resource lies in the exploitation of parallelism. Software parallelization falls generally into two broad categories--"true parallel" and high-throughput computing. This chapter focuses on the latter of these two types of parallelism. With high-throughput computing, users can run many copies of their software at the same time across many different computers. This technique for achieving parallelism is powerful in its ability to provide high degrees of parallelism, yet simple in its conceptual implementation. This chapter covers various patterns of high-throughput computing usage and the skills and techniques necessary to take full advantage of them. By utilizing numerous examples and sample codes and scripts, we hope to provide the reader not only with a deeper understanding of the principles behind high-throughput computing, but also with a set of tools and references that will prove invaluable as she explores software parallelism with her own software applications and research.

  19. Flow Cytometric and Cytokine ELISpot Approaches To Characterize the Cell-Mediated Immune Response in Ferrets following Influenza Virus Infection.

    PubMed

    DiPiazza, Anthony; Richards, Katherine; Batarse, Frances; Lockard, Laura; Zeng, Hui; García-Sastre, Adolfo; Albrecht, Randy A; Sant, Andrea J

    2016-09-01

    Influenza virus infections represent a significant socioeconomic and public health burden worldwide. Although ferrets are considered by many to be ideal for modeling human responses to influenza infection and vaccination, efforts to understand the cellular immune response have been severely hampered by a paucity of standardized procedures and reagents. In this study, we developed flow cytometric and T cell enzyme-linked immunosorbent spot (ELISpot) approaches to characterize the leukocyte composition and antigen-specific T cell response within key lymphoid tissues following influenza virus infection in ferrets. Through a newly designed and implemented set of serological reagents, we used multiparameter flow cytometry to directly quantify the frequency of CD4(+) and CD8(+) T cells, Ig(+) B cells, CD11b(+) myeloid-derived cells, and major histocompatibility complex (MHC) class II-positive antigen-presenting cells (APCs) both prior to and after intranasal infection with A/California/04/09 (H1N1). We found that the leukocyte composition was altered at 10 days postinfection, with notable gains in the frequency of T cells and myeloid cells within the draining lymph node. Furthermore, these studies revealed that the antigen specificity of influenza virus-reactive CD4 and CD8 T cells was very broad, with recognition of the viral HA, NA, M1, NS1, and NP proteins, and that total reactivity to influenza virus postinfection represented approximately 0.1% of the circulating peripheral blood mononuclear cells (PBMC). Finally, we observed distinct patterns of reactivity between individual animals, suggesting heterogeneity at the MHC locus in ferrets within commercial populations, a finding of considerable interest in efforts to move the ferret model forward for influenza vaccine and challenge studies. Ferrets are an ideal animal model to study transmission, diseases, and vaccine efficacies of respiratory viruses because of their close anatomical and physiological resemblances to

  20. The evaluation of histo-blood group ABO typing by flow cytometric and PCR-amplification of specific alleles analyses and their application in clinical laboratories.

    PubMed

    Aki, Kensaku; Izumi, Azusa; Hosoi, Eiji

    2012-01-01

    ABO antigens are oligosaccharide antigens, and are widely distributed on red blood and tissue cells as well as in saliva and body fluid. Therefore, these antigens are important not only for blood transfusion, but also for tissue cell and organ transplantations. Also, blood, hair, and seminal fluid are important sources of evidence at crime scenes, and these antigens are some of the most important markers for personal identification in forensic investigations. Here, we describe the development and use of quantitative analysis of A, B, and H antigens on red blood cells by employing flow cytometric analysis and the ABO genotyping method based on PCR-amplification of specific alleles (PASA) within DNA, especially from blood and saliva. In this study, flow cytometric analysis could be used to compare the differences between the expression of A and/or B and H antigens on red blood cells with various phenotypes, and the PASA method was able to determine the genotype of the type cisA(2)B(3) pedigree using only DNA extracted from saliva. These analysis methods are simple and useful for judging the ABO blood group system and genotyping, and are used widely throughout research and clinical laboratories and forensic fields.

  1. Usefulness of Flow Cytometric Mepacrine Uptake/Release Combined with CD63 Assay in Diagnosis of Patients with Suspected Platelet Dense Granule Disorder.

    PubMed

    Cai, Huili; Mullier, François; Frotscher, Birgit; Briquel, Marie-Elisabeth; Toussaint, Marie; Massin, Frédéric; Lecompte, Thomas; Latger-Cannard, Véronique

    2016-04-01

    Dense granule disorder is one of the most common platelet abnormalities, resulting from dense granule deficiency or secretion defect. This study was aimed to evaluate the clinical usefulness of the flow cytometric combination of mepacrine uptake/release assay and CD63 expression detection in the management of patients with suspected dense granule disorder. Over a period of 5 years, patients with abnormal platelet aggregation and/or reduced adenosine triphosphate (ATP) secretion suggestive of dense granule disorder were consecutively enrolled. The flow cytometric assays were systematically performed to further investigate dense granule functionality. Among the 26 included patients, 18 cases showed impaired mepacrine uptake/release and reduced CD63 expression on activated platelets, consistent with δ-storage pool deficiency (SPD). Another seven patients showed decrease in mepacrine release and CD63 expression but mepacrine uptake was normal, indicating secretion defect rather than δ-SPD. Unfortunately, ATP secretion could not be measured in 7 out of the 26 patients due to insufficient sample and/or severe thrombocytopenia. This test combination provides a rapid and effective method to detect the heterogeneous abnormalities of platelet dense granule by distinguishing between storage and release defects. This combination is particularly advantageous for severely thrombocytopenic patients and pediatric patients in which only minimal sample is required.

  2. Mantle cell lymphoma, blastoid variant, diagnosed on the basis of cytomorphology and flow cytometric immunophenotyping of the lymph node aspirate and peripheral blood.

    PubMed Central

    Lee, Mi-Ja; Kee, Keun-Hong; Jeon, Ho-Jong

    2002-01-01

    Mantle cell lymphoma, blastoid variant (B-MCL), is a very rare type of non-Hodgkin's lymphoma exhibiting an aggressive clinical course. We describe a case of B-MCL showing generalized lymphadenopathy and leukemic conversion in a 62-yr-old man. The case was diagnosed and subclassified as B-MCL on the basis of cyto-morphology and immunophenotype. Microscopic examination of the peripheral blood (PB) showed a spectrum of cells ranging from small mature lymphocytes to medium- and large-sized lymphocytes with blast-like chromatin and prominent nucleoli. The lymphoma cells were monoclonal B cells with moderately intense surface IgM. They were CD5 positive, cyclin D1 positive, CD10 negative, and CD23 negative. The flow cytometric immunophenotyping and DNA ploidy analysis of the PB and material obtained by aspiration cytology supported the diagnosis of B-MCL. These findings underline the utility of aspiration cytology in diagnosing B-MCL when cytomorphologic examination is combined with flow cytometric analysis of immuno-phenotype and demonstration of proliferation markers. PMID:11961299

  3. Mantle cell lymphoma, blastoid variant, diagnosed on the basis of cytomorphology and flow cytometric immunophenotyping of the lymph node aspirate and peripheral blood.

    PubMed

    Lee, Mi-Ja; Kee, Keun-Hong; Jeon, Ho-Jong

    2002-04-01

    Mantle cell lymphoma, blastoid variant (B-MCL), is a very rare type of non-Hodgkin's lymphoma exhibiting an aggressive clinical course. We describe a case of B-MCL showing generalized lymphadenopathy and leukemic conversion in a 62-yr-old man. The case was diagnosed and subclassified as B-MCL on the basis of cyto-morphology and immunophenotype. Microscopic examination of the peripheral blood (PB) showed a spectrum of cells ranging from small mature lymphocytes to medium- and large-sized lymphocytes with blast-like chromatin and prominent nucleoli. The lymphoma cells were monoclonal B cells with moderately intense surface IgM. They were CD5 positive, cyclin D1 positive, CD10 negative, and CD23 negative. The flow cytometric immunophenotyping and DNA ploidy analysis of the PB and material obtained by aspiration cytology supported the diagnosis of B-MCL. These findings underline the utility of aspiration cytology in diagnosing B-MCL when cytomorphologic examination is combined with flow cytometric analysis of immuno-phenotype and demonstration of proliferation markers.

  4. The G6PD flow-cytometric assay is a reliable tool for diagnosis of G6PD deficiency in women and anaemic subjects.

    PubMed

    Bancone, Germana; Kalnoky, Michael; Chu, Cindy S; Chowwiwat, Nongnud; Kahn, Maria; Malleret, Benoit; Wilaisrisak, Pornpimon; Rénia, Laurent; Domingo, Gonzalo J; Nosten, Francois

    2017-08-29

    Glucose-6-phosphate dehydrogenase (G6PD) activity is essential for redox equilibrium of red blood cells (RBCs) and, when compromised, the RBCs are more susceptible to haemolysis. 8-aminoquinolines (primaquine and tafenoquine) are used for the radical curative treatment of Plasmodium vivax malaria and can cause haemolysis in G6PD deficient subjects. Haemolytic risk is dependent on treatment dose and patient G6PD status but ultimately it correlates with the number of G6PD deficient RBCs. The G6PD spectrophotometric assay reliably identifies deficient subjects but is less reliable in heterozygous females, especially when other blood conditions are present. In this work we analysed samples with a range of G6PD phenotypes and haematologic conditions from 243 healthy volunteers of Asian or African-American heritage using both the spectrophotomeric assay and the G6PD flow-cytometric assay. Overall 18.5% of subjects (29.3% of Asian females) presented with anaemia, associated with decreased RBCs volume (MCV) and reticulocytosis; the flow-cytometric assay showed good correlation with the spectrophotometric assay (Pearson's r 0.918-0.957) and was less influenced by haemoglobin concentration, number of RBCs and number of reticulocytes. This resulted in more precise quantification of the number of G6PD deficient RBCs and presumably higher predictive power of drug induced haemolytic risk.

  5. Web-based visual analysis for high-throughput genomics.

    PubMed

    Goecks, Jeremy; Eberhard, Carl; Too, Tomithy; Nekrutenko, Anton; Taylor, James

    2013-06-13

    Visualization plays an essential role in genomics research by making it possible to observe correlations and trends in large datasets as well as communicate findings to others. Visual analysis, which combines visualization with analysis tools to enable seamless use of both approaches for scientific investigation, offers a powerful method for performing complex genomic analyses. However, there are numerous challenges that arise when creating rich, interactive Web-based visualizations/visual analysis applications for high-throughput genomics. These challenges include managing data flow from Web server to Web browser, integrating analysis tools and visualizations, and sharing visualizations with colleagues. We have created a platform simplifies the creation of Web-based visualization/visual analysis applications for high-throughput genomics. This platform provides components that make it simple to efficiently query very large datasets, draw common representations of genomic data, integrate with analysis tools, and share or publish fully interactive visualizations. Using this platform, we have created a Circos-style genome-wide viewer, a generic scatter plot for correlation analysis, an interactive phylogenetic tree, a scalable genome browser for next-generation sequencing data, and an application for systematically exploring tool parameter spaces to find good parameter values. All visualizations are interactive and fully customizable. The platform is integrated with the Galaxy (http://galaxyproject.org) genomics workbench, making it easy to integrate new visual applications into Galaxy. Visualization and visual analysis play an important role in high-throughput genomics experiments, and approaches are needed to make it easier to create applications for these activities. Our framework provides a foundation for creating Web-based visualizations and integrating them into Galaxy. Finally, the visualizations we have created using the framework are useful tools for high-throughput

  6. Microfabricated high-throughput electronic particle detector.

    PubMed

    Wood, D K; Requa, M V; Cleland, A N

    2007-10-01

    We describe the design, fabrication, and use of a radio frequency reflectometer integrated with a microfluidic system, applied to the very high-throughput measurement of micron-scale particles, passing in a microfluidic channel through the sensor region. The device operates as a microfabricated Coulter counter [U.S. Patent No. 2656508 (1953)], similar to a design we have described previously, but here with significantly improved electrode geometry as well as including electronic tuning of the reflectometer; the two improvements yielding an improvement by more than a factor of 10 in the signal to noise and in the diametric discrimination of single particles. We demonstrate the high-throughput discrimination of polystyrene beads with diameters in the 4-10 microm range, achieving diametric resolutions comparable to the intrinsic spread of diameters in the bead distribution, at rates in excess of 15 x 10(6) beads/h.

  7. High-throughput TILLING for functional genomics.

    PubMed

    Till, Bradley J; Colbert, Trenton; Tompa, Rachel; Enns, Linda C; Codomo, Christine A; Johnson, Jessica E; Reynolds, Steven H; Henikoff, Jorja G; Greene, Elizabeth A; Steine, Michael N; Comai, Luca; Henikoff, Steven

    2003-01-01

    Targeting-induced local lesions in genomes (TILLING) is a general strategy for identifying induced point mutations that can be applied to almost any organism. Here, we describe the basic methodology for high-throughput TILLING. Gene segments are amplified using fluorescently tagged primers, and products are denatured and reannealed to form heteroduplexes between the mutated sequence and its wild-type counterpart. These heteroduplexes are substrates for cleavage by the endonuclease CEL I. Following cleavage, products are analyzed on denaturing polyacrylamide gels using the LI-COR DNA analyzer system. High-throughput TILLING has been adopted by the Arabidopsis TILLING Project (ATP) to provide allelic series of point mutations for the general Arabidopsis community.

  8. High-throughput TILLING for Arabidopsis.

    PubMed

    Till, Bradley J; Colbert, Trenton; Codomo, Christine; Enns, Linda; Johnson, Jessica; Reynolds, Steven H; Henikoff, Jorja G; Greene, Elizabeth A; Steine, Michael N; Comai, Luca; Henikoff, Steven

    2006-01-01

    Targeting induced local lesions in genomes (TILLING) is a general strategy for identifying induced point mutations that can be applied to almost any organism. In this chapter, we describe the basic methodology for high-throughput TILLING. Gene segments are amplified using fluorescently tagged primers, and products are denatured and reannealed to form heteroduplexes between the mutated sequence and its wild-type counterpart. These heteroduplexes are substrates for cleavage by the endonuclease CEL I. Following cleavage, products are analyzed on denaturing polyacrylamide gels using the LI-COR DNA analyzer system. High-throughput TILLING has been adopted by the Arabidopsis TILLING Project (ATP) to provide allelic series of point mutations for the general Arabidopsis community.

  9. The use of flow cytometric applications to measure the effects of PAHs on growth, membrane integrity, and relative lipid content of the benthic diatom, Nitzschia brevirostris.

    PubMed

    Croxton, April N; Wikfors, Gary H; Schulterbrandt-Gragg, Richard D

    2015-02-15

    This laboratory study measured the direct effects of three polycyclic aromatic hydrocarbon (PAH) compounds (naphthalene, pyrene, and benzo(a)pyrene) upon cell growth, membrane integrity, and BODIPY-stained lipid fluorescence intensity of the benthic diatom Nitzschia brevirostris using flow cytometry as an analysis tool. Previous field and laboratory studies have reported reductions in algal populations following PAH exposure, but specific, functional responses of the microalgae to these pollutants could not be revealed by cell numbers alone. Using flow-cytometric measurements, we confirmed that maximal cell densities in PAH-exposed diatom cultures were significantly lower compared to controls; however, we also discovered increases in lipids and cells with compromised membranes in PAH-exposed cultures. These results highlight new tools for measuring the direct effects of organic pollutants upon the physiology of taxa comprising microphytobenthic communities important in estuarine food webs. Published by Elsevier Ltd.

  10. Optimization of Methanotrophic Growth and Production of Poly(3-Hydroxybutyrate) in a High-Throughput Microbioreactor System

    PubMed Central

    Criddle, Craig S.

    2015-01-01

    Production of poly(3-hydroxybutyrate) (P3HB) from methane has economic and environmental advantages over production by agricultural feedstock. Identification of high-productivity strains and optimal growth conditions is critical to efficient conversion of methane to polymer. Current culture conditions, including serum bottles, shake flasks, and agar plates, are labor-intensive and therefore insufficient for systematic screening and isolation. Gas chromatography, the standard method for analysis of P3HB content in bacterial biomass, is also incompatible with high-throughput screening. Growth in aerated microtiter plates coupled with a 96-well Nile red flow-cytometric assay creates an integrated microbioreactor system for high-throughput growth and analysis of P3HB-producing methanotrophic cultures, eliminating the need for individual manipulation of experimental replicates. This system was tested in practice to conduct medium optimization for P3HB production in pure cultures of Methylocystis parvus OBBP. Optimization gave insight into unexpected interactions: for example, low calcium concentrations significantly enhanced P3HB production under nitrogen-limited conditions. Optimization of calcium and copper concentrations in the growth medium increased final P3HB content from 18.1% to 49.4% and P3HB concentration from 0.69 g/liter to 3.43 g/liter while reducing doubling time from 10.6 h to 8.6 h. The ability to culture and analyze thousands of replicates with high mass transfer in completely mixed culture promises to streamline medium optimization and allow the detection and isolation of highly productive strains. Applications for this system are numerous, encompassing analysis of biofuels and other lipid inclusions, as well as analysis of heterotrophic and photosynthetic systems. PMID:25956771

  11. High Throughput Determination of Tetramine in Drinking ...

    EPA Pesticide Factsheets

    Report The sampling and analytical procedure (SAP) presented herein, describes a method for the high throughput determination of tetramethylene disulfotetramine in drinking water by solid phase extraction and isotope dilution gas chromatography/mass spectrometry. This method, which will be included in the SAM, is expected to provide the Water Laboratory Alliance, as part of EPA’s Environmental Response Laboratory Network, with a more reliable and faster means of analyte collection and measurement.

  12. High-throughput in vivo vertebrate screening

    PubMed Central

    Pardo-Martin, Carlos; Chang, Tsung-Yao; Koo, Bryan Kyo; Gilleland, Cody L.; Wasserman, Steven C.; Yanik, Mehmet Fatih

    2010-01-01

    We demonstrate a high-throughput platform for cellular-resolution in vivo pharmaceutical and genetic screens on zebrafish larvae. The system automatically loads animals from reservoirs or multiwell plates, and positions and orients them for high-speed confocal imaging and laser manipulation of both superficial and deep organs within 19 seconds without damage. We show small-scale test screening of retinal axon guidance mutants and neuronal regeneration assays in combination with femtosecond laser microsurgery. PMID:20639868

  13. High-throughput neuro-imaging informatics.

    PubMed

    Miller, Michael I; Faria, Andreia V; Oishi, Kenichi; Mori, Susumu

    2013-01-01

    This paper describes neuroinformatics technologies at 1 mm anatomical scale based on high-throughput 3D functional and structural imaging technologies of the human brain. The core is an abstract pipeline for converting functional and structural imagery into their high-dimensional neuroinformatic representation index containing O(1000-10,000) discriminating dimensions. The pipeline is based on advanced image analysis coupled to digital knowledge representations in the form of dense atlases of the human brain at gross anatomical scale. We demonstrate the integration of these high-dimensional representations with machine learning methods, which have become the mainstay of other fields of science including genomics as well as social networks. Such high-throughput facilities have the potential to alter the way medical images are stored and utilized in radiological workflows. The neuroinformatics pipeline is used to examine cross-sectional and personalized analyses of neuropsychiatric illnesses in clinical applications as well as longitudinal studies. We demonstrate the use of high-throughput machine learning methods for supporting (i) cross-sectional image analysis to evaluate the health status of individual subjects with respect to the population data, (ii) integration of image and personal medical record non-image information for diagnosis and prognosis.

  14. High-throughput neuro-imaging informatics

    PubMed Central

    Miller, Michael I.; Faria, Andreia V.; Oishi, Kenichi; Mori, Susumu

    2013-01-01

    This paper describes neuroinformatics technologies at 1 mm anatomical scale based on high-throughput 3D functional and structural imaging technologies of the human brain. The core is an abstract pipeline for converting functional and structural imagery into their high-dimensional neuroinformatic representation index containing O(1000–10,000) discriminating dimensions. The pipeline is based on advanced image analysis coupled to digital knowledge representations in the form of dense atlases of the human brain at gross anatomical scale. We demonstrate the integration of these high-dimensional representations with machine learning methods, which have become the mainstay of other fields of science including genomics as well as social networks. Such high-throughput facilities have the potential to alter the way medical images are stored and utilized in radiological workflows. The neuroinformatics pipeline is used to examine cross-sectional and personalized analyses of neuropsychiatric illnesses in clinical applications as well as longitudinal studies. We demonstrate the use of high-throughput machine learning methods for supporting (i) cross-sectional image analysis to evaluate the health status of individual subjects with respect to the population data, (ii) integration of image and personal medical record non-image information for diagnosis and prognosis. PMID:24381556

  15. Characterization and Purification of Neoplastic Cells of Nodular Lymphocyte Predominant Hodgkin Lymphoma from Lymph Nodes by Flow Cytometry and Flow Cytometric Cell Sorting.

    PubMed

    Fromm, Jonathan R; Thomas, Anju; Wood, Brent L

    2017-02-01

    We report the flow cytometric (FC) identification and characterization of lymphocyte predominant (LP) cells from tissues involved by nodular lymphocyte predominant Hodgkin lymphoma (NLPHL). First, we immunophenotyped the NLPHL cell line (DEV) confirming a germinal center immunophenotype, lack of expression of CD32 and CD58, and expression of CD54. Nineteen of 26 lymph nodes involved by NLPHL demonstrated a population with an LP immunophenotype (73%), which included expression of germinal center markers (CD75/Bcl-6-positive, CD32-weak/negative without CD10), a B-cell immunophenotype (CD19/CD20/CD40(+)), IgD and/or IgM expression (67%), and lack of programmed death-ligand 1/ligand 2. The LP cells demonstrated an adhesion macromolecule expression pattern distinct from Hodgkin and Reed-Sternberg cells of classical Hodgkin lymphoma (CHL) (uniform CD50 and variable CD58 for NLPHL; minimal CD50, bright CD58 expression for CHL). A two-tube consensus assay identified LP cells in all seven NLPHL cases examined and only one non-NLPHL case (94 cases evaluated). Finally, FC cell sorting studies confirm that FC-defined populations have an LP cytomorphology. Taken together, these findings demonstrate a two-tube consensus assay can be used to immunophenotype NLHPL with high specificity and sensitivity and rapidly purify LP cells for genetic studies. This study also confirms aneuploidy in LP cells, provides antigens that may be helpful in distinguishing NLPHL from CHL, and suggests that T cells interact less avidly with LP cells than with Hodgkin and Reed-Sternberg cells. Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  16. Complement resistance, as determined by viable count and flow cytometric methods, and its association with the presence of iss and the virulence of avian Escherichia coli.

    PubMed

    Nolan, Lisa K; Giddings, Catherine W; Horne, Shelley M; Doetkott, Curt; Gibbs, Penelope S; Wooley, Richard E; Foley, Steven L

    2002-01-01

    Previous work in our labs has shown that avian Escherichia coli virulence is correlated with resistance to complement. Also, our studies have revealed that the presence of the increased serum survival gene (iss), known to contribute to the complement resistance and virulence of mammalian E. coli, may predict the virulent nature of an avian E. coli isolate. This relationship warrants further research, but further clarification of the relationship among virulence, complement resistance, and iss sequences requires use of complement susceptibility assays. Such assays, unfortunately, are labor-intensive, expensive, and difficult to perform. In the present study, the results of two complement susceptibility assays for 20 E. coli isolates, 10 incriminated in avian colibacillosis and 10 from the intestinal tracts of apparently healthy birds, were compared in an attempt to determine if flow cytometric analysis was a reasonable alternative to a viable count assay. In addition, the virulence of these isolates for chick embryos was determined, and each isolate was examined for the presence of iss using amplification techniques. The flow cytometric method was found to be repeatable for most isolates, and its results showed moderate agreement with those obtained through viable counts. All intestinal isolates of healthy birds proved avirulent using the embryo lethality assay; however, not all isolates from sick birds were demonstrated to be virulent. Possible explanations of these results include that the methods originally used to isolate these organisms failed to detect the illness-inciting strains or that the virulence of these strains had declined following initial isolation. Additionally, we must consider the possibility that the embryo lethality assay of virulence used here might not be sensitive enough to detect differences between these two groups of isolates. Also, it should be noted that virulence assays, such as the one used here, fail to account for predisposing host

  17. Development of a flow cytometric bead immunoassay and its assessment as a possible aid to potency evaluation of enterotoxaemia vaccines.

    PubMed

    Buys, Angela; Macdonald, Raynard; Crafford, Jannie; Theron, Jacques

    2014-03-10

    Enterotoxaemia, an economically important disease of sheep, goats and calves, is caused by systemic effects of the epsilon toxin produced by the anaerobic bacterium Clostridium perfringens type D. The only practical means of controlling the occurrence of enterotoxaemia is to immunise animals by vaccination. The vaccine is prepared by deriving a toxoid from the bacterial culture filtrate and the potency of the vaccine is tested with the in vivo mouse neutralisation test (MNT). Due to ethical, economic and technical reasons, alternative in vitro assays are needed. In this study an indirect cytometric bead immunoassay (I-CBA) was developed for use in vaccine potency testing and the results were compared with those obtained using an indirect enzyme-linked immunosorbent assay (I-ELISA) and the MNT. Sera were collected from guinea pigs immunised with three different production batches of enterotoxaemia vaccine and the levels of anti-epsilon toxin antibodies were determined. Although the intra- and inter-assay variability was satisfactory, epsilon antitoxin levels determined by both the I-ELISA and indirect cytometric bead immunoassay (I-CBA) tests were higher than those of the MNT assay. In contrast to the MNT, all of the serum samples were identified as having antitoxin levels above the required minimum (not less than 5 U/mL). These results indicate that the respective in vitro tests in their current formats are not yet suitable alternatives to the in vivo MNT. The growing demand for a more humane, cost-effective and efficient method for testing the potency of enterotoxaemia vaccines, however, provides a strong impetus for further optimisation and standardisation of the I-CBA assay but further analytical research is required.

  18. High Throughput Screening For Hazard and Risk of Environmental Contaminants

    EPA Science Inventory

    High throughput toxicity testing provides detailed mechanistic information on the concentration response of environmental contaminants in numerous potential toxicity pathways. High throughput screening (HTS) has several key advantages: (1) expense orders of magnitude less than an...

  19. High Throughput Screening For Hazard and Risk of Environmental Contaminants

    EPA Science Inventory

    High throughput toxicity testing provides detailed mechanistic information on the concentration response of environmental contaminants in numerous potential toxicity pathways. High throughput screening (HTS) has several key advantages: (1) expense orders of magnitude less than an...

  20. High Throughput PBTK: Open-Source Data and Tools for ...

    EPA Pesticide Factsheets

    Presentation on High Throughput PBTK at the PBK Modelling in Risk Assessment meeting in Ispra, Italy Presentation on High Throughput PBTK at the PBK Modelling in Risk Assessment meeting in Ispra, Italy

  1. Flow cytometric method for in situ preparation of standard materials of a small defined number of microbial cells with colony-forming potentiality.

    PubMed

    Matsuoka, Hideaki; Nakano, Koichiro; Takatani, Norimasa; Yoshida, Tomonori; Igimi, Shizunobu; Saito, Mikako

    2014-01-01

    Standard materials of a small defined number of cells with colony-forming potentiality are essential for the rational validation of food microbiological methods. An in situ flow cytometric method using viable staining with 6-carboxyfluorescein diacetate (CFDA) and tryptic soy agar (TSA) was previously proposed and its feasibility was demonstrated with five strains. In this study, this method was applied to 16 strains to support its broad applicability. The cell sorting gate was previously determined based on the CFDA stainability alone. Now the structural properties of cells designated by forward and side-scattering intensities have been introduced as the second gating criteria. Under the optimum gate condition, 100 cells have been selected and sorted on TSA. Consequently, a 95% or higher colony-forming rate has been attained for every strain. A successful application to microaerophilic Campylobacter spp. is especially of great importance because it suggests further broader applicability.

  2. CD200 flow cytometric assessment and semiquantitative immunohistochemical staining distinguishes hairy cell leukemia from hairy cell leukemia-variant and other B-cell lymphoproliferative disorders.

    PubMed

    Pillai, Vinodh; Pozdnyakova, Olga; Charest, Karry; Li, Betty; Shahsafaei, Aliakbar; Dorfman, David M

    2013-10-01

    To evaluate CD200 expression in B-cell proliferative disorders. We analyzed 180 recent specimens of B-cell neoplasms for CD200 expression by flow cytometric immunophenotypic analysis, which is better able to assess relative intensity of staining than immunohistochemical staining. We found that hairy cell leukemia exhibits a high level of staining for CD200 in comparison to other B-cell lymphoproliferative disorders, including hairy cell leukemia-variant (HCL-V), marginal zone lymphoma, and lymphoplasmacytic lymphoma. We confirmed this observation by semiquantitative immunohistochemical staining. Assessment of the CD200 expression level is helpful to distinguish HCL from HCL-V and other B-cell lymphoproliferative disorders and in the differential diagnosis of B-cell neoplasms in general.

  3. Flow cytometric differentiation of abnormal and normal plasma cells in the bone marrow in patients with multiple myeloma and its precursor diseases.

    PubMed

    Tembhare, Prashant R; Yuan, Constance M; Venzon, David; Braylan, Raul; Korde, Neha; Manasanch, Elisabet; Zuchlinsky, Diamond; Calvo, Katherine; Kurlander, Roger; Bhutani, Manisha; Tageja, Nishant; Maric, Irina; Mulquin, Marcia; Roschewski, Mark; Kwok, Mary; Liewehr, David; Landgren, Ola; Stetler-Stevenson, Maryalice

    2014-03-01

    Flow cytometric (FC) enumeration of abnormal plasma cells (APCs) for diagnosis and prognostication of plasma cell dyscrasias (PCD) is challenging. We studied antigen expression in normal plasma cells (NPC) (N = 34) and APC in a series of unselected PCD (N = 59). NPC subpopulations often demonstrated CD19(-), CD20(+), CD45(-) or dim and CD56(+), an immunophenotype observed in PCD. However abnormal CD81 was only observed in APCs (APC detection sensitivity 95%; specificity 100%). We evaluated differences in antigen expression patterns among MGUS (N = 14), SMM (N = 35) and MM (N = 10), finding the combination of CD45 and CD56 helpful in differentiating MGUS from SMM and MM (p = 0.0002). Published by Elsevier Ltd.

  4. Simultaneous use of multiplex ligation-dependent probe amplification assay and flow cytometric DNA ploidy analysis in patients with acute leukemia.

    PubMed

    Reyes-Núñez, Virginia; Galo-Hooker, Evelyn; Pérez-Romano, Beatriz; Duque, Ricardo E; Ruiz-Arguelles, Alejandro; Garcés-Eisele, Javier

    2017-03-18

    The aim of this work was to simultaneously use multiplex ligation-dependent probe amplification (MLPA) assay and flow cytometric DNA ploidy analysis (FPA) to detect aneuploidy in patients with newly diagnosed acute leukemia. MLPA assay and propidium iodide FPA were used to test samples from 53 consecutive patients with newly diagnosed acute leukemia referred to our laboratory for immunophenotyping. Results were compared by nonparametric statistics. The combined use of both methods significantly increased the rate of detection of aneuploidy as compared to that obtained by each method alone. The limitations of one method are somehow countervailed by the other and vice versa. MPLA and FPA yield different yet complementary information concerning aneuploidy in acute leukemia. The simultaneous use of both methods might be recommended in the clinical setting. © 2017 Clinical Cytometry Society. © 2017 International Clinical Cytometry Society.

  5. Flow cytometric analysis with a fluorescently labeled formyl peptide receptor ligand as a new method to study the pharmacological profile of the histamine H2 receptor.

    PubMed

    Werner, Kristin; Kälble, Solveig; Wolter, Sabine; Schneider, Erich H; Buschauer, Armin; Neumann, Detlef; Seifert, Roland

    2015-10-01

    The histamine H2 receptor (H2R) is a Gs protein-coupled receptor. Its activation leads to increases in the second messenger adenosine-3',5'-cyclic monophosphate (cAMP). Presently, several systems are established to characterize the pharmacological profile of the H2R, mostly requiring radioactive material, animal models, or human blood cells. This prompted us to establish a flow cytometric analysis with a fluorescently labeled formyl peptide receptor (FPR) ligand in order to investigate the H2R functionally and pharmacologically. First, we stimulated U937 promonocytes, which mature in a cAMP-dependent fashion upon H2R activation, with histamine (HA) or selective H2R agonists and measured increases in cAMP concentrations by mass spectrometry. Next, indicative for the maturation of U937 promonocytes, we assessed the FPR expression upon incubation with HA or H2R agonists. FPR expression was measured either indirectly by formyl peptide-induced changes in intracellular calcium concentrations ([Ca(2+)]i) or directly with the fluorescein-labeled FPR ligand fNleLFNleYK-Fl. HA and H2R agonists concentration-dependently induced FPR expression, and potencies and efficacies of fMLP-induced increases in [Ca(2+)]i and FPR density correlated linearly. Accordingly, flow cytometric analysis of FPR expression constitutes a simple, inexpensive, sensitive, and reliable method to characterize the H2R pharmacologically. Furthermore, we evaluated FPR expression at the mRNA level. Generally, quantitative real-time polymerase chain reaction confirmed functional data. Additionally, our study supports the concept of functional selectivity of the H2R, since we observed dissociations in the efficacies of HA and H2R agonists in cAMP accumulation and FPR expression.

  6. Simplified flow cytometric immunophenotyping panel for multiple myeloma, CD56/CD19/CD138(CD38)/CD45, to differentiate neoplastic myeloma cells from reactive plasma cells.

    PubMed

    Jeong, Tae-Dong; Park, Chan-Jeoung; Shim, Hyoeun; Jang, Seongsoo; Chi, Hyun-Sook; Yoon, Dok Hyun; Kim, Dae-Young; Lee, Jung-Hee; Lee, Je-Hwan; Suh, Cheolwon; Lee, Kyoo Hyung

    2012-12-01

    Flow cytometric immunophenotyping has been used to identify neoplastic plasma cell populations in patients with multiple myeloma (MM). Previous reports have described the use of several antigens, including CD38, CD138, CD56, CD117, CD52, CD19 and CD45, to distinguish distinct populations of plasma cells. The aim of this study was to evaluate a simplified immunophenotyping panel for MM analysis. A total of 70 patients were enrolled in the study, 62 of which were newly diagnosed with MM (untreated), whereas the remaining 8 were undergoing bone marrow assessment as part of follow-up after treatment (treated). Treated cases included 3 patients with relapse and 5 patients with persistence of MM. Multiparametric flow cytometric immunophenotyping was performed using monoclonal antibodies against CD56, CD19, CD138 (CD38), and CD45. In differential counts, plasma cells in bone marrow (BM) accounted for 3.6-93.2% of the total nucleated cell count. The positive expression rates of CD56, CD19, CD138, and CD45 in neoplastic myeloma cells were 83.9%, 0%, 98.4%, and 37.1%, respectively, among the 62 untreated cases, and 75.0%, 0%, 87.5%, and 37.5%, respectively, among the 8 treated cases. CD19 expression of neoplastic plasma cells was negative in both untreated and treated cases. The simplified immunophenotyping panel, CD56/CD19/CD138(CD38)/CD45, is useful for distinguishing neoplastic myeloma cells from reactive plasma cells in clinical practice. In addition, CD19 represents the most valuable antigen for identifying neoplastic myeloma cells in patients with MM.

  7. Detection of P-glycoprotein with a rapid flow cytometric functional assay using Fluo-3: evaluation of sensitivity, specificity and feasibility in multiparametric analysis.

    PubMed

    Van Acker, K L; De Greef, C; Eggermont, J; Zhang, P; Vandenberghe, P; Boogaerts, M A

    1995-08-01

    The specificity and sensitivity of a flow cytometric assay simultaneously measuring expression and transport function of the multidrug resistance associated P-glycoprotein (Pgp) was evaluated. The monoclonal antibody (mAb), MRK16 was used to detect phenotypic Pgp expression while Fluo-3-AM was used as a fluorescent substrate in a Pgp functional transport assay. The specificity of the functional assay was examined in two vinblastine selected human leukemic cell lines (K562/VLB2.5 and CCRF-CEM/VLB50) with acquired Pgp overexpression. Downmodulation of Pgp function in these cell lines could be demonstrated with different substances (verapamil, vinblastine, trifluoperazine, cyclosporin A, progesterone and quinidine) and was proven to be consistently higher in the vinblastine selected cells than in their non-selected drug sensitive counterparts. Unexpectedly, modulator activity was also observed in drug sensitive K562 and CCRF-CEM cell lines despite the inability to detect Pgp in those cells by MRK16 flow cytometrically. Low level expression of the MDR1 gene encoding Pgp in sensitive K562 cells was however demonstrated with a sensitive RT-PCR procedure. The small effect of Pgp modulators in non-drug selected cells could therefore be attributed to low level basal expression of Pgp and illustrates the sensitivity of the functional assay. Also, the effect of various Pgp modulators on Pgp function was more pronounced in a subpopulation of Pgp expressing lymphocytes than in lymphocytes which did not express Pgp. Finally, a correlation was found between discrete variations in Pgp expression and Pgp function of CD4+ lymphocytes, underscoring the feasibility of the functional assay in a triple parametric procedure. The triple parametric assay holds promise to detect Pgp expression and function in clinical samples containing mixtures of malignant and non-malignant cells.

  8. Human Immunodeficiency Virus Type 1 Neutralization Measured by Flow Cytometric Quantitation of Single-Round Infection of Primary Human T Cells

    PubMed Central

    Mascola, John R.; Louder, Mark K.; Winter, Christine; Prabhakara, Ranjani; De Rosa, Stephen C.; Douek, Daniel C.; Hill, Brenna J.; Gabuzda, Dana; Roederer, Mario

    2002-01-01

    There is currently intensive research on the design of novel human immunodeficiency virus type 1 (HIV-1) vaccine immunogens that can elicit potent neutralizing antibodies. A prerequisite for comparing and optimizing these strategies is the ability to precisely measure neutralizing antibody responses. To this end, we sought to develop an assay that directly quantifies single-round HIV-1 infection of peripheral blood mononuclear cells (PBMC). Initial experiments demonstrated that essentially all productively infected PBMC could be identified by flow cytometric detection of intracellular p24 antigen (p24-Ag). After infection of PBMC with HIV-1, p24+ lymphocytes could be distinguished beginning 1 day postinfection, and the majority of CD8− T cells were p24-Ag positive by 3 to 4 days postinfection. To directly quantify first-round infection, we included a protease inhibitor in PBMC cultures. The resulting 2-day assay was highly sensitive and specific for the detection of HIV-1-infected PBMC. Serial dilutions of virus stocks demonstrated that the number of target cells infected was directly related to the amount of infectious virus input into the assay. In neutralization assays, the flow cytometric enumeration of first-round infection of PBMC provided quantitative data on the number of target cells infected and on the inactivation of infectious virus due to reaction with antibody. We also used this single-round assay to compare the percentage of cells expressing p24-Ag to the number of copies of HIV-1 gag per 100 PBMC. The precision and reproducibility of this assay will facilitate the measurement of HIV-1 neutralization, particularly incrementally improved neutralizing antibody responses generated by new candidate vaccines. PMID:11967298

  9. Japanese Society for Laboratory Hematology flow cytometric reference method of determining the differential leukocyte count: external quality assurance using fresh blood samples.

    PubMed

    Kawai, Y; Nagai, Y; Ogawa, E; Kondo, H

    2017-04-01

    To provide target values for the manufacturers' survey of the Japanese Society for Laboratory Hematology (JSLH), accurate standard data from healthy volunteers were needed for the five-part differential leukocyte count. To obtain such data, JSLH required an antibody panel that achieved high specificity (particularly for mononuclear cells) using simple gating procedures. We developed a flow cytometric method for determining the differential leukocyte count (JSLH-Diff) and validated it by comparison with the flow cytometric differential leukocyte count of the International Council for Standardization in Haematology (ICSH-Diff) and the manual differential count obtained by microscopy (Manual-Diff). First, the reference laboratory performed an imprecision study of JSLH-Diff and ICSH-Diff, as well as performing comparison among JSLH-Diff, Manual-Diff, and ICSH-Diff. Then two reference laboratories and seven participating laboratories performed imprecision and accuracy studies of JSLH-Diff, Manual-Diff, and ICSH-Diff. Simultaneously, six manufacturers' laboratories provided their own representative values by using automated hematology analyzers. The precision of both JSLH-Diff and ICSH-Diff methods was adequate. Comparison by the reference laboratory showed that all correlation coefficients, slopes and intercepts obtained by the JSLH-Diff, ICSH-Diff, and Manual-Diff methods conformed to the criteria. When the imprecision and accuracy of JSLH-Diff were assessed at seven laboratories, the CV% for lymphocytes, neutrophils, monocytes, eosinophils, and basophils was 0.5~0.9%, 0.3~0.7%, 1.7~2.6%, 3.0~7.9%, and 3.8~10.4%, respectively. More than 99% of CD45 positive leukocytes were identified as normal leukocytes by JSLH-Diff. When JSLH-Diff method were validated by comparison with Manual-Diff and ICSH-Diff, JSLH-Diff showed good performance as a reference method. © 2016 John Wiley & Sons Ltd.

  10. Clustering of High Throughput Gene Expression Data

    PubMed Central

    Pirim, Harun; Ekşioğlu, Burak; Perkins, Andy; Yüceer, Çetin

    2012-01-01

    High throughput biological data need to be processed, analyzed, and interpreted to address problems in life sciences. Bioinformatics, computational biology, and systems biology deal with biological problems using computational methods. Clustering is one of the methods used to gain insight into biological processes, particularly at the genomics level. Clearly, clustering can be used in many areas of biological data analysis. However, this paper presents a review of the current clustering algorithms designed especially for analyzing gene expression data. It is also intended to introduce one of the main problems in bioinformatics - clustering gene expression data - to the operations research community. PMID:23144527

  11. Automated High Throughput Drug Target Crystallography

    SciTech Connect

    Rupp, B

    2005-02-18

    The molecular structures of drug target proteins and receptors form the basis for 'rational' or structure guided drug design. The majority of target structures are experimentally determined by protein X-ray crystallography, which as evolved into a highly automated, high throughput drug discovery and screening tool. Process automation has accelerated tasks from parallel protein expression, fully automated crystallization, and rapid data collection to highly efficient structure determination methods. A thoroughly designed automation technology platform supported by a powerful informatics infrastructure forms the basis for optimal workflow implementation and the data mining and analysis tools to generate new leads from experimental protein drug target structures.

  12. High throughput screening technologies for ion channels

    PubMed Central

    Yu, Hai-bo; Li, Min; Wang, Wei-ping; Wang, Xiao-liang

    2016-01-01

    Ion channels are involved in a variety of fundamental physiological processes, and their malfunction causes numerous human diseases. Therefore, ion channels represent a class of attractive drug targets and a class of important off-targets for in vitro pharmacological profiling. In the past decades, the rapid progress in developing functional assays and instrumentation has enabled high throughput screening (HTS) campaigns on an expanding list of channel types. Chronologically, HTS methods for ion channels include the ligand binding assay, flux-based assay, fluorescence-based assay, and automated electrophysiological assay. In this review we summarize the current HTS technologies for different ion channel classes and their applications. PMID:26657056