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Sample records for high-throughput flow cytometric

  1. High-throughput flow cytometric screening of combinatorial chemistry bead libraries for proteomics and drug discovery

    NASA Astrophysics Data System (ADS)

    Leary, James F.; Reece, Lisa M.; Yang, Xian-Bin; Gorenstein, David

    2005-04-01

    For proteomics drug discovery applications, combinatorial microbead thioaptamer libraries (one thioaptamer sequence per bead) are being created by split synthesis method, creating a "proteomics library" of protein capture beads which can be analyzed by high-throughput screening methods in this case, flow cytometry and cell sorting. Thioaptamers, oligonucleotides with thiophosphate backbone substitutions, function like antibodies in terms of recognizing specific protein sequences but have a number of advantages over antibody libraries. These proteomics beads can then be analyzed by high-speed flow cytometry and sorted to single-bead level depending on relative fluorescence brightness of fluorescently-labeled proteins, or for a specific protein from all of the molecules of cell subpopulations being analyzed. The thioaptamer sequences on a given bead showing high affinity for that protein can then be sequenced. Alternatively, the protein-capturing beads can be analyzed by MALDI-TOF mass spectrometry for analysis of the bound proteins. The beads can be thought of as equivalent to single-element positions of a proteomics chip arrays but with the advantage of being able to much more rapidly analyze hundreds of millions of possible amino acid sequences/epitopes on the basis of thioaptamer sequence affinities to select single sequences of interest. Additionally, those beads can be manipulated and isolated at the single bead level by high-throughput flow cytometry/cell sorting for subsequent sequencing of the thioaptamer sequences.

  2. Flow Cytometric Analysis of BrdU Incorporation as a High-Throughput Method for Measuring Adult Neurogenesis in the Mouse

    PubMed Central

    Balu, Darrick T.; Hodes, Georgia E.; Hill, Tiffany E.; Ho, Nancy; Rahman, Zia; Bender, Corey N.; Ring, Robert H.; Dwyer, Jason M.; Rosenzweig-Lipson, Sharon; Hughes, Zoe A.; Schechter, Lee E.; Lucki, Irwin

    2009-01-01

    Introduction The generation of new neurons occurs throughout adulthood in discrete brain regions, and may be regulated by neuropsychiatric diseases and therapeutic drug treatments. Most current methods that study this process measure the labeling of newborn cells by 5-bromo-2-deoxyuridine (BrdU) using immunohistochemical methods followed by the microscopic counting of BrdU positive cells. This method is time consuming and labor intensive, typically taking several weeks to analyze. Methods Therefore, we characterized a method to measure BrdU incorporation in the adult mouse hippocampus in vivo by using flow cytometry, which normally allows analysis of data within a single day. Results The present study compared multiple BrdU dosing and loading protocols to determine a dosing strategy that produced the best signal to noise ratio. BrdU incorporation was also compared across different brain regions. The method was sensitive to a number of experimental disease manipulations. Induction of type-1 diabetes and depletion of norepinephrine reduced hippocampal cell proliferation. In contrast, chronic administration of electroconvulsive shock, a somatic treatment for depression, as well as chronic treatment with the antidepressant fluoxetine elevated hippocampal cell proliferation. This increase in cell proliferation with fluoxetine was detected as early as 14 days into treatment. Moreover, comparing measures of cell proliferation obtained by immunohistochemical and flow cytometric methods within the same animals were convergent and significantly correlated to each other. Flow cytometry was also sufficiently sensitive to quantify the survival of newly born cells. Discussion These experiments validate the utility of flow cytometry in analyzing hippocampal cell proliferation and survival in a reliable and high-throughput fashion. The speedy analysis afforded by flow cytometry lends itself to be utilized in novel drug discovery and physiology. PMID:19121403

  3. 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.

  4. A high-throughput method for detection of DNA in chloroplasts using flow cytometry

    PubMed Central

    Rowan, Beth A; Oldenburg, Delene J; Bendich, Arnold J

    2007-01-01

    Background The amount of DNA in the chloroplasts of some plant species has been shown recently to decline dramatically during leaf development. A high-throughput method of DNA detection in chloroplasts is now needed in order to facilitate the further investigation of this process using large numbers of tissue samples. Results The DNA-binding fluorophores 4',6-diamidino-2-phenylindole (DAPI), SYBR Green I (SG), SYTO 42, and SYTO 45 were assessed for their utility in flow cytometric analysis of DNA in Arabidopsis chloroplasts. Fluorescence microscopy and real-time quantitative PCR (qPCR) were used to validate flow cytometry data. We found neither DAPI nor SYTO 45 suitable for flow cytometric analysis of chloroplast DNA (cpDNA) content, but did find changes in cpDNA content during development by flow cytometry using SG and SYTO 42. The latter dye provided more sensitive detection, and the results were similar to those from the fluorescence microscopic analysis. Differences in SYTO 42 fluorescence were found to correlate with differences in cpDNA content as determined by qPCR using three primer sets widely spaced across the chloroplast genome, suggesting that the whole genome undergoes copy number reduction during development, rather than selective reduction/degradation of subgenomic regions. Conclusion Flow cytometric analysis of chloroplasts stained with SYTO 42 is a high-throughput method suitable for determining changes in cpDNA content during development and for sorting chloroplasts on the basis of DNA content. PMID:17381841

  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-Based Single Cell Deposition for High-Throughput Screening of Protein Libraries

    PubMed Central

    Kalber, Tammy; Badar, Adam; Lythgoe, Mark; Pule, Martin

    2015-01-01

    The identification and engineering of proteins having refined or novel characteristics is an important area of research in many scientific fields. Protein modelling has enabled the rational design of unique proteins, but high-throughput screening of large libraries is still required to identify proteins with potentially valuable properties. Here we report on the development and evaluation of a novel fluorescent activated cell sorting based screening platform. Single bacterial cells, expressing a protein library to be screened, are electronically sorted and deposited onto plates containing solid nutrient growth media in a dense matrix format of between 44 and 195 colonies/cm2. We show that this matrix format is readily applicable to machine interrogation (<30 seconds per plate) and subsequent bioinformatic analysis (~60 seconds per plate) thus enabling the high-throughput screening of the protein library. We evaluate this platform and show that bacteria containing a bioluminescent protein can be spectrally analysed using an optical imager, and a rare clone (0.5% population) can successfully be identified, picked and further characterised. To further enhance this screening platform, we have developed a prototype electronic sort stream multiplexer, that when integrated into a commercial flow cytometric sorter, increases the rate of colony deposition by 89.2% to 24 colonies per second. We believe that the screening platform described here is potentially the foundation of a new generation of high-throughput screening technologies for proteins. PMID:26536118

  7. Flow-Based Single Cell Deposition for High-Throughput Screening of Protein Libraries.

    PubMed

    Stowe, Cassandra; Pizzey, Arnold; Kalber, Tammy; Badar, Adam; Lythgoe, Mark; Pule, Martin

    2015-01-01

    The identification and engineering of proteins having refined or novel characteristics is an important area of research in many scientific fields. Protein modelling has enabled the rational design of unique proteins, but high-throughput screening of large libraries is still required to identify proteins with potentially valuable properties. Here we report on the development and evaluation of a novel fluorescent activated cell sorting based screening platform. Single bacterial cells, expressing a protein library to be screened, are electronically sorted and deposited onto plates containing solid nutrient growth media in a dense matrix format of between 44 and 195 colonies/cm2. We show that this matrix format is readily applicable to machine interrogation (<30 seconds per plate) and subsequent bioinformatic analysis (~60 seconds per plate) thus enabling the high-throughput screening of the protein library. We evaluate this platform and show that bacteria containing a bioluminescent protein can be spectrally analysed using an optical imager, and a rare clone (0.5% population) can successfully be identified, picked and further characterised. To further enhance this screening platform, we have developed a prototype electronic sort stream multiplexer, that when integrated into a commercial flow cytometric sorter, increases the rate of colony deposition by 89.2% to 24 colonies per second. We believe that the screening platform described here is potentially the foundation of a new generation of high-throughput screening technologies for proteins. PMID:26536118

  8. High-throughput single-microparticle imaging flow analyzer

    PubMed Central

    Goda, Keisuke; Ayazi, Ali; Gossett, Daniel R.; Sadasivam, Jagannath; Lonappan, Cejo K.; Sollier, Elodie; Fard, Ali M.; Hur, Soojung Claire; Adam, Jost; Murray, Coleman; Wang, Chao; Brackbill, Nora; Di Carlo, Dino; Jalali, Bahram

    2012-01-01

    Optical microscopy is one of the most widely used diagnostic methods in scientific, industrial, and biomedical applications. However, while useful for detailed examination of a small number (< 10,000) of microscopic entities, conventional optical microscopy is incapable of statistically relevant screening of large populations (> 100,000,000) with high precision due to its low throughput and limited digital memory size. We present an automated flow-through single-particle optical microscope that overcomes this limitation by performing sensitive blur-free image acquisition and nonstop real-time image-recording and classification of microparticles during high-speed flow. This is made possible by integrating ultrafast optical imaging technology, self-focusing microfluidic technology, optoelectronic communication technology, and information technology. To show the system’s utility, we demonstrate high-throughput image-based screening of budding yeast and rare breast cancer cells in blood with an unprecedented throughput of 100,000 particles/s and a record false positive rate of one in a million. PMID:22753513

  9. Flow Cytometric Findings in Hemophagocytic Lymphohistiocytosis

    PubMed Central

    McCall, Chad M.; Mudali, Shiyama; Arceci, Robert J.; Small, Donald; Fuller, Shirley; Gocke, Christopher D.; Vuica-Ross, Milena; Burns, Kathleen H.; Borowitz, Michael J.; Duffield, Amy S.

    2016-01-01

    Hemophagocytic lymphohistiocytosis (HLH) is an often fatal hyperinflammatory syndrome. HLH may be inherited, but it more commonly arises secondary to Epstein-Barr virus (EBV) or other infections, hematologic malignancies, or rheumatologic diseases. We identified 17 patients diagnosed with HLH who had flow cytometric analysis of peripheral blood or bone marrow performed at the time of diagnosis. Two patients had primary HLH, and the others had HLH secondary to EBV infection, hematologic malignancies, rheumatologic conditions, or tuberculosis. The marrow typically showed a reactive lymphocytosis and a marked left shift in myelopoiesis regardless of the etiology. Qualitative abnormalities were also found in several cases, including T-cell abnormalities in the majority of the EBV-associated HLH cases. While not specific, flow cytometric findings in HLH are different from the findings in uninvolved marrow samples, and care should be taken not to overinterpret immunophenotypic findings in these cases as indicative of a primary marrow disorder or lymphoma. PMID:22523218

  10. Development of a Human Whole Blood Screening Platform to Monitor JAK/STAT Signaling Using High-Throughput Flow Cytometry.

    PubMed

    Fereshteh, Mark P; Li, Xin; Li, Sha; Fan, Yi; Zhang, Rosemary; Farr, Glen A; Kolodin, Garrett; Lippy, Jonathan; Naglich, Joseph G; Schieven, Gary; Schweizer, Liang; Zhang, Litao

    2016-09-01

    Oral agents targeting Janus-associated kinases (JAKs) are promising new agents in clinical development. To better understand the relationship between JAK inhibition and biological outcome, compounds targeting JAKs were evaluated in peripheral human whole blood. To date, these analyses are low throughput and costly. Here, we developed a robust 384-well, high-throughput flow-based assay approach to screen small molecules for JAK/STAT signaling inhibition in human whole blood. This assay platform provides a highly sensitive analysis of signaling events in blood and facilitates measurement of target engagement. Further, the automation technologies and process optimizations developed here overcame sample integrity, handling, and multiparametric data analysis bottlenecks without affecting assay performance. Together these efforts dramatically increased sample throughput compared to conventional manual flow cytometric approaches and enabled development of novel JAK/STAT inhibitors. PMID:27142718

  11. Viscoelasticity as a Biomarker for High-Throughput Flow Cytometry

    PubMed Central

    Sawetzki, Tobias; Eggleton, Charles D.; Desai, Sanjay A.; Marr, David W.M.

    2013-01-01

    The mechanical properties of living cells are a label-free biophysical marker of cell viability and health; however, their use has been greatly limited by low measurement throughput. Although examining individual cells at high rates is now commonplace with fluorescence activated cell sorters, development of comparable techniques that nondestructively probe cell mechanics remains challenging. A fundamental hurdle is the signal response time. Where light scattering and fluorescence signatures are virtually instantaneous, the cell stress relaxation, typically occurring on the order of seconds, limits the potential speed of elastic property measurement. To overcome this intrinsic barrier to rapid analysis, we show here that cell viscoelastic properties measured at frequencies far higher than those associated with cell relaxation can be used as a means of identifying significant differences in cell phenotype. In these studies, we explore changes in erythrocyte mechanical properties caused by infection with Plasmodium falciparum and find that the elastic response alone fails to detect malaria at high frequencies. At timescales associated with rapid assays, however, we observe that the inelastic response shows significant changes and can be used as a reliable indicator of infection, establishing the dynamic viscoelasticity as a basis for nondestructive mechanical analogs of current high-throughput cell classification methods. PMID:24268140

  12. 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.

  13. Label-free high-throughput cell screening in flow

    PubMed Central

    Mahjoubfar, Ata; Chen, Claire; Niazi, Kayvan R.; Rabizadeh, Shahrooz; Jalali, Bahram

    2013-01-01

    Flow cytometry is a powerful tool for cell counting and biomarker detection in biotechnology and medicine especially with regards to blood analysis. Standard flow cytometers perform cell type classification both by estimating size and granularity of cells using forward- and side-scattered light signals and through the collection of emission spectra of fluorescently-labeled cells. However, cell surface labeling as a means of marking cells is often undesirable as many reagents negatively impact cellular viability or provide activating/inhibitory signals, which can alter the behavior of the desired cellular subtypes for downstream applications or analysis. To eliminate the need for labeling, we introduce a label-free imaging-based flow cytometer that measures size and cell protein concentration simultaneously either as a stand-alone instrument or as an add-on to conventional flow cytometers. Cell protein concentration adds a parameter to cell classification, which improves the specificity and sensitivity of flow cytometers without the requirement of cell labeling. This system uses coherent dispersive Fourier transform to perform phase imaging at flow speeds as high as a few meters per second. PMID:24049682

  14. FLOW CYTOMETRIC ANALYSIS OF THE CELLULAR TOXICITY TRIBUTYLTIN

    EPA Science Inventory

    Flow cytometric and light/fluorescence microscopic analyses indicate that tributyltin (TBT) alters the plasma membrane/cytoplasm complex of the murine erythroleukemic cell (MELC) in a dose dependent and time-dependent manner. he flow cytometric parameter axial light loss, a measu...

  15. FLOW CYTOMETRIC ANALYSIS OF THE CELLULAR TOXICITY OF TRIBUTYLIN

    EPA Science Inventory

    Flow cytometric and light/fluorescence microscopic analyses indicate that tributylin (TBT) alters the plasma membrane/cytoplasm complex of the murine erythrleukemic cell (MELC) in a dose-dependent and time-dependent manner. he flow cytometric parameter axial light loss, a measure...

  16. 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

  17. Flow cytometric evaluation of multicystic dysplastic kidneys.

    PubMed

    Jung, W H; Peters, C A; Mandell, J; Vawter, G F; Retik, A B

    1990-08-01

    The most appropriate management of the multicystic dysplastic kidney remains controversial. At issue is the long-term risk of the development of malignancy in the multicystic dysplastic kidney. The association between renal dysplasia and neoplasia has not been confirmed, with only 6 cases of malignancy reported. Nephroblastomatosis, a probable precursor of Wilms tumor, has been found in 5 to 7% of the cases of multicystic dysplastic kidney when specifically sought. In an attempt to determine whether a relationship exists between renal dysplasia and neoplasia in terms of abnormalities of cellular deoxyribonucleic acid content we performed flow cytometric evaluation on 30 formalin fixed, paraffin embedded archival specimens of multicystic dysplastic kidneys. None of the kidneys had evidence of malignancy. Nuclear deoxyribonucleic acid ploidy studies were performed on single dissociated nuclei prepared by the technique of McLemore and associates and stained with propidium iodide. All specimens demonstrated a diploid pattern of deoxyribonucleic acid, including 3 specimens with nephroblastomatosis or extensive papillary growth, and no specimen demonstrated a tetraploid or aneuploid pattern. The mean G0/G1 fraction was 85.94% (standard deviation 4.59) and the mean S/G2/M fraction was 12.54% (standard deviation 4.72). These findings do not support or negate the potential for neoplasm associated with multicystic dysplastic kidney, since a diploid deoxyribonucleic acid pattern does not eliminate the possibility of the future development of malignancy. PMID:2374213

  18. 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.

  19. flowCore: a Bioconductor package for high throughput flow cytometry

    PubMed Central

    Hahne, Florian; LeMeur, Nolwenn; Brinkman, Ryan R; Ellis, Byron; Haaland, Perry; Sarkar, Deepayan; Spidlen, Josef; Strain, Errol; Gentleman, Robert

    2009-01-01

    Background Recent advances in automation technologies have enabled the use of flow cytometry for high throughput screening, generating large complex data sets often in clinical trials or drug discovery settings. However, data management and data analysis methods have not advanced sufficiently far from the initial small-scale studies to support modeling in the presence of multiple covariates. Results We developed a set of flexible open source computational tools in the R package flowCore to facilitate the analysis of these complex data. A key component of which is having suitable data structures that support the application of similar operations to a collection of samples or a clinical cohort. In addition, our software constitutes a shared and extensible research platform that enables collaboration between bioinformaticians, computer scientists, statisticians, biologists and clinicians. This platform will foster the development of novel analytic methods for flow cytometry. Conclusion The software has been applied in the analysis of various data sets and its data structures have proven to be highly efficient in capturing and organizing the analytic work flow. Finally, a number of additional Bioconductor packages successfully build on the infrastructure provided by flowCore, open new avenues for flow data analysis. PMID:19358741

  20. Flow cytometric sexing of mammalian sperm.

    PubMed

    Garner, Duane L

    2006-03-15

    This review reexamines parameters needed for optimization of flow cytometric sexing mammalian sperm and updates the current status of sperm sexing for various species where this technology is currently being applied. Differences in DNA content have provided both a method to differentiate between these sex-determining gametes and a method to sort them that can be used for predetermining sex in mammals. Although the DNA content of all cells for each mammalian species is highly conserved, slight but measurable DNA content differences of sperm occur within species even among cattle breeds due to different sizes of Y-chromosomes. Most mammals produce flattened, oval-headed sperm that can be oriented within a sorter using hydrodynamic forces. Multiplying the percentage the difference in DNA content of the X- or Y-chromosome bearing sperm times the area of the flat profile of the sperm head gives a simple sorting index that suggests that bull and boar sperm are well suited for separation in a flow sorter. Successful sperm sexing of various species must take into account the relative susceptibilities of gametes to the stresses that occur during sexing. Sorting conditions must be optimized for each species to achieve acceptable sperm sexing efficiency, usually at 90% accuracy. In the commercial application of sperm sexing to cattle, fertility of sex-sorted bull sperm at 2 x 10(6)/dose remains at 70-80% of unsexed sperm at normal doses of 10 to 20 x 10(6) sperm. DNA content measurements have been used to identify the sex-chromosome bearing sperm populations with good accuracy in semen from at least 23 mammalian species, and normal-appearing offspring have been produced from sexed sperm of at least seven species. PMID:16242764

  1. 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

  2. An on-bacterium flow cytometric immunoassay for protein quantification.

    PubMed

    Lan, Wen-Jun; Lan, Wei; Wang, Hai-Yan; Yan, Lei; Wang, Zhe-Li

    2013-09-01

    The polystyrene bead-based flow cytometric immunoassay has been widely reported. However, the preparation of functional polystyrene bead is still inconvenient. This study describes a simple and easy on-bacterium flow cytometric immunoassay for protein quantification, in which Staphylococcus aureus (SAC) is used as an antibody-antigen carrier to replace the polystyrene bead. The SAC beads were prepared by carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling, paraformaldehyde fixation and antibody binding. Carcinoembryonic antigen (CEA) and cytokeratin-19 fragment (CYFRA 21-1) proteins were used as models in the test system. Using prepared SAC beads, biotinylated proteins, and streptavidin-phycoerythrin (SA-PE), the on-bacterium flow cytometric immunoassay was validated by quantifying CEA and CYFRA 21-1 in sample. Obtained data demonstrated a concordant result between the logarithm of the protein concentration and the logarithm of the PE mean fluorescence intensity (MFI). The limit of detection (LOD) in this immunoassay was at least 0.25 ng/ml. Precision and accuracy assessments appeared that either the relative standard deviation (R.S.D.) or the relative error (R.E.) was <10%. The comparison between this immunoassay and a polystyrene bead-based flow cytometric immunoassay showed a correlation coefficient of 0.998 for serum CEA or 0.996 for serum CYFRA 21-1. In conclusion, the on-bacterium flow cytometric immunoassay may be of use in the quantification of serum protein. PMID:23739299

  3. Automated high-dimensional flow cytometric data analysis

    PubMed Central

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

    2009-01-01

    Flow cytometric analysis allows rapid single cell interrogation of surface and intracellular determinants by measuring fluorescence intensity of fluorophore-conjugated reagents. The availability of new platforms, allowing detection of increasing numbers of cell surface markers, has challenged the traditional technique of identifying cell populations by manual gating and resulted in a growing need for the development of automated, high-dimensional analytical methods. We present a direct multivariate finite mixture modeling approach, using skew and heavy-tailed distributions, to address the complexities of flow cytometric analysis and to deal with high-dimensional cytometric data without the need for projection or transformation. We demonstrate its ability to detect rare populations, to model robustly in the presence of outliers and skew, and to perform the critical task of matching cell populations across samples that enables downstream analysis. This advance will facilitate the application of flow cytometry to new, complex biological and clinical problems. PMID:19443687

  4. Flow-Cytometric Isolation of Human Antibodies from a Nonimmune Saccharomyces cerevisiae Surface Display Library

    SciTech Connect

    Feldhaus, Michael ); Siegel, Robert W. ); Opresko, Lee ); Coleman, James R. ); Feldhaus, Jane M. ); Yeung, Yik A.; Cochran, Jennifer R.; Heinzelman, Peter; Colby, David; Swers, Jeffrey; Graff, Christilyn; Wiley, H Steven ); Wittrup, K D.

    2003-02-28

    A nonimmune library of 109 human antibody scFv fragments has been cloned and expressed on the surface of yeast, and nanomolar-affinity scFvs routinely obtained by magnetic bead screening and flow cytometric sorting. The yeast library can be amplified 1010-fold without measurable loss of clonal diversity, enabling effectively indefinite expansion of the library. The expression, stability, and antigen binding properties of more than 50 isolated scFv clones were assessed directly on the yeast cell surface by immunofluorescent labeling and flow cytometry, obviating separate subcloning, expression, and purification steps and thereby expediting the isolation of novel affinity reagents. The ability to use multiplex library screening demonstrates the utility of this approach for high throughput antibody isolation for proteomics applications.

  5. Label-free cell cycle analysis for high-throughput imaging flow cytometry

    PubMed Central

    Blasi, Thomas; Hennig, Holger; Summers, Huw D.; Theis, Fabian J.; Cerveira, Joana; Patterson, James O.; Davies, Derek; Filby, Andrew; Carpenter, Anne E.; Rees, Paul

    2016-01-01

    Imaging flow cytometry combines the high-throughput capabilities of conventional flow cytometry with single-cell imaging. Here we demonstrate label-free prediction of DNA content and quantification of the mitotic cell cycle phases by applying supervised machine learning to morphological features extracted from brightfield and the typically ignored darkfield images of cells from an imaging flow cytometer. This method facilitates non-destructive monitoring of cells avoiding potentially confounding effects of fluorescent stains while maximizing available fluorescence channels. The method is effective in cell cycle analysis for mammalian cells, both fixed and live, and accurately assesses the impact of a cell cycle mitotic phase blocking agent. As the same method is effective in predicting the DNA content of fission yeast, it is likely to have a broad application to other cell types. PMID:26739115

  6. Analysis of High-Throughput Flow Cytometry Data Using plateCore

    PubMed Central

    Strain, Errol; Hahne, Florian; Brinkman, Ryan R.; Haaland, Perry

    2009-01-01

    Flow cytometry (FCM) software packages from R/Bioconductor, such as flowCore and flowViz, serve as an open platform for development of new analysis tools and methods. We created plateCore, a new package that extends the functionality in these core packages to enable automated negative control-based gating and make the processing and analysis of plate-based data sets from high-throughput FCM screening experiments easier. plateCore was used to analyze data from a BD FACS CAP screening experiment where five Peripheral Blood Mononucleocyte Cell (PBMC) samples were assayed for 189 different human cell surface markers. This same data set was also manually analyzed by a cytometry expert using the FlowJo data analysis software package (TreeStar, USA). We show that the expression values for markers characterized using the automated approach in plateCore are in good agreement with those from FlowJo, and that using plateCore allows for more reproducible analyses of FCM screening data. PMID:19956418

  7. 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

  8. 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.

  9. 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.

  10. 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

  11. 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

  12. A fluorescent hydrogel-based flow cytometry high-throughput screening platform for hydrolytic enzymes.

    PubMed

    Pitzler, Christian; Wirtz, Georgette; Vojcic, Ljubica; Hiltl, Stephanie; Böker, Alexander; Martinez, Ronny; Schwaneberg, Ulrich

    2014-12-18

    Screening throughput is a key in directed evolution experiments and enzyme discovery. Here, we describe a high-throughput screening platform based on a coupled reaction of glucose oxidase and a hydrolase (Yersinia mollaretii phytase [YmPh]). The coupled reaction produces hydroxyl radicals through Fenton's reaction, acting as initiator of poly(ethyleneglycol)-acrylate-based polymerization incorporating a fluorescent monomer. As a consequence, a fluorescent hydrogel is formed around Escherichia coli cells expressing active YmPh. We achieve five times enrichment of active cell population through flow cytometry analysis and sorting of mixed populations. Finally, we validate the performance of the fluorescent polymer shell (fur-shell) technology by directed phytase evolution that yielded improved variants starting from a library containing 10(7) phytase variants. Thus, fur-shell technology represents a rapid and nonlaborious way of identifying the most active variants from vast populations, as well as a platform for generation of polymer-hybrid cells for biobased interactive materials. PMID:25525992

  13. 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.

  14. A Rapid, Multiplexed, High-Throughput Flow-Through Membrane Immunoassay: A Convenient Alternative to ELISA

    PubMed Central

    Ramachandran, Sujatha; Singhal, Mitra; McKenzie, Katherine G.; Osborn, Jennifer L.; Arjyal, Amit; Dongol, Sabina; Baker, Stephen G.; Basnyat, Buddha; Farrar, Jeremy; Dolecek, Christiane; Domingo, Gonzalo J.; Yager, Paul; Lutz, Barry

    2013-01-01

    This paper describes a rapid, high-throughput flow-through membrane immunoassay (FMIA) platform. A nitrocellulose membrane was spotted in an array format with multiple capture and control reagents for each sample detection area, and assay steps were carried out by sequential aspiration of sample and reagents through each detection area using a 96-well vacuum manifold. The FMIA provides an alternate assay format with several advantages over ELISA. The high surface area of the membrane permits high label concentration using gold labels, and the small pores and vacuum control provide rapid diffusion to reduce total assay time to ~30 min. All reagents used in the FMIA are compatible with dry storage without refrigeration. The results appear as colored spots on the membrane that can be quantified using a flatbed scanner. We demonstrate the platform for detection of IgM specific to lipopolysaccharides (LPS) derived from Salmonella Typhi. The FMIA format provides analytical results comparable to ELISA in less time, provides integrated assay controls, and allows compensation for specimen-to-specimen variability in background, which is a particular challenge for IgM assays. PMID:26835678

  15. Flow cytometric detection method for DNA samples

    DOEpatents

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

    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.

  16. 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.

  17. Flow cytometric detection of wild yeast in lager breweries.

    PubMed

    Jespersen, L; Lassen, S; Jakobsen, M

    1993-02-01

    A flow cytometric method for detection of wild yeast infections in breweries is reported. It is based on selective enrichment in Malt extract Yeast extract Glucose Peptone broth (MYGP) at 37 degrees C and in MYGP with 200 ppm CuSO4 at 25 degrees C, staining with a fluorochrome precursor and flow cytometry. In experiments with several types of wild yeast isolated from breweries and two different strains of lager yeast it has been possible to detect one wild yeast per 10(6) culture yeast after 48-72 h of incubation and, in some cases, after 24 h. PMID:8466805

  18. Development of a Robust Flow Cytometric Assay for Determining Numbers of Viable Bacteria

    PubMed Central

    Jepras, R. I.; Carter, J.; Pearson, S. C.; Paul, F. E.; Wilkinson, M. J.

    1995-01-01

    Several fluorescent probes were evaluated as indicators of bacterial viability by flow cytometry. The probes monitor a number of biological factors that are altered during loss of viability. The factors include alterations in membrane permeability, monitored by using fluorogenic substrates and fluorescent intercalating dyes such as propidium iodide, and changes in membrane potential, monitored by using fluorescent cationic and anionic potential-sensitive probes. Of the fluorescent reagents examined, the fluorescent anionic membrane potential probe bis-(1,3-dibutylbarbituric acid)trimethine oxonol [DiBAC(inf4)(3)] proved the best candidate for use as a general robust viability marker and is a promising choice for use in high-throughput assays. With this probe, live and dead cells within a population can be identified and counted 10 min after sampling. There was a close correlation between viable counts determined by flow cytometry and by standard CFU assays for samples of untreated cells. The results indicate that flow cytometry is a sensitive analytical technique that can rapidly monitor physiological changes of individual microorganisms as a result of external perturbations. The membrane potential probe DiBAC(inf4)(3) provided a robust flow cytometric indicator for bacterial cell viability. PMID:16535078

  19. 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.

  20. 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

  1. 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

    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

    2016-04-01

    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

  2. Uncovering Aberrant Mutant PKA Function with Flow Cytometric FRET.

    PubMed

    Lee, Shin-Rong; Sang, Lingjie; Yue, David T

    2016-03-29

    Biology has been revolutionized by tools that allow the detection and characterization of protein-protein interactions (PPIs). Förster resonance energy transfer (FRET)-based methods have become particularly attractive as they allow quantitative studies of PPIs within the convenient and relevant context of living cells. We describe here an approach that allows the rapid construction of live-cell FRET-based binding curves using a commercially available flow cytometer. We illustrate a simple method for absolutely calibrating the cytometer, validating our binding assay against the gold standard isothermal calorimetry (ITC), and using flow cytometric FRET to uncover the structural and functional effects of the Cushing-syndrome-causing mutation (L206R) on PKA's catalytic subunit. We discover that this mutation not only differentially affects PKAcat's binding to its multiple partners but also impacts its rate of catalysis. These findings improve our mechanistic understanding of this disease-causing mutation, while illustrating the simplicity, general applicability, and power of flow cytometric FRET. PMID:26997269

  3. Uncovering aberrant mutant PKA function with flow cytometric FRET

    PubMed Central

    Lee, Shin-Rong; Sang, Lingjie; Yue, David T.

    2016-01-01

    SUMMARY Biology has been revolutionized by tools that allow the detection and characterization of protein-protein interactions (PPI). Förster resonance energy transfer (FRET)-based methods have become particularly attractive as they allow quantitative studies of PPIs within the convenient and relevant context of living cells. We describe here an approach that allows the rapid construction of live-cell, FRET-based binding curves using a commercially-available flow cytometer. We illustrate a simple method for absolutely calibrating the cytometer, validate our binding assay against the gold-standard isothermal calorimetry (ITC), and use flow cytometric FRET to uncover the structural and functional effects of the Cushing syndrome-causing mutation (L206R) on PKA’s catalytic subunit. We discover that this mutation not only differentially affects PKAcat’s binding to its multiple partners, but also impacts its rate of catalysis. These findings improve our mechanistic understanding of this disease-causing mutation, while illustrating the simplicity, general applicability and power of flow cytometric FRET. PMID:26997269

  4. 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.

  5. Flow Cytometric Analysis of Immune Cells Within Murine Aorta.

    PubMed

    Gjurich, Breanne N; Taghavie-Moghadam, Parésa L; Galkina, Elena V

    2015-01-01

    The immune system plays a critical role in the modulation of atherogenesis at all stages of the disease. However, there are many technical difficulties when studying the immune system within murine aortas. Common techniques such as PCR and immunohistochemistry have answered many questions about the presence of immune cells and mediators of inflammation within the aorta yet many questions remain unanswered due to the limitations of these techniques. On the other hand, cumulatively the flow cytometry approach has propelled the immunology field forward but it has been challenging to apply this technique to aortic tissues. Here, we describe the methodology to isolate and characterize the immune cells within the murine aorta and provide examples of functional assays for aortic leukocytes using flow cytometry. The method involves the harvesting and enzymatic digestion of the aorta, extracellular and intracellular protein staining, and a subsequent flow cytometric analysis. PMID:26445788

  6. A Novel Flow Cytometric HTS Assay Reveals Functional Modulators of ATP Binding Cassette Transporter ABCB6

    PubMed Central

    Chavan, Hemantkumar; Young, Susan; Ma, Xiaochao; Waller, Anna; Garcia, Matthew; Perez, Dominique; Chavez, Stephanie; Strouse, Jacob J.; Haynes, Mark K.; Bologa, Cristian G.; Oprea, Tudor I.; Tegos, George P.; Sklar, Larry A.; Krishnamurthy, Partha

    2012-01-01

    ABCB6 is a member of the adenosine triphosphate (ATP)-binding cassette family of transporter proteins that is increasingly recognized as a relevant physiological and therapeutic target. Evaluation of modulators of ABCB6 activity would pave the way toward a more complete understanding of the significance of this transport process in tumor cell growth, proliferation and therapy-related drug resistance. In addition, this effort would improve our understanding of the function of ABCB6 in normal physiology with respect to heme biosynthesis, and cellular adaptation to metabolic demand and stress responses. To search for modulators of ABCB6, we developed a novel cell-based approach that, in combination with flow cytometric high-throughput screening (HTS), can be used to identify functional modulators of ABCB6. Accumulation of protoporphyrin, a fluorescent molecule, in wild-type ABCB6 expressing K562 cells, forms the basis of the HTS assay. Screening the Prestwick Chemical Library employing the HTS assay identified four compounds, benzethonium chloride, verteporfin, tomatine hydrochloride and piperlongumine, that reduced ABCB6 mediated cellular porphyrin levels. Validation of the identified compounds employing the hemin-agarose affinity chromatography and mitochondrial transport assays demonstrated that three out of the four compounds were capable of inhibiting ABCB6 mediated hemin transport into isolated mitochondria. However, only verteporfin and tomatine hydrochloride inhibited ABCB6’s ability to compete with hemin as an ABCB6 substrate. This assay is therefore sensitive, robust, and suitable for automation in a high-throughput environment as demonstrated by our identification of selective functional modulators of ABCB6. Application of this assay to other libraries of synthetic compounds and natural products is expected to identify novel modulators of ABCB6 activity. PMID:22808084

  7. FLOW CYTOMETRIC ANALYSIS OF THE CELLULAR TOXICITY OF TRIBUTYLTIN (JOURNAL VERSION)

    EPA Science Inventory

    Flow cytometric and light/fluorescence microscopic analyses indicate that tributyltin (TBT) alters the plasma membrane/cytoplasm complex of the murine erythroleukemic cell (MELC) in a dose-dependent and time-dependent manner. The flow cytometric parameter axial light loss, a meas...

  8. Flow cytometric detection of pathogenic E. coli in food.

    PubMed

    Raybourne, R B

    2001-05-01

    E. coli O157:H7 is one of the more important food pathogens, andrapid, quantitative methods to evaluate foods for the presence of this pathogen are needed. This unit provides exactly that: a very much simplified flow cytometric assay for detection of E. coli O157:H7 in a well established vehicle of infection, ground beef. The method uses commercially available FITC-conjugated specific antibody to this bacterial serotype. Sample preparation and bacterial enrichment procedures are described. Direct and indirect approaches for quantification of the number of bacteria are given. A key feature of the assay is the reduction in time compared with plate-counting methods; the tradeoff is a slight reduction in sensitivity. Particularly useful is the simultaneous inclusion of a spiked sample to ensure a positive control. In addition, the unit provides hints on sorting the organisms if desired. PMID:18770690

  9. 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.

  10. 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.

  11. 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. PMID:25074448

  12. 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. PMID:21116998

  13. Real Time Detection of Protein Trafficking with High Throughput Flow Cytometry (HTFC) and Fluorogen Activating Protein (FAP) Base Biosensor

    PubMed Central

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

    2014-01-01

    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, that 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 ~1,200 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 non-traditional 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. PMID:24510772

  14. High-throughput detection and quantification of mitochondrial fusion through imaging flow cytometry.

    PubMed

    Nascimento, Aldo; Lannigan, Joanne; Kashatus, David

    2016-08-01

    Mitochondria are highly dynamic organelles whose fusion and fission play an increasingly important role in a number of both normal and pathological cellular functions. Despite the increased interest in mitochondrial dynamics, robust, and quantitative methods to analyze mitochondrial fusion and fission activity in intact cells have not been developed. The current state-of-the art method to measure mitochondrial fusion activity is the polyethylene glycol (PEG) fusion assay in which cells expressing distinct mitochondrially-targeted fluorescent proteins (FPs) are fused together and mitochondrial fusion activity is determined by the rate at which color mixing occurs. Although this assay is useful, cell-cell fusion events are rare, and finding the number of fused cells required to generate statistically rigorous data is both tedious and time-consuming. Furthermore, the data-collection methods available for fluorescence microscopy lead to inherent selection biases that are difficult to control for. To that end, we have developed an unbiased and high-throughput method to detect, image, and analyze fused cells using the Amnis ImagestreamX™ MKII. With IDEAS™ software, we developed algorithms for identifying the fused cells (two nuclei within a single cell), distinguishing them from cell aggregates. Additionally, using the fluorescence localization of the mitochondrially-targeted fluorescent proteins (YFP and DsRed), we applied a modified co-localization algorithm to identify those cells that had a high co-localization score indicating mitochondrial fusion activity. These algorithms were tested using negative controls (FPs associated with fusion deficient mitochondria) and positive controls (cells expressing both FPs in the same mitochondria). Once validated these algorithms could be applied to test samples to evaluate the degree of mitochondrial fusion in cells with various genetic mutations. Ultimately, this new method is the first robust, high-throughput way to

  15. 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

  16. Reticulated platelets interfere with flow cytometric reticulocyte counts.

    PubMed

    Ivory, K; Sarria, B; Fairweather-Tait, S J; Hughes, D A

    2007-10-01

    As part of an iron absorption study, we needed to accurately count reticulocytes in the peripheral blood of healthy human volunteers before measuring their enrichment with stable iron isotopes given in an oral dose. Recent studies have suggested the usefulness of reticulocyte counting by flow cytometry, through a combination of differential light scatter and measurement of the stoichiometric binding of thiazole orange (TO) to RNA within the maturing erythrocyte. Using this method we set out to improve the precision of our quantitative analysis by counting more cells, as reticulocytes normally comprise <2% of the red cell population. To ensure exclusion of other cell types, we identified WBCs and platelets with CD16+CD45- allophycocyanin and CD61- phycoerythrin, respectively. After removal of CD16(+) CD45(+) TO(+) WBCs and CD61(+) TO(-) platelets from analysis, the remaining cells were a combination of CD61(-) TO(-) erythrocytes, CD61(-) TO(+) reticulocytes and CD61(+) TO(+) reticulated platelets. Reticulocyte counts were lower after exclusion of CD61(+) TO(+) cells from analysis. They were similarly lower when erythrocyte precursors were positively identified through their glycophorin A expression and TO uptake. We conclude that it is necessary to exclude reticulated platelets from flow cytometric reticulocyte analysis. PMID:17824916

  17. Flow cytometric analysis of circulating microparticles in plasma.

    PubMed

    Orozco, Aaron F; Lewis, Dorothy E

    2010-06-01

    Microparticles, which include exosomes, micro-vesicles, apoptotic bodies and apoptotic microparticles, are small (0.05 - 3 mum in diameter), membranous vesicles that can contain DNA, RNA, miRNA, intracellular proteins and express extracellular surface markers from the parental cells. They can be secreted from intracellular multivesicular bodies or released from the surface of blebbing membranes. Circulating microparticles are abundant in the plasma of normal individuals and can be derived from circulating blood cells such as platelets, red blood cells and leukocytes as well as from tissue sources, such as endothelial and placental tissues. Elevated levels of microparticles are associated with various diseases such as thrombosis (platelet microparticles), congestive heart failure (endothelial microparticles), breast cancer patients (leukocyte microparticles) and women with preeclampsia (syncytiotrophoblast microparticles). Although microparticles can be detected by microscopy, enzyme-linked immunoassays and functional assays, flow cytometry is the preferred method because of the ability to quantitate (fluorescent bead- or flow rate-based method) and because of polychromatic capabilities. However, standardization of pre-analytical and analytical modus operandi for isolating, enumerating and fluorescent labeling of microparticles remains a challenge. The primary focus of this article is to review the preliminary steps required to optimally study circulating in vivo microparticles which include: 1) centrifugation speed used, 2) quantitation of microparticles before antibody labeling, 3) levels of fluorescence intensity of antibody-labeled microparticles, 4) polychromatic flow cytometric analysis of microparticle sub-populations and 5) use of polyclonal antibodies designed for Western blotting for flow cytometry. These studies determine a roadmap to develop microparticles as biomarkers for a variety of conditions. PMID:20235276

  18. 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.

  19. 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

  20. 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. PMID:26858703

  1. Carcinoma of the anal canal and flow cytometric DNA analysis.

    PubMed Central

    Scott, N. A.; Beart, R. W.; Weiland, L. H.; Cha, S. S.; Lieber, M. M.

    1989-01-01

    Using flow cytometric DNA analysis of paraffin embedded tissue, DNA histograms were successfully obtained from the anal cancers of 117 patients. DNA diploid patterns were given by 82 cancers (70%) and DNA non-diploid patterns by 35 cancers (30%): 15 DNA aneuploid, 20 DNA tetraploid. Well differentiated squamous cell cancers were mainly DNA diploid, while a larger proportion of poorly differentiated and small cell cancers were DNA non-diploid. The large majority of stage A cancers were DNA diploid. A greater proportion of tumours that had invaded through the anal sphincter or had lymph node metastases or distant spread were DNA non-diploid. Prognosis was slightly poorer for patients with DNA non-diploid cancers when compared to patients with DNA diploid tumours (P = 0.08) and significantly poorer for individuals with DNA aneuploid anal cancers (P = 0.037). However, in a multivariate analysis model, the DNA ploidy pattern of an anal cancer was not of independent prognostic significance alongside tumour histology and tumour stage. PMID:2803916

  2. 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.

  3. COMPARISON OF CELLULAR AND NUCLEAR FLOW CYTOMETRIC TECHNIQUES FOR DISCRIMINATING APOPTOTIC SUBPOPULATIONS

    EPA Science Inventory

    We compared cellular flow cytometric methods employing carboxyfluorescein (CF), Hoechst 33342, and Hoechst 33258 with a nuclear method in their ability to discriminate apoptotic subpopulations in rat thymocyte cultures exposed to dexamethasone r tributyltin. n the nuclear techniq...

  4. 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.

  5. 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. PMID:24793853

  6. Flow Cytometric Analysis of Marine Bacteria with Hoechst 33342 †

    PubMed Central

    Monger, Bruce C.; Landry, Michael R.

    1993-01-01

    We investigated the accuracy and precision of flow cytometric (FCM) estimates of bacterial abundances using 4′, 6-diamidino-2-phenylindole (DAPI) and Hoechst 33342 (HO342, a bisbenzamide derivative) on paraformaldehyde-fixed seawater samples collected from two stations near Oahu, Hawaii. The accuracy of FCM estimates was assessed against direct counts by using epifluorescence microscopy. DAPI and HO342 differ in two aspects of their chemistry that make HO342 better suited for staining marine heterotrophic bacteria for FCM analysis. These differences are most important in studies of open-ocean ecosystems that require dual-beam FCM analysis to clearly separate heterotrophic bacterial populations from populations of photosynthetic Prochlorococcus spp. Bacterial populations were easier to distinguish from background fluorescence when stained with HO342 than when stained with DAPI, because HO342 has a higher relative fluorescence quantum yield. A substantially higher coefficient of variation of blue fluorescence, which was probably due to fluorescent complexes formed by DAPI with double-stranded RNA, was observed for DAPI-stained populations. FCM estimates averaged 2.0 and 12% higher than corresponding epifluorescence microscopy direct counts for HO342 and DAPI-stained samples, respectively. A paired-sample t test between FCM estimates and direct counts found no significant difference for HO342-stained samples but a significant difference for DAPI-stained samples. Coefficients of variation of replicate FCM abundance estimates ranged from 0.63 to 2.9% (average, 1.5%) for natural bacterial concentrations of 6 × 105 to 15 × 105 cells ml-1. PMID:16348898

  7. 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.

  8. 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

  9. Method for flow cytometric monitoring of Renibacterium salmoninarum inactivation.

    PubMed

    Pascho, R J; Ongerth, J E

    2000-07-14

    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 10(6) 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 10(6) 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 < or = 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 < or = 0.27), there was a correlation between these estimates and the duration of exposure in the reduced-Rs assessment (r2 > or = 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

  10. 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. PMID:19296591

  11. Dynamic Characterization of Growth and Gene Expression Using High-throughput Automated Flow cytometry

    PubMed Central

    Zuleta, Ignacio A.; Aranda-Díaz, Andrés; Li, Hao; El-Samad, Hana

    2014-01-01

    Cells adjust to changes in environmental conditions using complex regulatory programs. These cellular programs are the result of an intricate interplay between gene expression, cellular growth rate, and protein degradation fluxes. New technologies that enable simultaneous and time-resolved measurements of these variables are necessary to dissect cellular homeostatic strategies. Here, we report the development of a novel automated flow-cytometry robotic setup that enables real-time measurement of precise and simultaneous relative growth and protein synthesis rates of multiplexed microbial populations across many conditions. These measurements generate quantitative profiles of dynamically-evolving protein synthesis and degradation rates. We demonstrate this setup in the context of gene regulation of the unfolded protein response (UPR) and uncover a dynamic and complex landscape of gene expression, growth dynamics, and proteolysis following perturbations. PMID:24608180

  12. High throughput screening of scFv antibodies against viral hemorrhagic septicaemia virus by flow cytometry.

    PubMed

    Zhou, Yao; Xie, Zhi-Gang

    2015-07-01

    Viral hemorrhagic septicaemia (VHS) is an economically important disease that affects salmon and trout worldwide. In this study, a recombinant single chain variable fragment (scFv) antibody library derived from rainbow trout immunized with purified viral hemorrhagic septicaemia virus (VHSV) was constructed. The library was subjected to three rounds of screening by flow cytometry (FCM) against VHSV through a bacteria display technology, resulting in the enrichment of scFv. Four scFv clones with different fluorescence intensity were obtained by colony pick up at random following three rounds of screening. The isolated scFv antibodies were expressed and purified. Relative affinity assay showed the four clones had different sensitivity to VHSV, in accordance with FCM. The potential use of the selected VHSV-specific scFv antibodies was demonstrated by the successful application in Western blotting assay, ELISA and immunofluorescence antibody test (IFAT), and one of the isolated scFv molecular showed excellent in vitro and in vivo blocking activities against VHSV. scFv isolated in this study can be promising diagnostic and/or therapeutic reagents for VHS. This study provides powerful strategies for screening antibodies against new diseases. PMID:25813596

  13. Optofluidic time-stretch imaging - an emerging tool for high-throughput imaging flow cytometry.

    PubMed

    Lau, Andy K S; Shum, Ho Cheung; Wong, Kenneth K Y; Tsia, Kevin K

    2016-05-10

    Optical imaging is arguably the most effective tool to visualize living cells with high spatiotemporal resolution and in a nearly noninvasive manner. Driven by this capability, state-of-the-art cellular assay techniques have increasingly been adopting optical imaging for classifying different cell types/stages, and thus dissecting the respective cellular functions. However, it is still a daunting task to image and characterize cell-to-cell variability within an enormous and heterogeneous population - an unmet need in single-cell analysis, which is now widely advocated in modern biology and clinical diagnostics. The challenge stems from the fact that current optical imaging technologies still lack the practical speed and sensitivity for measuring thousands to millions of cells down to the single-cell precision. Adopting the wisdom in high-speed fiber-optics communication, optical time-stretch imaging has emerged as a completely new optical imaging concept which is now proven for ultrahigh-throughput optofluidic single-cell imaging, at least 1-2 orders-of-magnitude higher (up to ∼100 000 cells per second) compared to the existing imaging flow cytometers. It also uniquely enables quantification of intrinsic biophysical markers of individual cells - a largely unexploited class of single-cell signatures that is known to be correlated with the overwhelmingly investigated biochemical markers. With the aim of reaching a wider spectrum of experts specializing in cellular assay developments and applications, this paper highlights the essential basics of optical time-stretch imaging, followed by reviewing the recent developments and applications of optofluidic time-stretch imaging. We will also discuss the current challenges of this technology, in terms of providing new insights in basic biology and enriching the clinical diagnostic toolsets. PMID:27099993

  14. Identification of neutrophil surface marker changes in health and inflammation using high-throughput screening flow cytometry.

    PubMed

    Lakschevitz, Flavia S; Hassanpour, Siavash; Rubin, Ayala; Fine, Noah; Sun, Chunxiang; Glogauer, Michael

    2016-03-15

    Neutrophils are the most abundant white blood cell and are an essential component of the innate immune system. A complete cataloguing of cell surface markers has not been undertaken for neutrophils isolated from circulation as well as healthy and inflamed tissues. To identify cell-surface markers specific to human neutrophils, we used high-throughput flow cytometry to screen neutrophil populations isolated from blood and oral rinses from healthy and chronic periodontitis patients against a panel of 374 known cluster of differentiation (CD) antibodies. This screen identified CD11b, CD16, and CD66b as markers that are consistently expressed on neutrophils independent of the cell location, level of activation and disease state. Cell sorting against CD11b, CD16 and CD66b allowed for the enrichment of mature neutrophils, yielding neutrophil populations with up to 99% purity. These findings suggest an ideal surface marker set for isolating mature neutrophils from humans. The screen also demonstrated that tissue neutrophils from chronically inflamed tissue display a unique surface marker set compared to tissue neutrophils present in healthy, non-inflamed tissues. PMID:26970376

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

    PubMed

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

    2014-09-01

    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

  16. 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

  17. High-throughput measurement of the long excited-state lifetime of quantum dots in flow cytometry

    NASA Astrophysics Data System (ADS)

    Dahal, Eshan; Cao, Ruofan; Jenkins, Patrick; Houston, Jessica P.

    2014-03-01

    The long fluorescence lifetime of quantum dots (QDs) is not often utilized in high-throughput bioassays, despite of the potential for the lifetime to be an optimum parameter for multiplexing with spectrally overlapping excitable species that have short fluorescence lifetimes. The limitation of currently available instruments that can rapidly resolve complex decay kinetics of QDs contributes to this dearth. Therefore work in our laboratory is focused on developing unique and reliable frequency-domain flow cytometry (FDFC) systems as well as QDs applications where fluorescence dynamics are exploited. In this paper we demonstrate both by simulation and experimental validation, the viability of rapidly capturing the fluorescence lifetime of QDs from single QDs-labeled cells and microspheres by employing a home-built FDFC system. With FDFC theory we simulated measurements of long-lived QDs decays and evaluated the potential to discriminate multi-exponential decay profiles of QDs from typical cellular autofluorescence lifetimes. Our FDFC simulation work included calculations of fluorescence phase-shifts at multiple modulation frequencies extracted from square wave modulation signals (i.e. similar to heterodyning frequency-domain spectroscopy). Experimental work to support the result from our simulations involved acquiring measurements from real samples and processing them for multi-frequency phase shifts. Additionally the average excited-state lifetimes of QDs (streptavidin conjugated CdSe/Zns and oleic acid coated CdSxSe1-x/ZnS) measured were found to be greater than 15 ns. The average lifetime results were consistent with published literature values as well as verified with independent time domain measurements. This work opens the possibility of developing powerful bioassays using FDFC based on the long fluorescence lifetime of QDs.

  18. Flow cytometric determination of the photoinduced toxicity of anthracene to the green alga selenastrum capricornutum

    SciTech Connect

    Gala, W.R.; Giesy, J.P. . Dept. of Fisheries and Wildlife)

    1994-05-01

    Certain PAHs are photosensitizers and in the presence of solar radiation can cause toxicity to aquatic plants and animals. The photoinduced toxicity of anthracene to the green alga Selenastrum capricornutum was assessed by the use of flow cytometry to measure cell size, cellular chlorophyll concentration, and cell viability. Anthracene was slightly toxic in the absence of UV-A radiation. The detection of the direct toxicity of anthracene in this study at a concentration of 19 [mu]g/L anthracene resulted from the use of sensitive flow cytometric measures. There was a significant interaction between anthracene and UV-A radiation, which, in combination, caused significant toxic effects on Selenastrum capricornutum. The most sensitive flow cytometric measure of toxicity was the stress index (SI), which was predictive of longer term effects on cell growth. The 28-h EC50 and EC10 and for the SI for Selenastrum capricornutum were 16.1 and 8.3 [mu]g/L anthracene, respectively, at 125 [mu]W/cm[sup 2] UV-A. All combinations for anthracene and UV-A that inhibited algal growth also caused a significantly greater number of nonviable cells. The flow cytometric methods used in this study proved to be sensitive, predictive measures of the direct and photo-induced toxicity of anthracene and UV-A radiation to Selenastrum capricornutum.

  19. Intracellular Flow Cytometric Measurement of Extracellular Matrix Components in Porcine Intervertebral Disc Cells

    PubMed Central

    Flagler, Daniel J.; Huang, Chun-Yuh; Yuan, Tai-Yi; Lu, Zhongmin; Cheung, Herman S.; Gu, Wei Yong

    2009-01-01

    The objective of this study was to develop and demonstrate the utility of a novel method of evaluating intracellular levels of extracellular matrix (ECM) components in intervertebral disc (IVD) cells using flow cytometry. By using this method, this study discriminated between cell populations in porcine IVD and examined the response of IVD cells to monolayer cultures, a traditional method of cell expansion, by measuring phenotypic attributes of ECM component production. It was found that monolayer cultures affected collagen production of IVD cells while there were differences in collagen type II production between the cells isolated from the annulus fibrosus (AF) and nucleus pulposus (NP) regions of IVD. Size distributions of fresh and cultured cells were also presented while the relationships between cell size and intracellular collagen level revealed heterogeneous cell populations in AF and NP regions. Furthermore, this study showed that the intracellular collagen signals of IVD cells were significantly enhanced by the treatments of Brefeldin-A and ascorbic acid. This suggests that Brefeldin-A and ascorbic acid could be used to increase the sensitivity of flow cytometric analysis on intracellular collagen levels by maximizing collagen accumulation inside cells. Since a unique feature of the flow cytometric screening tool is the ability to discriminate between various cell populations in a single sample, the flow cytometric method developed in this study may have the potential to identify specific collagen-producing cell populations from tissues or cell cultures. PMID:20161070

  20. 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.

  1. 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.

  2. 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.

  3. Flow cytometric cell-based assay to preselect antibody constructs for radionuclide conjugation.

    PubMed

    Ingargiola, M; Dittfeld, C; Runge, R; Zenker, M; Heldt, J-M; Steinbach, J; Cordes, N; Baumann, M; Kotzerke, J; Kunz-Schughart, L A

    2012-10-01

    Radiolabeled antibodies (Abs) are an attractive tool for targeting and delivering particle emitters for therapy or imaging applications. The labeling of Abs with metal radionuclides requires chelating agents and can cause loss of binding to their ligands. The aim of the present approach was to design an easy-handling flow cytometric cell-based assay to evaluate Ab-binding capacity of conjugates of the therapeutic Ab Cetuximab and to verify the most promising candidate in a competitive radioactive binding experiment. The final setup for flow cytometric assessment of cellular binding capacities of epidermal growth factor receptor (EGFR)/ErbB1-directed Ab conjugates is based on (a) the selection of a robust cell line model (b) the definition of nonsaturated staining concentrations for the unconjugated reference Ab Cetuximab plus implementation of a reasonable isotype control, and (c) the calculation of relative Ab affinities based on the flow cytometric data. Two (FaDu, SAS) out of the three cell lines with different total and cell surface expression levels of EGFR turned out to be adequate models but the application of one cell line was sufficient to estimate reduced binding capacities of conjugates relative to Cetuximab. Only 1/11 conjugate Abs exhibited a fluorescence signal comparable to unconjugated Cetuximab and was applied for radiolabeling with Yttrium-90. Unaltered binding affinity of this conjugate was proven in a competitive radioactive Ab-binding study. We conclude that the flow cytometric assay is reliable and that the relative binding capacity of Cetuximab is neither affected by covalent modification with CHX-A"-DTPA (N-[(R)-2-Amino-3-(p-isothiocyanato-phenyl) propyl]-trans-(S,S)-cyclohexane-1,2-diamine-N,N,N',N",N"-pentaacetic acid) with a final chelator-to-Ab ratio of 5 nor by subsequent radiolabeling. [(90)Y]Y-CHX-A"-DTPA-Cetuximab thus qualifies for preclinical treatment testing as a prerequisite for therapeutic application. PMID:22930585

  4. 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.

  5. Fluoresceinated phosphoethanolamine for flow-cytometric measurement of lipid peroxidation.

    PubMed

    Maulik, G; Kassis, A I; Savvides, P; Makrigiorgos, G M

    1998-10-01

    A new lipophilic fluorescein probe (fluor-DHPE) has been identified that can assay lipid peroxidation in mammalian cells on a cell-by-cell or selected-cell-subpopulation basis by flow cytometry. Application of this approach requires that the fluorescent probe be nonexchangeable among cells. Fluorescein is an appropriate fluorophore, since its fluorescence matches the specifications of common flow cytometers and the compound loses its fluorescence upon reaction with peroxyl radicals. Upon examination of four lipophilic derivatives of fluorescein, fluor-DHPE was found to be the only probe that was nonexchangeable among labeled and unlabeled rat RBC for at least 24 h. The exposure of fluor-DHPE-labeled RBC to benzoyl peroxide followed by mixing the sample with RBC unexposed to peroxide led to a decrease in fluorescence. Furthermore, the flow cytometer could clearly select the subpopulation of cells undergoing lipid peroxidation from those cells that were not. Fluor-DHPE-labeled-RBC obtained from rats and exposed to cumene hydroperoxide also displayed a gradual decrease in fluorescence. This decrease was preventable by either regulation of the vitamin E content in the animal diet or in vitro supplementation of cells with vitamin E. We conclude that fluor-DHPE is a stable and nonexchangeable probe for monitoring lipid peroxidation in cell subpopulations by flow cytometry. PMID:9801063

  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. PMID:27084117

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

    PubMed

    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

  10. Flow cytometric measurement of immunoglobulin E to natural latex proteins.

    PubMed Central

    Kwittken, P L; Pawlowski, N A; Sweinberg, S K; Douglas, S D; Campbell, D E

    1994-01-01

    Immediate hypersensitivity to natural latex (NL) occurs in sensitized individuals after repeated exposure to products or devices containing NL components. Since allergic reactions to NL proteins are quite frequent and may be quite serious, diagnostic assays are needed to identify individuals at risk. A number of latex proteins have been considered the major antigens, but they have been incompletely characterized. There is no standard material available for skin testing. In vitro diagnostic tests, such as the radioallergosorbent test (RAST), are time consuming and their sensitivity and specificity remain to be proven. We have developed a rapid microsphere-based, fluorescence-activated flow cytometry assay for the measurement of NL protein-specific human immunoglobulin E and have compared it with both the enzyme-linked immunosorbent assay and radioallergosorbent test methods. By using the total purified NL protein fraction isolated from raw ammoniated NL sap as the antigen, the flow cytometry assay was both sensitive and specific for the detection of NL protein-specific human immunoglobulin E in the sera of sensitized pediatric patients. PMID:7496945

  11. Intra- and interboar variability in flow cytometric sperm sex sorting.

    PubMed

    Alkmin, Diego V; Parrilla, Inmaculada; Tarantini, Tatiana; Parlapan, Laura; Del Olmo, David; Vazquez, Juan M; Martinez, Emilio A; Roca, Jordi

    2014-08-01

    To improve the efficiency of porcine sperm sex sorting using flow cytometry, the aims of the present study were to determine the relevance of inter- and intraboar variability in sperm sortability and to evaluate the significance of ejaculate semen characteristics in such variability. In addition, the variability among boars in the ability of sex-sorted spermatozoa to survive liquid storage at 15 °C to 17 °C was also evaluated. In total, 132 ejaculates collected from 67 boars of different breeds that were housed at an artificial insemination center were used in three experiments. X- and Y-chromosome-bearing sperm were simultaneously separated according to the Beltsville sperm-sorting technology using a high-speed flow cytometer. In the first experiment, interboar variability in the ability of the ejaculated spermatozoa to undergo the flow-based sex-sorting procedure was observed; the ejaculates of nearly 15% of the boars (n = 67) did not exhibit well-defined X- and Y-chromosome-bearing spermatozoa peaks in the histogram, and the ejaculate sperm concentration demonstrated good predictive value for explaining this variation, as indicated by the area under the receiver operating characteristics curve (0.88, P < 0.001). In the second experiment, a certain degree of intraboar variability was observed only in the boars that showed poor sperm sortability (measured according to the presence or not a well-defined split together with sperm sortability parameters) in the first ejaculate (n = 3). In contrast, boars classified as having good sperm sortability in the first ejaculate (n = 5) maintained this condition in five ejaculates collected over the subsequent 5 months. In the third experiment, sex-sorted spermatozoa from boars with good sperm sortability (n = 5) remained viable and motile (above 70% in all boars) after 48 hours of storage at 15 °C to 17 °C, which may facilitate the commercial application of sex-sorted spermatozoa in swine artificial insemination programs

  12. 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

  13. Flow cytometric detection of Cryptosporidium oocysts in human stool samples.

    PubMed Central

    Valdez, L M; Dang, H; Okhuysen, P C; Chappell, C L

    1997-01-01

    Cryptosporidium parvum is an important pathogen that causes diarrhea in virtually all human populations. Improved diagnostic methods are needed to understand the risk factors, modes of transmission, and impact of cryptosporidiosis. In the present study, we fluorescently labeled and counted C. parvum oocysts by flow cytometry (FC) and developed a simple and efficient method of processing human stool samples for FC analysis. Formed stool (suspended in phosphate-buffered saline) from an asymptomatic, healthy individual was seeded with known concentrations of oocysts, and oocysts were labeled with a cell wall-specific monoclonal antibody and detected by FC. The method described herein resulted in a mean oocyst recovery rate of 45% +/- 16% (median, 42%), which consistently yielded a fourfold increase in sensitivity compared to direct fluorescent-antibody assay of seeded stool samples. However, in many instances, FC detected as few as 10(3) oocysts per ml. Thus, FC provides a reproducible and sensitive method for C. parvum oocyst detection. PMID:9230372

  14. 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)

  15. Flow cytometric detection of oxidative DNA damage in fish spermatozoa exposed to cadmium - Short communication.

    PubMed

    Nagy, Szabolcs; Kakasi, Balázs; Bercsényi, Miklós

    2016-03-01

    The aim of the present pilot study was to apply a flow cytometric assay, the so-called OxyDNA test, to determine the level of oxidative DNA damage in fish spermatozoa exposed to different concentrations (0.01-10,000 mg/L) of cadmium. Milt was collected from three randomly selected Prussian carp (Carassius auratus gibelio) males. Oxidative DNA damage was assessed with the OxyDNA kit and using flow cytometry. The ratio of OxyDNA-positive events increased significantly at higher cadmium concentrations. The results indicate that direct contact of fish spermatozoa with cadmium-polluted water initiates genotoxic damage. PMID:26919149

  16. 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.

  17. Collection, isolation, and flow cytometric analysis of human endocervical samples.

    PubMed

    Juno, Jennifer A; Boily-Larouche, Genevieve; Lajoie, Julie; Fowke, Keith R

    2014-01-01

    Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female genital tract (FGT). Because heterosexual transmission now represents the dominant mechanism of HIV transmission, and given the continual spread of sexually transmitted infections (STIs), it is critical to understand the interplay between host and pathogen at the genital mucosa. The substantial gaps in knowledge around FGT immunity are partially due to the difficulty in successfully collecting and processing mucosal samples. In order to facilitate studies with sufficient sample size, collection techniques must be minimally invasive and efficient. To this end, a protocol for the collection of cervical cytobrush samples and subsequent isolation of cervical mononuclear cells (CMC) has been optimized. Using ex vivo flow cytometry-based immunophenotyping, it is possible to accurately and reliably quantify CMC lymphocyte/monocyte population frequencies and phenotypes. This technique can be coupled with the collection of cervical-vaginal lavage (CVL), which contains soluble immune mediators including cytokines, chemokines and anti-proteases, all of which can be used to determine the anti- or pro-inflammatory environment in the vagina. PMID:25045942

  18. Amplified flow-cytometric separation-free fluorescence immunoassays

    SciTech Connect

    Saunders, G.C.; Jett, J.H.; Martin, J.C.

    1985-12-01

    An equilibrium-type competitive-binding fluorescence immunoassay with high sensitivity and excellent precision is described that obviates separation of free from bound label. In the assay relatively large (10 microns diameter) antibody-coated non-fluorescent particles and very small (0.10 micron diameter) antigen-coated fluorescent latex particles are used. Soluble nonlabeled antigen competes with antigen on the microspheres for antibody binding sites on the larger spheres. After equilibrium is attained, the fluorescence distribution of 5000 of the large spheres is measured in a flow cytometer. The mean values for the fluorescence distribution obtained from samples containing known concentrations of soluble antigen are used to construct a standard displacement curve. In a prototype assay for the antigen horseradish peroxidase, a sensitivity of 10(-12) mol/L has been achieved. Undiluted serum can be assayed without loss of sensitivity. Preliminary experiments also indicate that double-antibody sandwich-type assays of very high sensitivity (10(-14) mol/L) are also possible when this dual-sphere concept is exploited.

  19. A flow cytometric approach to assess phytoplankton respiration.

    PubMed

    Grégori, Gérald; Denis, Michel; Lefèvre, Dominique; Beker, Beatriz

    2002-01-01

    Microbial respiration in the ocean is considered as the major process representative of the organic matter biological oxidation. The corresponding metabolic CO2 production was estimated to be about 22 Pg C y(-1). However, the in situ respiration rate is generally too low (by several orders of magnitude) to be accessible to the available direct measurement methods. Some fluorescent probes, such as DiOC6(3) (Molecular Probes, USA) have been shown to be very sensitive to changes in the proton electrochemical potential difference (DeltamuH+), characterising mitochondrial and plasmic membranes bearing the cell respiratory system in eukaryotic and prokaryotic cells respectively. In mitochondria, DeltamuH+ is linked to the flux of oxygen uptake by a linear relationship. To our knowledge, no such relationship has been established in the case of whole marine cells. In the present work, we addressed the dark respiration rate of the Chlorophyceae Dunaliella tertiolecta (Butcher) in axenic cultures, both directly by using a highly sensitive oxygraph (Oroboros) and by staining cells with DiOC6(3). We found and standardized a linear relationship between oxygen uptake by D. tertiolecta and its green fluorescence induced by DiOC6(3), enabling the determination by flow cytometry of the respiration rate of D. tertiolecta. PMID:12815298

  20. Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples

    PubMed Central

    Juno, Jennifer A.; Boily-Larouche, Genevieve; Lajoie, Julie; Fowke, Keith R.

    2014-01-01

    Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female genital tract (FGT). Because heterosexual transmission now represents the dominant mechanism of HIV transmission, and given the continual spread of sexually transmitted infections (STIs), it is critical to understand the interplay between host and pathogen at the genital mucosa. The substantial gaps in knowledge around FGT immunity are partially due to the difficulty in successfully collecting and processing mucosal samples. In order to facilitate studies with sufficient sample size, collection techniques must be minimally invasive and efficient. To this end, a protocol for the collection of cervical cytobrush samples and subsequent isolation of cervical mononuclear cells (CMC) has been optimized. Using ex vivo flow cytometry-based immunophenotyping, it is possible to accurately and reliably quantify CMC lymphocyte/monocyte population frequencies and phenotypes. This technique can be coupled with the collection of cervical-vaginal lavage (CVL), which contains soluble immune mediators including cytokines, chemokines and anti-proteases, all of which can be used to determine the anti- or pro-inflammatory environment in the vagina. PMID:25045942

  1. Flow cytometric measurement of microparticles: pitfalls and protocol modifications.

    PubMed

    Shah, Mona D; Bergeron, Angela L; Dong, Jing-Fei; López, José A

    2008-08-01

    Upon activation, many cells shed components of their plasma membranes as microparticles. Depending on the methods of preparation and analyses, microparticle counts may vary significantly between laboratories, making data analyses and clinical correlations challenging. To assess how variations in sample preparation affect microparticle measurements, blood samples from 13 healthy, adult volunteers were labeled with Annexin V, cell-specific antibodies, and antibodies against tissue factor (TF). Data were acquired and analysed using an EPICS XL-MCL flow cytometer. Annexin V(+) monocyte-, platelet-, endothelial-, or erythrocyte-derived microparticles accounted for 10.4%, 38.5%, 43.8%, and 7.3% of the total number of microparticles (13.7 +/- 3.0 x 10(3)/ml of whole blood), respectively. A similar distribution of cell types was seen for TF(+) microparticles (6.3 +/- 2.6 x 10(3)/ml of whole blood). No statistical difference was noted in microparticle distribution using either 19- or 21-gauge needles. Elevated levels of platelet- and erythrocyte-derived microparticles were detected in heparin and PPACK-anticoagulated samples as compared to samples anticoagulated with ACD or sodium citrate (P < 0.05, student's t-test). Additional centrifugation was critical for removing platelet contamination, which significantly affected microparticle counts. Finally, Annexin V(+) and TF(+) microparticles were significantly reduced upon sample storage at low temperatures. Microparticle levels are significantly affected by variations in sample preparation and storage. These results illustrate the need to standardize assay protocols in order to obtain consistent measurements. Our studies further optimize sample preparation for microparticle detection. PMID:18791943

  2. 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. PMID:18638144

  3. 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

  4. 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

  5. Validation of a flow cytometric acridine orange micronuclei methodology in rats.

    PubMed

    Criswell, K A; Krishna, G; Zielinski, D; Urda, G A; Juneau, P; Bulera, S; Bleavins, M R

    2003-07-25

    Our laboratory has previously reported a flow cytometric acridine orange method for detection of micronucleus (MN) in the rat using cyclophosphamide as a test compound. To replace the manual method of scoring and satisfy Good Laboratory Practice (GLP) requirements, an extensive validation of the flow method was required. Therefore, manual scoring and flow cytometric determination of MN were compared using vincristine, chlorambucil, methotrexate, and doxorubicin compounds known to induce MN formation with various mechanisms of action. 1,2-Dimethylhydrazine (1,2-DH), a compound with negative or equivocal MN findings also was evaluated. The flow method consistently demonstrated dose- and time-dependent responses for MN production at all concentrations of vincristine, methotrexate, clorambucil, and doxorubicin. In contrast, manual scoring of slides failed to detect an increase in MN at the lowest doses of doxorubicin (1mg/kg) at 24 or 48 h, and methotrexate at 48 h, or any dose of methotrexate (50, 100, or 250 mg/kg) at 24h. Additionally, a dose-response for methotrexate at 48 h, and chlorambucil at 24 h were missed using manual scoring. For 1,2-DH, the flow method showed a low level (< 1.4-fold) increase in MN at all doses and times. In contrast, the manual method showed five-seven-fold increases at 24 h, but a < two-fold increase at 48 h in the highest dose only. These data may suggest that the flow method has a greater sensitivity and possibly accuracy than manual scoring. Significant decreases in polychromatic erythrocytes (PCE) were seen using both methods at approximately the same dose for all compounds. However, absolute flow cytometric PCE values were consistently higher than manual. Additional cytotoxicity parameters obtained by the flow method allowed a more complete assessment of cytotoxicity than PCE alone. Furthermore, data reported here combined with improved throughput, shortened data turnaround and reporting times, and possibly better precision due to

  6. A rapid, simple and sensitive flow cytometric system for detection of Plasmodium falciparum.

    PubMed

    Saito-Ito, A; Akai, Y; He, S; Kimura, M; Kawabata, M

    2001-11-01

    We have established a rapid, simple and sensitive flow cytometric system for the detection of Plasmodium falciparum that involves lysing erythrocytes and staining parasites at the same time using a newly developed hemolysing and staining solution containing dodecyl methyl ammonium chloride and acridine orange. In this system, freed parasites of P. falciparum could be plotted separately from erythrocyte ghosts, white blood cells and platelets on the two-dimensional scattergram of forward-angle light scatter and green fluorescence by flow cytometry with an argon laser. It took only 2-3 min per sample to obtain the scattergram and analyze the data, including the time of sample preparation for flow cytometric analysis. Sample preparation with this method does not require any difficult handling procedures. The threshold of parasite detection was almost equal to that of microscopic examination for cultured P. falciparum. The results of drug-susceptibility assays using this system were also almost identical to those obtained using microscopic examination. In this system, parasites at different erythrocytic stages could be easily distinguished. This system must prove useful and practical for basic laboratory studies of P. falciparum including those requiring the differential measurement of parasites at specific erythrocytic stages. PMID:11719111

  7. Flow cytometric analysis of micronuclei in mammalian cell cultures: past, present and future.

    PubMed

    Avlasevich, Svetlana; Bryce, Steven; De Boeck, Marlies; Elhajouji, Azeddine; Van Goethem, Freddy; Lynch, Anthony; Nicolette, John; Shi, Jing; Dertinger, Stephen

    2011-01-01

    The relative simplicity of the in vitro micronucleus (MNvit) endpoint has made it amenable to several automated scoring approaches. Flow cytometry is one such scoring platform that has been successfully employed. This review describes the origins of the MNvit assay, as well as the evolution and properties of flow cytometry-based scoring systems. While the current state-of-the-art methods acquire micronucleus (MN) frequency data very efficiently, it is becoming clear that they also endow the assay with high information content. For instance, simultaneous with MN frequency determinations, several additional endpoints are acquired that provide insights into cytotoxicity, cell cycle perturbations and, in the event of MN induction, information about genotoxic mode of action. This review concludes with a discussion regarding data gaps and also recommendations for additional work that is needed to more fully realise the potential of flow cytometric MNvit scoring. PMID:21164196

  8. Use of Flow Cytometric Methods to Quantify Protein-Protein Interactions

    PubMed Central

    Blazer, Levi L.; Roman, David L.; Muxlow, Molly R.; Neubig, Richard R.

    2010-01-01

    A method is described for the quantitative analysis of protein-protein interactions using the Flow Cytometry Protein Interaction Assay (FCPIA). This method is based upon immobilizing protein on a polystyrene bead, incubating these beads with a fluorescently labeled binding partner, and assessing the sample for bead-associated fluorescence in a flow cytometer. This method can be used to calculate protein-protein interaction affinities or to perform competition experiments with unlabeled binding partners or small molecules. Examples described in this protocol highlight the use of this assay in the quantification of the affinity of binding partners of the Regulator of G-Protein Signaling protein, RGS19, in either a saturation or competition format. An adaptation of this method that is compatible for High Throughput screening is also provided. PMID:20069525

  9. Flow cytometric determination of leukemia-associated marker combinations for the study of minimal residual disease.

    PubMed

    Babusíková, O; Glasová, M; Kusenda, J; Koníková, E; Mésárosová, A

    1994-01-01

    To study the minimal residual disease in acute leukemia patients we used some marker combinations related either to the simultaneous surface membrane and cytoplasmic marker expression, or to the expression of atypical marker combinations, that are absent or extremely rare in normal hematopoietic cells. We investigated to which extent flow cytometric analysis of leukemia-associated marker combination may contribute to sensitive follow-up in patients with acute leukemia. For this purpose dilution experiments were performed, in which artificial mixtures of normal peripheral blood lymphocytes and leukemia cells from a patient with leukemia-associated phenotype were prepared and analyzed for double positive cells. Our results showed that the sensitivity of double color immunofluorescence assay was 3 in 10(4) cells. Sequential studies of residual disease were evaluated in five acute leukemia patients with leukemia-associated markers combinations at diagnosis. In three of them morphologic relapse was preceded by the immunologic detection of small amounts of leukemia cells, while in two other cases, in which no double positive cells for leukemia-associated markers were found, patients are still in hematologic remission. This approach to the study of minimal residual disease could be valuable in monitoring the efficiency of chemotherapy, as well as in evaluating the quality control of bone marrow before autografting. Furthermore, flow cytometric approach can efficiently complete other methods, which are used for more exact definition of remission in acute leukemia patients. PMID:7870213

  10. 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.

  11. Flow cytometric characterization of microglia in the offspring of PolyI:C treated mice.

    PubMed

    Manitz, Marie Pierre; Plümper, Jennifer; Demir, Seray; Ahrens, Maike; Eßlinger, Manuela; Wachholz, Simone; Eisenacher, Martin; Juckel, Georg; Friebe, Astrid

    2016-04-01

    The neuropathology of schizophrenia has been reported to be closely associated with microglial activation. In a previous study, using the prenatal PolyI:C schizophrenia animal model, we showed an increase in cell numbers and a reduction in microglial branching in 30-day-old PolyI:C descendants, which suggests that there is microglial activation during adolescence. To provide more information about the activation state, we aimed to examine the expression levels of Iba1, which was reported to be up-regulated in activated microglia. We used a flow cytometric approach and investigated CD11b and CD45, two additional markers for the identification of microglial cells. We demonstrated that intracellular staining against Iba1 can be used as a reliable flow cytometric method for identification of microglial cells. Prenatal PolyI:C treatment had long-term effects on CD11b and CD45 expression. It also resulted in a trend towards increased Iba1 expression. Imbalance in CD11b, CD45, and Iba1 expression might contribute to impaired synaptic surveillance and enhanced activation/inflammatory activity of microglia in adult offspring. PMID:26872595

  12. Flow Cytometric Method for the Detection of Flavonoids in Cell Lines.

    PubMed

    Grootaert, Charlotte; Gonzales, Gerard Bryan; Vissenaekens, Hanne; Van de Wiele, Tom; Raes, Katleen; Smagghe, Guy; Van Camp, John

    2016-09-01

    Here, we describe an easy-to-use flow cytometric method using diphenylboric acid 2-amino ethyl ester (DPBA) stain for the detection of flavonoids in cells from human/animal origin. Flavonoid bioavailability and bioactivity depend on structure, conjugation and the cell type to which they are presented. We have studied cellular uptake of five flavonoids with different structures and conjugation forms. First, parameters including fixation method, technical and batch variability, and concentration were optimized. Second, uptake of two aglycones-quercetin and hesperetin-and their corresponding glycosides-rutin and hesperidin-in Caco-2 cells was compared. Third, the aglycone quercetin, glycoside rutin, and glucuronide baicalin were added to the Caco-2, HepG2, and CHO-K1 cell lines at 1, 10, and 20 µM concentrations and cellular uptake was measured after 1, 4, and 7 h. We conclude that quercetin was taken up by cells in a dose-dependent way, and that HepG2 cells had the highest uptake factors, followed by CHO-K1 and Caco-2 cells. Confocal microscopy showed cell type-dependent localization of quercetin in the cell membrane and cytoplasm. No uptake of flavonoid glycosides was detected. This flow cytometric method can be used for future research unravelling mechanisms behind flavonoid bioactivity in health and disease at the cellular level. PMID:27280551

  13. High-throughput inertial particle focusing in a curved microchannel: Insights into the flow-rate regulation mechanism and process model.

    PubMed

    Xiang, Nan; Yi, Hong; Chen, Ke; Sun, Dongke; Jiang, Di; Dai, Qing; Ni, Zhonghua

    2013-01-01

    In this work, we design and fabricate a miniaturized spiral-shaped microchannel device which can be used for high-throughput particle/cell ordering, enrichment, and purification. To probe into the flow rate regulation mechanism, an experimental investigation is carried out on the focusing behaviors of particles with significantly different sizes in this device. A complete picture of the focusing position shifting process is unfolded to clarify the confusing results obtained from flow regimes with different dominant forces in past research. Specifically, with the increase of the flow rate, particles are observed to first move towards the inner wall under the dominant inertial migration, then stabilize at a specific position and finally shift away from the inner wall due to the alternation of the dominant force. Novel phenomena of focusing instability, co-focusing, and focusing position interchange of differently sized particles are also observed and investigated. Based on the obtained experimental data, we develop and validate, for the first time, a five-stage model of the particle focusing process with increasing flow rate for interpreting particle behaviors in terms of the competition between inertial lift and Dean drag forces. These new experimental findings and the proposed process model provide an important supplement to the existing mechanism of inertial particle flow and enable more flexible and precise particle manipulation. Additionally, we examine the focusing behaviors of bioparticles with a polydisperse size distribution to validate the explored mechanisms and thus help realize efficient enrichment and purification of these particles. PMID:24404049

  14. 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.

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

    PubMed

    Sartipi, Sina; Jansma, Harrie; Bosma, Duco; Boshuizen, Bart; Makkee, Michiel; Gascon, Jorge; Kapteijn, Freek

    2013-12-01

    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. PMID:24387446

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

    NASA Astrophysics Data System (ADS)

    Sartipi, Sina; Jansma, Harrie; Bosma, Duco; Boshuizen, Bart; Makkee, Michiel; Gascon, Jorge; Kapteijn, Freek

    2013-12-01

    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.

  17. Flow Cytometric Analysis of T, B, and NK Cells Antigens in Patients with Mycosis Fungoides

    PubMed Central

    Yazıcı, Serkan; Bülbül Başkan, Emel; Budak, Ferah; Oral, Barbaros; Adim, Şaduman Balaban; Ceylan Kalin, Zübeyde; Özkaya, Güven; Aydoğan, Kenan; Saricaoğlu, Hayriye; Tunali, Şükran

    2015-01-01

    We retrospectively analyzed the clinicopathological correlation and prognostic value of cell surface antigens expressed by peripheral blood mononuclear cells in patients with mycosis fungoides (MF). 121 consecutive MF patients were included in this study. All patients had peripheral blood flow cytometry as part of their first visit. TNMB and histopathological staging of the cases were retrospectively performed in accordance with International Society for Cutaneous Lymphomas/European Organization of Research and Treatment of Cancer (ISCL/EORTC) criteria at the time of flow cytometry sampling. To determine prognostic value of cell surface antigens, cases were divided into two groups as stable and progressive disease. 17 flow cytometric analyses of 17 parapsoriasis (PP) and 11 analyses of 11 benign erythrodermic patients were included as control groups. Fluorescent labeled monoclonal antibodies were used to detect cell surface antigens: T cells (CD3+, CD4+, CD8+, TCRαβ+, TCRγδ+, CD7+, CD4+CD7+, CD4+CD7−, and CD71+), B cells (HLA-DR+, CD19+, and HLA-DR+CD19+), NKT cells (CD3+CD16+CD56+), and NK cells (CD3−CD16+CD56+). The mean value of all cell surface antigens was not statistically significant between parapsoriasis and MF groups. Along with an increase in cases of MF stage statistically significant difference was found between the mean values of cell surface antigens. Flow cytometric analysis of peripheral blood cell surface antigens in patients with mycosis fungoides may contribute to predicting disease stage and progression. PMID:26788525

  18. High-Throughput Proteomics

    NASA Astrophysics Data System (ADS)

    Zhang, Zhaorui; Wu, Si; Stenoien, David L.; Paša-Tolić, Ljiljana

    2014-06-01

    Mass spectrometry (MS)-based high-throughput proteomics is the core technique for large-scale protein characterization. Due to the extreme complexity of proteomes, sophisticated separation techniques and advanced MS instrumentation have been developed to extend coverage and enhance dynamic range and sensitivity. In this review, we discuss the separation and prefractionation techniques applied for large-scale analysis in both bottom-up (i.e., peptide-level) and top-down (i.e., protein-level) proteomics. Different approaches for quantifying peptides or intact proteins, including label-free and stable-isotope-labeling strategies, are also discussed. In addition, we present a brief overview of different types of mass analyzers and fragmentation techniques as well as selected emerging techniques.

  19. High throughput continuous cryopump

    SciTech Connect

    Foster, C.A.

    1986-01-01

    A cryocondensation pump with a unique regeneration mechanism that allows continuous operation has been constructed and tested. The pump features a device referred to as the ''Snail'' which removes the cryofrost layer as it is moved over the pumping surfaces. A forepump pumps the sublimed gas generated inside the Snail. The compression ratio of the pump is the ratio of the cryopump speed to the leakage conductance of the Snail. Deuterium had been pumped continuously at 30 torr.L/s at a speed of 2000 L/s and a compression ratio of 100. The pump, being all metal sealed and free of lubricating fluids, has many potential applications where untraclean high throughput pumping is desirable. Since the pump regenerates on a time scale of 60 seconds, the inventory in the pump is minimized - an important consideration when pumping radioactive materials such as tritium. Test data and a videotape of the Snail removing the cryofrost will be shown.

  20. 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.

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

    PubMed

    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

  2. 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

  3. 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.

  4. 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

  5. A Flow Cytometric Method for Rapid Selection of Novel Industrial Yeast Hybrids

    PubMed Central

    Bell, P. J. L.; Deere, D.; Shen, J.; Chapman, B.; Bissinger, P. H.; Attfield, P. V.; Veal, D. A.

    1998-01-01

    We rapidly produced and isolated novel yeast hybrids by using two-color flow cytometric cell sorting. We labeled one parent strain with a fluorescent green stain and the other parent with a fluorescent orange stain, and hybrids were selected based on their dual orange and green fluorescence. When this technique was applied to the production of hybrids by traditional mating procedures, more than 96% of the isolates were hybrids. When it was applied to rare mating, three hybrids were identified among 50 isolates enriched from a population containing 2 × 106 cells. This technology is not dependent on genetic markers and has applications in the development of improved industrial yeast strains. PMID:9572934

  6. Flow cytometric detection of altered signaling in myelodysplastic syndrome and cytopenia.

    PubMed

    Gao, Juehua; Swaminathan, Suchitra; Pai, Navin; Johnson, Zachary; Chen, Yi-Hua; Peterson, LoAnn; Goolsby, Charles

    2015-12-01

    Multiparameter flow cytometric analysis allows for precise evaluation of growth factor stimulated intracellular signaling in distinct immunophenotype defined hematopoetic populations. Our analysis of intracellular phosphoprotein in response to major hematopoietic growth factors or cytokines showed several interesting findings. Although there was no characteristic signaling abnormality that was diagnostic for MDS, MDS cases were often associated with more signaling aberrancies involving more cellular populations. Higher than average response in the CD34(+)CD117(+) progenitor cells to Flt3 ligand and stem cell factor stimulation was frequently associated with high risk features or disease progression in MDS. Although preliminary results hint an adverse prognostic role of dysregulated FLT3 pathway in MDS cases, whether this observation adds independent prognostic value to the existing prognostic system needs to be further explored in future prospective studies. PMID:26410459

  7. 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.

  8. 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. PMID:22833391

  9. 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

  10. 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. PMID:25808433

  11. Capillary Flow Layer-by-Layer: A Microfluidic Platform for the High-Throughput Assembly and Screening of Nanolayered Film Libraries

    PubMed Central

    2015-01-01

    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. PMID:24836460

  12. High throughput optical scanner

    DOEpatents

    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.

  13. 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. PMID:21641435

  14. 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

  15. Flow cytometry-assisted quantification of γH2AX expression has potential as a rapid high-throughput biodosimetry tool.

    PubMed

    Achel, Daniel G; Serafin, Antonio M; Akudugu, John M

    2016-08-01

    Large-scale radiological events require immediate and accurate estimates of doses received by victims, and possibly the first responders, to assist in treatment decisions. Although there are numerous efforts worldwide to develop biodosimetric tools to adequately handle triage needs during radiological incidents, such endeavours do not seem to actively involve sub-Saharan Africa which currently has a significant level of nuclear-related activity. To initiate a similar interest in Africa, ex vivo radiation-induced γH2AX expression in peripheral blood lymphocytes from fourteen healthy donors was assessed using flow cytometry. While the technique shows potential for use as a rapid high-throughput biodosimetric tool for radiation absorbed doses up to 5 Gy, significant inter-individual differences in γH2AX expression emerged. Also, female donors exhibited higher levels of γH2AX expression than their male counterparts. To address these shortcomings, gender-based in-house dose-response curves for γH2AX induction in lymphocytes 2, 4, and 6 h after X-ray irradiation are proposed for the South African population. The obtained results show that γH2AX is a good candidate biomarker for biodosimetry, but might need some refinement and validation through further studies involving a larger cohort of donors. PMID:27262315

  16. 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

    2016-09-01

    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. PMID:27538013

  17. Model for high-throughput screening of drug immunotoxicity--study of the anti-microbial G1 over peritoneal macrophages using flow cytometry.

    PubMed

    Tenorio-Borroto, Esvieta; Peñuelas-Rivas, Claudia G; Vásquez-Chagoyán, Juan C; Castañedo, Nilo; Prado-Prado, Francisco J; García-Mera, Xerardo; González-Díaz, Humberto

    2014-01-24

    Quantitative Structure-Activity (mt-QSAR) techniques may become an important tool for prediction of cytotoxicity and High-throughput Screening (HTS) of drugs to rationalize drug discovery process. In this work, we train and validate by the first time mt-QSAR model using TOPS-MODE approach to calculate drug molecular descriptors and Linear Discriminant Analysis (LDA) function. This model correctly classifies 8258 out of 9000 (Accuracy = 91.76%) multiplexing assay endpoints of 7903 drugs (including both train and validation series). Each endpoint correspond to one out of 1418 assays, 36 molecular and cellular targets, 46 standard type measures, in two possible organisms (human and mouse). After that, we determined experimentally, by the first time, the values of EC50 = 21.58 μg/mL and Cytotoxicity = 23.6% for the anti-microbial/anti-parasite drug G1 over Balb/C mouse peritoneal macrophages using flow cytometry. In addition, the model predicts for G1 only 7 positive endpoints out 1251 cytotoxicity assays (0.56% of probability of cytotoxicity in multiple assays). The results obtained complement the toxicological studies of this important drug. This work adds a new tool to the existing pool of few methods useful for multi-target HTS of ChEMBL and other libraries of compounds towards drug discovery. PMID:24445280

  18. 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. PMID:27110975

  19. A new spreadsheet method for the analysis of bivariate flow cytometric data

    PubMed Central

    Tzircotis, George; Thorne, Rick F; Isacke, Clare M

    2004-01-01

    Background A useful application of flow cytometry is the investigation of cell receptor-ligand interactions. However such analyses are often compromised due to problems interpreting changes in ligand binding where the receptor expression is not constant. Commonly, problems are encountered due to cell treatments resulting in altered receptor expression levels, or when cell lines expressing a transfected receptor with variable expression are being compared. To overcome this limitation we have developed a Microsoft Excel spreadsheet that aims to automatically and effectively simplify flow cytometric data and perform statistical tests in order to provide a clearer graphical representation of results. Results To demonstrate the use and advantages of this new spreadsheet method we have investigated the binding of the transmembrane adhesion receptor CD44 to its ligand hyaluronan. In the first example, phorbol ester treatment of cells results in both increased CD44 expression and increased hyaluronan binding. By applying the spreadsheet method we effectively demonstrate that this increased ligand binding results from receptor activation. In the second example we have compared AKR1 cells transfected either with wild type CD44 (WT CD44) or a mutant with a truncated cytoplasmic domain (CD44-T). These two populations do not have equivalent receptor expression levels but by using the spreadsheet method hyaluronan binding could be compared without the need to generate single cell clones or FACS sorting the cells for matching CD44 expression. By this method it was demonstrated that hyaluronan binding requires a threshold expression of CD44 and that this threshold is higher for CD44-T. However, at high CD44-T expression, binding was equivalent to WT CD44 indicating that the cytoplasmic domain has a role in presenting the receptor at the cell surface in a form required for efficient hyaluronan binding rather than modulating receptor activity. Conclusion Using the attached

  20. Flow cytometric analysis of micronuclei in rat peripheral blood: An interlaboratory reproducibility study.

    PubMed

    Kasamoto, Sawako; Mukai, Daisuke; Masumori, Shoji; Suzuki, Kenichiro; Tanaka, Ryota; Torous, Dorothea K; Yamate, Jyoji; Hayashi, Makoto

    2014-03-01

    In anticipation of proposed OECD guideline changes that may include increasing the number of reticulocytes scored for micronuclei, an inter-laboratory reproducibility study of the rat peripheral blood micronucleus assay was performed using flow cytometry. In this experiment, male Sprague-Dawley (SD) rats were treated with the model clastogen cyclophosphamide (CP: 5, 10 or 15mg/kg) by a single oral administration. As controls, rats were treated with physiological saline (solvent) in the same manner as for the model clastogen. Peripheral blood was collected from each rat 48h after the treatment. The blood samples were prepared at the Public Interest Incorporated Foundation, BioSafety Research Center (BSRC) in duplicate using the rat MicroFlow(PLUS) Kit. After fixation, one replicate set of samples was shipped to Litron Laboratories, and each sample was analyzed by flow cytometry at the two laboratories. In addition, the frequency of micronucleated reticulocytes (MNRETs) was determined at the BSRC by microscopic analysis using supravital acridine orange (AO) staining. The reproducibility of micronucleated reticulocyte frequencies analyzed by microscopy and flow cytometry showed good correlation (r(2)=0.84). The frequencies of micronucleated reticulocytes analyzed by flow cytometry at the two independent laboratories showed good concordance (r(2)=0.97). The data indicate that the flow cytometric micronucleus analysis method is a good alternative to manual microscopic analysis. Flow cytometry allows groups to readily score 5000 or even 20,000 RETs in a matter of minutes compared to manual analysis. This results in increased reliability of the assay by achieving better statistical power. PMID:24548793

  1. 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.

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

    PubMed Central

    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. PMID:25673909

  3. Relationship of flow cytometric sperm integrity assessments with boar fertility performance under optimized field conditions.

    PubMed

    Broekhuijse, M L W J; Šoštarić, E; Feitsma, H; Gadella, B M

    2012-12-01

    The number of intact and functional spermatozoa in semen can be assessed with flow cytometry and is believed to relate to male fertility. The aim of this study was to examine whether currently used sperm integrity assessments with flow cytometry correlate with field fertility data obtained for boar semen. For this purpose, 20 boars were followed for a 20-wk period (with a total average production of 33 ejaculates per boar) and the obtained fertility results (farrowing rate and number of piglets born) of commercial artificial insemination doses made from these ejaculates were recorded. Fertility results were corrected for farm, sow, boar, and semen-related parameters. From the same semen samples, sperm cell integrity was assessed with respect to DNA and to membrane integrity, acrosome intactness and responsiveness, and mitochondrial potential using established flow cytometric assays. This was done on freshly produced semen and on semen stored for up to 15 d. Remarkably, none of the individual membrane integrity variables was significantly related to fertility results. In contrast, the amount of DNA damage as assessed at 7 to 10 d and at 14 to 15 d of semen storage related to farrowing rate (P = 0.0400) and total number of piglets born (P = 0.0310), respectively. Therefore, the degree of DNA damage in stored boar semen samples may be a useful factor to evaluate semen as an indicator for litter size and farrowing rate. PMID:23255815

  4. Expression of oestrogen and progesterone receptors in gastric cancer: a flow cytometric study.

    PubMed

    Karat, D; Brotherick, I; Shenton, B K; Scott, D; Raimes, S A; Griffin, S M

    1999-06-01

    Increased expression of oestrogen (ER) and progesterone (PR) receptors have been reported in gastric adenocarcinoma, although results have been variable. Immunohistochemical staining methodologies, in particular in the detection of ER, have been inconsistent with many tumours being classified ER-negative. In this study we have used flow cytometry to quantify expression of ER and PR in gastric adenocarcinoma and examine their relationships with established prognostic indicators. Cytokeratin-positive cells obtained from tumour biopsies of 50 patients with gastric cancer and ten control patients were labelled with biotinylated ER or PR antibodies followed by streptavidin PE. Flow cytometry was seen to increase the detection of ER levels in gastric cancer with more receptor-positive patients in this study than in results published to date. We believe this is related to the sensitivity of the flow cytometric assay with the detection of small shifts in ER level detected using cytokeratin gating. On analysis, the data showed no significant correlations with tumour stage and grade, and no differences were seen between normal mucosa and gastric cancer samples. PMID:10376983

  5. 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.

  6. CUBIC: a three-dimensional colored projection of Consort 30 generated trivariate flow cytometric data.

    PubMed

    Greimers, R; Rongy, A M; Schaaf-Lafontaine, N; Boniver, J

    1991-01-01

    The CUBIC program displays three-dimensional colored dot plots of flow cytometric trivariate data collected by unmodified commercial instruments (FACScan flow cytometer, FACS 440 cell sorter). Assuming a bimodal distribution of the fluorescence intensity of the cells, the eight theoretical subpopulations involved in a three-color fluorescence histogram are clearly localized in the 3-D space by colored dots that are clustered near each corner of a cubic frame. Rotation, tilting, and zoom functions are available. Table look-up is not needed. CUBIC was illustrated by two experiments: 1) three-color immunofluorescence of antigens on human lymphocytes using monoclonal antibodies conjugated either to fluorescein (FITC), to R-phycoerythrin (PE), or to biotin revealed by a streptavidin coupled to a PE-Texas red tandem conjugate (TC); 2) two-color immunofluorescence of CD4 and CD8 antigens on thymocytes of healthy or preleukemic mice correlated to the DNA content quantified by 7-amino-actinomycin D (7-AAD). The three fluorescences were excited by a single argon-ion laser emitting at 488 nm. PMID:1764980

  7. Flow-cytometric method for simultaneous analysis of mouse lung epithelial, endothelial, and hematopoietic lineage cells.

    PubMed

    Singer, Benjamin D; Mock, Jason R; D'Alessio, Franco R; Aggarwal, Neil R; Mandke, Pooja; Johnston, Laura; Damarla, Mahendra

    2016-05-01

    Flow cytometry is a powerful tool capable of simultaneously analyzing multiple parameters on a cell-by-cell basis. Lung tissue preparation for flow cytometry requires creation of a single-cell suspension, which often employs enzymatic and mechanical dissociation techniques. These practices may damage cells and cause cell death that is unrelated to the experimental conditions under study. We tested methods of lung tissue dissociation and sought to minimize cell death in the epithelial, endothelial, and hematopoietic lineage cellular compartments. A protocol that involved flushing the pulmonary circulation and inflating the lung with Dispase, a bacillus-derived neutral metalloprotease, at the time of tissue harvest followed by mincing, digestion in a DNase and collagenase solution, and filtration before staining with fluorescent reagents concurrently maximized viable yields of epithelial, endothelial, and hematopoietic lineage cells compared with a standard method that did not use enzymes at the time of tissue harvest. Flow cytometry identified each population-epithelial (CD326(+)CD31(-)CD45(-)), endothelial (CD326(-)CD31(+)CD45(-)), and hematopoietic lineage (CD326(-)CD31(-)CD45(+))-and measured cellular viability by 7-aminoactinomycin D (7-AAD) staining. The Dispase method permitted discrimination of epithelial vs. endothelial cell death in a systemic lipopolysaccharide model of increased pulmonary vascular permeability. We conclude that application of a dissociative enzyme solution directly to the cellular compartments of interest at the time of tissue harvest maximized viable cellular yields of those compartments. Investigators could employ this dissociation method to simultaneously harvest epithelial, endothelial, and hematopoietic lineage and other lineage-negative cells for flow-cytometric analysis. PMID:26944088

  8. 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.

  9. 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. PMID:26099800

  10. 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. PMID:8789270

  11. 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

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

    PubMed

    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

  13. Flow cytometric probing of mitochondrial function in equine peripheral blood mononuclear cells

    PubMed Central

    Cassart, Dominique; Fett, Thomas; Sarlet, Michaël; Baise, Etienne; Coignoul, Freddy; Desmecht, Daniel

    2007-01-01

    Background The morphopathological picture of a subset of equine myopathies is compatible with a primary mitochondrial disease, but functional confirmation in vivo is still pending. The cationic dye JC-1 exhibits potential-dependent accumulation in mitochondria that is detectable by a fluorescence shift from green to orange. As a consequence, mitochondrial membrane potential can be optically measured by the orange/green fluorescence intensity ratio. A flow cytometric standardized analytic procedure of the mitochondrial function of equine peripheral blood mononuclear cells is proposed along with a critical appraisal of the crucial questions of technical aspects, reproducibility, effect of time elapsed between blood sampling and laboratory processing and reference values. Results The JC-1-associated fluorescence orange and green values and their ratio were proved to be stable over time, independent of age and sex and hypersensitive to intoxication with a mitochondrial potential dissipator. Unless time elapsed between blood sampling and laboratory processing does not exceed 5 hours, the values retrieved remain stable. Reference values for clinically normal horses are given. Conclusion Whenever a quantitative measurement of mitochondrial function in a horse is desired, blood samples should be taken in sodium citrate tubes and kept at room temperature for a maximum of 5 hours before the laboratory procedure detailed here is started. The hope is that this new test may help in confirming, studying and preventing equine myopathies that are currently imputed to mitochondrial dysfunction. PMID:17903245

  14. 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. PMID:23826393

  15. A rapid procedure for flow cytometric DNA analysis in cultures of normal and transformed epidermal cells.

    PubMed

    Tennenbaum, T; Giloh, H; Fusenig, N E; Kapitulnik, J

    1988-06-01

    A simple, rapid, and highly reproducible procedure for flow cytometric DNA analysis has been adapted for studying cell cycle kinetics in epidermal cell cultures. The preparation of cell nuclei and their staining with the fluorescent dye propidium iodide were performed directly on the culture dish, without prior suspension and fixation of the cells. Singly dispersed nuclei were produced by mild trypsinization of cells in the presence of the nonionic detergent Nonidet P-40 and spermine. The culture dishes could be kept frozen for prolonged periods of time before trypsinization and staining, without affecting either the recovery of nuclei or the cell cycle distribution profiles. This remarkable stability of cell nuclei greatly simplified the analysis of multiple samples in cell cycle kinetic studies. This method was used to analyze the cell cycle distribution in cultures of normal and transformed mouse epidermal cells, human colon carcinoma cells, primary bovine aortic endothelial cells, and fibroblastic and myogenic cell lines. This procedure should be very useful in studying growth kinetics, differentiation, and transformation of epidermal as well as other adherent cell types. PMID:2453587

  16. LY303366 exhibits rapid and potent fungicidal activity in flow cytometric assays of yeast viability.

    PubMed

    Green, L J; Marder, P; Mann, L L; Chio, L C; Current, W L

    1999-04-01

    LY303366 is a semisynthetic analog of the antifungal lipopeptide echinocandin B that inhibits (1,3)-beta-D-glucan synthase and exhibits efficacy in animal models of human fungal infections. In this study, we utilized flow cytometric analysis of propidium iodide uptake, single-cell sorting, and standard microbiological plating methods to study the antifungal effect of LY303366 on Saccharomyces cerevisiae and Candida albicans. Our data indicate that an initial 5-min pulse treatment with LY303366 caused yeasts to take up propidium iodide and lose their ability to grow. Amphotericin B and cilofungin required longer exposure periods (30 and 180 min, respectively) and higher concentrations to elicit these fungicidal effects. These two measurements of fungicidal activity by LY303366 were highly correlated (r > 0.99) in concentration response and time course experiments. As further validation, LY303366-treated yeasts that stained with propidium iodide were unable to grow in single-cell-sorted cultures. Our data indicate that LY303366 is potent and rapidly fungicidal for actively growing yeasts. The potency and rapid action of this new fungicidal compound suggest that LY303366 may be useful for antifungal therapy. PMID:10103187

  17. A novel flow cytometric-based method to measure kinase inhibition in sputum from COPD subjects

    PubMed Central

    Nicholson, G C; Holloway, R A; Leaker, B R; Kilty, I; Salganik, M; Tan, L; Barnes, P J; Donnelly, L E

    2016-01-01

    Introduction Janus kinases (JAKs) regulate inflammatory gene expression through phosphorylation of signal transducer and activator of transcription (STAT) proteins. Expression of STAT proteins is increased in chronic obstructive pulmonary disease (COPD), and may be involved in driving chronic inflammation. Oral JAK inhibitors are effective as anti-inflammatory therapy but exhibit dose-limiting adverse effects. Development of inhaled compounds would be enhanced by robust biomarkers that directly reflect the anti-inflammatory and pharmacological activity in the lung. Methods A novel flow cytometry assay was developed to measure STAT1 phosphorylation in sputum inflammatory cells. The standard sputum processing method was refined to improve sputum cell viability. The flow cytometric assay was used to assess the reproducibility of the measurement of STAT1 phosphorylation and the in vitro activity of a pan JAK-inhibitor on three separate visits in patients with COPD. Results Upregulation of STAT1 phosphorylation was measured following in vitro IFNγ stimulation of sputum macrophages (stimulated/unstimulated ratio 1.57; p<0.00001). Upregulation was inhibited following in vitro preincubation with a pan JAK-inhibitor (inhibited+stimulated/unstimulated ratio 0.97). STAT1 phosphorylation activity could only be measured in macrophages. Conclusions Sputum from patients with COPD can be used to reproducibly measure phospho-STAT expression in sputum macrophages. The flow cytometry-based method can be used to evaluate kinase inhibitors in vitro and subsequently in ex vivo studies. The assay is particularly useful for the assessment of inhaled compounds where whole blood assays may not be relevant. PMID:27403320

  18. High Content Flow Cytometric Micronucleus Scoring Method is Applicable to Attachment Cell Lines

    PubMed Central

    Bryce, Steven M.; Shi, Jing; Nicolette, John; Diehl, Marilyn; Sonders, Paul; Avlasevich, Svetlana; Raja, Sarojini; Bemis, Jeffrey C.; Dertinger, Stephen D.

    2009-01-01

    A flow cytometric method for analyzing suspension cell cultures for micronucleus content has been previously reported [Environ. Molec. Mutagen. 47 (2006) 56–66]. The experiments described herein were undertaken to evaluate the compatibility of this method (In Vitro MicroFlow®) with attachment cells. Initially, CHO-K1 cells were studied in nine independent experiments using mitomycin C and cyclophosphamide. The results demonstrated the effectiveness of the cell processing procedure, and also provided historical control data that were useful for setting criteria for making positive calls. Subsequently, CHO-K1 cells were treated with methyl methanesulfonate, mitomycin C, etoposide, vinblastine sulfate, dexamethasone, and sodium chloride. Whereas the four genotoxicants were each observed to increase micronucleus frequencies, the non-genotoxicants induced no such response up to cytotoxic concentrations. Following this initial work, inter-laboratory transferability was evaluated across three sites using a common cell staining and analysis protocol for CHO-K1 or V79 cells that had been treated with the ten chemicals listed in Annex 3 of the OECD Draft Proposal for a New Guideline 487: In Vitro Mammalian Cell Micronucleus Test. With the exception of benzo[a]pyrene at one site, each laboratory observed increased micronucleus frequencies for the genotoxicants, whereas no significant induction occurred with the non-genotoxicants. Interestingly, the method appeared to distinguish between genotoxic modes of action, as only aneugens increased the average micronucleus fluorescence intensity and the frequency of hypodiploid nuclei. Collectively, these data suggest that flow cytometry is capable of providing reliable micronucleus counts, and that additional information is obtained that appears to discern genotoxic modes of action. PMID:19950402

  19. 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

  20. 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

  1. Genome Size Variation among and within Camellia Species by Using Flow Cytometric Analysis

    PubMed Central

    Zhang, Qun-Jie; Gao, Li-Zhi

    2013-01-01

    Background The genus Camellia, belonging to the family Theaceae, is economically important group in flowering plants. Frequent interspecific hybridization together with polyploidization has made them become taxonomically “difficult taxa”. The DNA content is often used to measure genome size variation and has largely advanced our understanding of plant evolution and genome variation. The goals of this study were to investigate patterns of interspecific and intraspecific variation of DNA contents and further explore genome size evolution in a phylogenetic context of the genus. Methodology/Principal Findings The DNA amount in the genus was determined by using propidium iodide flow cytometry analysis for a total of 139 individual plants representing almost all sections of the two subgenera, Camellia and Thea. An improved WPB buffer was proven to be suitable for the Camellia species, which was able to counteract the negative effects of secondary metabolite and generated high-quality results with low coefficient of variation values (CV) <5%. Our results showed trivial effects on different tissues of flowers, leaves and buds as well as cytosolic compounds on the estimation of DNA amount. The DNA content of C. sinensis var. assamica was estimated to be 1C = 3.01 pg by flow cytometric analysis, which is equal to a genome size of about 2940 Mb. Conclusion Intraspecific and interspecific variations were observed in the genus Camellia, and as expected, the latter was larger than the former. Our study suggests a directional trend of increasing genome size in the genus Camellia probably owing to the frequent polyploidization events. PMID:23724111

  2. Flow cytometric analysis of circulating platelet-monocyte aggregates in whole blood: methodological considerations.

    PubMed

    Harding, Scott A; Din, Jehangir N; Sarma, Jaydeep; Jessop, Alasdair; Weatherall, Mark; Fox, Keith A A; Newby, David E

    2007-08-01

    Platelet-monocyte aggregates are increasingly being used to quantify platelet activation. The variables that influence platelet-monocyte aggregates have not been well defined. We sought to determine the effect of blood collection, handling and processing techniques on detected levels of platelet-monocyte aggregates using a flow cytometric assay. Whole blood was labelled with anti-CD14-PE and anti-CD42a-FITC. Thereafter, samples were fixed and red cells lysed. Analysis was performed with the flow cytometer initially triggering on light scatter and then on FL-2 to identify CD14-PE positive monocytes. Platelet-monocyte aggregates were defined as monocytes positive for CD42a. The effect of collection, handling and processing techniques on this assay were assessed. Anticoagulation with heparin (20.1 +/- 2.0%), PPACK (16.8 +/- 1.9%), sodium citrate (12.3 +/- 1.6%) and EDTA (9.5 +/- 1.0%) resulted in markedly different levels of platelet-monocyte aggregation (P < 0.0001). Platelet-monocyte aggregation was higher in samples obtained from intravenous cannulae compared to those obtained by venepuncture (20.9 +/- 3.9% vs.13.8 +/- 2.4%, P = 0.03). For every 10 minutes of delay prior to processing platelet-monocyte aggregates increased by 2.8% (P = 0.0001) in PPACK anticoagulated blood and 1.7% (P = 0.01) in citrate anticoagulated blood. Erythrocyte lysis together with fixation does not affect platelet-monocyte aggregation. Platelet-monocyte aggregates remained stable over 24 hours when fixed and stored at 4 degrees C. Multiple handling and processing factors may affect platelet-monocyte aggregation. We recommend the measurement of platelet-monocyte aggregates on samples collected by direct venepuncture, using a direct thrombin inhibitor as the anticoagulant and minimising the time delay before sample fixation. PMID:17721630

  3. 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.

  4. A histological and flow cytometric study of dog brain endothelial cell injuries in delayed radiation necrosis

    SciTech Connect

    Yamaguchi, N.; Yamashima, T.; Yamashita, J. )

    1991-04-01

    The pathogenesis of delayed cerebral radiation necrosis was studied histologically and biochemically in 25 dogs with special attention to vascular endothelial cell injuries. The dogs were sacrificed 3 to 30 months after irradiation with a single dose of 15 Gy to the head. Brain specimens were appropriately fixed for light and electron microscopic studies, and capillary endothelial cells were isolated for flow cytometric study. The endothelial cells were stained with acridine orange, then the cell ratios in the reproductive phase (S + G2 + M) were investigated with flow cytometry. Thereafter, Feulgen hydrolysis and computer analysis of the hydrolysis curves were performed to examine the qualitative changes in deoxyribonucleic acid (DNA) of endothelial cells after irradiation. Under light microscopy, spongy degeneration with small cell infiltration was observed, especially in the frontal white matter, at 6 months after irradiation. At 9 months, necrotic foci appeared and developed until 15 months after irradiation. Blood vessels around the necrotic area showed luminal narrowing with endothelial hyperplasia and proliferation. At 30 months, no fresh necrotic lesions were observed. Under electron microscopy, endothelial cells of capillaries and small vessels around the necrotic area showed an increase of pinocytosis, and in the nuclei there was an increase of infoldings and euchromatin. The cell ratios in the reproductive phase were 14.5% to 23.3% (maximum at 9 months) in the irradiated group compared to 6.4% in the control group. The rate constant of apurinic acid production, a parameter correlating with DNA transcriptional activity, was minimum at 3 months and maximum at 9 months after irradiation. The data suggest that impairment of the microcirculation plays an important role in the pathogenesis of delayed radiation necrosis.

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

    PubMed

    Gauthier, Julie D; Jenkins, Jill A; La Peyre, Jerome F

    2004-06-01

    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. PMID:15270084

  6. 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

  7. Excimer fluorescence compared to depolarization in the flow cytometric characterization of lateral membrane mobility in platelets

    NASA Astrophysics Data System (ADS)

    Rothe, Gregor; Schaefer, Buerk; Wimmer, Martin S.; Schmitz, Gerd

    1998-04-01

    An altered cellular membrane fluidity secondary to changes of cholesterol metabolism is a potentially important mechanism in the pathogenesis of atherosclerosis. Especially in blood platelets an increased sensitivity for stimulation dependent aggregation which is a risk factor for thrombosis has been experimentally linked to disorders of lipid and lipoprotein metabolism. The goal of this study was the development of a flow cytometric assay for the direct analysis of cellular membrane microviscosity in correlation to activation associated phenotypic changes of platelets in vitro. The analysis of fluorescence polarization following the staining of hydrophobic lipid regions of cell membranes with the fluorescent dye 1,6-diphenyl-1,3,5-hexatriene (DPH) is a well established method for the analysis of membrane fluidity. The extent of fluorescence anisotropy dependent on the rotational mobility of this fluorochrome is indirectly proportional to the microviscosity of the stained membrane subcompartment. In this study, an alternative and more simple method based on the diffusion dependent excimer formation of pyrenedecanoic acid (PDA) (J. Immunol. Methods 96:225-31, 1987) was characterized in comparison to the DPH method as a reference. Human platelets showed a rapid uptake of both DPH and PDA resulting in the staining primarily of the plasma membrane after up to 30 min of incubation. Staining analyzed at 351 nm excitation resulted in a saturation of the depolarization coefficient of DPH at 20 (mu) M but an increase of the excimer to monomer ratio of PDA with increasing dye concentration. A 'membrane fluidity coefficient' which saturated at 5 (mu) M PDA was calculated as the excimer fluorescence divided through the square of monomer fluorescence thereby correcting for the influence of dye concentration on excimer formation. The temperature dependent changes of membrane viscosity were further used as a model for the comparison of both methods. Cells analyzed at temperatures

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

    PubMed

    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

  9. Flow cytometric characterization of rat thymus cells in a radiation-dominated model of combined injury

    SciTech Connect

    Kaffenberger, W.; Gruber, D.F.; MacVittie, T.J.

    1988-05-01

    Thymuses of rats that had been: a) gamma-irradiated (500 cGy whole-body radiation (R)), or b) thermally injured (20% BSA dorsal, scald burn (TI)), or c) combined injured (irradiation followed by burn (CI)) were studied for involution and recovery processes after sublethal treatments. The expression of surface antigens on thymic cells before and after injuries was evaluated using the monoclonal antibodies (mcAB) MRC OX4, MRC OX7, MRC OX8, W3/13 HLK, and W3/25 and flow cytometric analysis. Thymic cellularity decreased to less than 1% of normal (N), age-matched rats by 4 days after R or CI. Recovery reached 60% to 70% of N by 28 days post treatments. TI caused a biphasic thymic recovery pattern with nadirs of 40% of N on days 7 and 21. Recovery at day 28 was similar to that after R and CI. Expression of OX7, OX8, W3/13, and W3/25 antigens all reached nadirs of 40% of N by day 4 after R and CI. Recovery of antigen expression, except for W3/25, was near completion by day 7 after R and CI. Changes in antigen expression after TI were less pronounced for all mcAB tested. Decreases in labeling of thymocytes with the helper T-cell marker, W3/25, observed after TI, could not be correlated with elevated expressions of the suppressor/cytotoxic T-lymphocyte antigen, OX8. Variations in relative labeling of nonlymphoid thymic cells with OX4 (Ia-antigen) reflected the disappearance and recovery of radiosensitive lymphoid thymocytes. The similarity of results after R and CI demonstrate that the model of CI is radiation-dominated. The addition of burn injury to radiation trauma had no synergistically damaging effect on the parameters studied.

  10. Influence of storage time on functional capacity of flow cytometrically sex-sorted boar spermatozoa.

    PubMed

    Parrilla, Inmaculada; Vazquez, Juan M; Gil, Maria A; Caballero, Ignacio; Almiñana, Carmen; Roca, Jordi; Martinez, Emilio A

    2005-07-01

    Sex-sorting of boar spermatozoa is an emerging biotechnology, still considered suboptimal owing to the slowness of the process, which requires long sorting periods to obtain an adequate number of spermatozoa to perform a non-surgical insemination. This period involves storage of sorted cells that could impair their functional capacity. Here, we have studied how the storage of sex-sorted boar spermatozoa affects their functional capacity. Sorted spermatozoa were assessed at various times (0, 2, 5h or 10h) during storage after sorting and compared with diluted and unsorted spermatozoa for sperm motility patterns, plasma membrane and acrosomal integrity and their ability to penetrate homologous IVM oocytes. Sex-sorted sperm motility and membrane integrity only decreased significantly (p<0.05) by the end of the storage period (10h) compared to unsorted spermatozoa. Sperm velocity, ALH and Dance increased significantly (p<0.05), immediately post-sorting, returning to unsorted sperm values during storage. Acrosome integrity was not seriously affected by the sorting process, but decreased (p<0.05) during storage after sorting. Sorted spermatozoa stored 2h after sorting did not differ from unsorted in penetration rates and numbers of spermatozoa per oocyte, reaching the highest (p<0.05) penetration rates and sperm numbers per oocyte, when co-cultured for 6 or more hours. Non-storage or storage for 5h or 10h negatively (p<0.05) affected sperm penetration ability. In conclusion, although flow cytometrically sex-sorted spermatozoa are able to maintain motility, viability and acrosomal integrity at optimal levels until 10h of storage after sorting, fertilizing ability is maintained only over shorter storage times (<5h). PMID:15935845

  11. 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

  12. Phagocytosis of viable Candida albicans by alveolar macrophages: flow cytometric quantification.

    PubMed

    Rosseau, S; Seeger, W; Pralle, H; Lohmeyer, J

    1994-08-01

    The phagocytic capacity of blood leukocytes may be assessed by flow cytometric techniques using fluorochrome-labeled particles including viable microorganisms. Application of this approach to alveolar macrophages (AM) is hampered or even rendered impossible by the strong autofluorescence of this cell type, superimposing the fluorescence intensity of the labeled phagocytic targets. Viable Candida albicans were loaded with the membrane-permeable fluorescent dye carboxy-seminaphtorhodafluor 2/acetoxymethylester (carboxy-SNARF 2-AM), which is cleaved intracellularly to generate the membrane-impermeable derivative carboxy-SNARF 2. Fluorescence was excited with the 488-nm line of an argon-ion laser, and the emission peak at 633 nm was used for quantification of dye-associated fluorescence. Rabbit and human AM were labeled with fluorescein isothiocyanate-coupled monoclonal mouse anti-macrophage antibodies. After coincubation of macrophages and yeast, 4% paraformaldehyde plus 0.5% EDTA in phosphate-buffered saline was used to stop the phagocytic process and detach adherent yeast from the AM surface. Macrophages loaded with yeast displayed a shift from monochromatic (green) to dual (green and red) fluorescence. The percentage of yeast-positive AM and red fluorescence intensity of phagocytosing macrophages were quantified. Yeast opsonization with serum or anti-Candida immunoglobulins was a prerequisite for phagocytosis. Under optimized conditions (0.5-10% serum; 60 min yeast-AM incubation; yeast-AM ratio 8:1 to 12:1), 71-91% of the AM were involved in the phagocytic process. Yeast engulfment was completely inhibited by N-ethylmaleimide and iodoacetic acid.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8074245

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

    PubMed Central

    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. PMID:24926290

  14. A Novel High-Throughput Viscometer.

    PubMed

    Deshmukh, Suraj; Bishop, Matthew T; Dermody, Daniel; Dietsche, Laura; Kuo, Tzu-Chi; Mushrush, Melissa; Harris, Keith; Zieman, Jonathan; Morabito, Paul; Orvosh, Brian; Patrick, Don

    2016-07-11

    A novel, rapid, parallel, and high-throughput system for measuring viscosity of materials under different conditions of shear rate, temperature, time, etc., has been developed. This unique system utilizes the transient flow of a complex fluid through pipettes. This approach offers significant practical advantages over microfluidic-based devices for viscosity screening: no cleanup is required, the method is high throughput (<1 h for 100 samples), and only small sample volumes (<1 mL) are used. This paper details for the first time the experimental and modeling efforts to implement this mass- and pressure-based viscosity measurement concept as a robust viscosity estimation tool. This approach is very well-suited for viscosity measurements in high-throughput formulation workflows, as it is rapid and parallel and operates directly on samples in various microtiter plate formats. We present systematic experimental observations together with numerical and analytical modeling approaches to characterize instrument capabilities and limitations. The complex transient flow of fluids through these pipettes leads to data-rich pressure profiles. Numerical and analytical modeling is then used to extract viscosity and other rheological parameters from these pressure profiles. We have successfully utilized this viscosity screening tool for a multitude of complex fluids including oils, paints, solvents, and detergents. PMID:27259016

  15. FLOW CYTOMETRIC DETECTION AND SIZING OF FLUORESCENT PARTICLES DEPOSITED AT A SEWAGE OUTFALL SITE

    EPA Science Inventory

    A suspension of fluorescent pigment particles (total mass, 120 kg) was injected over a period of several hours into a sewage outfall discharging into Salem Sound, MA. low cytometric analysis was successfully used to identify, quantify, and size the fluorescent pigment particles i...

  16. Genotoxic mode of action predictions from a multiplexed flow cytometric assay and a machine learning approach.

    PubMed

    Bryce, Steven M; Bernacki, Derek T; Bemis, Jeffrey C; Dertinger, Stephen D

    2016-04-01

    Several endpoints associated with cellular responses to DNA damage as well as overt cytotoxicity were multiplexed into a miniaturized, "add and read" type flow cytometric assay. Reagents included a detergent to liberate nuclei, RNase and propidium iodide to serve as a pan-DNA dye, fluorescent antibodies against γH2AX, phospho-histone H3, and p53, and fluorescent microspheres for absolute nuclei counts. The assay was applied to TK6 cells and 67 diverse reference chemicals that served as a training set. Exposure was for 24 hrs in 96-well plates, and unless precipitation or foreknowledge about cytotoxicity suggested otherwise, the highest concentration was 1 mM. At 4- and 24-hrs aliquots were removed and added to microtiter plates containing the reagent mix. Following a brief incubation period robotic sampling facilitated walk-away data acquisition. Univariate analyses identified biomarkers and time points that were valuable for classifying agents into one of three groups: clastogenic, aneugenic, or non-genotoxic. These mode of action predictions were optimized with a forward-stepping process that considered Wald test p-values, receiver operator characteristic curves, and pseudo R(2) values, among others. A particularly high performing multinomial logistic regression model was comprised of four factors: 4 hr γH2AX and phospho-histone H3 values, and 24 hr p53 and polyploidy values. For the training set chemicals, the four-factor model resulted in 94% concordance with our a priori classifications. Cross validation occurred via a leave-one-out approach, and in this case 91% concordance was observed. A test set of 17 chemicals that were not used to construct the model were evaluated, some of which utilized a short-term treatment in the presence of a metabolic activation system, and in 16 cases mode of action was correctly predicted. These initial results are encouraging as they suggest a machine learning strategy can be used to rapidly and reliably predict new chemicals

  17. Flow Cytometric Analysis of Protective T-Cell Response Against Pulmonary Coccidioides Infection.

    PubMed

    Hung, Chiung-Yu; Wozniak, Karen L; Cole, Garry T

    2016-01-01

    The incidence of systemic fungal infections has increased throughout the world, spurring much interest in developing effective vaccines. Coccidioidomycosis, also known as San Joaquin Valley fever, is a potentially life-threatening respiratory mycosis. A vaccine against Coccidioides infection would contribute significantly to the well-being of the approx. 30 million residents in the Southwestern USA as well as the multitude of travelers who annually visit the endemic regions. We have applied a live, attenuated vaccine (∆T) to explore the nature of vaccine immunity in mice after intranasal challenge with a potentially lethal dose of Coccidioides spores. Coccidioides spores are airborne and highly infectious for mammalian hosts and classified as a biosafety level 3 agent. T cells are critical in the development of protective immunity against a variety of microorganisms as well as the development of autoimmune disease and allergic responses. Profiles of cytokines detected in lung homogenates of ∆T-vaccinated mice were indicative of a mixed Th1, Th2, and Th17 immune response. We have developed an intracellular cytokine staining and flow cytometric (ICS) technique to measure activated CD4(+) and CD8(+) T cells and IFN-γ-, IL-4-, IL-5-, and IL-17A-producing T cells in the lungs of mice that are challenged with a potentially lethal dose of Coccidioides spores. The numbers of pulmonary Th1 and Th17 cells during the first 2 weeks post-challenge showed a progressive increase in vaccinated mice and corresponded with reduction of fungal burden. In this protocol, we describe the methodology for culture and isolation of the live, attenuated ΔT spores of Coccidioides used to vaccinate mice, preparation of pulmonary cells, and staining protocol for cell surface markers and intracellular cytokines. This is the most reliable and robust procedure to measure frequencies and numbers of each selected T-cell subsets in lungs of vaccinated versus control mice and can be readily

  18. 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.

  19. 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.

  20. High-throughput tetrad analysis.

    PubMed

    Ludlow, Catherine L; Scott, Adrian C; Cromie, Gareth A; Jeffery, Eric W; Sirr, Amy; May, Patrick; Lin, Jake; Gilbert, Teresa L; Hays, Michelle; Dudley, Aimée M

    2013-07-01

    Tetrad analysis has been a gold-standard genetic technique for several decades. Unfortunately, the need to manually isolate, disrupt and space tetrads has relegated its application to small-scale studies and limited its integration with high-throughput DNA sequencing technologies. We have developed a rapid, high-throughput method, called barcode-enabled sequencing of tetrads (BEST), that uses (i) a meiosis-specific GFP fusion protein to isolate tetrads by FACS and (ii) molecular barcodes that are read during genotyping to identify spores derived from the same tetrad. Maintaining tetrad information allows accurate inference of missing genetic markers and full genotypes of missing (and presumably nonviable) individuals. An individual researcher was able to isolate over 3,000 yeast tetrads in 3 h, an output equivalent to that of almost 1 month of manual dissection. BEST is transferable to other microorganisms for which meiotic mapping is significantly more laborious. PMID:23666411

  1. High-throughput continuous cryopump

    SciTech Connect

    Foster, C.A.

    1986-01-01

    A cryopump with a unique method of regeneration which allows continuous operation at high throughput has been constructed and tested. Deuterium was pumped continuously at a throughput of 30 Torr.L/s at a speed of 2000 L/s and a compression ratio of 200. Argon was pumped at a throughput of 60 Torr.L/s at a speed of 1275 L/s. To produce continuous operation of the pump, a method of regeneration that does not thermally cycle the pump is employed. A small chamber (the ''snail'') passes over the pumping surface and removes the frost from it either by mechanical action with a scraper or by local heating. The material removed is topologically in a secondary vacuum system with low conductance into the primary vacuum; thus, the exhaust can be pumped at pressures up to an effective compression ratio determined by the ratio of the pumping speed to the leakage conductance of the snail. The pump, which is all-metal-sealed and dry and which regenerates every 60 s, would be an ideal system for pumping tritium. Potential fusion applications are for mpmp limiters, for repeating pneumatic pellet injection lines, and for the centrifuge pellet injector spin tank, all of which will require pumping tritium at high throughput. Industrial applications requiring ultraclean pumping of corrosive gases at high throughput, such as the reactive ion etch semiconductor process, may also be feasible.

  2. Flow cytometric and immunohistochemical detection of in vivo BrdU-labeled cells in mouse fat depots.

    PubMed

    Staszkiewicz, Jaroslaw; Gimble, Jeffrey; Cain, Courtney; Dietrich, Marilyn; Burk, David; Kirk-Ballard, Heather; Gawronska-Kozak, Barbara

    2009-01-16

    This study has determined the natural frequency and localization of progenitor/stem cells within fat depots in situ based on their ability to retain DNA nucleotide label (BrdU). Neonate and mature male C57BL6/J mice were injected intraperitoneally with BrdU- and label-retaining cells (LRC) were quantified in fat depots by immunohistochemical, immunofluorescent, and flow cytometric methods. In neonates, LRC constituted 27% of the cells in inguinal fat (iWAT) and 65% in interscapular brown fat (BAT) after Day 10 and 26% of the cells in epididymal fat (eWAT) after Day 28. After 52 days, the LRC accounted for 0.72% of iWAT, 0.53% of eWAT and 1.05% of BAT, respectively. The BrdU-labeled cells localized to two areas: single cells distributed among adipocytes or those adjacent to the blood vessels wall. In mature C57BL6/J mice, flow cytometric analysis determined that a majority of the LRC were also positive for stem cell antigen-1 (Sca-1). PMID:19056354

  3. Flow cytometric and immunohistochemical detection of in vivo BrdU-labeled cells in mouse fat depots

    PubMed Central

    Staszkiewicz, Jaroslaw; Gimble, Jeffrey; Cain, Courtney; Dietrich, Marilyn; Burk, David; Kirk-Ballard, Heather; Gawronska-Kozak, Barbara

    2009-01-01

    This study has determined the natural frequency and localization of progenitor/stem cells within fat depots in situ based on their ability to retain DNA nucleotide label (BrdU). Neonate and mature male C57BL6/J mice were injected intraperitoneally with BrdU- and label-retaining cells (LRC) were quantified in fat depots by immunohistochemical, immunofluorescent, and flow cytometric methods. In neonates, LRC constituted 27% of the cells in inguinal fat (iWAT) and 65% in interscapular brown fat (BAT) after Day 10 and 26% of the cells in epididymal fat (eWAT) after Day 28. After 52 days, the LRC accounted for 0.72% of iWAT, 0.53% of eWAT and 1.05% of BAT, respectively. The BrdU-labeled cells localized to two areas: single cells distributed among adipocytes or those adjacent to the blood vessels wall. In mature C57BL6/J mice, flow cytometric analysis determined that a majority of the LRC were also positive for stem cell antigen-1 (Sca-1). PMID:19056354

  4. 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

  5. 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.

  6. 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.

  7. 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

  8. Multiplexing high-content flow (HCF) and quantitative high-throughput screening (qHTS) to identify compounds capable of decreasing cell viability, activating caspase 3/7, expressing annexin V, and changing mitochondrial membrane integrity.

    PubMed

    Mathews, Lesley A; Keller, Jonathan M; McKnight, Crystal; Michael, Sam; Shinn, Paul; Liu, Dongbo; Staudt, Louis M; Thomas, Craig J; Ferrer, Marc

    2013-01-01

    High-content flow (HCF) screening systems, such as the iQue Screener and HTFC Screening System from IntelliCyt, have facilitated the implementation of flow cytometry assays for high-throughput screening. HCF screening systems enable the use of smaller sample volumes and multiplexed assays to simultaneously assess different cellular parameters from a single well. This becomes invaluable when working with cells or compounds that are available in limited quantities or when conducting large-scale screens. When assays can be miniaturized to a 384- or 1536-well microplate format, it is possible to implement dose-response-based high-throughput screens, also known as quantitative HTS or qHTS. This article describes how qHTS at the new National Center for Advancing Translational Science (NCATS) has been systematically coupled with the HTFC Screening System and Multimetric Apoptosis Screening Kit from IntelliCyt to biologically validate active compounds from primary cell proliferation screens using a model of diffuse large B cell lymphoma (DLBCL). PMID:24391083

  9. A Protocol for the Comprehensive Flow Cytometric Analysis of Immune Cells in Normal and Inflamed Murine Non-Lymphoid Tissues

    PubMed Central

    Yu, Yen-Rei A.; O’Koren, Emily G.; Hotten, Danielle F.; Kan, Matthew J.; Kopin, David; Nelson, Erik R.; Que, Loretta; Gunn, Michael D.

    2016-01-01

    Flow cytometry is used extensively to examine immune cells in non-lymphoid tissues. However, a method of flow cytometric analysis that is both comprehensive and widely applicable has not been described. We developed a protocol for the flow cytometric analysis of non-lymphoid tissues, including methods of tissue preparation, a 10-fluorochrome panel for cell staining, and a standardized gating strategy, that allows the simultaneous identification and quantification of all major immune cell types in a variety of normal and inflamed non-lymphoid tissues. We demonstrate that our basic protocol minimizes cell loss, reliably distinguishes macrophages from dendritic cells (DC), and identifies all major granulocytic and mononuclear phagocytic cell types. This protocol is able to accurately quantify 11 distinct immune cell types, including T cells, B cells, NK cells, neutrophils, eosinophils, inflammatory monocytes, resident monocytes, alveolar macrophages, resident/interstitial macrophages, CD11b- DC, and CD11b+ DC, in normal lung, heart, liver, kidney, intestine, skin, eyes, and mammary gland. We also characterized the expression patterns of several commonly used myeloid and macrophage markers. This basic protocol can be expanded to identify additional cell types such as mast cells, basophils, and plasmacytoid DC, or perform detailed phenotyping of specific cell types. In examining models of primary and metastatic mammary tumors, this protocol allowed the identification of several distinct tumor associated macrophage phenotypes, the appearance of which was highly specific to individual tumor cell lines. This protocol provides a valuable tool to examine immune cell repertoires and follow immune responses in a wide variety of tissues and experimental conditions. PMID:26938654

  10. 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

  11. Flow cytometric two-color staining technique for simultaneous determination of human erythrocyte membrane antigen and intracellular malarial DNA.

    PubMed

    Pattanapanyasat, K; Webster, H K; Udomsangpetch, R; Wanachiwanawin, W; Yongvanitchit, K

    1992-01-01

    A novel fixative and permeabilization method is described which allows simultaneous flow cytometric detection of red blood cell membrane antigen and intracellular malaria parasites. To illustrate the method, red blood cells from patients with paroxysmal nocturnal hemoglobinuria were infected with Plasmodium falciparum and maintained in synchronous red blood cell culture. The infected red blood cells were immunolabeled with antibodies directed to the complement regulatory protein decay-accelerating factor (DAF) followed by subsequent fixations in paraformaldehyde and then glutaraldehyde in phosphate-buffered saline. Finally, DNA of the intraerythrocytic parasites was stained with propidium iodide. Using this technique, cellular morphology was well preserved, no cell aggregation was observed, and high-quality indirect immunofluorescence and parasite DNA staining were obtained with negligible nonspecific labelling. Simultaneous measurement of parasite DNA and red blood cell membrane determinants makes possible the investigation of alterations of red cell membrane proteins in association with development of intracellular malaria parasites. PMID:1372210

  12. A Flow Cytometric Analysis of the Inhibition of Platelet Reactivity Due to Nitrite Reduction by Deoxygenated Erythrocytes

    PubMed Central

    Akrawinthawong, Krittapoom; Park, Ji Won; Piknova, Barbora; Sibmooh, Nathawut; Fucharoen, Suthat; Schechter, Alan N.

    2014-01-01

    Nitric oxide (NO), a small gas molecule, has long been known to be a potent inhibitor of platelet function but the physiological and pathological implications of platelet inhibition by NO have not been well clarified. We recently showed that the addition of nitrite to platelet-rich plasma in the presence of erythrocytes could inhibit platelet aggregation and this inhibitory effect of nitrite + erythrocytes was enhanced by deoxygenation of erythrocytes as measured by P-selectin expression and cGMP production. In order to study the nitrite effect on platelets at different oxygen levels, we used the flow cytometric assays to detect platelet membrane surface markers upon activation. The P-selectin and activated gpIIb/IIIa expression on platelet membranes in response to ADP, collagen and thrombin stimulation was measured at various hematocrit and oxygen levels. Nitrite (0.1 to 1.0 μM) significantly decreased the percentage of these surface markers on the platelet membrane at the hematocrit values above 23% and oxygen levels lower than 49 mmHg. The inhibitory effect of nitrite was augmented by increasing hematocrit values and decreasing oxygen saturation. C-PTIO (an NO scavenger) prevented the platelet inhibition by nitrite + erythrocytes whereas the inhibitors of NO synthase and xanthine oxidoreductase had no effect. These results support the proposal that circulating nitrite decreases platelet reactivity in the presence of partially deoxygenated erythrocytes through its reduction to NO, which may also explain certain differences between arterial and venous thrombosis and support directly the role of deoxyhemoglobin in this process. We believe that our flow cytometric assays offer a possibility to identify the individual molecular process involved in these effects. PMID:24642865

  13. 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

  14. High-throughput Tetrad Analysis

    PubMed Central

    Ludlow, Catherine L.; Scott, Adrian C.; Cromie, Gareth A.; Jeffery, Eric W.; Sirr, Amy; May, Patrick; Lin, Jake; Gilbert, Teresa L.; Hays, Michelle; Dudley, Aimée M.

    2013-01-01

    Tetrad analysis has been a gold standard genetic technique for several decades. Unfortunately, the manual nature of the process has relegated its application to small-scale studies and limited its integration with rapidly evolving DNA sequencing technologies. We have developed a rapid, high-throughput method, called Barcode Enabled Sequencing of Tetrads (BEST), that replaces the manual processes of isolating, disrupting and spacing tetrads. BEST uses a meiosis-specific GFP fusion protein to isolate tetrads by fluorescence-activated cell sorting and molecular barcodes that are read during genotyping to identify spores derived from the same tetrad. Maintaining tetrad information allows accurate inference of missing genetic markers and full genotypes of missing (and presumably nonviable) individuals. By removing the bottleneck of manual dissection, hundreds or even thousands of tetrads can be isolated in minutes. We demonstrate the approach in Saccharomyces cerevisiae, but BEST is readily transferable to microorganisms in which meiotic mapping is significantly more laborious. PMID:23666411

  15. High frequency of circulating HBcAg-specific CD8 T cells in hepatitis B infection: a flow cytometric analysis

    PubMed Central

    Matsumura, S; Yamamoto, K; Shimada, N; Okano, N; Okamoto, R; Suzuki, T; Hakoda, T; Mizuno, M; Higashi, T; Tsuji, T

    2001-01-01

    Viral antigen-specific T cells are important for virus elimination. We studied the hepatitis B virus (HBV)-specific T cell response using flow cytometry. Three phases of HBV infection were studied: Group A, HBeAg (+) chronic hepatitis; Group B, HBeAb (+) HBV carrier after seroconversion; and Group C, HBsAb (+) phase. Peripheral T cells were incubated with recombinant HB core antigen (HBcAg), and intracytoplasmic cytokines were analysed by flow cytometry. HBcAg-specific CD4 and CD8 T cells were identified in all three groups and the number of IFN-γpositive T cells was greater than TNF-α-positive T cells. The frequency of IFN-γ-positive CD4 and CD8 T cells was highest in Group C, compared with Groups A and B. No significant difference in the HBcAg-specific T cell response was observed between Group A and Group B. The HBcAg-specific CD8 T cell response was diminished by CD4 depletion, addition of antibody against human leucocyte antigen (HLA) class I, class II or CD40L. Cytokine-positive CD8 T cells without HBcAg stimulation were present at a high frequency (7 of 13 cases) in Group B, but were rare in other groups. HBcAg-specific T cells can be detected at high frequency by a sensitive flow cytometric analysis, and these cells are important for controlling HBV replication. PMID:11472405

  16. 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. PMID:16998868

  17. 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.

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

    PubMed

    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

  19. 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

    2016-01-01

    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. PMID:27584036

  20. Screening of promoters from rhizosphere metagenomic DNA using a promoter-trap vector and flow cytometric cell sorting.

    PubMed

    Lee, Se Hee; Kim, Jeong Myeong; Lee, Hyo Jung; Jeon, Che Ok

    2011-02-01

    We constructed a facilitative and efficient promoter-trap vector, pCM-EGFP, for capturing and analyzing functional promoters from environmental DNA. The pCM-EGFP vector showed good chloramphenicol sensitivity and no enhanced green fluorescent protein (EGFP) gene expression. Promoter libraries were constructed for screening promoters responding to naringenin, a key molecule released from plant roots. After electroporation, E. coli transformants were incubated in LB broth containing chloramphenicol (10 μg/ml) to select against transformants with no cloned promoter. E. coli cells were sorted using flow cytometry without naringenin, and then sorted again with high fluorescence after incubation in LB broth with naringenin (1 mM) at 28 °C for 12 h. The inducible properties of approximately 400 sorted cells were evaluated, with most cells showing only strong EGFP gene expression without inducible properties. Two clones (5-4E and 15-3D) displayed naringenin inducibility, and both contained a promoter bounded by a TetR-family regulator. The regulator knock-out mutant of the 5-4E clone lost its ability to be induced by naringenin. In conclusion, the pCM-EGFP vector may be used as an efficient promoter-trap vector and a combination of the vector with flow cytometric cell sorting was demonstrated to be an useful method for screening promoters responding to specific conditions or inducers. PMID:21259288

  1. 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.

  2. 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. PMID:26828697

  3. 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

  4. 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. PMID:24830140

  5. Development and Application of Flow-Cytometric Techniques for Analyzing and Sorting Endospore-Forming Clostridia▿ †

    PubMed Central

    Tracy, Bryan P.; Gaida, Stefan M.; Papoutsakis, Eleftherios T.

    2008-01-01

    The study of microbial heterogeneity at the single-cell level is a rapidly growing area of research in microbiology and biotechnology due to its significance in pathogenesis, environmental biology, and industrial biotechnologies. However, the tools available for efficiently and precisely probing such heterogeneity are limited for most bacteria. Here we describe the development and application of flow-cytometric (FC) and fluorescence-assisted cell-sorting techniques for the study of endospore-forming bacteria. We show that by combining FC light scattering (LS) with nucleic acid staining, we can discriminate, quantify, and enrich all sporulation-associated morphologies exhibited by the endospore-forming anaerobe Clostridium acetobutylicum. Using FC LS analysis, we quantitatively show that clostridial cultures commonly perform multiple rounds of sporulation and that sporulation is induced earlier by the overexpression of Spo0A, the master regulator of endospore formers. To further demonstrate the power of our approach, we employed FC LS analysis to generate compelling evidence to challenge the long-accepted view in the field that the clostridial cell form is the solvent-forming phenotype. PMID:18931289

  6. Effects of dietary fish oil and vitamin E supplementation on canine lymphocyte proliferation evaluated using a flow cytometric technique.

    PubMed

    LeBlanc, Casey J; Dietrich, Marilyn A; Horohov, David W; Bauer, John E; Hosgood, Giselle; Mauldin, Glenna E

    2007-10-15

    Lymphocyte proliferation and peripheral blood mononuclear cell (PBMC) production of PGE(2) were assayed in 15 healthy dogs fed a basal diet supplemented with either sunflower oil (Group Sunflower oil), sunflower oil and menhaden fish oil (Group Fish oil), or sunflower oil and menhaden fish oil plus alpha-tocopherol acetate for 12 weeks (Group Fish oil + E). Lymphocyte proliferation was determined by a flow cytometric technique utilizing the fluorochrome carboxyfluorescein diacetate succinimidyl ester (CFSE). The PBMC supernatant PGE(2) concentration was assayed using a competitive enzyme-linked immunoassay. Group Fish oil had a significant decrease in lymphocyte proliferation at week 12. PBMC production of PGE(2) was decreased in all three groups but only significantly reduced in groups receiving fish oil supplementation. Based on these results, this level of fish oil supplementation appears to suppress the lymphoproliferative response in healthy, young dogs but this response can be attenuated by high levels of dietary vitamin E supplementation. Furthermore, fish oil-induced reduction in lymphocyte proliferation appears to manifest through a PGE(2)-independent mechanism and is not associated with increased lipid peroxidation. PMID:17658617

  7. Flow cytometric characterization of rat thymus cells in a radiation-dominated model of combined injury. Scientific report

    SciTech Connect

    Kaffenberger, K.; Gruber, D.F.; MacVittie, T.J.

    1988-01-01

    Thymuses of rats that had been: (a) gamma-irradiated 500 cGy whole-body radiation (R), or (b) thermally injured 20% BSA dorsal, scald burn (TI), or c) combined injured irradiation followed by burn (CI) were studied for involution and recovery processes after sublethal treatments. The expression of surface antigens on thymic cells before and after injuries was evaluated using the monoclonal antibodies and flow cytometric analysis. Thymic cellularity decreased to less than 1% of normal (N), age-matched rats by 4 days after R or CI. Recovery reached 60% to 70% of N by 28 days post treatments. Expression of OX7, OX8, W3/13, and W3/25 antigens all reached nadirs of 40% of N by day 4 after R and CI. Recovery of antigen expression, except for W3/25, was near completion by day 7 after R and CI. Changes in antigen expression after TI were less pronounced for all mcAB tested. Variations in relative labeling of nonlymphoid thymic cells with OX4 (Ia-antigen) reflected the disappearance and recovery of radiosensitive lymphoid thymocytes. The similarity of results after R and CI demonstrate that the model of CI is radiation-dominated. The addition of burn injury to radiation trauma had no synergistically damaging effect on the parameters studied.

  8. Prognostic factors in anal squamous carcinoma: a multivariate analysis of clinical, pathological and flow cytometric parameters in 235 cases.

    PubMed

    Shepherd, N A; Scholefield, J H; Love, S B; England, J; Northover, J M

    1990-06-01

    Clinical, pathological and flow cytometric parameters have been analysed by univariate and multivariate analysis to define those parameters of important prognostic influence in 235 cases of surgically treated squamous carcinoma of the anus and perianal skin. Patients had been treated by anorectal excision (166 patients) or by local excision (69). Analyses were carried out on five data sets--the two surgical subgroups, two groups distinguished by site of tumour and on all 235 patients. Univariate analysis showed many parameters to be of prognostic influence, although histological typing of tumours into the more common histological subtypes was of no prognostic value. Parameters of independent prognostic significance in multivariate analysis were those indicating depth of spread, inguinal lymph node involvement and DNA-ploidy. In this study the subdivision of the rarer types of anal canal tumour, such as mucoepidermoid carcinoma, microcystic squamous carcinoma and small cell anaplastic carcinoma, was relevant confirming that these tumours have a poor prognosis. It is now felt that surgery should not be employed as primary treatment in most cases of anal cancer and the results of this study have to be interpreted with caution when applied to patients treated with radiotherapy with or without chemotherapy. Nevertheless, our findings suggest that the most useful prognostic information can be gleaned from accurate clinical staging and an assessment of DNA-ploidy status. PMID:2376397

  9. 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

    2015-01-01

    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

  10. 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

  11. Quantitative analysis of cultured thymic reticulo-epithelial cells labelled by different antibodies: a flow cytometric study.

    PubMed Central

    Fabien, N; Auger, C; Bonnard, M; Andreoni, C; Rigal, D; Monier, J C

    1989-01-01

    Quantitative measurements of cultured human and murine thymic, and human thymoma reticuloepithelial cells (REC), immunolabeled by different antibodies (Ab) (TE3, TE4, anti-HTLV p19(p19), lu5, K11 and Aks) and by thymic hormones (thymulin and thymosin alpha 1 (Ta1)) within these cells, were performed using a flow cytometric technique. The anti-keratin polyclonal Ab labeled nearly the whole human or murine population. The p19 monoclonal Ab (MoAb), specific for the subcortical/medullary thymic regions, labelled 37-77% of the human REC. The TE3 MoAb, specific for the cortical region, labelled 54-83% of the REC. These percentages suggest that the cultured thymic REC (TREC) had markers of both regions together and therefore that these markers are not absolutely specific to determine their subcortical/medullary or cortical thymic origin. For the three populations there were more cells containing Ta1 than thymulin. The overlap of the percentage of labelled cells suggests that the same cell could synthesize the two hormones and that these hormones could be localized within the TE3 positive cells. PMID:2649289

  12. Flow-cytometric evaluation of lymphocyte subpopulations in synchronously developing Schistosoma mansoni egg and Sephadex bead pulmonary granulomas.

    PubMed Central

    Remick, D. G.; Chensue, S. W.; Hiserodt, J. C.; Higashi, G. I.; Kunkel, S. L.

    1988-01-01

    Synchronous models of T-cell-mediated and foreign body granulomas were induced in mice by intravenous embolization of Schistosoma mansoni eggs and Sephadex beads, respectively. The authors then performed flow-cytometric analysis of lymphocytes from dispersed granulomas, spleens, and peripheral blood at 4, 8, 16, and 32 days corresponding to the induction, growth, and maintenance, and resolution of these lesions. Lymphocytes were identified on the basis of light scatter characteristics, and the nature of the cells was confirmed by cell sorting and electron-microscopic examination. Lymphocyte subpopulations were characterized with antibodies to lymphocyte surface markers, specifically Ig, Thy 1.2, Lyt 1, Lyt 2, and L3T4. Natural killer cells were identified with anti-asialo GM1. Egg-induced granulomas had more lymphocytes of all phenotypes at all time points. Surprisingly, there was a significant number of cells staining positive for asialo GM1. On Day 16 after embolization there was a greater percentage of helper T cells, as defined by positive staining with L3T4, in the egg model, compared with the bead model. There was no obvious shift of lymphocytes from either the blood or spleen into the granuloma. These data confirm the importance of T cells in the direct participation of granulomatous inflammation, and the large numbers of asialo GM1-positive cells suggest a role for natural killer cells. Images Figure 4 PMID:2451888

  13. 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.

  14. Flow cytometric assessment of chicken T cell-mediated immune responses after Newcastle disease virus vaccination and challenge.

    PubMed

    Dalgaard, T S; Norup, L R; Pedersen, A R; Handberg, K J; Jørgensen, P H; Juul-Madsen, H R

    2010-06-17

    The objective of this study was to use flow cytometry to assess chicken T cell-mediated immune responses. In this study two inbred genetic chicken lines (L130 and L133) were subjected to two times vaccination against Newcastle disease (ND) and a subsequent challenge by ND virus (NDV) infection. Despite a delayed NDV-specific antibody response to vaccination, L133 appeared to be better protected than L130 in the subsequent infection challenge as determined by the presence of viral genomes. Peripheral blood was analyzed by flow cytometry and responses in vaccinated/challenged birds were studied by 5-color immunophenotyping as well as by measuring the proliferative capacity of NDV-specific T cells after recall stimulation. Immunophenotyping identified L133 as having a significantly lower CD4/CD8 ratio and a lower frequency of gammadelta T cells than L130 in the peripheral T cell compartment. Furthermore, peripheral lymphocytes from L133 exhibited a significantly higher expression of CD44 and CD45 throughout the experiment. Interestingly, also vaccine-induced differences were observed in L133 as immune chickens had a significantly higher CD45 expression on their lymphocytes than the naïve controls. Immune chickens from both lines had a significantly higher frequency of circulating gammadelta T cells than the naïve controls both after vaccination and challenge. Finally, the proliferative capacity of peripheral CD4+ and CD8+ cells specific for NDV was addressed 3 weeks after vaccination and 1 week after infection and found to be significantly higher in L133 than in L130 at both sampling times. In conclusion, we found the applied flow cytometric methods very useful for the study of chicken T cell biology. PMID:20434546

  15. 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. PMID:22573481

  16. FLOW CYTOMETRIC COMPARISON OF THE EFFECTS OF TRIALKYTING ON THE MURINE ERYTHROLEUKEMIC CELL

    EPA Science Inventory

    Cellular effects of exposure to tributyltin (TBT), triethyltin (TET), or trimethyltin (TMT) were investigated by flow cytometry employing the murine erythroleukemic cell (MELC) as a model cellular system. Cell viability was investigated by the carboxyfluorescein diacetate (CFDA) ...

  17. Sources of variation in flow cytometric analysis of aquatic species sperm: The effect of cryoprotectants on flow cytometry scatter plots and subsequent population gating.

    PubMed

    Daly, Jonathan; Tiersch, Terrence R

    2012-12-11

    The use of fluorescent staining and flow cytometry to assess sperm quality in aquatic species has increased over the past decade, but comparisons among studies are difficult or impossible due to variation in application, analysis, and reporting of protocols and data.The goal of the present study was to determine the effect of exposure to two cryoprotectants commonly used for cryopreservation of sperm from aquatic species on the accuracy of flow cytometric assessment of sperm quality.Membrane integrity of zebrafish (Danio rerio) sperm exposed to 10% and 20%methanol and dimethyl sulfoxide (DMSO)in 300 mOsm kg(-1) Hanks' balanced salt solution (HBSS) or calcium-free HBSSwas determined using SYBR 14/propidium iodide staining. Both cryoprotectants significantly affected forward-scatter and side-scatter characteristics of sperm samples, resulting in significant changes in the number of total and gated events, and in the number and percentage of intact cells. These results indicate that it cannot be assumed that the approach to flow cytometric analysis of fresh sperm will be applicable to cryoprotectant-treated or cryopreserved sperm. In total, we document examples of five potentially interacting factors that produce errors of 5 to 50% each, resulting in underestimates and overestimates of total and intact sperm (actual numbers and percentages) in the presence of the two most commonly used cryoprotectants at the concentrations used most often for cryopreservation of sperm from aquatic species. This study provides methods to reduce or eliminate these errors and recommendations necessary for standardization and reporting. PMID:23175587

  18. 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. PMID:27302040

  19. 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...

  20. FLOW CYTOMETRIC DISCRIMINATION OF MITOTIC NUCLEI BY RIGHT-ANGLE LIGHT SCATTER (JOURNAL VERSION)

    EPA Science Inventory

    Flow cytometry has been used to demonstrate alterations in protein, RNA, and DNA content of cells as they traverse the cell cycle. Employing fluorescein isothiocynate (FITC) to stain protein and propidium iodide (PI) to stain nucleic acids, multiple regions within the G1 and G2 p...

  1. FLOW CYTOMETRIC ANALYSIS OF EFFECTS OF 1,3-DINITROBENZENE ON RAT SPERMATOGENESIS

    EPA Science Inventory

    Exposure of 100-d old rats to 1,3-dinitiobenzene (m-DNB) at dosages up to 48 mg/kg resulted in disruption of spermatogenesis as measured by flow cytometry (FCM) of acridine orange-stained sperm and testis cells. ne day (d 1) after a single exposure to 48 mg/kg m-DNB. CM measureme...

  2. FLOW CYTOMETRIC ANALYSIS OF MOUSE SPERMATOGENIC FUNCTION FOLLOWING EXPOSURE TO ETHYLNITROSOUREA

    EPA Science Inventory

    The effects of the mutagenic agent ethylnitrosourea (ENU) on spermatogenic function and sperm chromatin structure were studied by flow cytometry and the results compared with sperm head morphology measurements. Groups of mice received daily exposures ranging from 0 to 75 mg/kg bo...

  3. IMPROVED FLOW CYTOMETRIC ASSAY FOR SOMATIC MUTATIONS AT THE GLYCOPHORIN A LOCUS IN HUMANS

    EPA Science Inventory

    An improved method has been developed for the glycophorin A assay for somatic cell mutations in humans. he new assay, named the "BR6" assay, can be performed on a commercially available, single-beam flow cytometer, in contrast to the previously described 1W1 assay that required a...

  4. 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

  5. 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. PMID:24081566

  6. High Throughput Sequencing Analysis of the Immunoglobulin Heavy Chain Gene from Flow-Sorted B Cell Sub-Populations Define the Dynamics of Follicular Lymphoma Clonal Evolution.

    PubMed

    Carlotti, Emanuela; Wrench, David; Rosignoli, Guglielmo; Marzec, Jacek; Sangaralingam, Ajanthah; Hazanov, Lena; Michaeli, Miri; Hallam, Simon; Chaplin, Tracy; Iqbal, Sameena; Calaminici, Maria; Young, Bryan; Mehr, Ramit; Campbell, Peter; Fitzgibbon, Jude; Gribben, John G

    2015-01-01

    Understanding the dynamics of evolution of Follicular Lymphoma (FL) clones during disease progression is important for monitoring and targeting this tumor effectively. Genetic profiling of serial FL biopsies and examples of FL transmission following bone marrow transplant suggest that this disease may evolve by divergent evolution from a common ancestor cell. However where this ancestor cell resides and how it evolves is still unclear. The analysis of the pattern of somatic hypermutation of the immunoglobulin gene (Ig) is traditionally used for tracking the physiological clonal evolution of B cells within the germinal center and allows to discriminate those cells that have just entered the germinal center and display features of ancestor cells from those B cells that keep re-circulating across different lymphoid organs. Here we investigated the pattern of somatic hypermutation of the heavy chain of the immunoglobulin gene (IgH-VH) in 4 flow-sorted B cells subpopulations belonging to different stages of differentiation, from sequential lymph node biopsies of cases displaying diverse patterns of evolution, using the GS-FLX Titanium sequencing platform. We observed an unexpectedly high level of clonality, with hundreds of distinct tumor subclones in the different subpopulations from the same sample, the majority detected at a frequency <10-2. By using a lineage trees analysis we observed in all our FL and t-FL cases that the oligoclonal FL population was trapped in a narrow intermediate stage of maturation that maintains the capacity to undergo SHM, but was unable to further differentiate. The presence of such a complex architecture highlights challenges currently encountered in finding a cure for this disease. PMID:26325507

  7. Flow injection combined with ICP-MS for accurate high throughput analysis of elemental impurities in pharmaceutical products according to USP <232>/<233>.

    PubMed

    Fischer, Lisa; Zipfel, Barbara; Koellensperger, Gunda; Kovac, Jessica; Bilz, Susanne; Kunkel, Andrea; Venzago, Cornel; Hann, Stephan

    2014-07-01

    New guidelines of the United States Pharmacopeia (USP), European Pharmacopeia (EP) and international organization (ICH, International Conference on Harmonization) regulating elemental impurity limits in pharmaceuticals seal the end of unspecific analysis of metal(oid)s as outlined in USP <231> and EP 2.4.8. Chapter USP <232> and EP 5.20 as well as drafts from ICH Q3D specify both daily doses and concentration limits of metallic impurities in pharmaceutical final products and in active pharmaceutical ingredients (API) and excipients. In chapters USP <233> and EP 2.4.20 method implementation, validation and quality control during the analytical process are described. By contrast with the--by now--applied methods, substance specific quantitative analysis features new basic requirements, further, significantly lower detection limits ask for the necessity of a general changeover of the methodology toward sensitive multi element analysis by ICP-AES and ICP-MS, respectively. A novel methodological approach based on flow injection analysis and ICP-SFMS/ICP-QMS for the quick and accurate analysis of Cd, Pb, As, Hg, Ir, Os, Pd, Pt, Rh, Ru, Cr, Mo, Ni, V, Cu, Mn, Fe and Zn in drug products by prior dilution, dissolution or microwave assisted closed vessel digestion according to the regulations is presented. In comparison to the acquisition of continuous signals, this method is advantageous with respect to the unprecedented high sample throughput due to a total analysis time of approximately 30s and the low sample consumption of below 50 μL, while meeting the strict USP demands on detection/quantification limits, precision and accuracy. PMID:24667566

  8. High Throughput Sequencing Analysis of the Immunoglobulin Heavy Chain Gene from Flow-Sorted B Cell Sub-Populations Define the Dynamics of Follicular Lymphoma Clonal Evolution

    PubMed Central

    Carlotti, Emanuela; Wrench, David; Rosignoli, Guglielmo; Marzec, Jacek; Sangaralingam, Ajanthah; Hazanov, Lena; Michaeli, Miri; Hallam, Simon; Chaplin, Tracy; Iqbal, Sameena; Calaminici, Maria; Young, Bryan; Mehr, Ramit; Campbell, Peter; Fitzgibbon, Jude; Gribben, John G.

    2015-01-01

    Understanding the dynamics of evolution of Follicular Lymphoma (FL) clones during disease progression is important for monitoring and targeting this tumor effectively. Genetic profiling of serial FL biopsies and examples of FL transmission following bone marrow transplant suggest that this disease may evolve by divergent evolution from a common ancestor cell. However where this ancestor cell resides and how it evolves is still unclear. The analysis of the pattern of somatic hypermutation of the immunoglobulin gene (Ig) is traditionally used for tracking the physiological clonal evolution of B cells within the germinal center and allows to discriminate those cells that have just entered the germinal center and display features of ancestor cells from those B cells that keep re-circulating across different lymphoid organs. Here we investigated the pattern of somatic hypermutation of the heavy chain of the immunoglobulin gene (IgH-VH) in 4 flow-sorted B cells subpopulations belonging to different stages of differentiation, from sequential lymph node biopsies of cases displaying diverse patterns of evolution, using the GS-FLX Titanium sequencing platform. We observed an unexpectedly high level of clonality, with hundreds of distinct tumor subclones in the different subpopulations from the same sample, the majority detected at a frequency <10−2. By using a lineage trees analysis we observed in all our FL and t-FL cases that the oligoclonal FL population was trapped in a narrow intermediate stage of maturation that maintains the capacity to undergo SHM, but was unable to further differentiate. The presence of such a complex architecture highlights challenges currently encountered in finding a cure for this disease. PMID:26325507

  9. Proliferation markers Ki-67 and p105 in soft-tissue lesions. Correlation with DNA flow cytometric characteristics.

    PubMed Central

    Swanson, S. A.; Brooks, J. J.

    1990-01-01

    Frozen tissue immunoreactivity with Ki-67, a monoclonal antibody that recognizes a nuclear antigen in nonresting or proliferating cells, was compared to DNA flow cytometry results (from fresh tissue) in a diverse group of 60 soft-tissue lesions. Both DNA index and Ki-67 score were independently reported to be associated with grade and prognosis in sarcomas, but no direct comparison of these two variables was made. It was attempted to measure proliferative activity in fixed paraffin-embedded tissues immunohistochemically in a subset of lesions using an antibody to another nuclear proliferation antigen, p105. Lesions were given a grade according to lesion category (reactive, 1; benign, 2; low-grade malignant, 3; and high-grade malignant, 4). Ki-67 reactivity correlated relatively well with this grading system (r = 0.59); benign lesions usually exhibited a low Ki-67 score and malignant lesions usually but not always exhibited a high score. For example, some malignant fibrous histiocytomas contained only rare positive cells. Some disparity between Ki-67 score and grade and within histologic types indicates some independence from these features, a fact that may be important when correlation with prognosis is performed. However Ki-67 did not correlate well with flow data such as percentage S phase (r = 0.30), percentage S + G2M phases (r = 0.37), or DNA index (r = 0.39). This probably is due to the fact that Ki-67 also marks cells in the G1 phase, whereas these are excluded in flow data analyses. Anti-p105 highlighted almost all nuclei in all cases tested, including fibromatosis, and did not correlate with Ki-67 score, histologic grade or DNA flow cytometric data. Results with p105 could not be favorably affected by titration experiments. It is reasonable to conclude that the Ki-67 score is a variable related to but independent of histologic grade, histologic type, and DNA flow values. Whether it is prognostically important in human sarcomas, as has been suggested

  10. Flow cytometric evaluation of sperm parameters in relation to fertility potential.

    PubMed

    Gillan, Lindsay; Evans, Gareth; Maxwell, W M C

    2005-01-15

    Most laboratory methods used to evaluate semen quality have not correlated highly with fertilizing capacity. The discovery of a variety of fluorochromes and compounds conjugated to fluorescent probes has enabled a more widespread analysis of sperm attributes, and in conjunction with the flow cytometer, permit the evaluation of a large number of spermatozoa. A number of characteristics of sperm integrity, viability and function can be assessed by flow cytometry. The DNA status of spermatozoa has been determined using the metachromatic properties of acridine orange (AO). AO staining, when used in the sperm chromatin structure assay (SCSA), correlates with fertility in a number of species. DNA fragmentation can also be assessed using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, which identifies DNA strand breaks by labeling free 3'-OH termini with modified nucleotides. The status of the sperm acrosome can be determined using fluorescently labeled lectins and LysoTracker Green DND-26, a fluorescent acidotropic probe. Capacitation status has been observed through calcium-mediated changes using chlortetracycline (CTC) or by changes in membrane fluidity monitored by the binding of the fluorescent amphiphilic probe, Merocyanine 540. Fluorescently labeled annexin-V, C6NBD and Ro-09-0198 can also be used to detect changes in membrane phospholipid distribution. Cell viability can be determined using the propensity of propidium iodide (PI), ethidium homodimer-1 (EthD-1) or Yo-Pro-1 to permeate damaged membranes. These are generally more adaptable to clinical flow cytometry than the bisbenzimide membrane impermeable stain, Hoechst 33258, which excites in the ultraviolet range and requires UV laser equipment. Mitochondrial function can be determined using rhodamine 123 (R123) and MitoTracker Green FM (MITO) and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1). Flow cytometry is a tool that may be used

  11. Validation of a High Throughput Methodology to Assess the Effects of Biomaterials on Dendritic Cell Phenotype

    PubMed Central

    Kou, Peng Meng; Babensee, Julia E.

    2012-01-01

    A variety of combination products composed of biomaterials and biologics have been developed for tissue regeneration or vaccine delivery. The host immune response to the immunogenic biological components in such products may be modulated by the biomaterial component. Distinct biomaterials have been shown to differentially affect the maturation of dendritic cells (DCs). DCs are professional antigen-presenting cells (APCs) that bridge innate and adaptive immunity and play a central role in inducing immunity or initiating immune tolerance. However, the biomaterials systems used to study DC response thus far have been insufficient to draw a clear conclusion as to which biomaterial properties are the key to controlling DC phenotype. In this study, we developed a 96-well filter plate-based high throughput (HTP) methodology to assess DC maturation upon biomaterial treatment. Equivalent biomaterial effects on DC phenotype were measured using the conventional flow cytometric and filter plate method, which validated the HTP methodology. This methodology will be used to screen a large number of biomaterials simultaneously and to draw correlations between material properties and DC phenotype, thereby providing biomaterial design criteria and immunomodulatory strategies for both tissue engineering and vaccine delivery applications. PMID:20097314

  12. Flow cytometric analysis to detect pathogens in bacterial cell mixtures using semiconductor quantum dots.

    PubMed

    Hahn, Megan A; Keng, Peter C; Krauss, Todd D

    2008-02-01

    Compared to a common green organic dye, semiconductor quantum dots (QDs) composed of CdSe/ZnS core/shell bioconjugates display brighter fluorescence intensities, lower detection thresholds, and better accuracy in analyzing bacterial cell mixtures composed of pathogenic E. coli O157:H7 and harmless E. coli DH5alpha using flow cytometry. For the same given bacterial mixture, QDs display fluorescence intensity levels that are approximately 1 order of magnitude brighter compared to the analogous experiments that utilize the standard dye fluorescein isothiocyanate. Detection limits are lowest when QDs are used as the fluorophore label for the pathogenic E. coli O157:H7 serotype: limits of 1% O157:H7 in 99% DH5alpha result, corresponding to 106 cells/mL, which is comparable to other developing fluorescence-based techniques for pathogen detection. Finally, utilizing QDs to label E. coli O157:H7 in cell mixtures results in greater accuracy and more closely approaches the ideal fluorophore for pathogen detection using flow cytometry. With their broader absorption spectra and narrower emission spectra than organic dyes, QDs can make vast improvements in the field of flow cytometry, where single-source excitation and simultaneous detection of multicolor species without complicating experimental setups or data analysis is quite advantageous for analyzing heterogeneous cell mixtures, both for prokaryotic pathogen detection and for studies on eukaryotic cell characteristics. PMID:18186615

  13. Flow cytometric detection of spontaneous apoptosis in human breast cancer using the TUNEL-technique.

    PubMed

    Ehemann, Volker; Sykora, Jaromir; Vera-Delgado, Jorge; Lange, Adelheid; Otto, Herwart F

    2003-05-01

    Microscopic detection of structural alterations is the most reliable method to identify apoptotic cells, which however, does not allow any correlation with cell cycle phases. Discrimination of individual cells within solid human tumors undergoing apoptotic death is possible by flow cytometry where apoptotic cells appear in a hypodiploid sub G0/1-peak as a consequence of partial DNA loss. To refer induction of apoptosis to cell cycle phases we adopted the terminal deoxynucleotidyl transferase nick-end-labelling (TUNEL) technique to flow cytometry which enables the detection of cellular DNA content and DNA fragmentation by multiparametric analysis. One thousand seven hundred human breast carcinomas were screened. In 40 cases (2.3%) of 1700 carcinomas we detected a hypodiploid sub -G0/1 apoptotic peak. The spontaneous apoptotic fractions within individual tumors ranged between 1.5 and 25%. A correlation (r(2)=0.78) was found between apoptotic cells in sub-G0/1-peak measured by DNA-cytometry and TUNEL positive cells measured by multiparametric cytometry, because TUNEL reaction signed also cells with strand breaks. High proliferation indices correspond well (r(2)=0.807) with the increased amount of TUNEL positive cells. Multiparametric flow cytometry for the combined determination of DNA-content and DNA-fragmentation by TUNEL offers not only the advantage of a higher apoptosis sensitivity but also enables the quantification of DNA fragmentation related to any cell cycle phase. PMID:12706866

  14. 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. PMID:22922462

  15. Detection and quantification of circulating immature platelets: agreement between flow cytometric and automated detection.

    PubMed

    Ibrahim, Homam; Nadipalli, Srinivas; Usmani, Saba; DeLao, Timothy; Green, LaShawna; Kleiman, Neal S

    2016-07-01

    Immature platelets-also termed reticulated platelets (RP)-are platelets newly released into the circulation, and have been associated with a variety of pathological thrombotic events. They can be assessed by flow cytometry after staining with thiazole orange (TO) or by using a module added to a fully automated analyzer that is currently in wide clinical use and expressed as a fraction of the total platelet count (IPF). We sought to assess the correlation and agreement between these two methods. IPF was measured using Sysmex XE 2100-and at the same time point- we used TO staining and flow cytometry to measure RP levels. Two different gates were used for the flow cytometry method, 1 and 0.5 %. Measurements from the automated analyzer were then compared separately to measurements performed using each gate. Agreement between methods was assessed using Bland-Altman method. Pearson's correlation coefficient was also calculated. 129 subjects were enrolled and stratified into 5 groups: (1) Healthy subjects, (2) End stage renal disease, (3) Chronic stable coronary artery disease, (4) Post Coronary artery bypass surgery, (5) Peripheral thrombocytopenia. Median IPF levels were increased for patients in groups 2, 3, 4 and 5 (4.0, 4.7, 4.3, and 8.3 % respectively) compared to healthy subjects (2.5 %) p = 0.0001. Although the observed correlation between the two methods tended to be good in patients with high IPF values (i.e., group 5), the overall observed correlation was poor (Pearson's correlation coefficient r = 0.27). Furthermore, there was poor agreement between the two methods in all groups. Despite the good correlation that was observed between the two methods at higher IPF values, the lack of agreement was significant. PMID:26831482

  16. Fluorescent brighteners: novel stains for the flow cytometric analysis of microorganisms.

    PubMed

    Davey, H M; Kell, D B

    1997-08-01

    Flow cytometry is a rapid method for measuring the optical properties of individual cells. The technique has found great utility in the study of mammalian cells, but microbiological applications have been more limited. We here show that UV-excited fluorescent whitening agents, in particular Tinopal CBS-X, are effective stains for both vegetative microbial cells and for spores of Gram-positive bacteria. Pretreatment of samples with ethanol speeds the staining process. Under favourable conditions, Tinopal CBS-X may be used to discriminate among organisms, a fact that may be useful when screening for a target microorganism against a high biological background. PMID:9266751

  17. 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

  18. 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. PMID:27142297

  19. Automated classification of patients with chronic lymphocytic leukemia and immunocytoma from flow cytometric three-color immunophenotypes.

    PubMed

    Valet, G K; Höffkes, H G

    1997-12-15

    The goal of this study was the discrimination between chronic lymphocytic leukemia (B-CLL), clinically more aggressive lymphoplasmocytoid immunocytoma (LP-IC) and other low-grade non-Hodgkin's lymphomas (NHL) of the B-cell type by automated analysis of flow cytometric immunophenotypes CD45/14/20, CD4/8/3, kappa/CD19/5, lambda/CD19/5 and CD10/23/19 from peripheral blood and bone marrow aspirate leukocytes using the multiparameter classification program CLASSIF1. The immunophenotype list mode files were exhaustively evaluated by combined lymphocyte, monocyte, and granulocyte (LMG) analysis. The results were introduced into databases and automatically classified in a standardized way. The resulting triple matrix classifiers are laboratory and instrument independent, error tolerant, and robust in the classification of unknown test samples. Practically 100% correct individual patient classification was achievable, and most manually unclassifiable patients were unambiguously classified. It is of interest that the single lambda/CD19/5 antibody triplet provided practically the same information as the full set of the five antibody triplets. This demonstrates that standardized classification can be used to optimize immunophenotype panels. On-line classification of test samples is accessible on the Internet: http://www.biochem.mpg.de/valet/leukaem1.html Immunophenotype panels are usually devised for the detection of the frequency of abnormal cell populations. As shown by computer classification, most the highly discriminant information is, however, not contained in percentage frequency values of cell populations, but rather in total antibody binding, antibody binding ratios, and relative antibody surface density parameters of various lymphocyte, monocyte, and granulocyte cell populations. PMID:9440819

  20. Analysis of informativeness of immunohistochemical and flow cytometric methods for estrogen receptor α assessment.

    PubMed

    Bogush, T A; Dudko, E A; Rodionova, M V; Bogush, E A; Kirsanov, V J; Rodionov, V V; Vorotnikov, I K

    2015-01-01

    Informative capacity analysis of immunohistochemistry (IHC) and flow cytometry (FCM) in the assessment of estrogen receptor α (ERα) expression in breast cancer tissue was performed. Similar frequencies of expression were shown by both methods: 27% of ERα-negative and 73% ERα-positive cases. However, IHC evaluation detected low levels in only 20% of ERα-positive cases, whereas low levels of ERα detected by FCM were 2 times more often (48%). Moreover, FCM revealed positive expression (23-60%) in 33% of IHC ERα-negative cases. Among IHC ER-positive cases, zero ERα expression was detected by FCM in 12.5%. The approaches to minimize errors in routine clinical determination of the estrogen receptor status were proposed. PMID:26728725

  1. 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.

  2. High speed flow cytometric detection of rare glycophorin A mutations in human blood cells

    SciTech Connect

    Langlois, R.G.; Engh, G. van den )

    1993-01-01

    The glycophorin A (GPA) assay utilizes immunofluorescent labeling and flow cytometry to measure the frequency of peripheral erythrocytes with mutant phenotypes, presumably due to mutations in erythroid precursor cells. Analysis of 5 [times] 10[sup 6] cells/assay is used to enumerate variant erythrocytes that occur at a frequency of 3-10 [times] 10[sup [minus]6] in unexposed donors. Extension of this assay to human reticulocytes requires detection of variants that occur at frequencies as low as 3 [times] 10[sup [minus]8]. The authors have used high speed data acquisition and cell classification electronics to perform 3-color analysis at rates up to 20,000 cells/s. High speed analysis of up to 10[sup 8] cells/assay has been used to enumerate GPA-variant reticulocytes in normal donors.

  3. Flow cytometric determination of intracellular or secreted IFNgamma for the quantification of antigen reactive T cells.

    PubMed

    Asemissen, A M; Nagorsen, D; Keilholz, U; Letsch, A; Schmittel, A; Thiel, E; Scheibenbogen, C

    2001-05-01

    The detection of antigen-induced IFNgamma secretion at the single cell level can be used to identify and enumerate antigen-reactive T cells from peripheral blood. This study was performed to analyze the suitability of T cell enumeration by flow cytometry in comparison with the ELISPOT assay. Peripheral blood mononuclear cell (PBMC) samples from six HLA-A2+ healthy subjects were analysed for the frequency of influenza-reactive CD8+ T cells by flow cytometry detecting either intracellular IFNgamma (IC-FC) or secreted IFNgamma (S-FC). All samples were also analysed by IFNgamma ELISPOT assay. The frequency of influenza peptide-reactive T cells determined by IC-FC was 0.01 to 0.34% of CD8+ T cells and by ELISPOT assay 0.02 to 0.23% of CD8+ T cells (n=6 subjects) with a high inter-assay reproducibility and a close correlation between the assays (r=0.77, P<0.001). Little or no IFNgamma production was observed in unstimulated PBMC samples using either the IC-FC or the ELISPOT assay. In contrast, using S-FC large numbers of IFNgamma-secreting CD8+ T cells (0.37% to 5.55%, n=6 subjects) were detected in unstimulated PBMC. The frequency of influenza-reactive CD8+ T cells (0.57-5.19%, n=6 subjects) determined by S-FC did not correlate with the values from the IC-FC or ELISPOT assays. This comparative study shows the suitability of the determination of frequencies of antigen reactive T cells in PBMC by IC-FC. The advantage of IC-FC is the possibility to phenotype simultaneously antigen-reactive T cells. PMID:11292486

  4. Flow-cytometric analysis of T-lymphocyte subsets in sinistral and dextral patients with gingivitis.

    PubMed

    Orbak, Recep; Canakçi, Varol; Erciyas, Kamile; Kaya, Hasan

    2003-01-01

    The aim of this study was to determine whether there was any change in T-lymphocyte subsets in sinistral and dextral patients with gingivitis. The study was carried out on 36 patients (16 males and 20 females) with gingivitis. The age of the patients ranged from 16 to 25 (mean age = 18.50 +/- 3.85). Patients were divided into two equal groups according to their right or left hand use. Being right- or left-handed was determined with Edinburgh Handedness Inventory (Oldfield). At the beginning of the study, gingival index (GI-Löe-Silness) and plaque index (PI-Silness-Löe) scores were recorded in order to assess the gingival tissue health in patients. At the same time, the biopsy samples were taken from the gingival pocket wall tissues at sites of gingivitis. Then, CD4+ and CD8+ lymphocyte and CD4/CD8 ratio values were determined using flow-cytometry in the biopsy samples. The two groups were compared by using Student's t-test. The normal value in peripheral blood of CD4+ lymphocyte and that of CD8+ lymphocyte are 25-29% and 19-48%, respectively. According to flow cytometry findings, in both sinistrality and dexterity with gingivitis, CD+ and CD8+ lymphocyte values were under the normal value while the CD4/CD8 rate was within normal distribution interval. CD4+ lymphocyte values observed in the sinistral patients were found to be lower than those in the dextral patients. The difference between the CD8+ lymphocyte values in left-handed patients and that obtained in right-handed patients was not found to be statistically significant while the difference between the CD4+ lymphocyte values in left-handed patients and that obtained in right-handed patients was found to be statistically significant (p < .05). In addition, the difference between the CD4/CD8 rate obtained in left-handed patients and that obtained in right-handed patients was found to be statistically very significant (p < .001). Consequently, these findings suggested CD4+ lymphocyte value and CD4/CD8 rate was

  5. 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.

  6. Flow cytometric analysis of platelet activation under calcium ion-chelating conditions.

    PubMed

    Nishioka, T; Yokota, M; Tsuda, I; Tatsumi, N

    2002-04-01

    Platelet activation and aggregation results in factitious counting and sizing in routine haematology testing. In this study, the possibility of platelet activation in anticoagulated solutions was examined. Whole blood was examined using an automated counter and a flow cytometer before and after strong vortex agitation. Blood treated with ethylenediaminetetraacetic acid (EDTA) exhibited platelet activation both pre- and postagitation but activated platelets did not cause platelet aggregation. With sodium citrate, platelets were only minimally activated both pre- and postagitation. Heparin-treated blood exhibited minimal platelet activation preagitation, but agitation resulted in strong platelet activation and aggregation. Platelet size was increased by agitation in blood with EDTA and with sodium citrate, in association with significant increases in mean platelet volume (MPV) and platelet distribution width (PDW), but MPV and PDW were significantly higher in EDTA solution than in sodium citrate solution. Change in platelet size was observed even in the presence of EDTA, indicating that careful sampling and processing are needed in the collection of specimens. Specimens obtained from patients with EDTA-dependent pseudothrombocytopenia exhibited the same level of activation as controls, although platelets exhibited aggregation in such specimens. In conclusion, platelet activation involving platelet size change can occur in the absence of calcium ions in blood treated with EDTA. PMID:11985558

  7. Flow cytometric lifetime-based cell viability assay using propidium iodide

    NASA Astrophysics Data System (ADS)

    Steinkamp, John A.; Lehnert, Bruce E.; Lehnert, Nancy M.

    1999-05-01

    Assays which discriminate and enumerate dying or dead cells are important in various types of cellular studies. In many instances, there is a need to identify dead cells that interfere with fluorescent probes which are used to measure functional and physiological properties in viable cells. For example, dead cells can introduce analytical errors arising from (1) nonspecific uptake of fluorescent probes, leading to erroneous percentages of positive labeled cells, (2) increased autofluorescence, and (3) altered antigen expression. The ability to detect dead cells is also of importance in determining the effectiveness of cytotoxic agents. Propidium iodide (PPI) exclusion, which is analogous to the non- fluorescent trypan blue dye test for viability, is used extensively in flow cytometry assays. However, the use of PI can potentially limit the application of additional fluorescent probes due to spectral overlap of the probe with PI. In this report we present phase-resolved fluorescence studies on rat and murine thymus cells labeled with phycoerythrin-antiThy 1.1 and phycoerythrin/Texas Red-antiThy 1.2 immunofluorescence markers, respectively, and PI. Overlapping emission spectra are resolved based on differences in fluorescence lifetimes of the probes and PI. These studies demonstrate a new lifetime-based viability method for use in analysis of immunofluorescent probes and for assaying the dynamics of cell killing.

  8. Complex karyotypes in flow cytometrically DNA-diploid squamous cell carcinomas of the head and neck.

    PubMed Central

    Akervall, J.; Jin, Y.; Baldetorp, B.; Mertens, F.; Wennerberg, J.

    1998-01-01

    In squamous cell carcinoma of the head and neck (SCCHN), DNA ploidy as determined by flow cytometry (FCM) has been found to yield prognostic information but only for tumours at oral sites. Cytogenetic findings have indicated complex karyotype to be a correlate of poor clinical outcome. In the present study, 73 SCCHN were investigated with the two techniques. Aneuploid cell populations were identified in 49 (67%) cases by FCM but in only 21 (29%) cases by cytogenetic analysis. The chromosome index (CI), calculated as the mean chromosome number divided by 46, was compared with the respective DNA index (DI) obtained by FCM in 15 tumours, non-diploid according to both techniques, DI being systematically 12% higher than CI in this subgroup. Eight (33%) of the 24 tumours diploid according to FCM had complex karyotypes, three of the tumours being cytogenetically hypodiploid, three diploid and two non-diploid. The findings in the present study may partly explain the low prognostic value of ploidy status as assessed by FCM that has been observed in SCCHN. In addition, we conclude that FCM yields information of the genetic changes that is too unspecific, and that cytogenetic analysis shows a high rate of unsuccessful investigations, thus diminishing the value of the two methods as prognostic factors in SCCHN. Images Figure 1 PMID:9569043

  9. 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.

  10. 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