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Sample records for cell based assays

  1. Cell-based Assays to Identify Inhibitors of Viral Disease

    Science.gov (United States)

    Green, Neil; Ott, Robert D.; Isaacs, Richard J.; Fang, Hong

    2009-01-01

    Background Antagonizing the production of infectious virus inside cells requires drugs that can cross the cell membrane without harming host cells. Objective It is therefore advantageous to establish intracellular potency of anti-viral drug candidates early in the drug-discovery pipeline. Methods To this end, cell-based assays are being developed and employed in high-throughput drug screening, ranging from assays that monitor replication of intact viruses to those that monitor activity of specific viral proteins. While numerous cell-based assays have been developed and investigated, rapid counter screens are also needed to define the specific viral targets of identified inhibitors and to eliminate nonspecific screening hits. Results/Conclusions Here, we describe the types of cell-based assays being used in antiviral drug screens and evaluate the equally important counter screens that are being employed to reach the full potential of cell-based high-throughput screening. PMID:19750206

  2. High content cell-based assay for the inflammatory pathway

    Science.gov (United States)

    Mukherjee, Abhishek; Song, Joon Myong

    2015-07-01

    Cellular inflammation is a non-specific immune response to tissue injury that takes place via cytokine network orchestration to maintain normal tissue homeostasis. However chronic inflammation that lasts for a longer period, plays the key role in human diseases like neurodegenerative disorders and cancer development. Understanding the cellular and molecular mechanisms underlying the inflammatory pathways may be effective in targeting and modulating their outcome. Tumor necrosis factor alpha (TNF-α) is a pro-inflammatory cytokine that effectively combines the pro-inflammatory features with the pro-apoptotic potential. Increased levels of TNF-α observed during acute and chronic inflammatory conditions are believed to induce adverse phenotypes like glucose intolerance and abnormal lipid profile. Natural products e. g., amygdalin, cinnamic acid, jasmonic acid and aspirin have proven efficacy in minimizing the TNF-α induced inflammation in vitro and in vivo. Cell lysis-free quantum dot (QDot) imaging is an emerging technique to identify the cellular mediators of a signaling cascade with a single assay in one run. In comparison to organic fluorophores, the inorganic QDots are bright, resistant to photobleaching and possess tunable optical properties that make them suitable for long term and multicolor imaging of various components in a cellular crosstalk. Hence we tested some components of the mitogen activated protein kinase (MAPK) pathway during TNF-α induced inflammation and the effects of aspirin in HepG2 cells by QDot multicolor imaging technique. Results demonstrated that aspirin showed significant protective effects against TNF-α induced cellular inflammation. The developed cell based assay paves the platform for the analysis of cellular components in a smooth and reliable way.

  3. Quantitative comparison between microfluidic and microtiter plate formats for cell-based assays.

    Science.gov (United States)

    Yin, Huabing; Pattrick, Nicola; Zhang, Xunli; Klauke, Norbert; Cordingley, Hayley C; Haswell, Steven J; Cooper, Jonathan M

    2008-01-01

    In this paper, we compare a quantitative cell-based assay measuring the intracellular Ca2+ response to the agonist uridine 5'-triphosphate in Chinese hamster ovary cells, in both microfluidic and microtiter formats. The study demonstrates that, under appropriate hydrodynamic conditions, there is an excellent agreement between traditional well-plate assays and those obtained on-chip for both suspended immobilized cells and cultured adherent cells. We also demonstrate that the on-chip assay, using adherent cells, provides the possibility of faster screening protocols with the potential for resolving subcellular information about local Ca2+ flux.

  4. Heat-transfer-method-based cell culture quality assay through cell detection by surface imprinted polymers.

    Science.gov (United States)

    Eersels, Kasper; van Grinsven, Bart; Khorshid, Mehran; Somers, Veerle; Püttmann, Christiane; Stein, Christoph; Barth, Stefan; Diliën, Hanne; Bos, Gerard M J; Germeraad, Wilfred T V; Cleij, Thomas J; Thoelen, Ronald; De Ceuninck, Ward; Wagner, Patrick

    2015-02-17

    Previous work has indicated that surface imprinted polymers (SIPs) allow for highly specific cell detection through macromolecular cell imprints. The combination of SIPs with a heat-transfer-based read-out technique has led to the development of a selective, label-free, low-cost, and user-friendly cell detection assay. In this study, the breast cancer cell line ZR-75-1 is used to assess the potential of the platform for monitoring the quality of a cell culture in time. For this purpose, we show that the proposed methodology is able to discriminate between the original cell line (adherent growth, ZR-75-1a) and a descendant cell line (suspension growth, ZR-75-1s). Moreover, ZR-75-1a cells were cultured for a prolonged period of time and analyzed using the heat-transfer method (HTM) at regular time intervals. The results of these experiments demonstrate that the thermal resistance (Rth) signal decays after a certain number of cell culture passages. This can likely be attributed to a compromised quality of the cell culture due to cross-contamination with the ZR-75-1s cell line, a finding that was confirmed by classical STR DNA profiling. The cells do not express the same functional groups on their membrane, resulting in a weaker bond between cell and imprint, enabling cell removal by mechanical friction, provided by flushing the measuring chamber with buffer solution. These findings were further confirmed by HTM and illustrate that the biomimetic sensor platform can be used as an assay for monitoring the quality of cell cultures in time.

  5. High content screening for G protein-coupled receptors using cell-based protein translocation assays

    DEFF Research Database (Denmark)

    Grånäs, Charlotta; Lundholt, Betina Kerstin; Heydorn, Arne;

    2005-01-01

    the capability to probe GPCR function at the cellular level with better resolution than has previously been possible, and offer practical strategies for more definitive selectivity evaluation and counter-screening in the early stages of drug discovery. The potential of cell-based translocation assays for GPCR...... will continue to be valuable discovery tools, the most exciting developments in the field involve cell-based assays for GPCR function. Some cell-based discovery strategies, such as the use of beta-arrestin as a surrogate marker for GPCR function, have already been reduced to practice, and have been used...... as valuable discovery tools for several years. The application of high content cell-based screening to GPCR discovery has opened up additional possibilities, such as direct tracking of GPCRs, G proteins and other signaling pathway components using intracellular translocation assays. These assays provide...

  6. Dendritic cell-based in vitro assays for vaccine immunogenicity

    OpenAIRE

    Vandebriel, Rob J.; Hoefnagel, Marcel H. N.

    2012-01-01

    Dendritic cells (DC) are pivotal in the induction of adaptive immune responses because they can activate naive T-cells. Moreover, they steer these adaptive immune responses by integrating various stimuli, such as from different pathogen associated molecular patterns and the cytokine milieu. Immature DC are very well capable of ingesting protein antigens, whereas mature DC are efficient presenters of peptides to naive T cells. Human DC can be readily cultured from peripheral blood mononuclear ...

  7. Functional screening with a live cell imaging-based random cell migration assay.

    Science.gov (United States)

    van Roosmalen, Wies; Le Dévédec, Sylvia E; Zovko, Sandra; de Bont, Hans; van de Water, Bob

    2011-01-01

    Cell migration, essential in cancer progression, is a complex process comprising a number of spatiotemporally regulated and well-coordinated mechanisms. In order to study (random) cell migration in the context of responses to various external cues (such as growth factors) or intrinsic cell signaling, a number of different tools and approaches have been developed. In order to unravel the key pathways and players involved in the regulation of (cancer) cell migration, a systematical mapping of the players/pathways is required. For this purpose, we developed a cell migration assay based on automatic high-throughput microscopy screen. This approach allows for screening of hundreds of genes, e.g., those encoding various kinases and phosphatases but can also be used for screening of drugs libraries. Moreover, we have developed an automatic analysis pipeline comprising of (a) automatic data acquisition (movie) and (b) automatic analysis of the acquired movies of the migrating cells. Here, we describe various facets of this approach. Since cell migration is essential in progression of cancer metastasis, we describe two examples of experiments performed on highly motile (metastatic) cancer cells.

  8. Comparison of cell-based and PCR-based assays as methods for measuring infectivity of Tulane virus.

    Science.gov (United States)

    Shan, Lei; Yang, David; Wang, Dapeng; Tian, Peng

    2016-05-01

    In this study, we used Tulane virus (TV) as a surrogate for HuNoV to evaluate for correlation between two cell-based assays and three PCR-based assays. Specifically, the cell-based plaque and TCID50 assays measure for infectious virus particles, while the PCR-based RNase exposure, porcine gastric mucin in-situ-capture qRT-PCR (PGM-ISC-qRT-PCR), and antibody in-situ-capture qRT-PCR (Ab-ISC-qRT-PCR) assays measure for an amplicon within encapsidated viral genome. Ten batches of viral stocks ranging from 3.41 × 10(5) to 6.67 × 10(6) plaque forming units (PFUs) were used for side by side comparison with PFU as a reference. The results indicate that one PFU was equivalent to 6.69 ± 2.34 TCID50 units, 9.75 ± 10.87 RNase-untreated genomic copies (GCs), 2.87 ± 3.05 RNase-treated GCs, 0.07 ± 0.07 PGM-ISC-qRT-PCR GCs, and 0.52 ± 0.39 Ab-ISC-qRT-PCR GCs. We observed that while the cell-based assays were consistent with each other, the TCID50 assay was more sensitive than the plaque assay. In contrast, the PCR-based assays were not always consistent with the cell-based assays. The very high variations in GCs as measured by both ISC-RT-qPCR assays made them difficult to correlate against the relatively small variations (<20-fold) in the PFUs or TCID50 units as measured by the cell-based assays.

  9. Tracking the Invasion of Small Numbers of Cells in Paper-Based Assays with Quantitative PCR.

    Science.gov (United States)

    Truong, Andrew S; Lochbaum, Christian A; Boyce, Matthew W; Lockett, Matthew R

    2015-11-17

    Paper-based scaffolds are an attractive material for culturing mammalian cells in a three-dimensional environment. There are a number of previously published studies, which utilize these scaffolds to generate models of aortic valves, cardiac ischemia and reperfusion, and solid tumors. These models have largely relied on fluorescence imaging and microscopy to quantify cells in the scaffolds. We present here a polymerase chain reaction (PCR)-based method, capable of quantifying multiple cell types in a single culture with the aid of DNA barcodes: unique sequences of DNA introduced to the genome of individual cells or cell types through lentiviral transduction. PCR-based methods are highly specific and are amenable to high-throughput and multiplexed analyses. To validate this method, we engineered two different breast cancer lines to constitutively express either a green or red fluorescent protein. These cells lines allowed us to directly compare the ability of fluorescence imaging (of the fluorescent proteins) and qPCR (of the unique DNA sequences of the fluorescent proteins) to quantify known numbers of cells in the paper based-scaffolds. We also used both methods to quantify the distribution of these breast cell lines in homotypic and heterotypic invasion assays. In the paper-based invasion assays, a single sheet of paper containing cells suspended in a hydrogel was sandwiched between sheets of paper containing only hydrogel. The stack was incubated, and the cells invaded the adjacent layers. The individual sheets of the invasion assay were then destacked and the number of cells in each layer quantified. Our results show both methods can accurately detect cell populations of greater than 500 cells. The qPCR method can repeatedly and accurately detect as few as 50 cells, allowing small populations of highly invasive cells to be detected and differentiated from other cell types.

  10. A Neutralizing Antibody Assay Based on a Reporter of Antibody-Dependent Cell-Mediated Cytotoxicity.

    Science.gov (United States)

    Wu, Yuling; Li, Jia J; Kim, Hyun Jun; Liu, Xu; Liu, Weiyi; Akhgar, Ahmad; Bowen, Michael A; Spitz, Susan; Jiang, Xu-Rong; Roskos, Lorin K; White, Wendy I

    2015-11-01

    Benralizumab is a humanized anti-IL5 receptor α (IL5Rα) monoclonal antibody (mAb) with enhanced (afucosylation) antibody-dependent cell-mediated cytotoxicity (ADCC) function. An ADCC reporter cell-based neutralizing antibody (NAb) assay was developed and characterized to detect NAb against benralizumab in human serum to support the clinical development of benralizumab. The optimal ratio of target cells to effector cells was 3:1. Neither parental benralizumab (fucosylated) nor benralizumab Fab resulted in ADCC activity, confirming the requirement for ADCC activity in the NAb assay. The serum tolerance of the cells was determined to be 2.5%. The cut point derived from normal and asthma serum samples was comparable. The effective range of benralizumab was determined, and 35 ng/mL [80% maximal effective concentration (EC80)] was chosen as the standard concentration to run in the assessment of NAb. An affinity purified goat anti-benralizumab polyclonal idiotype antibody preparation was shown to have NAb since it inhibited ADCC activity in a dose-dependent fashion. The low endogenous concentrations of IL5 and soluble IL5 receptor (sIL5R) did not demonstrate to interfere with the assay. The estimated assay sensitivities at the cut point were 1.02 and 1.10 μg/mL as determined by the surrogate neutralizing goat polyclonal and mouse monoclonal anti-drug antibody (ADA) controls, respectively. The assay can detect NAb (at 2.5 μg/mL) in the presence of 0.78 μg/mL benralizumab. The assay was not susceptible to non-specific matrix effects. This study provides an approach and feasibility of developing an ADCC cell-based NAb assay to support biopharmaceuticals with an ADCC function. PMID:26205082

  11. Multilayer-based lab-on-a-chip systems for perfused cell-based assays

    Science.gov (United States)

    Klotzbach, Udo; Sonntag, Frank; Grünzner, Stefan; Busek, Mathias; Schmieder, Florian; Franke, Volker

    2014-12-01

    A novel integrated technology chain of laser-microstructured multilayer foils for fast, flexible, and low-cost manufacturing of lab-on-a-chip devices especially for complex cell and tissue culture applications, which provides pulsatile fluid flow within physiological ranges at low media-to-cells ratio, was developed and established. Initially the microfluidic system is constructively divided into individual layers, which are formed by separate foils or plates. Based on the functional boundary conditions and the necessary properties of each layer, their corresponding foils and plates are chosen. In the third step, the foils and plates are laser microstructured and functionalized from both sides. In the fourth and last manufacturing step, the multiple plates and foils are joined using different bonding techniques like adhesive bonding, welding, etc. This multilayer technology together with pneumatically driven micropumps and valves permits the manufacturing of fluidic structures and perfusion systems, which spread out above multiple planes. Based on the established lab-on-a-chip platform for perfused cell-based assays, a multilayer microfluidic system with two parallel connected cell culture chambers was successfully implemented.

  12. A cell-based high-throughput screening assay for radiation susceptibility using automated cell counting

    International Nuclear Information System (INIS)

    Radiotherapy is one of the mainstays in the treatment for cancer, but its success can be limited due to inherent or acquired resistance. Mechanisms underlying radioresistance in various cancers are poorly understood and available radiosensitizers have shown only modest clinical benefit. There is thus a need to identify new targets and drugs for more effective sensitization of cancer cells to irradiation. Compound and RNA interference high-throughput screening technologies allow comprehensive enterprises to identify new agents and targets for radiosensitization. However, the gold standard assay to investigate radiosensitivity of cancer cells in vitro, the colony formation assay (CFA), is unsuitable for high-throughput screening. We developed a new high-throughput screening method for determining radiation susceptibility. Fast and uniform irradiation of batches up to 30 microplates was achieved using a Perspex container and a clinically employed linear accelerator. The readout was done by automated counting of fluorescently stained nuclei using the Acumen eX3 laser scanning cytometer. Assay performance was compared to that of the CFA and the CellTiter-Blue homogeneous uniform-well cell viability assay. The assay was validated in a whole-genome siRNA library screening setting using PC-3 prostate cancer cells. On 4 different cancer cell lines, the automated cell counting assay produced radiation dose response curves that followed a linear-quadratic equation and that exhibited a better correlation to the results of the CFA than did the cell viability assay. Moreover, the cell counting assay could be used to detect radiosensitization by silencing DNA-PKcs or by adding caffeine. In a high-throughput screening setting, using 4 Gy irradiated and control PC-3 cells, the effects of DNA-PKcs siRNA and non-targeting control siRNA could be clearly discriminated. We developed a simple assay for radiation susceptibility that can be used for high-throughput screening. This will aid

  13. Live-cell luciferase assay of drug resistant cells

    OpenAIRE

    sprotocols

    2015-01-01

    To date, multiplexing cell-based assay is essential for high-throughput screening of molecular targets. Measuring multiple parameters of a single sample increases consistency and decrease time and cost of assay. Functional assay of living cell is useful as a first step of multiplexing assay, because live-cell assay allows following second assay using cell lysate or stained cell. However, live-cell assay of drug resistant cells that are highly activated of drug efflux mechanisms is sometimes u...

  14. Paper-based assay for red blood cell antigen typing by the indirect antiglobulin test.

    Science.gov (United States)

    Yeow, Natasha; McLiesh, Heather; Guan, Liyun; Shen, Wei; Garnier, Gil

    2016-07-01

    A rapid and simple paper-based elution assay for red blood cell antigen typing by the indirect antiglobulin test (IAT) was established. This allows to type blood using IgG antibodies for the important blood groups in which IgM antibodies do not exist. Red blood cells incubated with IgG anti-D were washed with saline and spotted onto the paper assay pre-treated with anti-IgG. The blood spot was eluted with an elution buffer solution in a chromatography tank. Positive samples were identified by the agglutinated and fixed red blood cells on the original spotting area, while red blood cells from negative samples completely eluted away from the spot of origin. Optimum concentrations for both anti-IgG and anti-D were identified to eliminate the washing step after the incubation phase. Based on the no-washing procedure, the critical variables were investigated to establish the optimal conditions for the paper-based assay. Two hundred ten donor blood samples were tested in optimal conditions for the paper test with anti-D and anti-Kell. Positive and negative samples were clearly distinguished. This assay opens up new applications of the IAT on paper including antibody detection and blood donor-recipient crossmatching and extends its uses into non-blood typing applications with IgG antibody-based diagnostics. Graphical abstract A rapid and simple paper-based assay for red blood cell antigen typing by the indirect antiglobulin test. PMID:27185543

  15. Implementation and Use of State-of-the-Art, Cell-Based In Vitro Assays.

    Science.gov (United States)

    Langer, Gernot

    2016-01-01

    The impressive advances in the generation and interpretation of functional omics data have greatly contributed to a better understanding of the (patho-)physiology of many biological systems and led to a massive increase in the number of specific targets and phenotypes to investigate in both basic and applied research. The obvious complexity revealed by these studies represents a major challenge to the research community and asks for improved target characterisation strategies with the help of reliable, high-quality assays. Thus, the use of living cells has become an integral part of many research activities because the cellular context more closely represents target-specific interrelations and activity patterns. Although still predominant, the use of traditional two-dimensional (2D) monolayer cell culture models has been gradually complemented by studies based on three-dimensional (3D) spheroid (Sutherland 1988) and other 3D tissue culture systems (Santos et al. 2012; Matsusaki et al. 2014) in an attempt to employ model systems more closely representing the microenvironment of cells in the body. Hence, quite a variety of state-of-the-art cell culture models are available for the generation of novel chemical probes or the identification of starting points for drug development in translational research and pharma drug discovery. In order to cope with these information-rich formats and their increasing technical complexity, cell-based assay development has become a scientific research topic in its own right and is used to ensure the provision of significant, reliable and high-quality data outlasting any discussions related to the current "irreproducibility epidemic" (Dolgin 2014; Prinz et al. 2011; Schatz 2014). At the same time the use of cells in microplate assay formats has become state of the art and greatly facilitates rigorous cell-based assay development by providing the researcher with the opportunity to address the multitude of factors affecting the actual

  16. Human cell chips: adapting DNA microarray spotting technology to cell-based imaging assays.

    Directory of Open Access Journals (Sweden)

    Traver Hart

    Full Text Available Here we describe human spotted cell chips, a technology for determining cellular state across arrays of cells subjected to chemical or genetic perturbation. Cells are grown and treated under standard tissue culture conditions before being fixed and printed onto replicate glass slides, effectively decoupling the experimental conditions from the assay technique. Each slide is then probed using immunofluorescence or other optical reporter and assayed by automated microscopy. We show potential applications of the cell chip by assaying HeLa and A549 samples for changes in target protein abundance (of the dsRNA-activated protein kinase PKR, subcellular localization (nuclear translocation of NFkappaB and activation state (phosphorylation of STAT1 and of the p38 and JNK stress kinases in response to treatment by several chemical effectors (anisomycin, TNFalpha, and interferon, and we demonstrate scalability by printing a chip with approximately 4,700 discrete samples of HeLa cells. Coupling this technology to high-throughput methods for culturing and treating cell lines could enable researchers to examine the impact of exogenous effectors on the same population of experimentally treated cells across multiple reporter targets potentially representing a variety of molecular systems, thus producing a highly multiplexed dataset with minimized experimental variance and at reduced reagent cost compared to alternative techniques. The ability to prepare and store chips also allows researchers to follow up on observations gleaned from initial screens with maximal repeatability.

  17. A simple, versatile and sensitive cell-based assay for prions from various species.

    Directory of Open Access Journals (Sweden)

    Zaira E Arellano-Anaya

    Full Text Available Detection and quantification of prion infectivity is a crucial step for various fundamental and applied aspects of prion research. Identification of cell lines highly sensitive to prion infection led to the development of cell-based titration procedures aiming at replacing animal bioassays, usually performed in mice or hamsters. However, most of these cell lines are only permissive to mouse-adapted prions strains and do not allow titration of prions from other species. In this study, we show that epithelial RK13, a cell line permissive to mouse and bank vole prion strains and to natural prion agents from sheep and cervids, enables a robust and sensitive detection of mouse and ovine-derived prions. Importantly, the cell culture work is strongly reduced as the RK13 cell assay procedure designed here does not require subcultivation of the inoculated cultures. We also show that prions effectively bind to culture plastic vessel and are quantitatively detected by the cell assay. The possibility to easily quantify a wider range of prions, including rodent experimental strains but also natural agents from sheep and cervids, should prompt the spread of cell assays for routine prion titration and lead to valuable information in fundamental and applied studies.

  18. Intra-laboratory validation of a human cell based in vitro angiogenesis assay for testing angiogenesis modulators

    OpenAIRE

    TimoYlikomi; JukkaUotila

    2011-01-01

    The developed standardized human cell based in vitro angiogenesis assay was intra-laboratory pre-validated to verify that the method is reliable and relevant for routine testing of modulators of angiogenesis, e.g., pharmaceuticals and industrial chemicals. This assay is based on the earlier published method but it was improved and shown to be more sensitive and rapid than the previous assay. The performance of the assay was assessed by using six reference chemicals, which are widely used phar...

  19. Clausmarin A, Potential Immunosuppressant Revealed by Yeast-Based Assay and Interleukin-2 Production Assay in Jurkat T Cells.

    Directory of Open Access Journals (Sweden)

    Pitipreya Suauam

    Full Text Available Small-molecule inhibitors of Ca2+-signaling pathways are of medicinal importance, as exemplified by the immunosuppressants FK506 and cyclosporin A. Using a yeast-based assay devised for the specific detection of Ca2+-signaling inhibitors, clausmarin A, a previously reported terpenoid coumarin, was identified as an active substance. Here, we investigated the likely mechanism of clausmarin A action in yeast and Jurkat T-cells. In the presence of 100 mM CaCl2 in the growth medium of Ca2+-sensitive Δzds1 strain yeast, clausmarin A exhibited a dose-dependent alleviation of various defects due to hyperactivation of Ca2+ signaling, such as growth inhibition, polarized bud growth and G2 phase cell-cycle arrest. Furthermore, clausmarin A inhibited the growth of Δmpk1 (lacking the Mpk1 MAP kinase pathway but not Δcnb1 (lacking the calcineurin pathway strain, suggesting that clausmarin A inhibited the calcineurin pathway as presumed from the synthetic lethality of these pathways. Furthermore, clausmarin A alleviated the serious defects of a strain expressing a constitutively active form of calcineurin. In the human Jurkat T-cell line, clausmarin A exhibited a dose-dependent inhibition of IL-2 production and IL-2 gene transcription, as well as an inhibition of NFAT dephosphorylation. The effects of clausmarin A observed in both yeast and Jurkat cells are basically similar to those of FK506. Our study revealed that clausmarin A is an inhibitor of the calcineurin pathway, and that this is probably mediated via inhibition of calcineurin phosphatase activity. As such, clausmarin A is a potential immunosuppressant.

  20. Micromachined nanocalorimetric sensor for ultra-low-volume cell-based assays.

    Science.gov (United States)

    Johannessen, Erik A; Weaver, John M R; Bourova, Lenka; Svoboda, Petr; Cobbold, Peter H; Cooper, Jonathan M

    2002-05-01

    Current strategies for cell-based screening generally focus on the development of highly specific assays, which require an understanding of the nature of the signaling molecules and cellular pathways involved. In contrast, changes in temperature of cells provides a measure of altered cellular metabolism that is not stimulus specific and hence could have widespread applications in cell-based screening of receptor agonists and antagonists, as well as in the assessment of toxicity of new lead compounds. Consequently, we have developed a micromachined nanocalorimetric biological sensor using a small number of isolated living cells integrated within a subnanoliter format, which is capable of detecting 13 nW of generated power from the cells, upon exposure to a chemical or pharmaceutical stimulus. The sensor comprises a 10-junction gold and nickel thermopile, integrated on a silicon chip which was back-etched to span a 800-nm-thick membrane of silicon nitride. The thin-film membrane, which supported the sensing junctions of the thermoelectric transducer, gave the system a temperature resolution of 0.125 mK, a low heat capacity of 1.2 nJ mK(-1), and a rapid (unfiltered) response time of 12 ms. The application of the system in ultra-low-volume cell-based assays could provide a rapid endogenous screen. It offers important additional advantages over existing methods in that it is generic in nature, it does not require the use of recombinant cell lines or of detailed assay development, and finally, it can enable the use of primary cell lines or tissue biopsies. PMID:12033326

  1. GFP-based fluorescence assay for CAG repeat instability in cultured human cells.

    Directory of Open Access Journals (Sweden)

    Beatriz A Santillan

    Full Text Available Trinucleotide repeats can be highly unstable, mutating far more frequently than point mutations. Repeats typically mutate by addition or loss of units of the repeat. CAG repeat expansions in humans trigger neurological diseases that include myotonic dystrophy, Huntington disease, and several spinocerebellar ataxias. In human cells, diverse mechanisms promote CAG repeat instability, and in mice, the mechanisms of instability are varied and tissue-dependent. Dissection of mechanistic complexity and discovery of potential therapeutics necessitates quantitative and scalable screens for repeat mutation. We describe a GFP-based assay for screening modifiers of CAG repeat instability in human cells. The assay exploits an engineered intronic CAG repeat tract that interferes with expression of an inducible GFP minigene. Like the phenotypes of many trinucleotide repeat disorders, we find that GFP function is impaired by repeat expansion, in a length-dependent manner. The intensity of fluorescence varies inversely with repeat length, allowing estimates of repeat tract changes in live cells. We validate the assay using transcription through the repeat and engineered CAG-specific nucleases, which have previously been reported to induce CAG repeat instability. The assay is relatively fast and should be adaptable to large-scale screens of chemical and shRNA libraries.

  2. Cell-Based Assay Design for High-Content Screening of Drug Candidates.

    Science.gov (United States)

    Nierode, Gregory; Kwon, Paul S; Dordick, Jonathan S; Kwon, Seok-Joon

    2016-02-01

    To reduce attrition in drug development, it is crucial to consider the development and implementation of translational phenotypic assays as well as decipher diverse molecular mechanisms of action for new molecular entities. High-throughput fluorescence and confocal microscopes with advanced analysis software have simplified the simultaneous identification and quantification of various cellular processes through what is now referred to as highcontent screening (HCS). HCS permits automated identification of modifiers of accessible and biologically relevant targets and can thus be used to detect gene interactions or identify toxic pathways of drug candidates to improve drug discovery and development processes. In this review, we summarize several HCS-compatible, biochemical, and molecular biology-driven assays, including immunohistochemistry, RNAi, reporter gene assay, CRISPR-Cas9 system, and protein-protein interactions to assess a variety of cellular processes, including proliferation, morphological changes, protein expression, localization, post-translational modifications, and protein-protein interactions. These cell-based assay methods can be applied to not only 2D cell culture but also 3D cell culture systems in a high-throughput manner. PMID:26428732

  3. A Caco-2 cell-based quantitative antioxidant activity assay for antioxidants.

    Science.gov (United States)

    Wan, Hongxia; Liu, Dong; Yu, Xiangying; Sun, Haiyan; Li, Yan

    2015-05-15

    A Caco-2 cell-based antioxidant activity (CAA) assay for quantitative evaluation of antioxidants was developed by optimizing seeding density and culture time of Caco-2 cells, incubation time and concentration of fluorescent probe (2',7'-dichlorofluorescin diacetate, DCFH-DA), incubation way and incubation time of antioxidants (pure phytochemicals) and DCFH-DA with cells, and detection time of fluorescence. Results showed that the CAA assay was of good reproducibility and could be used to evaluate the antioxidant activity of antioxidants at the following conditions: seeding density of 5 × 10(4)/well, cell culture time of 24h, co-incubation of 60 μM DCFH-DA and pure phytochemicals with Caco-2 cells for 20 min and fluorescence recorded for 90 min. Additionally, a significant correlation was observed between CAA values and rat plasma ORAC values following the intake of antioxidants for selected pure phytochemicals (R(2) = 0.815, p < 0.01), demonstrating the good biological relevance of CAA assay.

  4. Identification of compounds that modulate retinol signaling using a cell-based qHTS assay.

    Science.gov (United States)

    Chen, Yanling; Sakamuru, Srilatha; Huang, Ruili; Reese, David H; Xia, Menghang

    2016-04-01

    In vertebrates, the retinol (vitamin A) signaling pathway (RSP) controls the biosynthesis and catabolism of all-trans retinoic acid (atRA), which regulates transcription of genes essential for embryonic development. Chemicals that interfere with the RSP to cause abnormal intracellular levels of atRA are potential developmental toxicants. To assess chemicals for the ability to interfere with retinol signaling, we have developed a cell-based RARE (Retinoic Acid Response Element) reporter gene assay to identify RSP disruptors. To validate this assay in a quantitative high-throughput screening (qHTS) platform, we screened the Library of Pharmacologically Active Compounds (LOPAC) in both agonist and antagonist modes. The screens detected known RSP agonists, demonstrating assay reliability, and also identified novel RSP agonists including kenpaullone, niclosamide, PD98059 and SU4312, and RSP antagonists including Bay 11-7085, LY294002, 3,4-Methylenedioxy-β-nitrostyrene, and topoisomerase inhibitors (camptothecin, topotecan, amsacrine hydrochloride, and idarubicin). When evaluated in the P19 pluripotent cell, these compounds were found to affect the expression of the Hoxa1 gene that is essential for embryo body patterning. These results show that the RARE assay is an effective qHTS approach for screening large compound libraries to identify chemicals that have the potential to adversely affect embryonic development through interference with retinol signaling. PMID:26820057

  5. Microbead agglutination based assays

    KAUST Repository

    Kodzius, Rimantas

    2013-01-21

    We report a simple and rapid room temperature assay for point-of-care (POC) testing that is based on specific agglutination. Agglutination tests are based on aggregation of microbeads in the presence of a specific analyte thus enabling the macroscopic observation. Such tests are most often used to explore antibody-antigen reactions. Agglutination has been used for protein assays using a biotin/streptavidin system as well as a hybridization based assay. The agglutination systems are prone to selftermination of the linking analyte, prone to active site saturation and loss of agglomeration at high analyte concentrations. We investigated the molecular target/ligand interaction, explaining the common agglutination problems related to analyte self-termination, linkage of the analyte to the same bead instead of different microbeads. We classified the agglutination process into three kinds of assays: a two- component assay, a three-component assay and a stepped three- component assay. Although we compared these three kinds of assays for recognizing DNA and protein molecules, the assay can be used for virtually any molecule, including ions and metabolites. In total, the optimized assay permits detecting analytes with high sensitivity in a short time, 5 min, at room temperature. Such a system is appropriate for POC testing.

  6. A Cell-Based Assay Reveals Nuclear Translocation of Intracellular Domains Released by SPPL Proteases.

    Science.gov (United States)

    Mentrup, Torben; Häsler, Robert; Fluhrer, Regina; Saftig, Paul; Schröder, Bernd

    2015-08-01

    During regulated intramembrane proteolysis (RIP) a membrane-spanning substrate protein is cleaved by an ectodomain sheddase and an intramembrane cleaving protease. A cytoplasmic intracellular domain (ICD) is liberated, which can migrate to the nucleus thereby influencing transcriptional regulation. Signal peptide peptidase-like (SPPL) 2a and 2b have been implicated in RIP of type II transmembrane proteins. Even though SPPL2a might represent a potential pharmacological target for treatment of B-cell-mediated autoimmunity, no specific and potent inhibitors for this enzyme are currently available. We report here on the first quantitative cell-based assay for measurement of SPPL2a/b activity. Demonstrating the failure of standard Gal4/VP16 reporter assays for SPPL2a/b analysis, we have devised a novel system employing β-galactosidase (βGal) complementation. This is based on detecting nuclear translocation of the proteolytically released substrate ICDs, which results in specific restoration of βGal activity. Utilizing this potentially high-throughput compatible new setup, we demonstrate nuclear translocation of the ICDs from integral membrane protein 2B (ITM2B), tumor necrosis factor (TNF) and CD74 and identify secreted frizzled-related protein 2 (SFRP2) as potential transcriptional downstream target of the CD74 ICD. We show that the presented assay is easily adaptable to other intramembrane proteases and therefore represents a valuable tool for the functional analysis and development of new inhibitors of this class of enzymes. PMID:25824657

  7. RT-qPCR-based microneutralization assay for human cytomegalovirus using fibroblasts and epithelial cells.

    Science.gov (United States)

    Wang, Xiao; Peden, Keith; Murata, Haruhiko

    2015-12-16

    Human cytomegalovirus (HCMV) is a leading cause of congenital infection that can result in serious disabilities in affected children. To facilitate HCMV vaccine development, a microscale neutralization assay based on reverse transcription quantitative PCR (RT-qPCR) was developed to quantify HCMV-neutralizing antibodies. Our approach relies on the generation of crude lysates from virus-infected cells that are amenable to direct analysis by RT-qPCR, thereby circumventing rate-limiting procedures associated with sample RNA extraction and purification. By serial passaging of the laboratory HCMV strain AD169 in epithelial cells (ARPE-19), a revertant virus with restored epithelial cell tropism, designated AD169(wt131), was obtained. AD169 and AD169(wt131) were evaluated in both epithelial cells (ARPE-19) and fibroblasts (MRC-5) by one-step RT-qPCR targeting the immediate-early gene IE1 transcript of HCMV. Expression kinetics indicated that RT-qPCR assessment could be conducted as early as 6h post-infection. Human serum samples (n=30) from healthy donors were tested for HCMV-specific IgG using a commercially available ELISA and for HCMV-neutralizing activity using our RT-qPCR-based neutralization assay. In agreement with the ELISA results, higher neutralizing activity was observed in the HCMV IgG seropositive group when compared with the HCMV IgG seronegative group. In addition, HCMV IgG seropositive human sera exhibited higher neutralizing titers using epithelial cells compared with using fibroblasts (geometric mean titers of 344 and 8 in ARPE-19 cells and MRC-5 cells, respectively). Our assay was robust to variation in input virus dose. In addition, a simple lysis buffer containing a non-ionic detergent was successfully demonstrated to be a less costly alternative to commercial reagents for cell-lysate preparation. Thus, our rapid HCMV neutralization assay may be a straightforward and flexible high-throughput tool for measuring antibody responses induced by vaccination

  8. Establishment of a cell-based assay to screen regulators for Klotho gene promoter

    Institute of Scientific and Technical Information of China (English)

    Zhi-liang XU; Hong GAO; Ke-qing OU-YANG; Shao-xi CAI; Ying-he HU

    2004-01-01

    AIM: To discover compounds which can regulate Klotho promoter activity. Klotho is an aging suppressor gene. A defect in Klotho gene expression in the mouse results in the phenotype similar to human aging. Recombinant Klotho protein improves age-associated diseases in animal models. It has been proposed that up-regulation of Klotho gene expression may have anti-aging effects. METHODS: Klotho promoter was cloned into a vector containing luciferase gene, and the reporter gene vector was transfected into HEK293 cells to make a stable cell line (HEK293/KL). A model for cellular aging was established by treating HEK293/KL cells with H2O2. These cells were treated with extracts from Traditional Chinese Medicines (TCMs). The luciferase activity was detected to identify compounds that can regulate Klotho promoter. RESULTS:The expression of luciferase in these cells was under control of Klotho promoter and down-regulated after H2O2 treatment The down-regulation of luciferase expression was H2O2 concentration-dependent with an IC50 at approximately 0.006 %. This result demonstrated that the Klotho gene promoter was regulated by oxidative stress. Using the cell-based reporter gene assay, we screened natural product extracts for regulation of Klotho gene promoter. Several extracts were identified that could rescue the H2O2effects and up-regulated Klotho promoter activity. CONCLUSION: A cell -based assay for high-throughput drug screening was established to identify compounds that regulate Klotho promoter activity, and several hits were discovered from natural products. Further characterization of these active extracts could help to investigate Klotho function and aging mechanisms.

  9. A Fluid Membrane-Based Soluble Ligand Display System for Live CellAssays

    Energy Technology Data Exchange (ETDEWEB)

    Nam, Jwa-Min; Nair, Pradeep N.; Neve, Richard M.; Gray, Joe W.; Groves, Jay T.

    2005-10-14

    Cell communication modulates numerous biological processes including proliferation, apoptosis, motility, invasion and differentiation. Correspondingly, there has been significant interest in the development of surface display strategies for the presentation of signaling molecules to living cells. This effort has primarily focused on naturally surface-bound ligands, such as extracellular matrix components and cell membranes. Soluble ligands (e.g. growth factors and cytokines) play an important role in intercellular communications, and their display in a surface-bound format would be of great utility in the design of array-based live cell assays. Recently, several cell microarray systems that display cDNA, RNAi, or small molecules in a surface array format were proven to be useful in accelerating high-throughput functional genetic studies and screening therapeutic agents. These surface display methods provide a flexible platform for the systematic, combinatorial investigation of genes and small molecules affecting cellular processes and phenotypes of interest. In an analogous sense, it would be an important advance if one could display soluble signaling ligands in a surface assay format that allows for systematic, patterned presentation of soluble ligands to live cells. Such a technique would make it possible to examine cellular phenotypes of interest in a parallel format with soluble signaling ligands as one of the display parameters. Herein we report a ligand-modified fluid supported lipid bilayer (SLB) assay system that can be used to functionally display soluble ligands to cells in situ (Figure 1A). By displaying soluble ligands on a SLB surface, both solution behavior (the ability to become locally enriched by reaction-diffusion processes) and solid behavior (the ability to control the spatial location of the ligands in an open system) could be combined. The method reported herein benefits from the naturally fluid state of the supported membrane, which allows

  10. Seropositivity rates of water channel protein 4 antibodies compared between a cell-based immunofluorescence assay and an enzyme-linked immunosorbent assay in neuromyelitis optica patients

    Institute of Scientific and Technical Information of China (English)

    Xiaoli Wu; Zhangyuan Liao; Jing Ye; Huiqing Dong; Chaodong Wang; Piu Chan

    2011-01-01

    A total of 66 samples (from 27 cases with neuromyelitis optica, 26 cases with multiple sclerosis, and 13 cases with optic neuritis) were tested for aquaporin-4 antibody by a cell-based immunofluorescence assay and an enzyme-linked immunosorbent assay.The sensitivities and specificities of the two assays were similar.We further analyzed an additional 68 patients and 93 healthy controls using the enzyme-linked immunosorbent assay.A Kappa test showed good consistency between the two methods in terms of detection of anti-aquaporin-4 antibody in the sera of neuromyelitis optica patients.No significant correlations were identified with onset age or disease duration, suggesting that aquaporin-4 antibody is a good marker for neuromyelitis optica.The enzyme-linked immunosorbent assay can be used for quantifying aquaporin-4 antibody concentrations and may be useful to dynamically monitor changes in the levels of aquaporin-4 antibody during disease duration.

  11. Aquaporin-4 autoantibodies in neuromyelitis optica spectrum disorders: comparison between tissue-based and cell-based indirect immunofluorescence assays

    Directory of Open Access Journals (Sweden)

    Chan Koon H

    2010-09-01

    Full Text Available Abstract Background Neuromyelitis optica spectrum disorders (NMOSD are severe central nervous system inflammatory demyelinating disorders (CNS IDD characterized by monophasic or relapsing, longitudinally extensive transverse myelitis (LETM and/or optic neuritis (ON. A significant proportion of NMOSD patients are seropositive for aquaporin-4 (AQP4 autoantibodies. We compared the AQP4 autoantibody detection rates of tissue-based indirect immunofluorescence assay (IIFA and cell-based IIFA. Methods Serum of Chinese CNS IDD patients were assayed for AQP4 autoantibodies by tissue-based IIFA using monkey cerebellum and cell-based IIFA using transfected HEK293 cells which express human AQP4 on their cell membranes. Results In total, 128 CNS IDD patients were studied. We found that 78% of NMO patients were seropositive for AQP4 autoantibodies by cell-based IIFA versus 61% by tissue-based IFA (p = 0.250, 75% of patients having relapsing myelitis (RM with LETM were seropositive by cell-based IIFA versus 50% by tissue-based IIFA (p = 0.250, and 33% of relapsing ON patients were seropositive by cell-based IIFA versus 22% by tissue-based IIFA (p = 1.000; however the differences were not statistically significant. All patients seropositive by tissue-based IIFA were also seropositive for AQP4 autoantibodies by cell-based IIFA. Among 29 NMOSD patients seropositive for AQP4 autoantibodies by cell-based IIFA, 20 (69% were seropositive by tissue-based IIFA. The 9 patients seropositive by cell-based IIFA while seronegative by tissue-based IIFA had NMO (3, RM with LETM (3, a single attack of LETM (1, relapsing ON (1 and a single ON attack (1. Among 23 NMO or RM patients seropositive for AQP4 autoantibodies by cell-based IIFA, comparison between those seropositive (n = 17 and seronegative (n = 6 by tissue-based IIFA revealed no differences in clinical and neuroradiological characteristics between the two groups. Conclusion Cell-based IIFA is slightly more sensitive

  12. A Cell-Based Fluorescent Assay to Detect the Activity of Shiga Toxin and Other Toxins That Inhibit Protein Synthesis

    Science.gov (United States)

    Escherichia coli O157:H7, a major cause of food-borne illness, produces Shiga toxins that block protein synthesis by inactivating the ribosome. In this chapter we describe a simple cell-based fluorescent assay to detect Shiga toxins and inhibitors of toxin activity. The assay can also be used to d...

  13. Identification of human dopamine D1-like receptor agonist using a cell-based functional assay

    Institute of Scientific and Technical Information of China (English)

    Nan JIANG; Ke-qing OU-YANG; Shao-xi CAI; Ying-he HU; Zhi-liang XU

    2005-01-01

    Aim: To establish a cell-based assay to screen human dopamine D1 and D5 receptor agonists against compounds from a natural product compound library.Methods: Synthetic responsive elements 6×cAMP response elements (CRE) and a mini promoter containing a TATA box were inserted into the pGL3 basic vector to generate the reporter gene construct pCRE/TA/Luci. CHO cells were co-transfected with the reporter gene construct and human D1 or D5 receptor cDNA in mammalian expression vectors. Stable cell lines were established for agonist screening. A natural product compound library from over 300 herbs has been established. The extracts from these herbs were used for human D1 and D5 receptor agonist screenings. Results: A number of extracts were identified that activated both D1 and D5 receptors. One of the herb extracts, SBG492, demonstrated distinct pharmacological characteristics with human D1 and D5 receptors.The EC50 values of SBG492 were 342.7 μg/mL for the D1 receptor and 31.7 μg/mL for the D5 receptor. Conclusion: We have established a cell-based assay for high-throughput drug screening to identify D 1-like receptor agonists from natural products. Several extracts that can active D1-like receptors were discovered.These compounds could be useful tools for studies on the functions of these receptors in the brain and could potentially be developed into therapeutic drugs for the treatment of central nervous system diseases.

  14. Novel patient cell-based HTS assay for identification of small molecules for a lysosomal storage disease.

    Directory of Open Access Journals (Sweden)

    Haifeng Geng

    Full Text Available Small molecules have been identified as potential therapeutic agents for lysosomal storage diseases (LSDs, inherited metabolic disorders caused by defects in proteins that result in lysosome dysfunctional. Some small molecules function assisting the folding of mutant misfolded lysosomal enzymes that are otherwise degraded in ER-associated degradation. The ultimate result is the enhancement of the residual enzymatic activity of the deficient enzyme. Most of the high throughput screening (HTS assays developed to identify these molecules are single-target biochemical assays. Here we describe a cell-based assay using patient cell lines to identify small molecules that enhance the residual arylsulfatase A (ASA activity found in patients with metachromatic leukodystrophy (MLD, a progressive neurodegenerative LSD. In order to generate sufficient cell lines for a large scale HTS, primary cultured fibroblasts from MLD patients were transformed using SV40 large T antigen. These SV40 transformed (SV40t cells showed to conserve biochemical characteristics of the primary cells. Using a specific colorimetric substrate para-nitrocatechol sulfate (pNCS, detectable ASA residual activity were observed in primary and SV40t fibroblasts from a MLD patient (ASA-I179S cultured in multi-well plates. A robust fluorescence ASA assay was developed in high-density 1,536-well plates using the traditional colorimetric pNCS substrate, whose product (pNC acts as "plate fluorescence quencher" in white solid-bottom plates. The quantitative cell-based HTS assay for ASA generated strong statistical parameters when tested against a diverse small molecule collection. This cell-based assay approach can be used for several other LSDs and genetic disorders, especially those that rely on colorimetric substrates which traditionally present low sensitivity for assay-miniaturization. In addition, the quantitative cell-based HTS assay here developed using patient cells creates an

  15. Development of a Cell-Based Functional Assay for the Detection of Clostridium botulinum Neurotoxin Types A and E

    Directory of Open Access Journals (Sweden)

    Uma Basavanna

    2013-01-01

    Full Text Available The standard procedure for definitive detection of BoNT-producing Clostridia is a culture method combined with neurotoxin detection using a standard mouse bioassay (MBA. The mouse bioassay is highly sensitive and specific, but it is expensive and time-consuming, and there are ethical concerns due to use of laboratory animals. Cell-based assays provide an alternative to the MBA in screening for BoNT-producing Clostridia. Here, we describe a cell-based assay utilizing a fluorescence reporter construct expressed in a neuronal cell model to study toxin activity in situ. Our data indicates that the assay can detect as little as 100 pM BoNT/A activity within living cells, and the assay is currently being evaluated for the analysis of BoNT in food matrices. Among available in vitro assays, we believe that cell-based assays are widely applicable in high-throughput screenings and have the potential to at least reduce and refine animal assays if not replace it.

  16. A Cell-based High-throughput Screening Assay for Farnesoid X Recepter Agonist

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective To develop a high-throughput screening assay for Farnesoid X receptor (FXR) agonists based on mammalian one-hybrid system (a chimera receptor gene system) for the purpose of identifying new lead compounds for dyslipidaemia drug from the chemical library. Methods cDNA encoding the human FXR ligand binding domain (LBD) was amplified by RT-PCR from a human liver total mRNA and fused to the DNA binding domain (DBD) of yeast GAL4 of pBIND to construct a GAL4-FXR (LBD) chimera expression plasmid. Five copies of the GAL4 DNA binding site were synthesized and inserted into upstream of the SV40 promoter of pGL3-promoter vector to construct a reporter plasmid pG5-SV40 Luc. The assay was developed by transient co-transfection with pG5-SV40 Luc reporter plasmid and pBIND-FXR-LBD (189-472) chimera expression plasmid. Results After optimization, CDCA, a FXR natural agonist, could induce expression of the luciferase gene in a dose-dependent manner, and had a signal/noise ratio of 10 and Z'factor value of 0.65. Conclusion A stable and sensitive cell-based high-throughput screening model can be used in high-throughput screening for FXR agonists from the synthetic and natural compound library.

  17. Demonstration of a visual cell-based assay for screening glucose transporter 4 translocation modulators in real time

    Indian Academy of Sciences (India)

    Maleppillil Vavachan Vijayakumar; Amrendra Kumar Ajay; Manoj Kumar Bhat

    2010-12-01

    Insulin-stimulated translocation of glucose transporter 4 (GLUT4) to cell membrane leading to glucose uptake is the rate-limiting step in diabetes. It is also a defined target of antidiabetic drug research. Existing GLUT4 translocation assays are based on time-consuming immunoassays and are hampered by assay variability and low sensitivity. We describe a real-time, visual, cell-based qualitative GLUT4 translocation assay using CHO-HIRc-myc-GLUT4eGFP cells that stably express myc- and eGFP-tagged GLUT4 in addition to human insulin receptor (HIRc). GLUT4 translocation is visualized by live cell imaging based on GFP fluorescence by employing a cooled charge-coupled device camera attached to a fluorescent microscope. This video imaging method and further quantitative analysis of GLUT4 on the cell membrane provide rapid and foolproof visual evidence that this method is suitable for screening GLUT4 translocation modulators.

  18. Rapid assessment of antibody-induced ricin neutralization by employing a novel functional cell-based assay.

    Science.gov (United States)

    Gal, Yoav; Alcalay, Ron; Sabo, Tamar; Noy-Porat, Tal; Epstein, Eyal; Kronman, Chanoch; Mazor, Ohad

    2015-09-01

    Ricin is one of the most potent and lethal toxins known against which there is no available antidote. Currently, the most promising countermeasures against the toxin are based on neutralizing antibodies elicited by active vaccination or administered passively. A cell-based assay is widely applied for the primary screening and evaluation of anti-ricin antibodies, yet such assays are usually time-consuming (18-72 h). Here, we report of a novel assay to monitor ricin activity, based on HeLa cells that stably express the rapidly-degraded ubiquitin-luciferase (Ub-FL, half-life of 2 min). Ricin-induced arrest of protein synthesis could be quantified within 3 to 6h post intoxication (IC90 of 300 and 100 ng/ml, respectively). Furthermore, by stabilizing the intracellular levels of Ub-FL in the last hour of the assay, a 3-fold increase in the assay sensitivity was attained. We applied this assay to monitor the efficacy of a ricin holotoxin-based vaccine by measuring the formation of neutralizing antibodies throughout the immunization course. The potency of anti-ricin monoclonal antibodies (directed to either subunit of the toxin) could also be easily and accurately measured in this assay format. Owing to its simplicity, this assay may be implemented for high-throughput screening of ricin-neutralizing antibodies and for identification of small-molecule inhibitors of the toxin, as well as other ribosome-inactivating toxins. PMID:26003675

  19. A Multi-Modality CMOS Sensor Array for Cell-Based Assay and Drug Screening.

    Science.gov (United States)

    Chi, Taiyun; Park, Jong Seok; Butts, Jessica C; Hookway, Tracy A; Su, Amy; Zhu, Chengjie; Styczynski, Mark P; McDevitt, Todd C; Wang, Hua

    2015-12-01

    In this paper, we present a fully integrated multi-modality CMOS cellular sensor array with four sensing modalities to characterize different cell physiological responses, including extracellular voltage recording, cellular impedance mapping, optical detection with shadow imaging and bioluminescence sensing, and thermal monitoring. The sensor array consists of nine parallel pixel groups and nine corresponding signal conditioning blocks. Each pixel group comprises one temperature sensor and 16 tri-modality sensor pixels, while each tri-modality sensor pixel can be independently configured for extracellular voltage recording, cellular impedance measurement (voltage excitation/current sensing), and optical detection. This sensor array supports multi-modality cellular sensing at the pixel level, which enables holistic cell characterization and joint-modality physiological monitoring on the same cellular sample with a pixel resolution of 80 μm × 100 μm. Comprehensive biological experiments with different living cell samples demonstrate the functionality and benefit of the proposed multi-modality sensing in cell-based assay and drug screening.

  20. Geosmin induces genomic instability in the mammalian cell microplate-based comet assay.

    Science.gov (United States)

    Silva, Aline Flor; Lehmann, Mauricio; Dihl, Rafael Rodrigues

    2015-11-01

    Geosmin (GEO) (trans-1,10-dimethyl-trans-9-decalol) is a metabolite that renders earthy and musty taste and odor to water. Data of GEO genotoxicity on mammalian cells are scarce in the literature. Thus, the present study assessed the genotoxicity of GEO on Chinese hamster ovary (CHO) cells in the microplate-based comet assay. The percent of tail DNA (tail intensity (TI)), tail moment (TM), and tail length (TL) were used as parameters for DNA damage assessment. The results demonstrated that concentrations of GEO of 30 and 60 μg/mL were genotoxic to CHO cells after 4- and 24-h exposure periods, in all parameters evaluated, such as TI, TM, and TL. Additionally, GEO 15 μg/mL was genotoxic in the three parameters only in the 24-h exposure time. The same was observed for GEO 7.5 μg/mL, which induced significant DNA damage observed as TI in the 24-h treatment. The results present evidence that exposure to GEO may be associated with genomic instability in mammalian cells.

  1. Cell-based assays and animal models for GPCR drug screening.

    Science.gov (United States)

    Takakura, Hideo; Hattori, Mitsuru; Tanaka, Miho; Ozawa, Takeaki

    2015-01-01

    The family of G protein-coupled receptors (GPCRs) remains a central focus of basic pharmacology and drug discovery efforts. Convenient methods to assess the efficacy of potentially therapeutic reagents for GPCRs are strongly required for high-throughput screening (HTS) assay. We recently developed a rapid, sensitive, and quantitative method for detecting potential chemicals that act on GPCRs using split luciferase complementation. In principle, this is based on the detection of interactions of GPCR with β-arrestin, which translocates to the activated GPCRs. This method can facilitate the construction of HTS systems in a multi-well plate format. Particularly, the method is compatible with single-cell imaging and animal models and even deeper tissues such as organs, because of its high sensitivity, suggesting that promising candidates from HTS assay can be moved easily to the next phase for additional analysis. This system can contribute to the effective evaluation of potentially therapeutic reagents and expedite the development of new drugs for GPCRs. PMID:25563190

  2. Intra-laboratory validation of a human cell based in vitro angiogenesis assay for testing angiogenesis modulators

    Directory of Open Access Journals (Sweden)

    Jertta-Riina Sarkanen

    2011-01-01

    Full Text Available The developed standardized human cell based in vitro angiogenesis assay was intra-laboratory validated to verify that the method is reliable and relevant for routine testing of modulators of angiogenesis e.g. pharmaceuticals and industrial chemicals. This assay is based on the earlier published method but it was improved and shown to be more sensitive and rapid than the previous assay. The performance of the assay was assessed by using 6 reference chemicals, which are widely used pharmaceuticals that inhibit angiogenesis: acetyl salicylic acid, erlotinib, 2-methoxyestradiol, levamisole, thalidomide, and anti-vascular endothelial growth factor. In the intra-laboratory validation, the sensitivity of the assay (upper and lower limits of detection and linearity of response in tubule formation, batch to batch variation in tubule formation between different Master cell bank batches, and precision as well as the reliability of the assay (reproducibility and repeatability were tested. The pre-set acceptance criteria for the intra-laboratory validation study were met. The relevance of the assay in man was investigated by comparing the effects of reference chemicals and their concentrations to the published human data. The comparison showed a good concordance, which indicates that this human cell based angiogenesis model predicts well the effects in man and has the potential to be used to supplement and/or replace of animal tests.

  3. A new cell-based assay to evaluate myogenesis in mouse myoblast C2C12 cells

    Energy Technology Data Exchange (ETDEWEB)

    Kodaka, Manami [Department of Medical Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Yang, Zeyu [Department of Medical Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Department of Ultrasound, Shengjing Hospital of China Medical University, Shenyang (China); Nakagawa, Kentaro; Maruyama, Junichi [Department of Medical Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Xu, Xiaoyin [Department of Medical Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Department of Breast Surgery, The Second Affiliated Hospital of Wenzhou Medical University, Wenzhou (China); Sarkar, Aradhan; Ichimura, Ayana [Department of Medical Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Nasu, Yusuke [Department of Breast Surgery, The Second Affiliated Hospital of Wenzhou Medical University, Wenzhou (China); Ozawa, Takeaki [Department of Chemistry, School of Science, The University of Tokyo, Tokyo (Japan); Iwasa, Hiroaki [Department of Medical Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Ishigami-Yuasa, Mari [Chemical Biology Screening Center, Tokyo Medical and Dental University, Tokyo (Japan); Ito, Shigeru [Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo (Japan); Kagechika, Hiroyuki [Chemical Biology Screening Center, Tokyo Medical and Dental University, Tokyo (Japan); Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo (Japan); and others

    2015-08-15

    The development of the efficient screening system of detecting compounds that promote myogenesis and prevent muscle atrophy is important. Mouse C2C12 cells are widely used to evaluate myogenesis but the procedures of the assay are not simple and the quantification is not easy. We established C2C12 cells expressing the N-terminal green fluorescence protein (GFP) and the C-terminal GFP (GFP1–10 and GFP11 cells). GFP1–10 and GFP11 cells do not exhibit GFP signals until they are fused. The signal intensity correlates with the expression of myogenic markers and myofusion. Myogenesis-promoting reagents, such as insulin-like growth factor-1 (IGF1) and β-guanidinopropionic acid (GPA), enhance the signals, whereas the poly-caspase inhibitor, z-VAD-FMK, suppresses it. GFP signals are observed when myotubes formed by GFP1–10 cells are fused with single nuclear GFP11 cells, and enhanced by IGF1, GPA, and IBS008738, a recently-reported myogenesis-promoting reagent. Fusion between myotubes formed by GFP1–10 and GFP11 cells is associated with the appearance of GFP signals. IGF1 and GPA augment these signals, whereas NSC23766, Rac inhibitor, decreases them. The conditioned medium of cancer cells suppresses GFP signals during myogenesis and reduces the width of GFP-positive myotubes after differentiation. Thus the novel split GFP-based assay will provide the useful method for the study of myogenesis, myofusion, and atrophy. - Highlights: • C2C12 cells expressing split GFP proteins show GFP signals when mix-cultured. • The GFP signals correlate with myogenesis and myofusion. • The GFP signals attenuate under the condition that muscle atrophy is induced.

  4. A new cell-based assay to evaluate myogenesis in mouse myoblast C2C12 cells

    International Nuclear Information System (INIS)

    The development of the efficient screening system of detecting compounds that promote myogenesis and prevent muscle atrophy is important. Mouse C2C12 cells are widely used to evaluate myogenesis but the procedures of the assay are not simple and the quantification is not easy. We established C2C12 cells expressing the N-terminal green fluorescence protein (GFP) and the C-terminal GFP (GFP1–10 and GFP11 cells). GFP1–10 and GFP11 cells do not exhibit GFP signals until they are fused. The signal intensity correlates with the expression of myogenic markers and myofusion. Myogenesis-promoting reagents, such as insulin-like growth factor-1 (IGF1) and β-guanidinopropionic acid (GPA), enhance the signals, whereas the poly-caspase inhibitor, z-VAD-FMK, suppresses it. GFP signals are observed when myotubes formed by GFP1–10 cells are fused with single nuclear GFP11 cells, and enhanced by IGF1, GPA, and IBS008738, a recently-reported myogenesis-promoting reagent. Fusion between myotubes formed by GFP1–10 and GFP11 cells is associated with the appearance of GFP signals. IGF1 and GPA augment these signals, whereas NSC23766, Rac inhibitor, decreases them. The conditioned medium of cancer cells suppresses GFP signals during myogenesis and reduces the width of GFP-positive myotubes after differentiation. Thus the novel split GFP-based assay will provide the useful method for the study of myogenesis, myofusion, and atrophy. - Highlights: • C2C12 cells expressing split GFP proteins show GFP signals when mix-cultured. • The GFP signals correlate with myogenesis and myofusion. • The GFP signals attenuate under the condition that muscle atrophy is induced

  5. Whole cell based electrical impedance sensing approach for a rapid nanotoxicity assay

    Energy Technology Data Exchange (ETDEWEB)

    Hondroulis, Evangelia; Liu Chang; Li Chenzhong, E-mail: licz@fiu.edu [Nanobioengineering/Bioelectronics Laboratory, Department of Biomedical Engineering, Florida International University, 10555 West Flagler Street, Miami, FL 33174 (United States)

    2010-08-06

    A whole cell based biosensor for rapid real-time testing of human and environmental toxicity of nanoscale materials is reported. Recent studies measuring nanoparticle cytotoxicity in vitro provide a final measurement of toxicity to a cell culture overlooking the ongoing cytotoxic effects of the nanoparticles over the desired timeframe. An array biosensor capable of performing multiple cytotoxicity assays simultaneously was designed to address the need for a consistent method to measure real-time assessments of toxicity. The impedimetric response of human lung fibroblasts (CCL-153) and rainbow trout gill epithelial cells (RTgill-W1) when exposed to gold and silver nanoparticles (AuNPs, AgNPs), single walled carbon nanotubes (SWCNTs) and cadmium oxide (CdO) was tested. Exposure to CdO particles exhibited the fastest rate of cytotoxicity and demonstrated the biosensor's ability to monitor toxicity instantaneously in real time. Advantages of the present method include shorter run times, easier usage, and multi-sample analysis leading to a method that can monitor the kinetic effects of nanoparticle toxicity continuously over a desired timeframe.

  6. Influenza virus assays based on virus‐inducible reporter cell lines

    Science.gov (United States)

    Li, Yunsheng; Larrimer, Audrey; Curtiss, Teresa; Kim, Jaekyung; Jones, Abby; Baird‐Tomlinson, Heather; Pekosz, Andrew; Olivo, Paul D.

    2009-01-01

    Background  Virus‐inducible reporter genes have been used as the basis of virus detection and quantitation assays for a number of viruses. A strategy for influenza A virus‐induction of a reporter gene was recently described. In this report, we describe the extension of this strategy to influenza B virus, the generation of stable cell lines with influenza A and B virus‐inducible reporter genes, and the use of these cells in various clinically relevant viral assays. Each of the cell lines described herein constitutively express an RNA transcript that contains a reporter gene coding region flanked by viral 5′‐ and 3′‐untranslated regions (UTR) and therefore mimics an influenza virus genomic segment. Upon infection of the cells with influenza virus the virus‐inducible reporter gene segment (VIRGS) is replicated and transcribed by the viral polymerase complex resulting in reporter gene expression. Findings  Reporter gene induction occurs after infection with a number of laboratory strains and clinical isolates of influenza virus including several H5N1 strains. The induction is dose‐dependent and highly specific for influenza A or influenza B viruses. Conclusions  These cell lines provide the basis of simple, rapid, and objective assays that involve virus quantitation such as determination of viral titer, assessment of antiviral susceptibility, and determination of antibody neutralization titer. These cell lines could be very useful for influenza virus researchers and vaccine manufacturers. PMID:21462401

  7. Development of a novel HAC-based "gain of signal" quantitative assay for measuring chromosome instability (CIN) in cancer cells.

    Science.gov (United States)

    Kim, Jung-Hyun; Lee, Hee-Sheung; Lee, Nicholas C O; Goncharov, Nikolay V; Kumeiko, Vadim; Masumoto, Hiroshi; Earnshaw, William C; Kouprina, Natalay; Larionov, Vladimir

    2016-03-22

    Accumulating data indicates that chromosome instability (CIN) common to cancer cells can be used as a target for cancer therapy. At present the rate of chromosome mis-segregation is quantified by laborious techniques such as coupling clonal cell analysis with karyotyping or fluorescence in situ hybridization (FISH). Recently, a novel assay was developed based on the loss of a non-essential human artificial chromosome (HAC) carrying a constitutively expressed EGFP transgene ("loss of signal" assay). Using this system, anticancer drugs can be easily ranked on by their effect on HAC loss. However, it is problematic to covert this "loss of signal" assay into a high-throughput screen to identify drugs and mutations that increase CIN levels. To address this point, we re-designed the HAC-based assay. In this new system, the HAC carries a constitutively expressed shRNA against the EGFP transgene integrated into human genome. Thus, cells that inherit the HAC display no green fluorescence, while cells lacking the HAC do. We verified the accuracy of this "gain of signal" assay by measuring the level of CIN induced by known antimitotic drugs and added to the list of previously ranked CIN inducing compounds, two newly characterized inhibitors of the centromere-associated protein CENP-E, PF-2771 and GSK923295 that exhibit the highest effect on chromosome instability measured to date. The "gain of signal" assay was also sensitive enough to detect increase of CIN after siRNA depletion of known genes controlling mitotic progression through distinct mechanisms. Hence this assay can be utilized in future experiments to uncover novel human CIN genes, which will provide novel insight into the pathogenesis of cancer. Also described is the possible conversion of this new assay into a high-throughput screen using a fluorescence microplate reader to characterize chemical libraries and identify new conditions that modulate CIN level. PMID:26943579

  8. Erythrocytes and cell line-based assays to evaluate the cytoprotective activity of antioxidant components obtained from natural sources.

    Science.gov (United States)

    Botta, Albert; Martínez, Verónica; Mitjans, Montserrat; Balboa, Elena; Conde, Enma; Vinardell, M Pilar

    2014-02-01

    Oxidative stress can damage cellular components including DNA, proteins or lipids, and may cause several skin diseases. To protect from this damage and addressing consumer's appeal to natural products, antioxidants obtained from algal and vegetal extracts are being proposed as antioxidants to be incorporated into formulations. Thus, the development of reliable, quick and economic in vitro methods to study the cytoactivity of these products is a meaningful requirement. A combination of erythrocyte and cell line-based assays was performed on two extracts from Sargassum muticum, one from Ulva lactuca, and one from Castanea sativa. Antioxidant properties were assessed in erythrocytes by the TBARS and AAPH assays, and cytotoxicity and antioxidant cytoprotection were assessed in HaCaT and 3T3 cells by the MTT assay. The extracts showed no antioxidant activity on the TBARS assay, whereas their antioxidant capacity in the AAPH assay was demonstrated. On the cytotoxicity assays, extracts showed low toxicity, with IC50 values higher than 200μg/mL. C. sativa extract showed the most favourable antioxidant properties on the antioxidant cytoprotection assays; while S. muticum and U. lactuca extracts showed a slight antioxidant activity. This battery of methods was useful to characterise the biological antioxidant properties of these natural extracts.

  9. Development and validation of a simple cell-based fluorescence assay for dipeptidyl peptidase 1 (DPP1) activity.

    Science.gov (United States)

    Thong, Bob; Pilling, James; Ainscow, Edward; Beri, Raj; Unitt, John

    2011-01-01

    Dipeptidyl peptidase 1 (DPP1) (EC 3.4.14.1; also known as cathepsin C, cathepsin J, dipeptidyl aminopeptidase, and dipeptidyl aminotransferase) is a lysosomal cysteinyl protease of the papain family involved in the intracellular degradation of proteins. Isolated enzyme assays for DPP1 activity using a variety of synthetic substrates such as dipeptide or peptide linked to amino-methyl-coumarin (AMC) or other fluorophores are well established. There is, however, no report of a simple whole-cell-based assay for measuring lysosomal DPP1 activity other than the use of flow cytometry (fluorescence-activated cell sorting) or the use of invasive activity-based probes or the production of physiological products such as neutrophil elastase. The authors investigated a number of DPP1 fluorogenic substrates that have the potential to access the lysosome and enable the measurement of DPP1 enzyme activity in situ. They describe the development and evaluation of a simple noninvasive fluorescence assay for measuring DPP1 activity in fresh or cryopreserved human THP-1 cells using the substrate H-Gly-Phe-AFC (amino-fluoro-coumarin). This cell-based fluorescence assay can be performed in a 96-well plate format and is ideally suited for determining the cell potency of potential DPP1 enzyme inhibitors.

  10. Estimating the wound healing ability of bioactive milk proteins using an optimized cell based assay

    DEFF Research Database (Denmark)

    Nyegaard, Steffen; Andreasen, Trine; Rasmussen, Jan Trige

    oxidoreductase along with minor whey constituents like osteopontin, EPV20 etc. The enterocyte migration rate is a key parameter in maintaining intestinal homeostasis and intestinal repair when recovering from infection or intestinal diseases like Crohns and ulcerative colitis. We developed a novel in vitro wound...... healing assay to determine the bioactive effects of various milk proteins using human small intestine cells grown on extracellular matrix. Silicone inserts are placed in a 96-well plate and enterocytes seeded around it, creating a monolayer with a cell free area. In current ongoing experiments, various...

  11. Cell-based optical assay for amyloid β-induced neuronal cell dysfunction using femtosecond-pulsed laser

    Science.gov (United States)

    Lee, Seunghee; Yoon, Jonghee; Choi, Chulhee

    2015-03-01

    Amyloid β-protein (Aβ) is known as a key molecule related to the pathogenesis of Alzheimer's disease (AD). Over time, the amyloid cascade disrupts essential function of mitochondria including Ca2+ homeostasis and reactive oxygen species (ROS) regulation, and eventually leads to neuronal cell death. However, there have been no methods that analyze and measure neuronal dysfuction in pathologic conditions quantitatively. Here, we suggest a cell-based optical assay to investigate neuronal function in AD using femtosecond-pulsed laser stimulation. We observed that laser stimulation on primary rat hippocampal neurons for a few microseconds induced intracellular Ca2+ level increases or produced intracellular ROS which was a primary cause of neuronal cell death depending on delivered energy. Although Aβ treatment alone had little effect on the neuronal morphologies and networks in a few hours, Aβ-treated neurons showed delayed Ca2+ increasing pattern and were more vulnerable to laser-induced cell death compared to normal neurons. Our results collectively indicate that femtosecond laser stimulation can be a useful tool to study neuronal dysfuction related to AD pathologies. We anticipate this optical method to enable studies in the early progression of neuronal impairments and the quantitative evaluation of drug effects on neurons in neurodegenerative diseases, including AD and Parkinson's disease in a preclinical study.

  12. [Development of a functional cell-based HTS assay for the identification mGluR4 modulators].

    Science.gov (United States)

    Zhang, Yaling; Bai, Yanqiu

    2009-03-01

    To identify metabotropic glutamate receptor 4 (mGluR4) modulators by Ca2+ influx assay, we developed the functional cell-based high throughput-screening (HTS) assay. The human mGluR4 cDNA was transfected into HEK-293 stably expressing promiscuous G-protein (Ga alpha15) cells. Recombinant stable mGluR4 cell line was selected under Zeocin and validated by Ca2+ influx assay. The assay was optimized on loading time of Fluo Calcium Indicator, Dimethyl sulfoxide (DMSO) tolerance and sodium hydroxide (NaOH) tolerance using agonist (L-Glutamic acid (L-Glu)) of mGluR4. The rank order of the agonist potency for the stable human mGluR4 cell line was L-(+)-2-Amino-4-phosphonobutyric acid (L-AP4) > L-Serine-O-phosphate (L-SOP) > L-Glu, and of the antagonist potency was (RS)-alpha-Methylserine-O-phosphate (MSOP) > (RS)-alpha-Methyl-4-phosphonophenylglycine (MPPG). Z' factor value of the cell line in 96- and 384-well plate format was 0.80 and 0.65. Our data indicate a successful development of functional human mGluR4 recombinant stable cell line that was suitable for high throughput screening to identify mGluR4 agonist/antagonist. PMID:19621591

  13. Development of a novel HAC-based "gain of signal" quantitative assay for measuring chromosome instability (CIN) in cancer cells

    OpenAIRE

    Kim, Jung Hyun; Lee, Hee Sheung; Lee, Nicholas C.O.; Goncharov, Nikolay V.; Kumeiko, Vadim; Masumoto, Hiroshi; Earnshaw, William C.; Kouprina, Natalay; Larionov, Vladimir

    2016-01-01

    Accumulating data indicates that chromosome instability (CIN) common to cancer cells can be used as a target for cancer therapy. At present the rate of chromosome mis-segregation is quantified by laborious techniques such as coupling clonal cell analysis with karyotyping or fluorescence in situ hybridization (FISH). Recently, a novel assay was developed based on the loss of a non-essential human artificial chromosome (HAC) carrying a constitutively expressed EGFP transgene ("loss of signal" a...

  14. White blood cell-based detection of asymptomatic scrapie infection by ex vivo assays.

    Directory of Open Access Journals (Sweden)

    Sophie Halliez

    Full Text Available Prion transmission can occur by blood transfusion in human variant Creutzfeldt-Jakob disease and in experimental animal models, including sheep. Screening of blood and its derivatives for the presence of prions became therefore a major public health issue. As infectious titer in blood is reportedly low, highly sensitive and robust methods are required to detect prions in blood and blood derived products. The objectives of this study were to compare different methods--in vitro, ex vivo and in vivo assays--to detect prion infectivity in cells prepared from blood samples obtained from scrapie infected sheep at different time points of the disease. Protein misfolding cyclic amplification (PMCA and bioassays in transgenic mice expressing the ovine prion protein were the most efficient methods to identify infected animals at any time of the disease (asymptomatic to terminally-ill stages. However scrapie cell and cerebellar organotypic slice culture assays designed to replicate ovine prions in culture also allowed detection of prion infectivity in blood cells from asymptomatic sheep. These findings confirm that white blood cells are appropriate targets for preclinical detection and introduce ex vivo tools to detect blood infectivity during the asymptomatic stage of the disease.

  15. Development of PDMS-based Microfluidic Device for Cell-based Assays

    Institute of Scientific and Technical Information of China (English)

    LI Chenuk-Wing; YANG Jun; TZANG Chi-Hung; YANG Meng-Su

    2004-01-01

    In a single step photolithography, muhi-level microfluidic device is fabricated by printing novel architectures on a film photomasks. The whole fabrication process is executed by classical PCB technology without the need to access clean room facilities. Different levels of protruding features on PCB master are produced by exposing a photomask with specifically arranged "windows and rims" architectures, followed by chemical wet etching. Poly(dimethylsiloxane)(PDMS) is then molded against the positive relief master to generate microfluidic device featured with multi-level sandbag structure and peripheral microchannels. This sandbag structure is an analog to traditional dam or weir for particle entrapment. The microstructure does not collapse when subjected to applied pressure, which is suitable for operation on elastic PDMS substrate.Typical immunocytochemcial staining assays were performed in the microdevice to demonstrate the applicability of the sandbag structure for cellular analysis. This simplified microfabrication process employs low-cost materials and minimal specialized equipment and can reproducibly produce mask lines with about 20 μm in width, which is sufficient for most microfluidic applications.

  16. Development of a high-throughput cell-based reporter assay for screening JAK3 inhibitors

    OpenAIRE

    Yin, Chang-Hong; Bach, Erika A.; Baeg, Gyeong-Hun

    2011-01-01

    JAK3 has become an ideal target for the therapeutic treatment of immune-related diseases, as well as for the prevention of organ allograft rejection. A number of JAK3 inhibitors have been identified by in vitro biochemical enzymatic assays, but the majority display significant off-target effects on JAK2. Therefore, there is an urgent need to develop new experimental approaches to identify compounds that specifically inhibit JAK3. Here, we showed that in 32D/IL-2Rβ cells, STAT5 becomes phospho...

  17. A cell-based fluorescent glucose transporter assay for SGLT2 inhibitor discovery

    OpenAIRE

    Yi Huan; Linyi Li; Quan Liu; Shuainan Liu; Zhufang Shen

    2013-01-01

    The sodium/glucose cotransporter 2 (SGLT2) is responsible for the majority of glucose reabsorption in the kidney, and currently, SGLT2 inhibitors are considered as promising hypoglycemic agents for the treatment of type 2 diabetes mellitus. By constructing CHO cell lines that stably express the human SGLT2 transmembrane protein, along with a fluorescent glucose transporter assay that uses 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]2-deoxyglucose (2-NBDG) as a glucose analog, we have develo...

  18. Lectin Conjugated Gold Nanoparticle-based Colorimetric Assay for Studying the Interactions of Antibiotic with Living Cell

    Institute of Scientific and Technical Information of China (English)

    WANG Jin-e; WANG Cheng-ke; LIU Dian-jun; WANG Zhen-xin

    2011-01-01

    The interactions of antibiotic with living cells were studied by iectin conjugated gold nanoparticles(GNPs)based colorimetric assay. Because of the high affinity of lectin for saccharides, the lectin conjugated GNPs are able to employ as indicators for monitoring the antibiotic induced changes of glycosyl complexes. The interactions of a well known antibiotic, tunicamycin, with two different cell lines, HeLa and SHG-44, were selected to establish this assay.In the presence of tunicamycin, the dose- and time-dependence on the decreasing of binding affinity of lectin conjugated GNPs with living cells were demonstrated by conventional microscopic and UV-Vis spectroscopic studies. The experimental result demonstrates that our approach can be used to identify antibiotic induced expression difference of glycosyl complexes on different cellular surfaces and determine drug activity quantitatively. For further confirming the capability of the GNP-based assay, the system was also studied by confocal laser scanning microscopy(CLSM)and classic flow cytometry(FCM) assay, and satisfactory results were obtained.

  19. An impedance-based cytotoxicity assay for real-time and label-free assessment of T-cell-mediated killing of adherent cells.

    Science.gov (United States)

    Peper, Janet Kerstin; Schuster, Heiko; Löffler, Markus W; Schmid-Horch, Barbara; Rammensee, Hans-Georg; Stevanović, Stefan

    2014-03-01

    The in vitro assessment of T-cell-mediated cytotoxicity plays an important and increasingly relevant role both in preclinical target evaluation and during immunomonitoring to accompany clinical trials employing targeted immunotherapies. For a long time, the gold standard for this purpose has been the chromium release assay (CRA). This end point assay, however, shows several disadvantages including the inevitable use of radioactivity. Based on electrical impedance measurements (using the xCELLigence system), we have established a label-free assay, facilitating the real-time monitoring of T-cell-mediated cytotoxicity. The coculture of peptide-specific T-cell lines with peptide-loaded target cells reproducibly led to a decrease in impedance due to induced apoptosis and detachment of target cells. Comparing our results to the standard CRA assay, we could demonstrate that impedance-based measurements show comparable results after short incubation periods (6h) but outperform the CRA both in reproducibility and sensitivity after prolonged incubation (24h), enabling the detection of target cell lysis with an effector to target ratio as low as 0.05:1. The impedance-based assay represents a valuable and highly sensitive tool for label-free real-time high throughput analysis of T-cell-mediated cytotoxicity.

  20. A cell-based pharmacokinetics assay for evaluating tubulin-binding drugs.

    Science.gov (United States)

    Wang, Yuwei; Liu, Jihua; Zhang, Jun; Wang, Liping; Chan, Jonathon; Wang, Hai; Jin, Yi; Yu, Lei; Grainger, David W; Ying, Wenbin

    2014-01-01

    Increasing evidence reveals that traditional pharmacokinetics parameters based on plasma drug concentrations are insufficient to reliably demonstrate accurate pharmacological effects of drugs in target organs or cells in vivo. This underscores the increasing need to improve the types and qualities of cellular pharmacokinetic information for drug preclinical screening and clinical efficacy assessments. Here we report a whole cell-based method to assess drugs that disturb microtubule dynamics to better understand different formulation-mediated intracellular drug release profiles. As proof of concept for this approach, we compared the well-known taxane class of anti-microtubule drugs based on paclitaxel (PTX), including clinically familiar albumin nanoparticle-based Abraxane™, and a polymer nanoparticle-based degradable paclitaxel carrier, poly(L-glutamic acid)-paclitaxel conjugate (PGA-PTX, also known as CT-2103) versus control PTX. This in vitro cell-based evaluation of PTX efficacy includes determining the cellular kinetics of tubulin polymerization, relative populations of cells under G2 mitotic arrest, cell proliferation and total cell viability. For these taxane tubulin-binding compounds, the kinetics of cell microtubule stabilization directly correlate with G2 arrest and cell proliferation, reflecting the kinetics and amounts of intracellular PTX release. Each individual cell-based dose-response experiment correlates with published, key therapeutic parameters and taken together, provide a comprehensive understanding of drug intracellular pharmacokinetics at both cellular and molecular levels. This whole cell-based evaluating method is convenient, quantitative and cost-effective for evaluating new formulations designed to optimize cellular pharmacokinetics for drugs perturbing tubulin polymerization as well as assisting in explaining drug mechanisms of action at cellular levels.

  1. A Simple, Versatile and Sensitive Cell-Based Assay for Prions from Various Species

    OpenAIRE

    Arellano-Anaya, Zaira E.; Jimmy Savistchenko; Jacinthe Mathey; Alvina Huor; Caroline Lacroux; Olivier Andréoletti; Didier Vilette

    2011-01-01

    Detection and quantification of prion infectivity is a crucial step for various fundamental and applied aspects of prion research. Identification of cell lines highly sensitive to prion infection led to the development of cell-based titration procedures aiming at replacing animal bioassays, usually performed in mice or hamsters. However, most of these cell lines are only permissive to mouse-adapted prions strains and do not allow titration of prions from other species. In this study, we show ...

  2. Development of a Highly Sensitive Cell-Based Assay for Detecting Botulinum Neurotoxin Type A through Neural Culture Media Optimization.

    Science.gov (United States)

    Hong, Won S; Pezzi, Hannah M; Schuster, Andrea R; Berry, Scott M; Sung, Kyung E; Beebe, David J

    2016-01-01

    Botulinum neurotoxin (BoNT) is the most lethal naturally produced neurotoxin. Due to the extreme toxicity, BoNTs are implicated in bioterrorism, while the specific mechanism of action and long-lasting effect was found to be medically applicable in treating various neurological disorders. Therefore, for both public and patient safety, a highly sensitive, physiologic, and specific assay is needed. In this paper, we show a method for achieving a highly sensitive cell-based assay for BoNT/A detection using the motor neuron-like continuous cell line NG108-15. To achieve high sensitivity, we performed a media optimization study evaluating three commercially available neural supplements in combination with retinoic acid, purmorphamine, transforming growth factor β1 (TGFβ1), and ganglioside GT1b. We found nonlinear combinatorial effects on BoNT/A detection sensitivity, achieving an EC50 of 7.4 U ± 1.5 SD (or ~7.9 pM). The achieved detection sensitivity is comparable to that of assays that used primary and stem cell-derived neurons as well as the mouse lethality assay.

  3. Fluorescence assay for glycan expression on living cancer cells based on competitive strategy coupled with dual-functionalized nanobiocomposites.

    Science.gov (United States)

    Fu, Ying; Lu, Danqin; Lin, Bin; Sun, Qianqian; Liu, Kai; Xu, Lili; Zhang, Shengping; Hu, Chen; Wang, Chuangui; Xu, Zhiai; Zhang, Wen

    2013-11-21

    Cell surface glycans are a class of sophisticated biomolecules related to cancer development and progression, and their analysis is of great significance for early cancer diagnosis and treatment. In this paper, we proposed a fluorescence assay to evaluate glycan expression on living cancer cells based on a competitive strategy coupled with dual-functionalized nanobiocomposites. The competitive assay was conducted between living cancer cells and thiomannosyl derivatives using concanavalin A (Con A)-modified electrode as the interaction platform. To impart fluorescence signaling ability to competitive derivatives, quantum dots (QDs) were anchored on BSA-protected Au nanoparticles, and thiomannosyl derivatives were further immobilized on the nanoparticle surface through Au-S binding. Due to the spacing between QDs and Au nanoparticles by BSA, the {QDs-Au-BSA-mannose} nanobiocomposites maintained the fluorescence of QDs and showed binding ability with the Con A-modified electrode. Au nanorods (AuNRs)-modified electrode was used as an effective substrate to immobilize Con A. This assay was successfully applied to the analysis of two cancer cells lines (A549 and QGY-7701). The method is simple and shows promise for the study of glycan expression on living cancer cells.

  4. Probing protein complexes inside living cells using a silicon nanowire-based pull-down assay

    Science.gov (United States)

    Choi, Sojoong; Kim, Hyunju; Kim, So Yeon; Yang, Eun Gyeong

    2016-06-01

    Most proteins perform their functions as interacting complexes. Here we propose a novel method for capturing an intracellular protein and its interacting partner out of living cells by utilizing intracellular access of antibody modified vertical silicon nanowire arrays whose surface is covered with a polyethylene glycol layer to prevent strong cell adhesion. Such a feature facilitates the removal of cells by simple washing, enabling subsequent detection of a pulled-down protein and its interacting partner, and further assessment of a drug-induced change in the interacting complex. Our new SiNW-based tool is thus suitable for authentication of protein networks inside living cells.Most proteins perform their functions as interacting complexes. Here we propose a novel method for capturing an intracellular protein and its interacting partner out of living cells by utilizing intracellular access of antibody modified vertical silicon nanowire arrays whose surface is covered with a polyethylene glycol layer to prevent strong cell adhesion. Such a feature facilitates the removal of cells by simple washing, enabling subsequent detection of a pulled-down protein and its interacting partner, and further assessment of a drug-induced change in the interacting complex. Our new SiNW-based tool is thus suitable for authentication of protein networks inside living cells. Electronic supplementary information (ESI) available: Materials, experimental methods and Fig. S1-S8. See DOI: 10.1039/c6nr00171h

  5. Development of a flow cytometry-based potency assay for measuring the in vitro immunomodulatory properties of mesenchymal stromal cells.

    Science.gov (United States)

    Ribeiro, Andreia; Ritter, Thomas; Griffin, Matthew; Ceredig, Rhodri

    2016-09-01

    Human bone marrow-derived mesenchymal stromal/stem cells (MSC) have well-documented modulatory effects on multiple immune cell types. Although these effects are linked to their therapeutic benefit in diverse diseases, a reliable, quantitative assay of the immunomodulatory potency of individual human MSC preparations is lacking. The aims of this study were to develop an optimised rapid turnaround, flow cytometry-based whole-blood assay to monitor MSC potency and to validate its application to MSC immunomodulation. A protocol for short-term LPS stimulation of anti-coagulated whole blood samples followed by combined surface CD45/CD14 and intracellular TNF-α staining was initially developed for analysis on a 4 colour desktop cytometer. Optimal monocyte activation was dependent on the presence of extracellular calcium ions thereby precluding the use of EDTA and sodium citrate as anticoagulants. Optimal assay conditions proved to be 1ng/mL ultrapure-LPS added to 10-fold diluted, heparin anti-coagulated whole blood incubated for 6h at 37°C. Under these conditions, addition of human bone marrow-derived MSC (hBM-MSC) from multiple donors resulted in a reproducible, dose-dependent inhibition of LPS-stimulated monocyte TNF-α expression. We conclude that this protocol represents a practical, quantitative assay of a clinically relevant functional effect of hBM-MSCs as well as other immunomodulatory agents. PMID:27451032

  6. Immune cell-based screening assay for response to anticancer agents: applications in pharmacogenomics

    Directory of Open Access Journals (Sweden)

    Frick A

    2015-02-01

    were generated using GraphPad Prism 6. Results: Phenotypes were quantified using flow cytometry, yielding interstrain variation for measured endpoints in different immune cells. The flow cytometry assays produced over 16,000 data points that were used to generate dose-response curves. The more targeted agents, BEZ-235 and selumetinib, were less toxic to immune cells than the anthracycline agents. The calculated heritability for the viability of immune cells was higher with anthracyclines than the novel agents, making them better suited for downstream genetic analysis. Conclusion: Using this approach, we identify cell lines of variable sensitivity to chemotherapeutic agents and aim to identify robust, replicable endpoints of cellular response to drugs that provide the starting point for identifying candidate genes and cellular toxicity pathways for future validation in human studies. Keywords: immunomodulation, cytotoxicity, chemotherapy, precision medicine

  7. A high-throughput fluorescence resonance energy transfer (FRET)-based endothelial cell apoptosis assay and its application for screening vascular disrupting agents

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Xiaoming; Fu, Afu [Division of Bioengineering, School of Chemical and Biomedical Engineering, Nanyang Technological University (Singapore); Luo, Kathy Qian, E-mail: kluo@ntu.edu.sg [Division of Bioengineering, School of Chemical and Biomedical Engineering, Nanyang Technological University (Singapore)

    2012-02-24

    Highlights: Black-Right-Pointing-Pointer An endothelial cell apoptosis assay using FRET-based biosensor was developed. Black-Right-Pointing-Pointer The fluorescence of the cells changed from green to blue during apoptosis. Black-Right-Pointing-Pointer This method was developed into a high-throughput assay in 96-well plates. Black-Right-Pointing-Pointer This assay was applied to screen vascular disrupting agents. -- Abstract: In this study, we developed a high-throughput endothelial cell apoptosis assay using a fluorescence resonance energy transfer (FRET)-based biosensor. After exposure to apoptotic inducer UV-irradiation or anticancer drugs such as paclitaxel, the fluorescence of the cells changed from green to blue. We developed this method into a high-throughput assay in 96-well plates by measuring the emission ratio of yellow fluorescent protein (YFP) to cyan fluorescent protein (CFP) to monitor the activation of a key protease, caspase-3, during apoptosis. The Z Prime factor for this assay was above 0.5 which indicates that this assay is suitable for a high-throughput analysis. Finally, we applied this functional high-throughput assay for screening vascular disrupting agents (VDA) which could induce endothelial cell apoptosis from our in-house compounds library and dioscin was identified as a hit. As this assay allows real time and sensitive detection of cell apoptosis, it will be a useful tool for monitoring endothelial cell apoptosis in living cell situation and for identifying new VDA candidates via a high-throughput screening.

  8. A high-throughput fluorescence resonance energy transfer (FRET)-based endothelial cell apoptosis assay and its application for screening vascular disrupting agents

    International Nuclear Information System (INIS)

    Highlights: ► An endothelial cell apoptosis assay using FRET-based biosensor was developed. ► The fluorescence of the cells changed from green to blue during apoptosis. ► This method was developed into a high-throughput assay in 96-well plates. ► This assay was applied to screen vascular disrupting agents. -- Abstract: In this study, we developed a high-throughput endothelial cell apoptosis assay using a fluorescence resonance energy transfer (FRET)-based biosensor. After exposure to apoptotic inducer UV-irradiation or anticancer drugs such as paclitaxel, the fluorescence of the cells changed from green to blue. We developed this method into a high-throughput assay in 96-well plates by measuring the emission ratio of yellow fluorescent protein (YFP) to cyan fluorescent protein (CFP) to monitor the activation of a key protease, caspase-3, during apoptosis. The Z′ factor for this assay was above 0.5 which indicates that this assay is suitable for a high-throughput analysis. Finally, we applied this functional high-throughput assay for screening vascular disrupting agents (VDA) which could induce endothelial cell apoptosis from our in-house compounds library and dioscin was identified as a hit. As this assay allows real time and sensitive detection of cell apoptosis, it will be a useful tool for monitoring endothelial cell apoptosis in living cell situation and for identifying new VDA candidates via a high-throughput screening.

  9. Human Pluripotent Stem Cell Based Developmental Toxicity Assays for Chemical Safety Screening and Systems Biology Data Generation.

    Science.gov (United States)

    Shinde, Vaibhav; Klima, Stefanie; Sureshkumar, Perumal Srinivasan; Meganathan, Kesavan; Jagtap, Smita; Rempel, Eugen; Rahnenführer, Jörg; Hengstler, Jan Georg; Waldmann, Tanja; Hescheler, Jürgen; Leist, Marcel; Sachinidis, Agapios

    2015-01-01

    Efficient protocols to differentiate human pluripotent stem cells to various tissues in combination with -omics technologies opened up new horizons for in vitro toxicity testing of potential drugs. To provide a solid scientific basis for such assays, it will be important to gain quantitative information on the time course of development and on the underlying regulatory mechanisms by systems biology approaches. Two assays have therefore been tuned here for these requirements. In the UKK test system, human embryonic stem cells (hESC) (or other pluripotent cells) are left to spontaneously differentiate for 14 days in embryoid bodies, to allow generation of cells of all three germ layers. This system recapitulates key steps of early human embryonic development, and it can predict human-specific early embryonic toxicity/teratogenicity, if cells are exposed to chemicals during differentiation. The UKN1 test system is based on hESC differentiating to a population of neuroectodermal progenitor (NEP) cells for 6 days. This system recapitulates early neural development and predicts early developmental neurotoxicity and epigenetic changes triggered by chemicals. Both systems, in combination with transcriptome microarray studies, are suitable for identifying toxicity biomarkers. Moreover, they may be used in combination to generate input data for systems biology analysis. These test systems have advantages over the traditional toxicological studies requiring large amounts of animals. The test systems may contribute to a reduction of the costs for drug development and chemical safety evaluation. Their combination sheds light especially on compounds that may influence neurodevelopment specifically. PMID:26132533

  10. Molecular design, synthesis and cell based HCV replicon assay of novel benzoxazole derivatives.

    Science.gov (United States)

    Ismail, M A H; Adel, M; Ismail, N S M; Abouzid, K A M

    2013-03-01

    Hepatitis C virus inhibitors based on benzoxazole scaffold were designed based on molecular modeling simulation study including docking into the NS5B polymerase active site. Several compounds showed significant high simulation docking scores relative to the assigned benzimidazole lead compound. The designed compounds were synthesized, structurally elucidated and their antiviral activity was evaluated through cell-based replicon in cultured Huh 5-2 cells. A number of the synthesized compounds showed significant inhibitory activity ranging from (52.2% inhibition up to 98% at<50 µg/mL). N-Benzyl-2-phenylbenzo[1,3]oxazole-5-carboxamide (8b) and N-Phenethyl-2-phenylbenzo[1,3] oxazole-5-carboxamide (8c) demonstrated genuine HCV inhibitory activity with EC50 values of 41.6 and 24.5 µg/mL respectively.

  11. Development of a quantitative, cell-based, high-content screening assay for epidermal growth factor receptor modulators

    Institute of Scientific and Technical Information of China (English)

    Jue WANG; Xin XIE

    2007-01-01

    Aim: To develop a robust, cell-based, high-content screening (HCS) assay based on receptor internalization for the identification of novel modulators of the epidermal growth factor receptor (EGFR). Methods: Agonist-induced receptor internalization is part of the signaling cascade of EGFR. Fluorescent-tagged epidermal growth factor (EGF) was used to visualize the internalized receptorligand complex. The fluorescent intracellular spots were detected and measured with an ArrayScan HCS reader. Compounds that can competitively bind to EGFR or interfere with EGFR internalization process would result in a reduced number and intensity of intracellular fluorescent spots. This assay was validated,optimized, and applied to a large-scale screening of a library containing 48 000 synthetic compounds. Results: The competition between fluorescent EGF and unlabeled EGF reveals the IC50 of unlabeled EGF is approximately 0.2 nmol/L,which is comparable with other published reports. Thirteen compounds with a relatively high degree of interference with EGFR internalization were identified.One of the compounds was proven to be agonist of the EGFR since it induced phosphorylation of the receptor and extracellular signal-regulated protein kinase (ERK). Conclusion: This automated, objective, and easy-to-use assay provided abundant information, quantitative results, and demonstrated the potential use of HCS methods in searching membrane receptor modulators.

  12. Utility value of a T-cell interferon-γ release assay based on recombinant Mycobacterium tuberculosis 11kD protein in the diagnosis of tuberculosis

    Institute of Scientific and Technical Information of China (English)

    张丽帆

    2014-01-01

    Objective To evaluate the diagnostic efficiency of a T-cell interferon-γrelease assay based on recombinant Mycobacterium tuberculosis(MTB)11kD protein for diagnosing tuberculosis.Methods This prospective study enrolled inpatients with suspected tuberculosis at PUMCH to examine the diagnostic sensitivity,specificity,predictive value(PV)and likelihood ratio(LR)of T-cell interferon-γrelease assays based on recombinant MTB-11kD

  13. Determination of cell survival after irradiation via clonogenic assay versus multiple MTT Assay - A comparative study

    OpenAIRE

    Buch Karl; Peters Tanja; Nawroth Thomas; Sänger Markus; Schmidberger Heinz; Langguth Peter

    2012-01-01

    Abstract For studying proliferation and determination of survival of cancer cells after irradiation, the multiple MTT assay, based on the reduction of a yellow water soluble tetrazolium salt to a purple water insoluble formazan dye by living cells was modified from a single-point towards a proliferation assay. This assay can be performed with a large number of samples in short time using multi-well-plates, assays can be performed semi-automatically with a microplate reader. Survival, the calc...

  14. A novel polymerase chain reaction (PCR based assay for authentication of cell lines or tissues from human, pig and chicken origin

    Directory of Open Access Journals (Sweden)

    MARIO GORENJAK

    2012-01-01

    Full Text Available A polymerase chain reaction based assay was developed for authentication of cell lines or tissues from human, pig and chicken origin. Specificity was achieved by species specific primer design targeting the mitochondrial D-loop sequence. Amplicon sizes were 114 bp, 169 bp and 645-648 bp for chicken, human and pig derived cell lines, respectively. Primers were tested for species specificity and non-specificity between haplogroups of the same organisms using BLAST tool and subsequently for cross amplification DNA extracted from human, chicken and pig venous blood as a positive control. Primers were also amplifying specific products in DNA extracted from individual cell line in both functional cell models and intentionally mixed cell lines consisting functional cell models. The PCR assay developed in this study represents a low-cost species specific end-point PCR based assay of the mitochondrial D-loop for the authentication of the cell line origin.

  15. Differentiation of Helicobacter pylori isolates based on lectin binding of cell extracts in an agglutination assay

    OpenAIRE

    Hynes, Sean; Hirmo, Siiri; Wadström, Torkel; Moran, Anthony P.

    1999-01-01

    Plant and animal lectins with various carbohydrate specificities were used to type 35 Irish clinical isolates of Helicobacter pylori and the type strain NCTC 11637 in a microtiter plate assay. Initially, a panel of eight lectins with the indicated primary specificities were used: Anguilla anguilla (AAA), Lotus tetragonolobus (Lotus A), and Ulex europaeus I (UEA I), specific for α-l-fucose; Solanum tuberosum (STA) and Triticum vulgaris (WGA), specific for β-N-acetylglucosamine; Glycine max (SB...

  16. High-Throughput Cell Toxicity Assays.

    Science.gov (United States)

    Murray, David; McWilliams, Lisa; Wigglesworth, Mark

    2016-01-01

    Understanding compound-driven cell toxicity is vitally important for all drug discovery approaches. With high-throughput screening (HTS) being the key strategy to find hit and lead compounds for drug discovery projects in the pharmaceutical industry [1], an understanding of the cell toxicity profile of hit molecules from HTS activities is fundamentally important. Recently, there has been a resurgence of interest in phenotypic drug discovery and these cell-based assays are now being run in HTS labs on ever increasing numbers of compounds. As the use of cell assays increases the ability to measure toxicity of compounds on a large scale becomes increasingly important to ensure that false hits are not progressed and that compounds do not carry forward a toxic liability that may cause them to fail at later stages of a project. Here we describe methods employed in the AstraZeneca HTS laboratory to carry out very large scale cell toxicity screening. PMID:27317000

  17. Single-cell multiple gene expression analysis based on single-molecule-detection microarray assay for multi-DNA determination

    International Nuclear Information System (INIS)

    Highlights: • A single-molecule-detection (SMD) microarray for 10 samples is fabricated. • The based-SMD microarray assay (SMA) can determine 8 DNAs for each sample. • The limit of detection of SMA is as low as 1.3 × 10−16 mol L−1. • The SMA can be applied in single-cell multiple gene expression analysis. - Abstract: We report a novel ultra-sensitive and high-selective single-molecule-detection microarray assay (SMA) for multiple DNA determination. In the SMA, a capture DNA (DNAc) microarray consisting of 10 subarrays with 9 spots for each subarray is fabricated on a silanized glass coverslip as the substrate. On the subarrays, the spot-to-spot spacing is 500 μm and each spot has a diameter of ∼300 μm. The sequence of the DNAcs on the 9 spots of a subarray is different, to determine 8 types of target DNAs (DNAts). Thus, 8 types of DNAts are captured to their complementary DNAcs at 8 spots of a subarray, respectively, and then labeled with quantum dots (QDs) attached to 8 types of detection DNAs (DNAds) with different sequences. The ninth spot is used to detect the blank value. In order to determine the same 8 types of DNAts in 10 samples, the 10 DNAc-modified subarrays on the microarray are identical. Fluorescence single-molecule images of the QD-labeled DNAts on each spot of the subarray are acquired using a home-made single-molecule microarray reader. The amounts of the DNAts are quantified by counting the bright dots from the QDs. For a microarray, 8 types of DNAts in 10 samples can be quantified in parallel. The limit of detection of the SMA for DNA determination is as low as 1.3 × 10−16 mol L−1. The SMA for multi-DNA determination can also be applied in single-cell multiple gene expression analysis through quantification of complementary DNAs (cDNAs) corresponding to multiple messenger RNAs (mRNAs) in single cells. To do so, total RNA in single cells is extracted and reversely transcribed into their cDNAs. Three types of c

  18. Cell Phone-based Lateral Flow Assay for Blood Biomarker Detection Project

    Data.gov (United States)

    National Aeronautics and Space Administration — The ability to integrate a sensor platform with a cell phone for health monitoring and disease diagnosis for astronauts in space exploration has the potential to be...

  19. Cell Phone-based Lateral Flow Assay for Blood Biomarker Detection Project

    Data.gov (United States)

    National Aeronautics and Space Administration — The ability to integrate a sensor platform with a cell phone for health monitoring and disease diagnosis for astronauts in space has the potential to be cost...

  20. Yellow fluorescent protein-based assay to measure GABA(A channel activation and allosteric modulation in CHO-K1 cells.

    Directory of Open Access Journals (Sweden)

    Teres Johansson

    Full Text Available The γ-aminobutyric acid A (GABA(A ion channels are important drug targets for treatment of neurological and psychiatric disorders. Finding GABA(A channel subtype selective allosteric modulators could lead to new improved treatments. However, the progress in this area has been obstructed by the challenging task of developing functional assays to support screening efforts and the generation of cells expressing functional GABA(A ion channels with the desired subtype composition. To address these challenges, we developed a yellow fluorescent protein (YFP-based assay to be able to study allosteric modulation of the GABA(A ion channel using cryopreserved, transiently transfected, assay-ready cells. We show for the first time how the MaxCyte STX electroporation instrument can be used to generate CHO-K1 cells expressing functional GABA(A α2β3γ2 along with a halide sensing YFP-H148Q/I152L (YFP-GABA(A2 cells. As a basis for a cell-based assay capable of detecting allosteric modulators, experiments with antagonist, ion channel blocker and modulators were used to verify GABA(A subunit composition and functionality. We found that the I(- concentration used in the YFP assay affected both basal quench of YFP and potency of GABA. For the first time the assay was used to study modulation of GABA with 7 known modulators where statistical analysis showed that the assay can distinguish modulatory pEC50 differences of 0.15. In conclusion, the YFP assay proved to be a robust, reproducible and inexpensive assay. These data provide evidence that the assay is suitable for high throughput screening (HTS and could be used to discover novel modulators acting on GABA(A ion channels.

  1. Proteasome Assay in Cell Lysates

    Science.gov (United States)

    Maher, Pamela

    2016-01-01

    The ubiquitin-proteasome system (UPS) mediates the majority of the proteolysis seen in the cytoplasm and nucleus of mammalian cells. As such it plays an important role in the regulation of a variety of physiological and pathophysiological processes including tumorigenesis, inflammation and cell death (Ciechanover, 2005; Kisselev and Goldberg, 2001). A number of recent studies have shown that proteasome activity is decreased in a variety of neurological disorders including Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis and stroke as well as during normal aging (Chung et al., 2001; Ciechanover and Brundin, 2003; Betarbet et al., 2005). This decrease in proteasome activity is thought to play a critical role in the accumulation of abnormal and oxidized proteins. Protein clearance by the UPS involves two sequential reactions. The first is the tagging of protein lysine residues with ubiquitin (Ub) and the second is the subsequent degradation of the tagged proteins by the proteasome. We herein describe an assay for the second of these two reactions (Valera et al., 2013). This assay uses fluorogenic substrates for each of the three activities of the proteasome: chymotrypsin-like activity, trypsin-like activity and caspase-like activity. Cleavage of the fluorophore from the substrate by the proteasome results in fluorescence that can be detected with a fluorescent plate reader.

  2. Establishment of a cell-based assay system for hepatitis C virus serine protease and its primary applications

    Institute of Scientific and Technical Information of China (English)

    Hong-Xia Mao; Shui-Yun Lan; Yun-Wen Hu; Li Xiang; Zheng-Hong Yuan

    2003-01-01

    AIM: To establish an efficient, sensitive, cell-based assay system for NS3 serine protease in an effort to study further the property of hepatitis C virus (HCV) and develop new antiviral agents.METHOOS: We constructed pCI-neo-NS3/4A-SEAP chimeric plasmid, in which the secreted alkaline phosphatase (SEAP) was fused in-frame to the downstream of NS4A/4B cleavage site. The protease activity of NS3 was reflected by the activity of SEAP in the culture media of transient or stable expression cells. Stably expressing cell lines were obtained by G418 selection. Pefabloc SC, a potent irreversible serine protease inhibitor, was used to treat the stably expressing cell lines to assess the system for screening NS3 inhibitors. To compare the activity of serine proteases from 1b and 1a, two chimeric clones were constructed and introduced into both transient and stable expression systems.RESULTS: The SEAP activity in the culture media could be detected in both transient and stable expression systems,and was apparently decreased after Pefabloc SC treatment.In both transient and stable systems, NS3/4A-SEAP chimeric gene from HCV genotype 1b produced higher SEAP activity in the culture media than that from 1a.CONCLUSION: The cell-based system is efficient and sensitive enough for detection and comparison of NS3 protease activity, and screening of anti-NS3 inhibitors. The functional difference between NS3/4A from 1a and 1b subtypes revealed by this system provides a clue for further investigations.

  3. Determination of cell survival after irradiation via clonogenic assay versus multiple MTT Assay - A comparative study

    International Nuclear Information System (INIS)

    For studying proliferation and determination of survival of cancer cells after irradiation, the multiple MTT assay, based on the reduction of a yellow water soluble tetrazolium salt to a purple water insoluble formazan dye by living cells was modified from a single-point towards a proliferation assay. This assay can be performed with a large number of samples in short time using multi-well-plates, assays can be performed semi-automatically with a microplate reader. Survival, the calculated parameter in this assay, is determined mathematically. Exponential growth in both control and irradiated groups was proven as the underlying basis of the applicability of the multiple MTT assay. The equivalence to a clonogenic survival assay with its disadvantages such as time consumption was proven in two setups including plating of cells before and after irradiation. Three cell lines (A 549, LN 229 and F 98) were included in the experiment to study its principal and general applicability

  4. Progress on the development of human in vitro dendritic cell based assays for assessment of the sensitizing potential of a compound

    International Nuclear Information System (INIS)

    Allergic contact dermatitis is the result of an adaptive immune response of the skin to direct exposure to an allergen. Since many chemicals are also allergens, European regulations require strict screening of all ingredients in consumer products. Until recently, identifying a potential allergen has completely relied on animal testing (e.g.: Local Lymph Node Assay). In addition to the ethical problems, both the 7th Amendment to the Cosmetics Directive and REACH have stimulated the development of alternative tests for the assessment of potential sensitizers. This review is aimed at summarising the progress on cell based assays, in particular dendritic cell based assays, being developed as animal alternatives. Primary cells (CD34+ derived dendritic cells, monocyte derived dendritic cells) as well as dendritic cell-like cell lines (THP-1, U-937, MUTZ-3, KG-1, HL-60, and K562) are extensively described along with biomarkers such as cell surface markers, cytokines, chemokines and kinases. From this review, it can be concluded that no single cell based assay nor single marker is yet able to distinguish all sensitizers from non-sensitizers in a test panel of chemicals, nor is it possible to rank the sensitizing potential of the test chemicals. This suggests that sensitivity and specificity may be increased by a tiered assay approach. Only a limited number of genomic and proteomic studies have been completed until now. Such studies have the potential to identify novel biomarkers for inclusion in future assay development. Although progress is promising, this review suggests that it may be difficult to meet the up and coming European regulatory deadlines.

  5. Inhibitors of alphavirus entry and replication identified with a stable Chikungunya replicon cell line and virus-based assays.

    Directory of Open Access Journals (Sweden)

    Leena Pohjala

    Full Text Available Chikungunya virus (CHIKV, an alphavirus, has recently caused epidemic outbreaks and is therefore considered a re-emerging pathogen for which no effective treatment is available. In this study, a CHIKV replicon containing the virus replicase proteins together with puromycin acetyltransferase, EGFP and Renilla luciferase marker genes was constructed. The replicon was transfected into BHK cells to yield a stable cell line. A non-cytopathic phenotype was achieved by a Pro718 to Gly substitution and a five amino acid insertion within non-structural protein 2 (nsP2, obtained through selection for stable growth. Characterization of the replicon cell line by Northern blotting analysis revealed reduced levels of viral RNA synthesis. The CHIKV replicon cell line was validated for antiviral screening in 96-well format and used for a focused screen of 356 compounds (natural compounds and clinically approved drugs. The 5,7-dihydroxyflavones apigenin, chrysin, naringenin and silybin were found to suppress activities of EGFP and Rluc marker genes expressed by the CHIKV replicon. In a concomitant screen against Semliki Forest virus (SFV, their anti-alphaviral activity was confirmed and several additional inhibitors of SFV with IC₅₀ values between 0.4 and 24 µM were identified. Chlorpromazine and five other compounds with a 10H-phenothiazinyl structure were shown to inhibit SFV entry using a novel entry assay based on a temperature-sensitive SFV mutant. These compounds also reduced SFV and Sindbis virus-induced cytopathic effect and inhibited SFV virion production in virus yield experiments. Finally, antiviral effects of selected compounds were confirmed using infectious CHIKV. In summary, the presented approach for discovering alphaviral inhibitors enabled us to identify potential lead structures for the development of alphavirus entry and replication phase inhibitors as well as demonstrated the usefulness of CHIKV replicon and SFV as biosafe surrogate

  6. Distinct gene expression responses of two anticonvulsant drugs in a novel human embryonic stem cell based neural differentiation assay protocol

    NARCIS (Netherlands)

    Schulpen, Sjors H. W.; de Jong, Esther; de la Fonteyne, Liset J. J.; de Klerk, Arja; Piersma, Aldert H.

    2015-01-01

    Hazard assessment of chemicals and pharmaceuticals is increasingly gaining from knowledge about molecular mechanisms of toxic action acquired in dedicated in vitro assays. We have developed an efficient human embryonic stem cell neural differentiation test (hESTn) that allows the study of the molecu

  7. Searching for anti-prion compounds: cell-based high-throughput in vitro assays and animal testing strategies.

    Science.gov (United States)

    Kocisko, David A; Caughey, Byron

    2006-01-01

    The transmissible spongiform encephalopathies (TSEs) or prion diseases are infectious neurodegenerative diseases of mammals. Protease-resistant prion protein (PrP-res) is only associated with TSEs and thus has been a target for therapeutic intervention. The most effective compounds known against scrapie in vivo are inhibitors of PrP-res in infected cells. Mouse neuroblastoma (N2a) cells have been chronically infected with several strains of mouse scrapie including RML and 22L. Also, rabbit epithelial cells that produce sheep prion protein in the presence of doxycycline (Rov9) have been infected with sheep scrapie. Here a high-throughput 96-well plate PrP-res inhibition assay is described for each of these scrapie-infected cell lines. With this dot-blot assay, thousands of compounds can easily be screened for inhibition of PrP-res formation. This assay is designed to find new PrP-res inhibitors, which may make good candidates for in vivo anti-scrapie testing. However, an in vitro assay can only suggest that a given compound might have in vivo anti-scrapie activity, which is typically measured as increased survival times. Methods for in vivo testing of compounds for anti-scrapie activity in transgenic mice, a much more lengthy and expensive process, are also discussed. PMID:17046661

  8. Distinct gene expression responses of two anticonvulsant drugs in a novel human embryonic stem cell based neural differentiation assay protocol.

    Science.gov (United States)

    Schulpen, Sjors H W; de Jong, Esther; de la Fonteyne, Liset J J; de Klerk, Arja; Piersma, Aldert H

    2015-04-01

    Hazard assessment of chemicals and pharmaceuticals is increasingly gaining from knowledge about molecular mechanisms of toxic action acquired in dedicated in vitro assays. We have developed an efficient human embryonic stem cell neural differentiation test (hESTn) that allows the study of the molecular interaction of compounds with the neural differentiation process. Within the 11-day differentiation protocol of the assay, embryonic stem cells lost their pluripotency, evidenced by the reduced expression of stem cell markers Pou5F1 and Nanog. Moreover, stem cells differentiated into neural cells, with morphologically visible neural structures together with increased expression of neural differentiation-related genes such as βIII-tubulin, Map2, Neurogin1, Mapt and Reelin. Valproic acid (VPA) and carbamazepine (CBZ) exposure during hESTn differentiation led to concentration-dependent reduced expression of βIII-tubulin, Neurogin1 and Reelin. In parallel VPA caused an increased gene expression of Map2 and Mapt which is possibly related to the neural protective effect of VPA. These findings illustrate the added value of gene expression analysis for detecting compound specific effects in hESTn. Our findings were in line with and could explain effects observed in animal studies. This study demonstrates the potential of this assay protocol for mechanistic analysis of specific compound-induced inhibition of human neural cell differentiation.

  9. Optimization of cell motility evaluation in scratch assay

    Directory of Open Access Journals (Sweden)

    Gotsulyak N. Ya.

    2014-05-01

    Full Text Available A scratch test is one of the most popular methods of classical cell migration assay in a monolayer culture. At the same time, the scratch assay has some disadvantages that can be easily corrected. Aim. Optimization the existent scratch assay on the base of detection of scratch wound surface area and the length of the field of observation which is more objective and less time consuming. Methods. Scratch assay. Results. The modification of scratch assay enables to perform measurement more accurately and rapidly. This approach is more simple and eliminates the main disadvantages of the classical method. Conclusions. The procedure of scratch wound width measurement calculated on the base of detection of cell free area and the length of the field of observation is more effective than the classical wound healing assay. It will be useful for the estimation of cell migration dynamics in monolayer culture for a wide range of live cell based experiments.

  10. Cell Migration and Invasion Assays as Tools for Drug Discovery

    Directory of Open Access Journals (Sweden)

    Keren I. Hulkower

    2011-03-01

    Full Text Available Cell migration and invasion are processes that offer rich targets for intervention in key physiologic and pathologic phenomena such as wound healing and cancer metastasis. With the advent of high-throughput and high content imaging systems, there has been a movement towards the use of physiologically relevant cell-based assays earlier in the testing paradigm. This allows more effective identification of lead compounds and recognition of undesirable effects sooner in the drug discovery screening process. This article will review the effective use of several principle formats for studying cell motility: scratch assays, transmembrane assays, microfluidic devices and cell exclusion zone assays.

  11. A plasmacytoid dendritic cell (CD123+/CD11c-) based assay system to predict contact allergenicity of chemicals

    International Nuclear Information System (INIS)

    A predictive allergenicity test system for assessing the contact allergenicity of chemicals is needed by the cosmetic and pharmaceutical industry to monitor product safety in the marketplace. Development of such non-animal alternative assay systems for skin sensitization and hazard identification has been pursued by policy makers and regulatory agencies. We investigated whether phenotypic and functional changes to a subset of dendritic cells (DC), plasmacytoid DC (pDC), could be used to identify contact allergens. To achieve this goal, normal human DC were generated from CD34+ progenitor cells and cryopreserved. Frozen DC were thawed and the pDC fraction (CD123+/CD11c-) was harvested using FACS sorting. The pDC were cultured, expanded, and exposed to chemical allergens (N = 26) or non-allergens (N = 22). Concentrations of each chemical that resulted in >50% viability was determined using FACS analysis of propidium iodide stained cells using pDC from 2 to 5 donors. Expression of the surface marker, CD86, which has been implicated in dendritic cell maturation, was used as a marker of allergenicity. CD86 expression increased (≥1.5-fold) for 25 of 26 allergens (sensitivity = 96%) but did not increase for 19 of 22 non-allergens (specificity = 86%). In a direct comparison to historical data for the regulatory approved, mouse local lymph node assay (LLNA) for 23 allergens and 22 non-allergens, the pDC method had sensitivity and specificity of 96% and 86%, respectively, while the sensitivity and specificity of the LLNA assay was 83% and 82%, respectively. In conclusion, CD86 expression in pDC appears to be a sensitive and specific indicator to identify contact allergenicity. Such an assay method utilizing normal human cells will be useful for high throughput screening of chemicals for allergenicity.

  12. A High Throughput Screening Assay for Anti-Mycobacterial Small Molecules Based on Adenylate Kinase Release as a Reporter of Cell Lysis.

    Directory of Open Access Journals (Sweden)

    Lauren Forbes

    Full Text Available Mycobacterium tuberculosis (Mtb is well-established to be one of the most important bacterial pathogens for which new antimicrobial therapies are needed. Herein, we describe the development of a high throughput screening assay for the identification of molecules that are bactericidal against Mycobacteria. The assay utilizes the release of the intracellular enzyme adenylate kinase into the culture medium as a reporter of mycobacterial cell death. We demonstrate that the assay is selective for mycobactericidal molecules and detects anti-mycobacterial activity at concentrations below the minimum inhibitory concentration of many molecules. Thus, the AK assay is more sensitive than traditional growth assays. We have validated the AK assay in the HTS setting using the Mtb surrogate organism M. smegmatis and libraries of FDA approved drugs as well as a commercially available Diversity set. The screen of the FDA-approved library demonstrated that the AK assay is able to identify the vast majority of drugs with known mycobactericidal activity. Importantly, our screen of the Diversity set revealed that the increased sensitivity of the AK assay increases the ability of M. smegmatis-based screens to detect molecules with relatively poor activity against M. smegmatis but good to excellent activity against Mtb.

  13. Reference cells and ploidy in the comet assay

    OpenAIRE

    Brunborg, Gunnar; Collins, Andrew; Graupner, Anne; Gutzkow, Kristine B.; Olsen, Ann-Karin

    2015-01-01

    In the comet assay single cells are analyzed with respect to their level of DNA damage. Discrimination of the individual cell or cell type based on DNA content, with concomitant scoring of the DNA damage, is useful since this may allow analysis of mixtures of cells. Different cells can then be characterized based on their ploidy, cell cycle stage, or genome size. We here describe two applications of such a cell type-specific comet assay: (i) Testicular cell suspensions, analyzed on the basis ...

  14. High throughput cell-based assay for identification of glycolate oxidase inhibitors as a potential treatment for Primary Hyperoxaluria Type 1

    Science.gov (United States)

    Wang, Mengqiao; Xu, Miao; Long, Yan; Fargue, Sonia; Southall, Noel; Hu, Xin; McKew, John C.; Danpure, Christopher J.; Zheng, Wei

    2016-01-01

    Glycolate oxidase (GO) and alanine:glyoxylate aminotransferase (AGT) are both involved in the peroxisomal glyoxylate pathway. Deficiency in AGT function causes the accumulation of intracellular oxalate and the primary hyperoxaluria type 1 (PH1). AGT enhancers or GO inhibitors may restore the abnormal peroxisomal glyoxylate pathway in PH1 patients. With stably transformed cells which mimic the glyoxylate metabolic pathway, we developed an indirect glycolate cytotoxicity assay in a 1,536-well plate format for high throughput screening. This assay can be used to identify compounds that reduce indirect glycolate-induced cytotoxicity by either enhancing AGT activity or inhibiting GO. A pilot screen of 4,096 known compounds identified two membrane permeable GO inhibitors: dichromate salt and colistimethate. We also developed a GO enzyme assay using the hydrogen peroxide-Amplex red reporter system. The IC50 values of potassium dichromate, sodium dichromate, and colistimethate sodium were 0.096, 0.108, and 2.3 μM in the GO enzyme assay, respectively. Further enzyme kinetic study revealed that both types of compounds inhibit GO activity by the mixed linear inhibition. Our results demonstrate that the cell-based assay and GO enzyme assay developed in this study are useful for further screening of large compound libraries for drug development to treat PH1. PMID:27670739

  15. A whole blood monokine-based reporter assay provides a sensitive and robust measurement of the antigen-specific T cell response

    Directory of Open Access Journals (Sweden)

    Bennett Sophia C

    2011-08-01

    Full Text Available Abstract Background The ability to measure T-cell responses to antigens is proving critical in the field of vaccine development and for understanding immunity to pathogens, allergens and self-antigens. Although a variety of technologies exist for this purpose IFNγ-ELISpot assays are widely used because of their sensitivity and simplicity. However, ELISpot assays cannot be performed on whole blood, and require relatively large volumes of blood to yield sufficient numbers of peripheral blood mononuclear cells. To address these deficiencies, we describe an assay that measures antigen-specific T cell responses through changes in monokine gene transcription. The biological amplification of the IFNγ signal generated by this assay provides sensitivity comparable to ELISpot, but with the advantage that responses can be quantified using small volumes of whole blood. Methods Whole blood or peripheral blood mononuclear cells (PBMCs from healthy controls and immunosuppressed recipients of solid organ transplants were incubated with peptide pools covering viral and control antigens or mitogen for 20 hours. Total RNA was extracted and reverse transcribed before amplification in a TaqMan qPCR reaction using primers and probes specific for MIG (CXCL9, IP-10 (CXCL10 and HPRT. The induction of MIG and IP-10 in response to stimuli was analysed and the results were compared with those obtained by ELISpot. Results Antigen-specific T cell responses can be measured through the induction of MIG or IP-10 gene expression in PBMCs or whole blood with results comparable to those achieved in ELISpot assays. The biological amplification generated by IFNγ-R signaling allows responses to be detected in as little as 25 μL of whole blood and enables the assay to retain sensitivity despite storage of samples for up to 48 hours prior to processing. Conclusions A monokine-based reporter assay provides a sensitive measure of antigen-specific T cell activation. Assays can be

  16. Matrix effects on a cell-based assay used for the detection of paralytic shellfish toxins in bivalve shellfish samples.

    Science.gov (United States)

    Aballay-Gonzalez, Ambbar; Ulloa, Viviana; Rivera, Alejandra; Hernández, Víctor; Silva, Macarena; Caprile, Teresa; Delgado-Rivera, Lorena; Astuya, Allisson

    2016-05-01

    Detecting marine biotoxins such as paralytic shellfish toxins (PSTs) is essential to ensuring the safety of seafood. The mouse bioassay is the internationally accepted method for monitoring PSTs, but technical and ethical issues have led to a search for new detection methods. The mouse neuroblastoma cell-based assay (Neuro-2a CBA) using ouabain and veratridine (O/V) has proven useful for the detection of PSTs. However, CBAs are sensitive to shellfish-associated matrix interferences. As the extraction method highly influences matrix interferences, this study compared three extraction protocols: Association of Official Analytical Chemists (AOAC) 2005.06, AOAC 2011.02 and an alternative liquid-liquid method. These methods were used to assess the matrix effect of extracts from four commercially important bivalve species (Chilean mussel, Magellan mussel, clam and Pacific oyster) in Neuro-2a CBA. Extracts from all three protocols caused a toxic effect in Neuro-2a cells (without O/V) when tested at a concentration of 25 mg of tissue-equivalent (TE) ml(-1). The greatest toxicity was obtained through the AOAC 2011.02 protocol, especially for the Chilean mussel and Pacific oyster extracts. Similar toxicity levels (less than 15%) were observed in all extracts at 3.1 mg TE ml(-1). When assessed in Neuro-2a CBA, AOAC 2005.06 extracts presented the lowest matrix interferences, while the highest interferences were observed for AOAC 2011.02 in Magellan mussel and clam extracts. Finally, the AOAC 2005.06 and alternative protocols were compared using Chilean mussel samples fortified with 40 and 80 µg STX per 100 g meat. The AOAC 2005.06 method demonstrated better results. In conclusion, the AOAC 2005.06 extracts exhibited the fewest interferences in the Neuro-2a CBA. Therefore, this extraction method should be considered for the implementation of Neuro-2a CBA as a high-throughput screening methodology for PST detection.

  17. Matrix effects on a cell-based assay used for the detection of paralytic shellfish toxins in bivalve shellfish samples.

    Science.gov (United States)

    Aballay-Gonzalez, Ambbar; Ulloa, Viviana; Rivera, Alejandra; Hernández, Víctor; Silva, Macarena; Caprile, Teresa; Delgado-Rivera, Lorena; Astuya, Allisson

    2016-05-01

    Detecting marine biotoxins such as paralytic shellfish toxins (PSTs) is essential to ensuring the safety of seafood. The mouse bioassay is the internationally accepted method for monitoring PSTs, but technical and ethical issues have led to a search for new detection methods. The mouse neuroblastoma cell-based assay (Neuro-2a CBA) using ouabain and veratridine (O/V) has proven useful for the detection of PSTs. However, CBAs are sensitive to shellfish-associated matrix interferences. As the extraction method highly influences matrix interferences, this study compared three extraction protocols: Association of Official Analytical Chemists (AOAC) 2005.06, AOAC 2011.02 and an alternative liquid-liquid method. These methods were used to assess the matrix effect of extracts from four commercially important bivalve species (Chilean mussel, Magellan mussel, clam and Pacific oyster) in Neuro-2a CBA. Extracts from all three protocols caused a toxic effect in Neuro-2a cells (without O/V) when tested at a concentration of 25 mg of tissue-equivalent (TE) ml(-1). The greatest toxicity was obtained through the AOAC 2011.02 protocol, especially for the Chilean mussel and Pacific oyster extracts. Similar toxicity levels (less than 15%) were observed in all extracts at 3.1 mg TE ml(-1). When assessed in Neuro-2a CBA, AOAC 2005.06 extracts presented the lowest matrix interferences, while the highest interferences were observed for AOAC 2011.02 in Magellan mussel and clam extracts. Finally, the AOAC 2005.06 and alternative protocols were compared using Chilean mussel samples fortified with 40 and 80 µg STX per 100 g meat. The AOAC 2005.06 method demonstrated better results. In conclusion, the AOAC 2005.06 extracts exhibited the fewest interferences in the Neuro-2a CBA. Therefore, this extraction method should be considered for the implementation of Neuro-2a CBA as a high-throughput screening methodology for PST detection. PMID:27002718

  18. Processing nanoparticles with A4F-SAXS for toxicological studies: Iron oxide in cell-based assays.

    Science.gov (United States)

    Knappe, Patrick; Boehmert, Linda; Bienert, Ralf; Kamutzki, Silvana; Karmutzki, Silvana; Niemann, Birgit; Lampen, Alfonso; Thünemann, Andreas F

    2011-07-01

    Nanoparticles are not typically ready-to-use for in vitro cell culture assays. Prior to their use in assays, powder samples containing nanoparticles must be dispersed, de-agglomerated, fractionated by size, and characterized with respect to size and size distribution. For this purpose we report exemplarily on polyphosphate-stabilized iron oxide nanoparticles in aqueous suspension. Fractionation and online particle size analysis was performed in a time-saving procedure lasting 50 min by combining asymmetrical flow field-flow fractionation (A4F) and small-angle X-ray scattering (SAXS). Narrowly distributed nanoparticle fractions with radii of gyration (R(g)) from 7 to 21 nm were obtained from polydisperse samples. The A4F-SAXS combination is introduced for the preparation of well-characterized sample fractions originating from a highly polydisperse system as typically found in engineered nanoparticles. A4F-SAXS processed particles are ready-to-use for toxicological studies. The results of preliminary tests of the effects of fractionated iron oxide nanoparticles with a R(g) of 15 nm on a human colon model cell line are reported.

  19. Comet assay on tetraploid yeast cells

    DEFF Research Database (Denmark)

    Rank, Jette; Syberg, Kristian; Jensen, Klara

    2009-01-01

    Tetraploid yeast cells (Saccharomyces cerevisiae) were used in the comet assay with the intention of developing a new, fast and easy assay for detecting environmental genotoxic agents without using higher organisms. Two DNA-damaging chemicals, H2O2 and acrylamide, together with wastewater from...... three municipal treatment plants were tested for their effect on the yeast-cell DNA. The main problem with using yeast in the comet assay is the necessity to degrade the cell wall. This was achieved by using Zymolase 100 T twice during the procedure, since Zymolase 20 T did not open the cell wall....... Analytical problems that arose due to the small amount of DNA in the yeast nuclei in haploid and diploid cells, which contain 13 Mbp and 26 Mbp DNA per cell, respectively, were solved by using tetraploid yeast cells (52 Mbp) instead. DNA damage was shown after exposure to H2O2 and acrylamide. The lowest dose...

  20. A novel cell-based duplex high-throughput screening assay combining fluorescent Ca(2+) measurement with homogeneous time-resolved fluorescence technology.

    Science.gov (United States)

    Kiss, László; Cselenyák, Attila; Varga, Ágnes; Visegrády, András

    2016-08-15

    Cell-based assays for G-protein-coupled receptor (GPCR) activation applied in high-throughput screening (HTS) monitor various readouts for second messengers or intracellular effectors. Recently, our understanding of diverging signaling pathways downstream of receptor activation and the capability of small molecules to selectively modulate signaling routes has increased substantially, underlining the importance of selecting appropriate readouts in cellular functional screens. To minimize the rate of false negatives in large-scale screening campaigns, it is crucial to maximize the chance of a ligand being detected, and generally applicable methods for detecting multiple analytes from a single well might serve this purpose. The few assays developed so far based on multiplexed GPCR readouts are limited to only certain applications and usually rely on genetic manipulations hindering screening in native or native-like cellular systems. Here we describe a more generally applicable and HTS-compatible homogeneous assay based on the combination of fluorometric detection of [Ca(2+)] with subsequent homogeneous time-resolved fluorescence (HTRF) cAMP readout in the same well. Besides describing development and validation of the assay, using a cell line recombinantly expressing the human PTH1 receptor screening of a small library is also presented, demonstrating the robustness and HTS compatibility of the novel paradigm. PMID:27235172

  1. A novel cell-based duplex high-throughput screening assay combining fluorescent Ca(2+) measurement with homogeneous time-resolved fluorescence technology.

    Science.gov (United States)

    Kiss, László; Cselenyák, Attila; Varga, Ágnes; Visegrády, András

    2016-08-15

    Cell-based assays for G-protein-coupled receptor (GPCR) activation applied in high-throughput screening (HTS) monitor various readouts for second messengers or intracellular effectors. Recently, our understanding of diverging signaling pathways downstream of receptor activation and the capability of small molecules to selectively modulate signaling routes has increased substantially, underlining the importance of selecting appropriate readouts in cellular functional screens. To minimize the rate of false negatives in large-scale screening campaigns, it is crucial to maximize the chance of a ligand being detected, and generally applicable methods for detecting multiple analytes from a single well might serve this purpose. The few assays developed so far based on multiplexed GPCR readouts are limited to only certain applications and usually rely on genetic manipulations hindering screening in native or native-like cellular systems. Here we describe a more generally applicable and HTS-compatible homogeneous assay based on the combination of fluorometric detection of [Ca(2+)] with subsequent homogeneous time-resolved fluorescence (HTRF) cAMP readout in the same well. Besides describing development and validation of the assay, using a cell line recombinantly expressing the human PTH1 receptor screening of a small library is also presented, demonstrating the robustness and HTS compatibility of the novel paradigm.

  2. Establishment and validation of whole-cell based fluorescence assays to identify anti-mycobacterial compounds using the Acanthamoeba castellanii-Mycobacterium marinum host-pathogen system.

    Directory of Open Access Journals (Sweden)

    Sébastien Kicka

    Full Text Available Tuberculosis is considered to be one of the world's deadliest disease with 2 million deaths each year. The need for new antitubercular drugs is further exacerbated by the emergence of drug-resistance strains. Despite multiple recent efforts, the majority of the hits discovered by traditional target-based screening showed low efficiency in vivo. Therefore, there is heightened demand for whole-cell based approaches directly using host-pathogen systems. The phenotypic host-pathogen assay described here is based on the monitoring of GFP-expressing Mycobacterium marinum during infection of the amoeba Acanthamoeba castellanii. The assay showed straight-forward medium-throughput scalability, robustness and ease of manipulation, demonstrating its qualities as an efficient compound screening system. Validation with a series of known antitubercular compounds highlighted the advantages of the assay in comparison to previously published macrophage-Mycobacterium tuberculosis-based screening systems. Combination with secondary growth assays based on either GFP-expressing D. discoideum or M. marinum allowed us to further fine-tune compound characterization by distinguishing and quantifying growth inhibition, cytotoxic properties and antibiotic activities of the compounds. The simple and relatively low cost system described here is most suitable to detect anti-infective compounds, whether they present antibiotic activities or not, in which case they might exert anti-virulence or host defense boosting activities, both of which are largely overlooked by classical screening approaches.

  3. Assay of mast cell mediators

    DEFF Research Database (Denmark)

    Rådinger, Madeleine; Jensen, Bettina M; Swindle, Emily;

    2015-01-01

    Mediator release from activated mast cells is a major initiator of the symptomology associated with allergic disorders such as anaphylaxis and asthma. Thus, methods to monitor the generation and release of such mediators have widespread applicability in studies designed to understand the processes...... regulating mast cell activation and for the identification of therapeutic approaches to block mast cell-driven disease. In this chapter, we discuss approaches used for the determination of mast cell degranulation, lipid-derived inflammatory mediator production, and cytokine/chemokine gene expression as well...

  4. Safety Evaluation of Chinese Medicine Injections with a Cell Imaging-Based Multiparametric Assay Revealed a Critical Involvement of Mitochondrial Function in Hepatotoxicity

    Directory of Open Access Journals (Sweden)

    Meng Wang

    2015-01-01

    Full Text Available The safety of herbal medicine products has been a widespread concern due to their complex chemical nature and lack of proper evaluation methods. We have adapted a sensitive and reproducible multiparametric cell-based high-content analysis assay to evaluate the hepatic-safety of four Chinese medicine injections and validated it with classical animal-based toxicity assays. Our results suggested that the reported hepatotoxicity by one of the drugs, Fufangkushen injection, could be attributed at least in part to the interference of mitochondrial function in human HepG2 cells by some of its constituents. This method should be useful for both preclinical screen in a drug discovery program and postclinical evaluation of herbal medicine preparations.

  5. Donor-specific cell-based assays in studying sensitivity to low-dose radiation: a population-based perspective

    Directory of Open Access Journals (Sweden)

    Dora eIl'yasova

    2014-11-01

    Full Text Available Currently, a linear no-threshold model is used to estimate health risks associated with exposure to low-dose radiation, a prevalent exposure in the general population, because the direct estimation from epidemiological studies suffers from uncertainty. This model has been criticized based on unique biology of low-dose radiation. Whether the departure from linearity is toward increased or decreased risk is intensely debated. We present an approach based on individual radiosensitivity testing and discuss how individual radiosensitivity can be assessed with the goal to develop a quantifiable measure of cellular response that can be conducted via high-throughput population testing.

  6. Comet assay on mice testicular cells

    Directory of Open Access Journals (Sweden)

    Anoop Kumar Sharma

    2015-05-01

    Full Text Available Heritable mutations may result in a variety of adverse outcomes including genetic disease in the offspring. In recent years the focus on germ cell mutagenicity has increased and the “Globally Harmonized System of Classification and Labelling of Chemicals (GHS” has published classification criteria for germ cell mutagens (Speit et al., 2009. The in vivo Comet assay is considered a useful tool for investigating germ cell genotoxicity. In the present study DNA strand breaks in testicular cells of mice were investigated. Different classes of chemicals were tested in order to evaluate the sensitivity of the comet assay in testicular cells. The chemicals included environmentally relevant substances such as Bisphenol A, PFOS and Tetrabrombisphenol A. Statistical power calculations will be presented to aid in the design of future Comet assay studies on testicular cells. Power curves were provided with different fold changes in % tail DNA, different number of cells scored and different number of gels (Hansen et al., 2014. An example is shown in Figure 1. A high throughput version of the Comet assay was used. Samples were scored with a fully automatic comet assay scoring system that provided faster scoring of randomly selected cells.

  7. Mitochondrial base excision repair assays

    DEFF Research Database (Denmark)

    Maynard, Scott; de Souza-Pinto, Nadja C; Scheibye-Knudsen, Morten;

    2010-01-01

    glycosylases, AP endonuclease, DNA polymerase (POLgamma in mitochondria) and DNA ligase. This article outlines procedures for measuring oxidative damage formation and BER in mitochondria, including isolation of mitochondria from tissues and cells, protocols for measuring BER enzyme activities, gene......The main source of mitochondrial DNA (mtDNA) damage is reactive oxygen species (ROS) generated during normal cellular metabolism. The main mtDNA lesions generated by ROS are base modifications, such as the ubiquitous 8-oxoguanine (8-oxoG) lesion; however, base loss and strand breaks may also occur....... Many human diseases are associated with mtDNA mutations and thus maintaining mtDNA integrity is critical. All of these lesions are repaired primarily by the base excision repair (BER) pathway. It is now known that mammalian mitochondria have BER, which, similarly to nuclear BER, is catalyzed by DNA...

  8. Reference cells and ploidy in the comet assay

    Directory of Open Access Journals (Sweden)

    Gunnar eBrunborg

    2015-02-01

    Full Text Available In the comet assay, single cells are analyzed with respect to their level of DNA damage. Discrimination of the individual cell or cell type based on DNA content, with concomitant scoring of the DNA damage, is useful since this may allow analysis of mixtures of cells. Different cells can then be characterized based on their ploidy, cell cycle stage, or genome size. We here describe two applications of such a cell type-specific comet assay: (i Testicular cell suspensions, analyzed on the basis of their ploidy during spermatogenesis; and (ii reference cells in the form of fish erythrocytes which can be included as internal standards to correct for inter-assay variations. With standard fluorochromes used in the comet assay, the total staining signal from each cell – whether damaged or undamaged – was found to be associated with the cell’s DNA content. Analysis of the fluorescence intensity of single cells is straightforward since these data are available in scoring systems based on image analysis. The analysis of testicular cell suspensions provides information on cell type specific composition, susceptibility to genotoxicants, and DNA repair. Internal reference cells, either untreated or carrying defined numbers of lesions induced by ionizing radiation, are useful for investigation of experimental factors that can cause variation in comet assay results, and for routine inclusion in experiments to facilitate standardization of methods and comparison of comet assay data obtained in different experiments or in different laboratories. They can also be used - in combination with a reference curve - to quantify the DNA lesions induced by a certain treatment. Fish cells of a range of genome sizes, both greater and smaller than human, are suitable for this purpose and they are inexpensive.

  9. Avicequinone C Isolated from Avicennia marina Exhibits 5α-Reductase-Type 1 Inhibitory Activity Using an Androgenic Alopecia Relevant Cell-Based Assay System

    OpenAIRE

    Ruchy Jain; Orawan Monthakantirat; Parkpoom Tengamnuay; Wanchai De-Eknamkul

    2014-01-01

    Avicennia marina (AM) exhibits various biological activities and has been traditionally used in Egypt to cure skin diseases. In this study, the methanolic heartwood extract of AM was evaluated for inhibitory activity against 5α-reductase (5α-R) [E.C.1.3.99.5], the enzyme responsible for the over-production of 5α-dihydrotestosterone (5α-DHT) causing androgenic alopecia (AGA). An AGA-relevant cell-based assay was developed using human hair dermal papilla cells (HHDPCs), the main regulator of ha...

  10. Cell Culture Assay for Human Noroviruses [response

    Energy Technology Data Exchange (ETDEWEB)

    Straub, Tim M.; Honer Zu Bentrup, Kerstin; Orosz Coghlan, Patricia; Dohnalkova, Alice; Mayer, Brooke K.; Bartholomew, Rachel A.; Valdez, Catherine O.; Bruckner-Lea, Cindy J.; Gerba, Charles P.; Abbaszadegan, Morteza A.; Nickerson, Cheryl A.

    2007-07-01

    We appreciate the comments provided by Leung et al., in response to our recently published article “In Vitro Cell Culture Infectivity Assay for Human Noroviruses” by Straub et al. (1). The specific aim of our project was to develop an in vitro cell culture infectivity assay for human noroviruses (hNoV) to enhance risk assessments when they are detected in water supplies. Reverse transcription (RT) qualitative or quantitative PCR are the primary assays for waterborne NoV monitoring. However, these assays cannot distinguish between infectious vs. non-infectious virions. When hNoV is detected in water supplies, information provided by our infectivity assay will significantly improve risk assessment models and protect human health, regardless of whether we are propagating NoV. Indeed, in vitro cell culture infectivity assays for the waterborne pathogen Cryptosporidium parvum that supplement approved fluorescent microscopy assays, do not result in amplification of the environmentally resistant hard-walled oocysts (2). However, identification of life cycle stages in cell culture provides evidence of infectious oocysts in a water supply. Nonetheless, Leung et al.’s assertion regarding the suitability of our method for the in vitro propagation of high titers of NoV is valid for the medical research community. In this case, well-characterized challenge pools of virus would be useful for developing and testing diagnostics, therapeutics, and vaccines. As further validation of our published findings, we have now optimized RT quantitative PCR to assess the level of viral production in cell culture, where we are indeed finding significant increases in viral titer. The magnitude and time course of these increases is dependent on both virus strain and multiplicity of infection. We are currently preparing a manuscript that will discuss these findings in greater detail, and the implications this may have for creating viral challenge pools

  11. Comet assay on mice testicular cells

    OpenAIRE

    Anoop Kumar Sharma

    2015-01-01

    Heritable mutations may result in a variety of adverse outcomes including genetic disease in the offspring. In recent years the focus on germ cell mutagenicity has increased and the “Globally Harmonized System of Classification and Labelling of Chemicals (GHS)” has published classification criteria for germ cell mutagens (Speit et al., 2009). The in vivo Comet assay is considered a useful tool for investigating germ cell genotoxicity. In the present study DNA strand breaks in testicular cel...

  12. Use of activity-based probes to develop high throughput screening assays that can be performed in complex cell extracts.

    Directory of Open Access Journals (Sweden)

    Edgar Deu

    Full Text Available BACKGROUND: High throughput screening (HTS is one of the primary tools used to identify novel enzyme inhibitors. However, its applicability is generally restricted to targets that can either be expressed recombinantly or purified in large quantities. METHODOLOGY AND PRINCIPAL FINDINGS: Here, we described a method to use activity-based probes (ABPs to identify substrates that are sufficiently selective to allow HTS in complex biological samples. Because ABPs label their target enzymes through the formation of a permanent covalent bond, we can correlate labeling of target enzymes in a complex mixture with inhibition of turnover of a substrate in that same mixture. Thus, substrate specificity can be determined and substrates with sufficiently high selectivity for HTS can be identified. In this study, we demonstrate this method by using an ABP for dipeptidyl aminopeptidases to identify (Pro-Arg2-Rhodamine as a specific substrate for DPAP1 in Plasmodium falciparum lysates and Cathepsin C in rat liver extracts. We then used this substrate to develop highly sensitive HTS assays (Z'>0.8 that are suitable for use in screening large collections of small molecules (i.e >300,000 for inhibitors of these proteases. Finally, we demonstrate that it is possible to use broad-spectrum ABPs to identify target-specific substrates. CONCLUSIONS: We believe that this approach will have value for many enzymatic systems where access to large amounts of active enzyme is problematic.

  13. A simple and rapid Hepatitis A Virus (HAV titration assay based on antibiotic resistance of infected cells: evaluation of the HAV neutralization potency of human immune globulin preparations

    Directory of Open Access Journals (Sweden)

    Kaplan Gerardo G

    2008-12-01

    Full Text Available Abstract Background Hepatitis A virus (HAV, the causative agent of acute hepatitis in humans, is an atypical Picornaviridae that grows poorly in cell culture. HAV titrations are laborious and time-consuming because the virus in general does not cause cytopathic effect and is detected by immunochemical or molecular probes. Simple HAV titration assays could be developed using currently available viral construct containing selectable markers. Results We developed an antibiotic resistance titration assay (ARTA based on the infection of human hepatoma cells with a wild type HAV construct containing a blasticidin (Bsd resistance gene. Human hepatoma cells infected with the HAV-Bsd construct survived selection with 2 μg/ml of blasticidin whereas uninfected cells died within a few days. At 8 days postinfection, the color of the pH indicator phenol red in cell culture media correlated with the presence of HAV-Bsd-infected blasticidin-resistant cells: an orange-to-yellow color indicated the presence of growing cells whereas a pink-to-purple color indicated that the cells were dead. HAV-Bsd titers were determined by an endpoint dilution assay based on the color of the cell culture medium scoring orange-to-yellow wells as positive and pink-to-purple wells as negative for HAV. As a proof-of-concept, we used the ARTA to evaluate the HAV neutralization potency of two commercially available human immune globulin (IG preparations and a WHO International Standard for anti-HAV. The three IG preparations contained comparable levels of anti-HAV antibodies that neutralized approximately 1.5 log of HAV-Bsd. Similar neutralization results were obtained in the absence of blasticidin by an endpoint dilution ELISA at 2 weeks postinfection. Conclusion The ARTA is a simple and rapid method to determine HAV titers without using HAV-specific probes. We determined the HAV neutralization potency of human IG preparations in 8 days by ARTA compared to the 14 days required by the

  14. A novel cell-based assay for inhibitory anti-muscarinic type 3 receptor antibodies in primary Sjögren's syndrome.

    Science.gov (United States)

    Bastian, Isabell; Gordon, Tom P; Jackson, Michael W

    2015-12-01

    Inhibitory autoantibodies acting at the muscarinic acetylcholine receptor type 3 (M3R) are postulated to mediate autonomic dysfunction, including decreased salivary and lacrimal gland output and extra-glandular manifestations, in patients with primary Sjögren's syndrome. However, the contention that anti-M3R antibodies are pathogenic in patients remains untested, due to a lack of assays both sophisticated enough to detect inhibitory anti-M3R antibodies yet suitable for screening large patient cohorts. In the current study, we have established a cell-based bioassay of M3R activity, based on dual transfection of the M3R and a luciferase reporter gene. The bioassay is capable of capturing real-time agonist-mediated signalling of the M3R, which is inhibited specifically by patient IgG that have previously been demonstrated to have anti-M3R activity. The assay can be run in multi-well culture plates, and analysed using simple luminescence readers. As such, the new bioassay incorporating M3R-mediated luciferase transduction is the first assay adaptable to common diagnostic platforms that is capable of determining the presence in patient serum of functionally active anti-M3R autoantibodies. The new bioassay should prove useful for large cohort screening studies aiming to correlate the presence in patients of inhibitory anti-M3R antibodies with symptoms of both glandular and extra-glandular autonomic dysfunction. PMID:26584897

  15. Identification of candidate agents active against N. ceranae infection in honey bees: establishment of a medium throughput screening assay based on N. ceranae infected cultured cells.

    Directory of Open Access Journals (Sweden)

    Sebastian Gisder

    Full Text Available Many flowering plants in both natural ecosytems and agriculture are dependent on insect pollination for fruit set and seed production. Managed honey bees (Apis mellifera and wild bees are key pollinators providing this indispensable eco- and agrosystem service. Like all other organisms, bees are attacked by numerous pathogens and parasites. Nosema apis is a honey bee pathogenic microsporidium which is widely distributed in honey bee populations without causing much harm. Its congener Nosema ceranae was originally described as pathogen of the Eastern honey bee (Apis cerana but jumped host from A. cerana to A. mellifera about 20 years ago and spilled over from A. mellifera to Bombus spp. quite recently. N. ceranae is now considered a deadly emerging parasite of both Western honey bees and bumblebees. Hence, novel and sustainable treatment strategies against N. ceranae are urgently needed to protect honey and wild bees. We here present the development of an in vitro medium throughput screening assay for the identification of candidate agents active against N. ceranae infections. This novel assay is based on our recently developed cell culture model for N. ceranae and coupled with an RT-PCR-ELISA protocol for quantification of N. ceranae in infected cells. The assay has been adapted to the 96-well microplate format to allow automated analysis. Several substances with known (fumagillin or presumed (surfactin or no (paromomycin activity against N. ceranae were tested as well as substances for which no data concerning N. ceranae inhibition existed. While fumagillin and two nitroimidazoles (metronidazole, tinidazole totally inhibited N. ceranae proliferation, all other test substances were inactive. In summary, the assay proved suitable for substance screening and demonstrated the activity of two synthetic antibiotics against N. ceranae.

  16. Identification of candidate agents active against N. ceranae infection in honey bees: establishment of a medium throughput screening assay based on N. ceranae infected cultured cells.

    Science.gov (United States)

    Gisder, Sebastian; Genersch, Elke

    2015-01-01

    Many flowering plants in both natural ecosytems and agriculture are dependent on insect pollination for fruit set and seed production. Managed honey bees (Apis mellifera) and wild bees are key pollinators providing this indispensable eco- and agrosystem service. Like all other organisms, bees are attacked by numerous pathogens and parasites. Nosema apis is a honey bee pathogenic microsporidium which is widely distributed in honey bee populations without causing much harm. Its congener Nosema ceranae was originally described as pathogen of the Eastern honey bee (Apis cerana) but jumped host from A. cerana to A. mellifera about 20 years ago and spilled over from A. mellifera to Bombus spp. quite recently. N. ceranae is now considered a deadly emerging parasite of both Western honey bees and bumblebees. Hence, novel and sustainable treatment strategies against N. ceranae are urgently needed to protect honey and wild bees. We here present the development of an in vitro medium throughput screening assay for the identification of candidate agents active against N. ceranae infections. This novel assay is based on our recently developed cell culture model for N. ceranae and coupled with an RT-PCR-ELISA protocol for quantification of N. ceranae in infected cells. The assay has been adapted to the 96-well microplate format to allow automated analysis. Several substances with known (fumagillin) or presumed (surfactin) or no (paromomycin) activity against N. ceranae were tested as well as substances for which no data concerning N. ceranae inhibition existed. While fumagillin and two nitroimidazoles (metronidazole, tinidazole) totally inhibited N. ceranae proliferation, all other test substances were inactive. In summary, the assay proved suitable for substance screening and demonstrated the activity of two synthetic antibiotics against N. ceranae.

  17. A faster, high resolution, mtPA-GFP-based mitochondrial fusion assay acquiring kinetic data of multiple cells in parallel using confocal microscopy.

    Science.gov (United States)

    Lovy, Alenka; Molina, Anthony J A; Cerqueira, Fernanda M; Trudeau, Kyle; Shirihai, Orian S

    2012-01-01

    Mitochondrial fusion plays an essential role in mitochondrial calcium homeostasis, bioenergetics, autophagy and quality control. Fusion is quantified in living cells by photo-conversion of matrix targeted photoactivatable GFP (mtPAGFP) in a subset of mitochondria. The rate at which the photoconverted molecules equilibrate across the entire mitochondrial population is used as a measure of fusion activity. Thus far measurements were performed using a single cell time lapse approach, quantifying the equilibration in one cell over an hour. Here, we scale up and automate a previously published live cell method based on using mtPAGFP and a low concentration of TMRE (15 nm). This method involves photoactivating a small portion of the mitochondrial network, collecting highly resolved stacks of confocal sections every 15 min for 1 hour, and quantifying the change in signal intensity. Depending on several factors such as ease of finding PAGFP expressing cells, and the signal of the photoactivated regions, it is possible to collect around 10 cells within the 15 min intervals. This provides a significant improvement in the time efficiency of this assay while maintaining the highly resolved subcellular quantification as well as the kinetic parameters necessary to capture the detail of mitochondrial behavior in its native cytoarchitectural environment. Mitochondrial dynamics play a role in many cellular processes including respiration, calcium regulation, and apoptosis. The structure of the mitochondrial network affects the function of mitochondria, and the way they interact with the rest of the cell. Undergoing constant division and fusion, mitochondrial networks attain various shapes ranging from highly fused networks, to being more fragmented. Interestingly, Alzheimer's disease, Parkinson's disease, Charcot Marie Tooth 2A, and dominant optic atrophy have been correlated with altered mitochondrial morphology, namely fragmented networks. Often times, upon fragmentation

  18. Comet assay as a predictive assay for radiosensitivity of two human brain tumor cell lines

    International Nuclear Information System (INIS)

    Micronucleus assay and comet assay were compared as a predictive assay for radiosensitivity of tumors. Two human brain tumor cell lines, Becker (derived from astrocytoma) and ONS76 (derived from medulloblastoma) were used. Colony methods as the gold standard showed ONS76 as radiosensitive and Becker as radioresistant cell lines. Micronucleus assay revealed no different radiosensitivity between them. With comet assay, Becker cells received irradiation showed less damage to the DNA and faster repair of the damage than ONS76 cells did. The results correlate with those from colony methods. Comet assay is simple and rapid method for clinical use and it has an advantage not to establish the primary culture. Moreover, the results of comet assay showed not only DNA damage but also repair from the damage. It is concluded that comet assay is a superior method than micronucleus assay and has a potent candidate for clinical predictive assay. (author)

  19. 21 CFR 864.7100 - Red blood cell enzyme assay.

    Science.gov (United States)

    2010-04-01

    ... enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity in... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Red blood cell enzyme assay. 864.7100 Section 864... kinase or 2,3-diphosphoglycerate. A red blood cell enzyme assay is used to determine the enzyme...

  20. Branched DNA-based Alu quantitative assay for cell-free plasma DNA levels in patients with sepsis or systemic inflammatory response syndrome.

    Science.gov (United States)

    Hou, Yan-Qiang; Liang, Dong-Yu; Lou, Xiao-Li; Zhang, Mei; Zhang, Zhen-huan; Zhang, Lu-rong

    2016-02-01

    Cell-free circulating DNA (cf-DNA) can be detected by various of laboratory techniques. We described a branched DNA-based Alu assay for measuring cf-DNA in septic patients. Compared to healthy controls and systemic inflammatory response syndrome (SIRS) patients, serum cf-DNA levels were significantly higher in septic patients (1426.54 ± 863.79 vs 692.02 ± 703.06 and 69.66 ± 24.66 ng/mL). The areas under the receiver operating characteristic curve of cf-DNA for normal vs sepsis and SIRS vs sepsis were 0.955 (0.884-1.025), and 0.856 (0.749-0.929), respectively. There was a positive correlation between cf-DNA and interleukin 6 or procalcitonin or Acute Physiology and Chronic Health Evaluation II. The cf-DNA concentration was higher in intensive care unit nonsurviving patients compared to surviving patients (2183.33 ± 615.26 vs 972.46 ± 648.36 ng/mL; P DNA-based Alu assays are feasible and useful to quantify serum cf-DNA levels. Increased cf-DNA levels in septic patients might complement C-reactive protein and procalcitonin in a multiple marker format. Cell-free circulating DNA might be a new marker in discrimination of sepsis and SIRS.

  1. AFBI assay - Aptamer Fluorescence Binding and Internalization assay for cultured adherent cells.

    Science.gov (United States)

    Thiel, William H; Giangrande, Paloma H

    2016-07-01

    The SELEX (Systematic Evolution of Ligands by Exponential Enrichment) process allows for the enrichment of DNA or RNA aptamers from a complex nucleic acid library that are specific for a target molecule. The SELEX process has been adapted from identifying aptamers in vitro using recombinant target protein to cell-based methodologies (Cell-SELEX), where the targets are expressed on the surface of cells. One major advantage of Cell-SELEX is that the target molecules are maintained in a native confirmation. Additionally, Cell-SELEX may be used to discover novel therapeutic biomarkers by performing selections on diseased versus healthy cells. However, a caveat to Cell-SELEX is that testing of single aptamers identified in the selection is laborious, time-consuming, and expensive. The most frequently used methods to screen for aptamer binding and internalization on cells are flow cytometry and quantitative PCR (qPCR). While flow cytometry can directly assess binding of a fluorescently-labeled aptamer to a target, it requires significant starting material and is not easily scalable. qPCR-based approaches are highly sensitive but have non-negligible experiment-to-experiment variability due to the number of sample processing steps. Herein we describe a cell-based aptamer fluorescence binding and internalization (AFBI) assay. This assay requires minimal reagents and has few experimental steps/manipulations, thereby allowing for rapid screening of many aptamers and conditions simultaneously and direct quantitation of aptamer binding and internalization. PMID:26972784

  2. A high-throughput three-dimensional cell migration assay for toxicity screening with mobile device-based macroscopic image analysis

    Science.gov (United States)

    Timm, David M.; Chen, Jianbo; Sing, David; Gage, Jacob A.; Haisler, William L.; Neeley, Shane K.; Raphael, Robert M.; Dehghani, Mehdi; Rosenblatt, Kevin P.; Killian, T. C.; Tseng, Hubert; Souza, Glauco R.

    2013-10-01

    There is a growing demand for in vitro assays for toxicity screening in three-dimensional (3D) environments. In this study, 3D cell culture using magnetic levitation was used to create an assay in which cells were patterned into 3D rings that close over time. The rate of closure was determined from time-lapse images taken with a mobile device and related to drug concentration. Rings of human embryonic kidney cells (HEK293) and tracheal smooth muscle cells (SMCs) were tested with ibuprofen and sodium dodecyl sulfate (SDS). Ring closure correlated with the viability and migration of cells in two dimensions (2D). Images taken using a mobile device were similar in analysis to images taken with a microscope. Ring closure may serve as a promising label-free and quantitative assay for high-throughput in vivo toxicity in 3D cultures.

  3. Development of a Recombinant Cell-Based Indirect Immunofluorescence Assay for the Determination of Autoantibodies against Soluble Liver Antigen in Autoimmune Hepatitis

    Directory of Open Access Journals (Sweden)

    Christiane Radzimski

    2013-01-01

    Full Text Available Autoantibodies against soluble liver antigen (SLA are specific markers for autoimmune hepatitis (AIH type 1. In contrast to the determination of other AIH-associated autoantibodies by indirect immunofluorescence assay (IFA, detection of anti-SLA relied up to now on ELISA or immunoblot based on bacterially expressed recombinant protein. In order to develop a complementary IFA substrate, SLA isoform 1 was recombinantly produced in the human cell line HEK293 and controlled by a rabbit hyperimmune serum against SLA. The recombinant cells were used in IFA (RC-IFA to analyze sera from 20 AIH patients with anti-SLA positivity predetermined by ELISA together with 80 controls (20 anti-SLA negative AIH, 15 primary biliary cirrhosis, 15 HCV, and 30 healthy blood donors. Using RC-IFA, anti-SLA was detected in all ELISA positive AIH sera but in none of the controls. Furthermore, a cytosolic fraction of HEK293 containing SLA was able to neutralize the autoantibodies in all positive sera in a dose-dependent manner. HEK293 cells expressing SLA are a valid substrate for the serodiagnosis of AIH relevant autoantibodies by IFA. In concert with cryosections of primate liver, rat kidney, rat liver, rat stomach, and HEp-2 cells, they enable the parallel determination of all autoantibodies associated with autoimmune liver diseases.

  4. Identification of small-molecule agonists of human relaxin family receptor 1 (RXFP1) by using a homogenous cell-based cAMP assay.

    Science.gov (United States)

    Chen, Catherine Z; Southall, Noel; Xiao, Jingbo; Marugan, Juan J; Ferrer, Marc; Hu, Xin; Jones, Raisa E; Feng, Shu; Agoulnik, Irina U; Zheng, Wei; Agoulnik, Alexander I

    2013-07-01

    The relaxin hormone is involved in a variety of biological functions, including female reproduction and parturition, as well as regulation of cardiovascular, renal, pulmonary, and hepatic functions. It regulates extracellular matrix remodeling, cell invasiveness, proliferation, differentiation, and overall tissue homeostasis. The G protein-coupled receptor (GPCR) relaxin family receptor 1 (RXFP1) is a cognate relaxin receptor that mainly signals through cyclic AMP second messenger. Although agonists of the receptor could have a wide range of pharmacologic utility, until now there have been no reported small-molecule agonists for relaxin receptors. Here, we report the development of a quantitative high-throughput platform for an RXFP1 agonist screen based on homogenous cell-based HTRF cyclic AMP (cAMP) assay technology. Two small molecules of similar structure were independently identified from a screen of more than 365 677 compounds. Neither compound showed activity in a counterscreen with HEK293T cells transfected with an unrelated GPCR vasopressin 1b receptor. These small-molecule agonists also demonstrated selectivity against the RXFP2 receptor, providing a basis for future medicinal chemistry optimization of selective relaxin receptor agonists. PMID:23212924

  5. Impedance sensor technology for cell-based assays in the framework of a high-content screening system

    International Nuclear Information System (INIS)

    Living cultured cells react to external influences, such as pharmaceutical agents, in an intricate manner due to their complex internal signal processing. Impedance sensing of cells on microelectrodes is a favored label-free technology to indicate cellular events, usually ascribed to morphologic alteration or changes in cellular adhesion, which is usually found in stand-alone systems that do not incorporate life support or additional sensor systems. However, only in symbiosis with metabolic activity sensing and picture documentation may a complete insight into cellular vitality be provided. This complement was created within the framework of an automated high-content screening system previously developed by our group, monitoring 24 cell culture chambers in parallel. The objective of this paper is the development of miniaturized electronics for impedance measurements and its system integration as a modular unit. In addition, it is shown how sensor electrodes were optimized by impedance matching such that spectroscopy and raw data analysis become feasible for every culture well. Undesired mechanical stress on cultured cells may arise from the medium and agent support system of the autonomous screening apparatus. This paper demonstrates how this hazard is treated with the simulation of microfluidics and impedance measurements. Physiological data are subsequently derived from the exemplary tumor cell line MCF-7 both during treatment with the agent doxorubicin and through the impact of natural killer cells. This correlates the information content of complex impedance spectra with cellular respiration as well as data from microscopy

  6. Cell Migration and Invasion Assays as Tools for Drug Discovery

    OpenAIRE

    Hulkower, Keren I.; Herber, Renee L.

    2011-01-01

    Cell migration and invasion are processes that offer rich targets for intervention in key physiologic and pathologic phenomena such as wound healing and cancer metastasis. With the advent of high-throughput and high content imaging systems, there has been a movement towards the use of physiologically relevant cell-based assays earlier in the testing paradigm. This allows more effective identification of lead compounds and recognition of undesirable effects sooner in the drug discovery screeni...

  7. Integration of Affinity Selection-Mass Spectrometry and Functional Cell-Based Assays to Rapidly Triage Druggable Target Space within the NF-κB Pathway.

    Science.gov (United States)

    Kutilek, Victoria D; Andrews, Christine L; Richards, Matthew P; Xu, Zangwei; Sun, Tianxiao; Chen, Yiping; Hashke, Andrew; Smotrov, Nadya; Fernandez, Rafael; Nickbarg, Elliott B; Chamberlin, Chad; Sauvagnat, Berengere; Curran, Patrick J; Boinay, Ryan; Saradjian, Peter; Allen, Samantha J; Byrne, Noel; Elsen, Nathaniel L; Ford, Rachael E; Hall, Dawn L; Kornienko, Maria; Rickert, Keith W; Sharma, Sujata; Shipman, Jennifer M; Lumb, Kevin J; Coleman, Kevin; Dandliker, Peter J; Kariv, Ilona; Beutel, Bruce

    2016-07-01

    The primary objective of early drug discovery is to associate druggable target space with a desired phenotype. The inability to efficiently associate these often leads to failure early in the drug discovery process. In this proof-of-concept study, the most tractable starting points for drug discovery within the NF-κB pathway model system were identified by integrating affinity selection-mass spectrometry (AS-MS) with functional cellular assays. The AS-MS platform Automated Ligand Identification System (ALIS) was used to rapidly screen 15 NF-κB proteins in parallel against large-compound libraries. ALIS identified 382 target-selective compounds binding to 14 of the 15 proteins. Without any chemical optimization, 22 of the 382 target-selective compounds exhibited a cellular phenotype consistent with the respective target associated in ALIS. Further studies on structurally related compounds distinguished two chemical series that exhibited a preliminary structure-activity relationship and confirmed target-driven cellular activity to NF-κB1/p105 and TRAF5, respectively. These two series represent new drug discovery opportunities for chemical optimization. The results described herein demonstrate the power of combining ALIS with cell functional assays in a high-throughput, target-based approach to determine the most tractable drug discovery opportunities within a pathway. PMID:26969322

  8. Cell-based assay for the detection of chemically induced cellular stress by immortalized untransformed transgenic hepatocytes

    Directory of Open Access Journals (Sweden)

    Vezzoni Paolo

    2004-03-01

    Full Text Available Abstract Background Primary hepatocytes, one of the most widely used cell types for toxicological studies, have a very limited life span and must be freshly derived from mice or even humans. Attempts to use stable cell lines maintaining the enzymatic pattern of liver cells have been so far unsatisfactory. Stress proteins (heat shock proteins, HSPs have been proposed as general markers of cellular injury and their use for environmental monitoring has been suggested. The aim of this work is to develop a bi-transgenic hepatocyte cell line in order to evaluate the ability of various organic and inorganic chemicals to induce the expression of the HSP70 driven reporter gene. We previously described transgenic mice (Hsp70/hGH secreting high levels of human Growth Hormone (hGH following exposure to toxic compounds in vivo and in vitro in primary cultures derived from different organs. In addition, we also reported another transgenic model (AT/cytoMet allowing the reproducible immortalization of untransformed hepatocytes retaining in vitro complex liver functions. Results The transgenic mouse line Hsp70/hGH was crossed with the AT/cytoMet transgenic strain permitting the reproducible immortalization of untransformed hepatocytes. From double transgenic animals we derived several stable hepatic cell lines (MMH-GH which showed a highly-differentiated phenotype as judged from the retention of epithelial cell polarity and the profile of gene expression, including hepatocyte-enriched transcription factors and detoxifying enzymes. In these cell lines, stresses induced by exposure to inorganic [Sodium Arsenite (NaAsO2 and Cadmium Chloride (CdCl2], and organic [Benzo(aPyrene (BaP, PentaChloroPhenol (PCP, TetraChloroHydroQuinone (TCHQ, 1-Chloro-2,4-DiNitro-Benzene (CDNB] compounds, specifically induced hGH release in the culture medium. Conclusions MMH-GH, an innovative model to evaluate the toxic potential of chemical and physical xenobiotics, provides a simple

  9. Avicequinone C isolated from Avicennia marina exhibits 5α-reductase-type 1 inhibitory activity using an androgenic alopecia relevant cell-based assay system.

    Science.gov (United States)

    Jain, Ruchy; Monthakantirat, Orawan; Tengamnuay, Parkpoom; De-Eknamkul, Wanchai

    2014-01-01

    Avicennia marina (AM) exhibits various biological activities and has been traditionally used in Egypt to cure skin diseases. In this study, the methanolic heartwood extract of AM was evaluated for inhibitory activity against 5α-reductase (5α-R) [E.C.1.3.99.5], the enzyme responsible for the over-production of 5α-dihydrotestosterone (5α-DHT) causing androgenic alopecia (AGA). An AGA-relevant cell-based assay was developed using human hair dermal papilla cells (HHDPCs), the main regulator of hair growth and the only cells within the hair follicle that are the direct site of 5α-DHT action, combined with a non-radioactive thin layer chromatography (TLC) detection technique. The results revealed that AM is a potent 5α-R type 1 (5α-R1) inhibitor, reducing the 5α-DHT production by 52% at the final concentration of 10 µg/mL. Activity-guided fractionation has led to the identification of avicequinone C, a furanonaphthaquinone, as a 5α-R1 inhibitor with an IC50 of 9.94 ± 0.33 µg/mL or 38.8 ± 1.29 µM. This paper is the first to report anti-androgenic activity through 5α-R1 inhibition of AM and avicequinone C. PMID:24858268

  10. Avicequinone C Isolated from Avicennia marina Exhibits 5α-Reductase-Type 1 Inhibitory Activity Using an Androgenic Alopecia Relevant Cell-Based Assay System

    Directory of Open Access Journals (Sweden)

    Ruchy Jain

    2014-05-01

    Full Text Available Avicennia marina (AM exhibits various biological activities and has been traditionally used in Egypt to cure skin diseases. In this study, the methanolic heartwood extract of AM was evaluated for inhibitory activity against 5α-reductase (5α-R [E.C.1.3.99.5], the enzyme responsible for the over-production of 5α-dihydrotestosterone (5α-DHT causing androgenic alopecia (AGA. An AGA-relevant cell-based assay was developed using human hair dermal papilla cells (HHDPCs, the main regulator of hair growth and the only cells within the hair follicle that are the direct site of 5α-DHT action, combined with a non-radioactive thin layer chromatography (TLC detection technique. The results revealed that AM is a potent 5α-R type 1 (5α-R1 inhibitor, reducing the 5α-DHT production by 52% at the final concentration of 10 µg/mL. Activity-guided fractionation has led to the identification of avicequinone C, a furanonaphthaquinone, as a 5α-R1 inhibitor with an IC50 of 9.94 ± 0.33 µg/mL or 38.8 ± 1.29 µM. This paper is the first to report anti-androgenic activity through 5α-R1 inhibition of AM and avicequinone C.

  11. A novel fluorescence-based cellular permeability assay.

    Science.gov (United States)

    Chandra, Ankur; Barillas, Samuel; Suliman, Ahmed; Angle, Niren

    2007-04-10

    Vascular permeability is a pathologic process in many disease states ranging from metastatic progression of malignancies to ischemia-reperfusion injury. In order to more precisely study tissue, and more specifically cell layer permeability, our goal was to create a fluorescence-based assay which could quantify permeability without radioactivity or electrical impedance measurements. Human aortic endothelial cells were grown in monolayer culture on Costar-Transwell clear polyester membrane 6-well cell culture inserts. After monolayer integrity was confirmed, vascular endothelial growth factor (VEGF(165)) at varying concentrations with a fixed concentration of yellow-green fluorescent 0.04 microm carboxylate-modified FluoSpheres microspheres were placed in the luminal chamber and incubated for 24 h. When stimulated with VEGF(165) at 20, 40, 80, and 100 ng/ml, this assay system was able to detect increases in trans-layer flux of 8.2+/-2.4%, 16.0+/-3.7%, 41.5+/-4.9%, and 58.6+/-10.1% for each concentration, respectively. This represents the first fluorescence-based permeability assay with the sensitivity to detect changes in the permeability of a cell layer to fluid flux independent of protein flux; as well as being simpler and safer than previous radioactive-and impedance-based permeability assays. With the application of this in vitro assay to a variety of pathologic conditions, both the dynamics and physiology relating to cellular permeability can be more fully investigated. PMID:16962665

  12. A sensitive fluorescence-based assay for monitoring GM2 ganglioside hydrolysis in live patient cells and their lysates.

    Science.gov (United States)

    Tropak, Michael B; Bukovac, Scott W; Rigat, Brigitte A; Yonekawa, Sayuri; Wakarchuk, Warren; Mahuran, Don J

    2010-03-01

    Enzyme enhancement therapy, utilizing small molecules as pharmacological chaperones, is an attractive approach for the treatment of lysosomal storage diseases that are associated with protein misfolding. However, pharmacological chaperones are also inhibitors of their target enzyme. Thus, a major concern with this approach is that, despite enhancing protein folding within, and intracellular transport of the functional mutant enzyme out of the endoplasmic reticulum, the chaperone will continue to inhibit the enzyme in the lysosome, preventing substrate clearance. Here we demonstrate that the in vitro hydrolysis of a fluorescent derivative of lyso-GM2 ganglioside, like natural GM2 ganglioside, is specifically carried out by the beta-hexosaminidase A isozyme, requires the GM2 activator protein as a co-factor, increases when the derivative is incorporated into anionic liposomes and follows similar Michaelis-Menten kinetics. This substrate can also be used to differentiate between lysates from normal and GM2 activator-deficient cells. When added to the growth medium of cells, the substrate is internalized and primarily incorporated into lysosomes. Utilizing adult Tay-Sachs fibroblasts that have been pre-treated with the pharmacological chaperone Pyrimethamine and subsequently loaded with this substrate, we demonstrate an increase in both the levels of mutant beta-hexosaminidase A and substrate-hydrolysis as compared to mock-treated cells. PMID:19917668

  13. Multiresidue Method for Analysis of β Agonists in Swine Urine by Enzyme Linked Receptor Assay Based on β2 Adrenergic Receptor Expressed in HEK293 Cells.

    Directory of Open Access Journals (Sweden)

    Jian Wang

    Full Text Available A novel enzyme-linked receptor assay (ELRA based on β2-adrenergic receptor (β2-AR has been developed for rapid and high-throughput detection of β-adrenergic agonists (β-agonists in urine. Human embryonic kidney cells (HEK293 were introduced as the expression system to enhance the functionality of the recombinant β2-AR, and the attempt to detect β-agonists in swine urine using such approaches was accomplished unprecedentedly. In this article, a recombinant porcine β2-AR was produced in the inner membrane of HEK293 cells and purified from crude membrane protein by nickel-nitrilotriacetic acid affinity chromatography. After activity identification, the recombinant receptor was used in the development of direct competitive ELRA. Several parameters such as blocking buffer and blocking process were optimized and the performance of the system was determined. The IC50 concentrations of clenbuterol, salbutamol, and ractopamine were 34, 53 and 63 μg/L, and the average recovery rates were 68.2%, 60.3% and 65.5%, respectively. ELRA based on β2-AR shows a series of advantages such as safety, easy operation, and high efficiency, making it promising for the rapid screening of β-agonists in animal urine.

  14. Biotinylation of the Fcγ receptor ectodomains by mammalian cell co-transfection: application to the development of a surface plasmon resonance-based assay.

    Science.gov (United States)

    Dorion-Thibaudeau, July; St-Laurent, Gilles; Raymond, Céline; De Crescenzo, Gregory; Durocher, Yves

    2016-02-01

    We here report the production of four biotinylated Fcγ receptor (FcγR) ectodomains and their subsequent stable capture on streptavidin-biosensor surfaces. For receptor biotinylation, we first describe an in-cell protocol based on the co-transfection of two plasmids corresponding to one of the FcγR ectodomains and the BirA enzyme in mammalian cells. This strategy is compared with a standard sequential in vitro enzymatic biotinylation with respect to biotinylation level and yield. Biotinylated FcγR ectodomains that have been prepared with both strategies are then compared by analytical ultracentrifugation and surface plasmon resonance (SPR) analyses. Overall, we demonstrate that in-cell biotinylation is an interesting alternative to standard biotinylation protocol, as it requires less purification steps while yielding higher titers. Finally, biotin-tagged FcγRs produced with the in-cell approach are successfully applied to the development of SPR-based assays to evaluate the impact of the glycosylation pattern of monoclonal antibodies on their interaction with CD16a and CD64. In that endeavor, we unambiguously observe that highly galactosylated trastuzumab (TZM-gal), non-glycosylated trastuzumab (TZM-NG), and reference trastuzumab are characterized by different kinetic profiles upon binding to CD16a and CD64 that had been captured at the biosensor surface via their biotin tag. More precisely, while TZM-NG binding to CD16a was not detected, TZM-gal formed a more stable complex with CD16a than our reference TZM. In contrast, both glycosylated TZM bound to captured CD64 in a stable and similar fashion, whereas the interaction of their non-glycosylated form with CD64 was characterized by a higher dissociation rate. PMID:26762306

  15. Comparative study of in vitro cell based assays versus in vivo toxicity tests to monitor environmental hazard of pesticides

    OpenAIRE

    Alañón Ribas, Maria del Pilar

    2006-01-01

    Carbofuran and chlorpyrifos are two well-known pesticides widely investigated, and its effects on different organisms have been previously reported in separate studies. For this reason were considered to be good model subtances, relevant from the environmental perspective. On the other hand, we selected this kind of compounds because they are used in many tones annually in agriculture and horticulture and they are significant especially in greenhouse-based production of vegetables and fruits ...

  16. Novel circulating peptide biomarkers for esophageal squamous cell carcinoma revealed by a magnetic bead-based MALDI-TOFMS assay.

    Science.gov (United States)

    Jia, Kun; Li, Wei; Wang, Feng; Qu, Haixia; Qiao, Yuanyuan; Zhou, Lanping; Sun, Yulin; Ma, Qingwei; Zhao, Xiaohang

    2016-04-26

    Esophageal squamous cell carcinoma (ESCC) is one of the most common malignant neoplasms worldwide. Patients are often diagnosed at advanced stages with poor prognosis due to the absence of obvious early symptoms. Here, we applied a high-throughput serum peptidome analysis to identify circulating peptide markers of ESCC. Weak cationic exchange magnetic beads coupled to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used for two-stage proteotypic peptide profiling in complex serum samples collected from 477 cancer patients and healthy controls. We established a genetic algorithm model containing three significantly differentially expressed peptides at 1,925.5, 2,950.6 and 5,900.0 Da with a sensitivity and specificity of 97.00% and 95.92% in the training set and 97.03% and 100.00% in the validation set, respectively. The model's diagnostic capability was significantly better than SCC-Ag and Cyfra 21-1, especially for early stage ESCC, with an achieved sensitivity of 96.94%. Subsequently, these peptides were identified as fragments of AHSG, TSP1 and FGA by linear ion trap-orbitrap hybrid tandem mass spectrometry. Notably, increased tissue and serum levels of TSP1 in ESCC were verified and correlated with disease progression. In addition, tissue TSP1 was an independent poor prognostic factor in ESCC. In conclusion, the newly established circulating peptide panel and identified proteins could serve as potential biomarkers for the early detection and diagnosis of ESCC. Nevertheless, a larger cohort will be required for further unequivocal validation of their clinical application. PMID:26993605

  17. Identification of Antiviral Agents Targeting Hepatitis B Virus Promoter from Extracts of Indonesian Marine Organisms by a Novel Cell-Based Screening Assay.

    Science.gov (United States)

    Yamashita, Atsuya; Fujimoto, Yuusuke; Tamaki, Mayumi; Setiawan, Andi; Tanaka, Tomohisa; Okuyama-Dobashi, Kaori; Kasai, Hirotake; Watashi, Koichi; Wakita, Takaji; Toyama, Masaaki; Baba, Masanori; de Voogd, Nicole J; Maekawa, Shinya; Enomoto, Nobuyuki; Tanaka, Junichi; Moriishi, Kohji

    2015-11-01

    The current treatments of chronic hepatitis B (CHB) face a limited choice of vaccine, antibody and antiviral agents. The development of additional antiviral agents is still needed for improvement of CHB therapy. In this study, we established a screening system in order to identify compounds inhibiting the core promoter activity of hepatitis B virus (HBV). We prepared 80 extracts of marine organisms from the coral reefs of Indonesia and screened them by using this system. Eventually, two extracts showed high inhibitory activity (>95%) and low cytotoxicity (66% to 77%). Solvent fractionation, column chromatography and NMR analysis revealed that 3,5-dibromo-2-(2,4-dibromophenoxy)-phenol (compound 1) and 3,4,5-tribromo-2-(2,4-dibromophenoxy)-phenol (compound 2), which are classified as polybrominated diphenyl ethers (PBDEs), were identified as anti-HBV agents in the extracts. Compounds 1 and 2 inhibited HBV core promoter activity as well as HBV production from HepG2.2.15.7 cells in a dose-dependent manner. The EC50 values of compounds 1 and 2 were 0.23 and 0.80 µM, respectively, while selectivity indexes of compound 1 and 2 were 18.2 and 12.8, respectively. These results suggest that our cell-based HBV core promoter assay system is useful to determine anti-HBV compounds, and that two PBDE compounds are expected to be candidates of lead compounds for the development of anti-HBV drugs. PMID:26561821

  18. Single cell multiplexed assay for proteolytic activity using droplet microfluidics.

    Science.gov (United States)

    Ng, Ee Xien; Miller, Miles A; Jing, Tengyang; Chen, Chia-Hung

    2016-07-15

    Cellular enzymes interact in a post-translationally regulated fashion to govern individual cell behaviors, yet current platform technologies are limited in their ability to measure multiple enzyme activities simultaneously in single cells. Here, we developed multi-color Förster resonance energy transfer (FRET)-based enzymatic substrates and use them in a microfluidics platform to simultaneously measure multiple specific protease activities from water-in-oil droplets that contain single cells. By integrating the microfluidic platform with a computational analytical method, Proteolytic Activity Matrix Analysis (PrAMA), we are able to infer six different protease activity signals from individual cells in a high throughput manner (~100 cells/experimental run). We characterized protease activity profiles at single cell resolution for several cancer cell lines including breast cancer cell line MDA-MB-231, lung cancer cell line PC-9, and leukemia cell line K-562 using both live-cell and in-situ cell lysis assay formats, with special focus on metalloproteinases important in metastasis. The ability to measure multiple proteases secreted from or expressed in individual cells allows us to characterize cell heterogeneity and has potential applications including systems biology, pharmacology, cancer diagnosis and stem cell biology. PMID:26995287

  19. Epithelial cells as alternative human biomatrices for comet assay

    OpenAIRE

    Rojas, Emilio; Lorenzo, Yolanda; Haug, Kristiane; Nicolaissen, Bjørn; Valverde, Mahara

    2014-01-01

    The comet assay is a valuable experimental tool aimed at mapping DNA damage in human cells in vivo for environmental and occupational monitoring, as well as for therapeutic purposes, such as storage prior to transplant, during tissue engineering, and in experimental ex vivo assays. Furthermore, due to its great versatility, the comet assay allows to explore the use of alternative cell types to assess DNA damage, such as epithelial cells. Epithelial cells, as specialized components of many org...

  20. Single cell kinase signaling assay using pinched flow coupled droplet microfluidics

    OpenAIRE

    Ramji, Ramesh; Wang, Ming; Bhagat, Ali Asgar S.; Tan Shao Weng, Daniel; Thakor, Nitish V.; Teck Lim, Chwee; Chen, Chia-Hung

    2014-01-01

    Droplet-based microfluidics has shown potential in high throughput single cell assays by encapsulating individual cells in water-in-oil emulsions. Ordering cells in a micro-channel is necessary to encapsulate individual cells into droplets further enhancing the assay efficiency. This is typically limited due to the difficulty of preparing high-density cell solutions and maintaining them without cell aggregation in long channels (>5 cm). In this study, we developed a short pinched flow channel...

  1. Identification of novel anti-hepatitis C virus agents by a quantitative high throughput screen in a cell-based infection assay.

    Science.gov (United States)

    Hu, Zongyi; Hu, Xin; He, Shanshan; Yim, Hyung Joon; Xiao, Jingbo; Swaroop, Manju; Tanega, Cordelle; Zhang, Ya-qin; Yi, Guanghui; Kao, C Cheng; Marugan, Juan; Ferrer, Marc; Zheng, Wei; Southall, Noel; Liang, T Jake

    2015-12-01

    Hepatitis C virus (HCV) poses a major health threat to the world. The recent development of direct-acting antivirals (DAAs) against HCV has markedly improved the response rate of HCV and reduced the side effects in comparison to the interferon-based therapy. Despite this therapeutic advance, there is still a need to develop new inhibitors that target different stages of the HCV life cycle because of various limitations of the current regimens. In this study, we performed a quantitative high throughput screening of the Molecular Libraries Small Molecule Repository (MLSMR) of ∼350,000 chemicals for novel HCV inhibitors using our previously developed cell-based HCV infection assay. Following confirmation and structural clustering analysis, we narrowed down to 158 compounds from the initial ∼3000 molecules that showed inhibitory activity for further structural and functional analyses. We were able to assign the majority of these compounds to specific stage(s) in the HCV life cycle. Three of them are direct inhibitors of NS3/4A protease. Most of the compounds appear to act on novel targets in HCV life cycle. Four compounds with novel structure and excellent drug-like properties, three targeting HCV entry and one targeting HCV assembly/secretion, were advanced for further development as lead hits. These compounds represent diverse chemotypes that are potential lead compounds for further optimization and may offer promising candidates for the development of novel therapeutics against HCV infection. In addition, they represent novel molecular probes to explore the complex interactions between HCV and the cells. PMID:26515788

  2. Application value of ATP based bioluminescence tumor chemosensitivity assay in the chemotherapy for hydrothorax caused by non-small cell lung cancer

    Institute of Scientific and Technical Information of China (English)

    Kaijian Le; Yuming Jia; Jing Wang; Maoqiong Jiang

    2013-01-01

    Objective: The aim of the study was to investigate the clinical value and application of ATP based bioluminescencetumor chemosensitivity assay (ATP-TCA) in the chemotherapy for hydrothorax caused by non-small cell lung cancer(NSCLC). Methods: Hydrothorax specimens from 120 NSCLC patients were analyzed by ATP-TCA and the most sensitivechemotherapeutic drugs were used in NSCLC patients (treatment group). At the same time, 56 NSCLC patients with hydrothoraxwere admitted in our Hospital (Department of Oncology, The No. 2 People's Hospital of Yibin, China) and given chemotherapywithout guidance of the ATP-TCA (control group). Before the third chemotherapeutic cycle, clinical outcomes wereanalyzed in the two groups. Results: Effective rate of hydrothorax in treatment group was 67%, while 46% in control group(P < 0.05). In refractory hydrothorax patients, they were 69% and 40% (P < 0.05), respectively. In vitro results correlated wellwith clinical outcomes (P < 0.01). Conclusion: Effective rate of chemotherapy for hydrothorax in NSCLC is higher in treatmentgroup than that in control group. ATP-TCA is especially helpful for refractory hydrothorax.

  3. Characterization of mesenchymal stromal cells: potency assay development.

    Science.gov (United States)

    Hematti, Peiman

    2016-04-01

    Based on their many different mechanisms of action, presumed immune-privileged status, and relative ease of production, mesenchymal stromal cells (MSCs) are under intensive clinical investigation for treating a wide range of degenerative, inflammatory, and immunologic disorders. Identification of relevant and robust potency assays is not only a regulatory requirement, but it is also the basis for producing and delivering a product that is consistent, safe, and ultimately an effective therapy. Although development of an appropriate potency assay is one of the most challenging issues in cell-based therapies, it is of paramount importance in the process of developing and testing cellular products. Regardless of the many different tissue sources and methods used in culture expansion of MSCs, they possess many of the same morphologic, cell surface markers, and differentiation characteristics. However, MSC products with similar phenotypic characteristics could still have major differences in their biologic and functional attributes. Understanding the different mechanisms of action and establishment of relevant potency assays is of pivotal importance in allowing investigators and regulatory agencies to compare MSCs used in different clinical trials. PMID:27079322

  4. Identification of Antiviral Agents Targeting Hepatitis B Virus Promoter from Extracts of Indonesian Marine Organisms by a Novel Cell-Based Screening Assay

    Directory of Open Access Journals (Sweden)

    Atsuya Yamashita

    2015-11-01

    Full Text Available The current treatments of chronic hepatitis B (CHB face a limited choice of vaccine, antibody and antiviral agents. The development of additional antiviral agents is still needed for improvement of CHB therapy. In this study, we established a screening system in order to identify compounds inhibiting the core promoter activity of hepatitis B virus (HBV. We prepared 80 extracts of marine organisms from the coral reefs of Indonesia and screened them by using this system. Eventually, two extracts showed high inhibitory activity (>95% and low cytotoxicity (66% to 77%. Solvent fractionation, column chromatography and NMR analysis revealed that 3,5-dibromo-2-(2,4-dibromophenoxy-phenol (compound 1 and 3,4,5-tribromo-2-(2,4-dibromophenoxy-phenol (compound 2, which are classified as polybrominated diphenyl ethers (PBDEs, were identified as anti-HBV agents in the extracts. Compounds 1 and 2 inhibited HBV core promoter activity as well as HBV production from HepG2.2.15.7 cells in a dose-dependent manner. The EC50 values of compounds 1 and 2 were 0.23 and 0.80 µM, respectively, while selectivity indexes of compound 1 and 2 were 18.2 and 12.8, respectively. These results suggest that our cell-based HBV core promoter assay system is useful to determine anti-HBV compounds, and that two PBDE compounds are expected to be candidates of lead compounds for the development of anti-HBV drugs.

  5. A Cell-Based Internalization and Degradation Assay with an Activatable Fluorescence-Quencher Probe as a Tool for Functional Antibody Screening.

    Science.gov (United States)

    Li, Yan; Liu, Peter Corbett; Shen, Yang; Snavely, Marshall D; Hiraga, Kaori

    2015-08-01

    For the development of therapeutically potent anti-cancer antibody drugs, it is often important to identify antibodies that internalize into cells efficiently, rather than just binding to antigens on the cell surface. Such antibodies can mediate receptor endocytosis, resulting in receptor downregulation on the cell surface and potentially inhibiting receptor function and tumor growth. Also, efficient antibody internalization is a prerequisite for the delivery of cytotoxic drugs into target cells and is critical for the development of antibody-drug conjugates. Here we describe a novel activatable fluorescence-quencher pair to quantify the extent of antibody internalization and degradation in the target cells. In this assay, candidate antibodies were labeled with a fluorescent dye and a quencher. Fluorescence is inhibited outside and on the surface of cells, but activated upon endocytosis and degradation of the antibody. This assay enabled the development of a process for rapid characterization of candidate antibodies potentially in a high-throughput format. By employing an activatable secondary antibody, primary antibodies in purified form or in culture supernatants can be screened for internalization and degradation. Because purification of candidate antibodies is not required, this method represents a direct functional screen to identify antibodies that internalize efficiently early in the discovery process. PMID:26024945

  6. Additive mixture effects of estrogenic chemicals in human cell-based assays can be influenced by inclusion of chemicals with differing effect profiles.

    Directory of Open Access Journals (Sweden)

    Richard Mark Evans

    Full Text Available A growing body of experimental evidence indicates that the in vitro effects of mixtures of estrogenic chemicals can be well predicted from the estrogenicity of their components by the concentration addition (CA concept. However, some studies have observed small deviations from CA. Factors affecting the presence or observation of deviations could include: the type of chemical tested; number of mixture components; mixture design; and assay choice. We designed mixture experiments that address these factors, using mixtures with high numbers of components, chemicals from diverse chemical groups, assays with different in vitro endpoints and different mixture designs and ratios. Firstly, the effects of mixtures composed of up to 17 estrogenic chemicals were examined using estrogenicity assays with reporter-gene (ERLUX and cell proliferation (ESCREEN endpoints. Two mixture designs were used: 1 a 'balanced' design with components present in proportion to a common effect concentration (e.g. an EC(10 and 2 a 'non-balanced' design with components in proportion to potential human tissue concentrations. Secondly, the individual and simultaneous ability of 16 potential modulator chemicals (each with minimal estrogenicity to influence the assay outcome produced by a reference mixture of estrogenic chemicals was examined. Test chemicals included plasticizers, phthalates, metals, PCBs, phytoestrogens, PAHs, heterocyclic amines, antioxidants, UV filters, musks, PBDEs and parabens. In all the scenarios tested, the CA concept provided a good prediction of mixture effects. Modulation studies revealed that chemicals possessing minimal estrogenicity themselves could reduce (negatively modulate the effect of a mixture of estrogenic chemicals. Whether the type of modulation we observed occurs in practice most likely depends on the chemical concentrations involved, and better information is required on likely human tissue concentrations of estrogens and of potential

  7. γδ T cell-mediated antibody-dependent cellular cytotoxicity with CD19 antibodies assessed by an impedance-based label-free real-time cytotoxicity assay

    Directory of Open Access Journals (Sweden)

    Ursula Jördis Eva Seidel

    2014-12-01

    Full Text Available γδ T cells are not MHC restricted, elicit cytotoxicity against various malignancies, are present in early post-transplant phases in novel stem cell transplantation (SCT strategies and have been shown to mediate antibody-dependent cellular cytotoxicity (ADCC with monoclonal antibodies (mAbs. These features make γδ T cells promising effector cells for antibody-based immunotherapy in pediatric patients with B-lineage acute lymphoblastic leukemia (ALL. To evaluate combination of human γδ T cells with CD19 antibodies for immunotherapy of B-lineage ALL, γδ T cells were expanded after a GMP-compliant protocol and ADCC of both primary and expanded γδ T cells with an Fc optimized CD19 antibody (4G7SDIE and a bispecific antibody with the specificities CD19 and CD16 (N19-C16 was evaluated in CD107a degranulation assays and intracellular cytokine staining (ICS. CD107a, TNF-α and IFN-γ expression of primary γδ T cells were significantly increased and correlated with CD16-expression of γδ T cells. γδ T cells highly expressed CD107a after expansion and no further increased expression by 4G7SDIE and N19-C16 was measured. Cytotoxicity of purified expanded γδ T cells targeting CD19-expressing cells was assessed in both europium-TDA release and in an impedance-based label-free method (using the xCELLigence system measuring γδ T cell lysis in real-time. Albeit in the 2 h end-point europium-TDA release assay no increased lysis was observed, in real-time xCELLigence assays both significant antibody-independent cytotoxicity and ADCC of γδ T cells were observed. The xCELLigence system outperformed the end-point europium-TDA release assay in sensitivity and allows drawing of conclusions to lysis kinetics of γδ T cells over prolonged periods of time periods. Combination of CD19 antibodies with primary as well as expanded γδ T cells exhibits an promising approach, which may enhance clinical outcome of patients with pediatric B-lineage ALL and

  8. γδ T Cell-Mediated Antibody-Dependent Cellular Cytotoxicity with CD19 Antibodies Assessed by an Impedance-Based Label-Free Real-Time Cytotoxicity Assay.

    Science.gov (United States)

    Seidel, Ursula Jördis Eva; Vogt, Fabian; Grosse-Hovest, Ludger; Jung, Gundram; Handgretinger, Rupert; Lang, Peter

    2014-01-01

    γδ T cells are not MHC restricted, elicit cytotoxicity against various malignancies, are present in early post-transplant phases in novel stem cell transplantation strategies and have been shown to mediate antibody-dependent cellular cytotoxicity (ADCC) with monoclonal antibodies (mAbs). These features make γδ T cells promising effector cells for antibody-based immunotherapy in pediatric patients with B-lineage acute lymphoblastic leukemia (ALL). To evaluate combination of human γδ T cells with CD19 antibodies for immunotherapy of B-lineage ALL, γδ T cells were expanded after a GMP-compliant protocol and ADCC of both primary and expanded γδ T cells with an Fc-optimized CD19 antibody (4G7SDIE) and a bi-specific antibody with the specificities CD19 and CD16 (N19-C16) was evaluated in CD107a-degranulation assays and intracellular cytokine staining. CD107a, TNFα, and IFNγ expression of primary γδ T cells were significantly increased and correlated with CD16-expression of γδ T cells. γδ T cells highly expressed CD107a after expansion and no further increased expression by 4G7SDIE and N19-C16 was measured. Cytotoxicity of purified expanded γδ T cells targeting CD19-expressing cells was assessed in both europium-TDA release and in an impedance-based label-free method (using the xCELLigence system) measuring γδ T cell lysis in real-time. Albeit in the 2 h end-point europium-TDA release assay no increased lysis was observed, in real-time xCELLigence assays both significant antibody-independent cytotoxicity and ADCC of γδ T cells were observed. The xCELLigence system outperformed the end-point europium-TDA release assay in sensitivity and allows drawing of conclusions to lysis kinetics of γδ T cells over prolonged periods of time periods. Combination of CD19 antibodies with primary as well as expanded γδ T cells exhibits a promising approach, which may enhance clinical outcome of patients with pediatric B-lineage ALL and requires clinical

  9. Validation of a Flow Cytometry Based Binding Assay for Evaluation of Monoclonal Antibody Recognizing EGF Receptor

    Science.gov (United States)

    Cedeño-Arias, Mercedes; Sánchez-Ramírez, Javier; Blanco-Santana, Rancés; Rengifo-Calzado, Enrique

    2011-01-01

    An ideal test used to characterize a product must be appropriate for the measurement of product quality, manufacturing consistency, product stability, and comparability studies. Flow cytometry has been successfully applied to the examination of antibodies and receptors on membrane surfaces; however, to date, the analytical validation of cytometry based assays is limited. Here we report on the validation of a flow cytometry-based assay used in the evaluation of nimotuzumab binding to cells over-expressing EGFR on cell surface. The assay was validated by examining, assay robustness, specificity, repeatability and intermediate precision. The assay was highly specific, robust for all studied factors except for cell fixation with 1% paraformaldehyde and met criteria for precision with RSD < 2%. In addition the assay has stability-indicating properties evidenced by the ability to detect changes in mAb degraded samples. Most importantly, the assay demonstrated to be useful for its intended use. PMID:21886904

  10. Blockade of Androgen Markers Using a Novel Betasitosterol, Thioctic Acid and Carnitine-containing Compound in Prostate and Hair Follicle Cell-based Assays.

    Science.gov (United States)

    Chen, Li; Wang, Jiaolong; Mouser, Glen; Li, Yan Chun; Marcovici, Geno

    2016-06-01

    Androgenetic alopecia (AGA) affects approximately 70% of men and 40% of women in an age-dependent manner and is partially mediated by androgen hormones. Benign prostatic hyperplasia (BPH) similarly affects 50% of the male population, rising by 10% each decade. Finasteride inhibits 5-alpha reductase (5AR) and is used to treat both disorders, despite offering limited clinical benefits accompanied by significant adverse side effects. Building on our previous work demonstrating the efficacy of naturally derived 5AR inhibitors (such as stigmasterol and beta sitosterol), we hypothesize that targeting 5AR as well as inflammatory pathways may yield improved efficacy in AGA and BPH. Here we address these dual pathomechanisms by examining the potency of a novel composition using in vitro assays of representative cell lines for AGA (hair follicle dermal papilla cells) and BPH (LNCaP prostate cells), respectively. Exposure of cells to the novel test composition down-regulated mRNA expression profiles characteristic of both disease processes, which outperformed finasteride. Changes in mRNA expression were corroborated at the protein level as assessed by western blotting. These studies provide proof of concept that novel, naturally derived compositions simultaneously targeting 5AR and inflammatory mediators may represent a rational approach to treating AGA and BPH. Copyright © 2016 John Wiley & Sons, Ltd.

  11. Blockade of Androgen Markers Using a Novel Betasitosterol, Thioctic Acid and Carnitine-containing Compound in Prostate and Hair Follicle Cell-based Assays.

    Science.gov (United States)

    Chen, Li; Wang, Jiaolong; Mouser, Glen; Li, Yan Chun; Marcovici, Geno

    2016-06-01

    Androgenetic alopecia (AGA) affects approximately 70% of men and 40% of women in an age-dependent manner and is partially mediated by androgen hormones. Benign prostatic hyperplasia (BPH) similarly affects 50% of the male population, rising by 10% each decade. Finasteride inhibits 5-alpha reductase (5AR) and is used to treat both disorders, despite offering limited clinical benefits accompanied by significant adverse side effects. Building on our previous work demonstrating the efficacy of naturally derived 5AR inhibitors (such as stigmasterol and beta sitosterol), we hypothesize that targeting 5AR as well as inflammatory pathways may yield improved efficacy in AGA and BPH. Here we address these dual pathomechanisms by examining the potency of a novel composition using in vitro assays of representative cell lines for AGA (hair follicle dermal papilla cells) and BPH (LNCaP prostate cells), respectively. Exposure of cells to the novel test composition down-regulated mRNA expression profiles characteristic of both disease processes, which outperformed finasteride. Changes in mRNA expression were corroborated at the protein level as assessed by western blotting. These studies provide proof of concept that novel, naturally derived compositions simultaneously targeting 5AR and inflammatory mediators may represent a rational approach to treating AGA and BPH. Copyright © 2016 John Wiley & Sons, Ltd. PMID:26990224

  12. Discriminating Different Cancer Cells Using a Zebrafish in Vivo Assay

    International Nuclear Information System (INIS)

    Despite the expanded understanding of tumor angiogenesis phenomenon and how it impacts cancer treatment outcomes, we have yet to develop a robust assay that can quickly, easily, and quantitatively measure tumor-induced angiogenesis. Since the zebrafish/tumor xenograft represents an emerging tool in this regard, the present study strives to capitalize on the ease, effectiveness, and the adaptability of this model to quantify tumor angiogenesis. In order to test a range of responses, we chose two different tumorigenic cell lines, the human non-small cell lung carcinoma (H1299) and the mouse lung adenocarcinoma (CL13). Non-tumorigenic 3T3-L1 cells served as negative control. The cells were grafted near to the perivitelline space of the zebrafish embryos and the angiogenic response was analyzed using whole-mount alkaline phosphatase (AP) vessel staining and fluorescence microscopy. Angiogenic activity was scored based on the length and number of the newly formed ectopic vessels and the percentage of embryos with ectopic vessels. At 2 day-post-implantation, we detected a significant increase in the length and number of ectopic vessels with H1299 cell implantation compared to CL13 cell transplantation, both are higher than 3T3-L1 control. We also observed a significantly higher percentage of embryos with ectopic vessels with H1299 and CL13 transplantation compared to the 3T3-L1 control, but this parameter is not as robust and reliable as measuring the length and number of ectopic vessels. Furthermore, the systemic exposure of zebrafish embryos to an anti-angiogenesis drug (PTK 787, inhibitor of vascular endothelial growth factor receptor tyrosine kinase) inhibited tumor-induced angiogenesis, suggesting that the assay can be used to evaluate anti-angiogenic drugs. This study implicates the feasibility of using zebrafish xenotransplantation to perform quantitative measurement of the angiogenic activity of cancer cells which can be further extended to measure cancer cell

  13. Discriminating Different Cancer Cells Using a Zebrafish in Vivo Assay

    Energy Technology Data Exchange (ETDEWEB)

    Moshal, Karni S.; Ferri-Lagneau, Karine F.; Haider, Jamil; Pardhanani, Pooja; Leung, TinChung, E-mail: tleung@nccu.edu [Biomedical/Biotechnology Research Institute, North Carolina Central University, North Carolina Research Campus, Nutrition Research Center, 500 Laureate Way, Kannapolis, NC 28081 (United States)

    2011-10-31

    Despite the expanded understanding of tumor angiogenesis phenomenon and how it impacts cancer treatment outcomes, we have yet to develop a robust assay that can quickly, easily, and quantitatively measure tumor-induced angiogenesis. Since the zebrafish/tumor xenograft represents an emerging tool in this regard, the present study strives to capitalize on the ease, effectiveness, and the adaptability of this model to quantify tumor angiogenesis. In order to test a range of responses, we chose two different tumorigenic cell lines, the human non-small cell lung carcinoma (H1299) and the mouse lung adenocarcinoma (CL13). Non-tumorigenic 3T3-L1 cells served as negative control. The cells were grafted near to the perivitelline space of the zebrafish embryos and the angiogenic response was analyzed using whole-mount alkaline phosphatase (AP) vessel staining and fluorescence microscopy. Angiogenic activity was scored based on the length and number of the newly formed ectopic vessels and the percentage of embryos with ectopic vessels. At 2 day-post-implantation, we detected a significant increase in the length and number of ectopic vessels with H1299 cell implantation compared to CL13 cell transplantation, both are higher than 3T3-L1 control. We also observed a significantly higher percentage of embryos with ectopic vessels with H1299 and CL13 transplantation compared to the 3T3-L1 control, but this parameter is not as robust and reliable as measuring the length and number of ectopic vessels. Furthermore, the systemic exposure of zebrafish embryos to an anti-angiogenesis drug (PTK 787, inhibitor of vascular endothelial growth factor receptor tyrosine kinase) inhibited tumor-induced angiogenesis, suggesting that the assay can be used to evaluate anti-angiogenic drugs. This study implicates the feasibility of using zebrafish xenotransplantation to perform quantitative measurement of the angiogenic activity of cancer cells which can be further extended to measure cancer cell

  14. Discriminating Different Cancer Cells Using a Zebrafish in Vivo Assay

    Directory of Open Access Journals (Sweden)

    Pooja Pardhanani

    2011-10-01

    Full Text Available Despite the expanded understanding of tumor angiogenesis phenomenon and how it impacts cancer treatment outcomes, we have yet to develop a robust assay that can quickly, easily, and quantitatively measure tumor-induced angiogenesis. Since the zebrafish/tumor xenograft represents an emerging tool in this regard, the present study strives to capitalize on the ease, effectiveness, and the adaptability of this model to quantify tumor angiogenesis. In order to test a range of responses, we chose two different tumorigenic cell lines, the human non-small cell lung carcinoma (H1299 and the mouse lung adenocarcinoma (CL13. Non-tumorigenic 3T3-L1 cells served as negative control. The cells were grafted near to the perivitelline space of the zebrafish embryos and the angiogenic response was analyzed using whole-mount alkaline phosphatase (AP vessel staining and fluorescence microscopy. Angiogenic activity was scored based on the length and number of the newly formed ectopic vessels and the percentage of embryos with ectopic vessels. At 2 day-post-implantation, we detected a significant increase in the length and number of ectopic vessels with H1299 cell implantation compared to CL13 cell transplantation, both are higher than 3T3-L1 control. We also observed a significantly higher percentage of embryos with ectopic vessels with H1299 and CL13 transplantation compared to the 3T3-L1 control, but this parameter is not as robust and reliable as measuring the length and number of ectopic vessels. Furthermore, the systemic exposure of zebrafish embryos to an anti-angiogenesis drug (PTK 787, inhibitor of vascular endothelial growth factor receptor tyrosine kinase inhibited tumor-induced angiogenesis, suggesting that the assay can be used to evaluate anti-angiogenic drugs. This study implicates the feasibility of using zebrafish xenotransplantation to perform quantitative measurement of the angiogenic activity of cancer cells which can be further extended to

  15. Epithelial cells as alternative human biomatrices for comet assay

    Directory of Open Access Journals (Sweden)

    Emilio eRojas

    2014-11-01

    Full Text Available The comet assay is a valuable experimental tool aimed at mapping DNA damage in human cells in vivo for environmental and occupational monitoring, as well as for therapeutic purposes, such as storage prior to transplant, during tissue engineering, and in experimental ex vivo assays. Furthermore, due to its great versatility, the comet assay allows to explore the use of alternative cell types to assess DNA damage, such as epithelial cells. Epithelial cells, as specialized components of many organs, have the potential to serve as biomatrices that can be used to evaluate genotoxicity and may also serve as early effect biomarkers. Furthermore, 80% of solid cancers are of epithelial origin, which points to the importance of studying DNA damage in these tissues. Indeed, studies including comet assay in epithelial cells have either clear clinical applications (lens and corneal epithelial cells or examine genotoxicity within human biomonitoring and in vitro studies. We here review improvements in determining DNA damage using the comet assay by employing lens, corneal, tear duct, buccal, and nasal epithelial cells. For some of these tissues invasive sampling procedures are needed. Desquamated epithelial cells must be obtained and dissociated prior to examination using the comet assay, and such procedures may induce varying amounts of DNA damage. Buccal epithelial cells require lysis enriched with proteinase K to obtain free nucleosomes.Over a thirty year period, the comet assay in epithelial cells has been litlle employed, however its use indicates that it could be an extraordinary tool not only for risk assessment, but also for diagnosis, prognosis of treatments and diseases.

  16. Mrassf1a-pap, a novel methylation-based assay for the detection of cell-free fetal DNA in maternal plasma.

    Directory of Open Access Journals (Sweden)

    Jessica M E van den Oever

    Full Text Available OBJECTIVES: RASSF1A has been described to be differentially methylated between fetal and maternal DNA and can therefore be used as a universal sex-independent marker to confirm the presence of fetal sequences in maternal plasma. However, this requires highly sensitive methods. We have previously shown that Pyrophosphorolysis-activated Polymerization (PAP is a highly sensitive technique that can be used in noninvasive prenatal diagnosis. In this study, we have used PAP in combination with bisulfite conversion to develop a new universal methylation-based assay for the detection of fetal methylated RASSF1A sequences in maternal plasma. METHODS: Bisulfite sequencing was performed on maternal genomic (gDNA and fetal gDNA from chorionic villi to determine differentially methylated regions in the RASSF1A gene using bisulfite specific PCR primers. Methylation specific primers for PAP were designed for the detection of fetal methylated RASSF1A sequences after bisulfite conversion and validated. RESULTS: Serial dilutions of fetal gDNA in a background of maternal gDNA show a relative percentage of ~3% can be detected using this assay. Furthermore, fetal methylated RASSF1A sequences were detected both retrospectively as well as prospectively in all maternal plasma samples tested (n = 71. No methylated RASSF1A specific bands were observed in corresponding maternal gDNA. Specificity was further determined by testing anonymized plasma from non-pregnant females (n = 24 and males (n = 21. Also, no methylated RASSF1A sequences were detected here, showing this assay is very specific for methylated fetal DNA. Combining all samples and controls, we obtain an overall sensitivity and specificity of 100% (95% CI 98.4%-100%. CONCLUSIONS: Our data demonstrate that using a combination of bisulfite conversion and PAP fetal methylated RASSF1A sequences can be detected with extreme sensitivity in a universal and sex-independent manner. Therefore, this assay could be of great

  17. A Versatile Cell Death Screening Assay Using Dye-Stained Cells and Multivariate Image Analysis.

    Science.gov (United States)

    Collins, Tony J; Ylanko, Jarkko; Geng, Fei; Andrews, David W

    2015-11-01

    A novel dye-based method for measuring cell death in image-based screens is presented. Unlike conventional high- and medium-throughput cell death assays that measure only one form of cell death accurately, using multivariate analysis of micrographs of cells stained with the inexpensive mix, red dye nonyl acridine orange, and a nuclear stain, it was possible to quantify cell death induced by a variety of different agonists even without a positive control. Surprisingly, using a single known cytotoxic agent as a positive control for training a multivariate classifier allowed accurate quantification of cytotoxicity for mechanistically unrelated compounds enabling generation of dose-response curves. Comparison with low throughput biochemical methods suggested that cell death was accurately distinguished from cell stress induced by low concentrations of the bioactive compounds Tunicamycin and Brefeldin A. High-throughput image-based format analyses of more than 300 kinase inhibitors correctly identified 11 as cytotoxic with only 1 false positive. The simplicity and robustness of this dye-based assay makes it particularly suited to live cell screening for toxic compounds.

  18. ApoHRP-based Assay to Measure Intracellular Regulatory Heme

    Science.gov (United States)

    Atamna, Hani; Brahmbhatt, Marmik; Atamna, Wafa; Shanower, Gregory A.; Dhahbi, Joseph M.

    2015-01-01

    The majority of the heme-binding proteins possess a “heme-pocket” that stably binds with heme. Usually known as housekeeping heme-proteins, they participate in a variety of metabolic reactions (e.g., catalase). Heme also binds with lower affinity to the “Heme-Regulatory Motifs” (HRM) in specific regulatory proteins. This type of heme binding is known as exchangeable or regulatory heme (RH). Heme binding to HRM proteins regulates their function (e.g., Bach1). Although there are well-established methods for assaying total cellular heme (e.g., heme-proteins plus RH), currently there is no method available for measuring RH independently from the total heme (TH). The current study describes and validates a new method to measure intracellular RH. The method is based on the reconstitution of apo-horseradish peroxidase (apoHRP) with heme to form holoHRP. The resulting holoHRP activity is then measured with a colorimetric substrate. The results show that apoHRP specifically binds RH but not with heme from housekeeping heme-proteins. The RH assay detects intracellular RH. Furthermore, using conditions that create positive (hemin) or negative (N-methyl protoporphyrin IX) controls for heme in normal human fibroblasts (IMR90), the RH assay shows that RH is dynamic and independent from TH. We also demonstrated that short-term exposure to subcytotoxic concentrations of lead (Pb), mercury (Hg), or amyloid-β(Aβ) significantly alters intracellular RH with little effect on TH. In conclusion the RH assay is an effective assay to investigate intracellular RH concentration and demonstrates that RH represents ~6% of total heme in IMR90 cells. PMID:25525887

  19. Monochromosomal hybrid cell assay for evaluating the genotoxicity of environmental chemicals

    Energy Technology Data Exchange (ETDEWEB)

    Sandhu, S.S.; Gudi, R.D.; Athwal, R.S.

    1988-12-01

    The development and utilization of a monochromosomal hybrid cell assay for detecting aneuploidy and chromosomal aberrations are described. The monochromosomal hybrid cell lines were produced by a two-step process involving transfer of a marker bacterial gene to a human chromosome and then by integration of that human chromosome into a mouse complement of chromosomes through microcell fusion. For chemically induced aneuploidy, the segregation of a single human chromosome among mouse chromosomes is used as a cytogenetic marker. The genetic assay for aneuploidy is based on the ability of the cells to grow in a medium that selects for the loss of the human chromosome. The assay for clastogenicity is based on survival of the cells after treatment with the chemicals in medium that selects for retention of the human chromosome but loss of its segment containing diphtheria toxin locus. The assays greatly simplify the detection of chromosomal aberrations induced by environmental factors at low-dose levels.

  20. Cytotoxicity and mitogenicity assays with real-time and label-free monitoring of human granulosa cells with an impedance-based signal processing technology intergrating micro-electronics and cell biology.

    Science.gov (United States)

    Oktem, Ozgur; Bildik, Gamze; Senbabaoglu, Filiz; Lack, Nathan A; Akin, Nazli; Yakar, Feridun; Urman, Defne; Guzel, Yilmaz; Balaban, Basak; Iwase, Akira; Urman, Bulent

    2016-04-01

    A recently developed technology (xCelligence) integrating micro-electronics and cell biology allows real-time, uninterrupted and quantitative analysis of cell proliferation, viability and cytotoxicity by measuring the electrical impedance of the cell population in the wells without using any labeling agent. In this study we investigated if this system is a suitable model to analyze the effects of mitogenic (FSH) and cytotoxic (chemotherapy) agents with different toxicity profiles on human granulosa cells in comparison to conventional methods of assessing cell viability, DNA damage, apoptosis and steroidogenesis. The system generated the real-time growth curves of the cells, and determined their doubling times, mean cell indices and generated dose-response curves after exposure to cytotoxic and mitogenic stimuli. It accurately predicted the gonadotoxicity of the drugs and distinguished less toxic agents (5-FU and paclitaxel) from more toxic ones (cisplatin and cyclophosphamide). This platform can be a useful tool for specific end-point assays in reproductive toxicology.

  1. Image classifiers for the cell transformation assay: a progress report

    Science.gov (United States)

    Urani, Chiara; Crosta, Giovanni F.; Procaccianti, Claudio; Melchioretto, Pasquale; Stefanini, Federico M.

    2010-02-01

    The Cell Transformation Assay (CTA) is one of the promising in vitro methods used to predict human carcinogenicity. The neoplastic phenotype is monitored in suitable cells by the formation of foci and observed by light microscopy after staining. Foci exhibit three types of morphological alterations: Type I, characterized by partially transformed cells, and Types II and III considered to have undergone neoplastic transformation. Foci recognition and scoring have always been carried visually by a trained human expert. In order to automatically classify foci images one needs to implement some image understanding algorithm. Herewith, two such algorithms are described and compared by performance. The supervised classifier (as described in previous articles) relies on principal components analysis embedded in a training feedback loop to process the morphological descriptors extracted by "spectrum enhancement" (SE). The unsupervised classifier architecture is based on the "partitioning around medoids" and is applied to image descriptors taken from histogram moments (HM). Preliminary results suggest the inadequacy of the HMs as image descriptors as compared to those from SE. A justification derived from elementary arguments of real analysis is provided in the Appendix.

  2. Evaluation of tetrazolium-based semiautomatic colorimetric assay for measurement of human antitumor cytotoxicity

    Energy Technology Data Exchange (ETDEWEB)

    Heo, D.S.; Park, J.G.; Hata, K.; Day, R.; Herberman, R.B.; Whiteside, T.L. (Univ. of Pittsburgh School of Medicine, PA (USA))

    1990-06-15

    A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based colorimetric assay was developed and compared with 51Cr release from different adherent tumor cell targets (human squamous cell carcinoma lines of the head and neck established in our laboratory, melanoma, and colorectal carcinoma) using 5-7-day human lymphokine-activated killer cells and monocyte-depleted peripheral blood lymphocytes as effectors. With adherent tumor cell targets, MTT colorimetry was more sensitive than the 51Cr release assay in measuring the antitumor activity of effectors: median, 4385 (range, 988-8144) versus median, 1061 (range, 582-7294) lytic units (the number of effector cells required to lyse 20% of 5 x 10(3) targets)/10(7) effectors (P less than 0.01). Background effects (without effector cells) were comparable in 4-h assays (9% versus 10%) between MTT colorimetry and 51Cr release. In 24-h assays, MTT colorimetry showed higher antitumor activity (70-100% versus 40-60% lysis at 1:1 effector:target cell ratio) but lower background effects (6% versus 38%) than 51Cr release assay. Thus, MTT colorimetry was more sensitive, did not use radiolabeled targets, required fewer effector cells, and was easier, less expensive, and better adaptable to serial monitoring of effector cell function in cancer patients. This colorimetric assay is especially well suited to adherent tumor cell targets. The use of adherent tumor cell monolayers, as opposed to trypsinized single cell suspensions, provides an opportunity to measure interactions of effector cells with enzymatically unaltered solid tumor targets. Because of the greater sensitivity of the colorimetric assay, the transformation of MTT data into lytic units, as commonly used for 51Cr release assays, required an adjustment to avoid the extrapolation based on the exponential fit equation.

  3. A sensitive real-time PCR based assay to estimate the impact of amino acid substitutions on the competitive replication fitness of human immunodeficiency virus type 1 in cell culture.

    Science.gov (United States)

    Liu, Yi; Holte, Sarah; Rao, Ushnal; McClure, Jan; Konopa, Philip; Swain, J Victor; Lanxon-Cookson, Erinn; Kim, Moon; Chen, Lennie; Mullins, James I

    2013-04-01

    Fixation of mutations in human immunodeficiency virus type 1 (HIV-1), such as those conferring drug resistance and immune escape, can result in a change in replication fitness. To assess these changes, a real-time TaqMan PCR detection assay and statistical methods for data analysis were developed to estimate sensitively relative viral fitness in competitive viral replication experiments in cell culture. Chimeric viruses with the gene of interest in an HIV-1NL4-3 backbone were constructed in two forms, vifA (native vif gene in NL4-3) and vifB (vif gene with six synonymous nucleotide differences from vifA). Subsequently, mutations of interest were introduced into the chimeric viruses in NL4-3VifA backbones, and the mutants were competed against the chimera with the isogenic viral sequence in the NL4-3VifB backbone in cell culture. In order to assess subtle fitness differences, culture supernatants were sampled longitudinally, and the viruses differentially quantified using vifA- and vifB-specific primers in real-time PCR assays. Based on an exponential net growth model, the growth rate of each virus was determined and the fitness cost of the mutation(s) distinguishing the two viruses represented as the net growth rate difference between the mutant and the native variants. Using this assay, the fitness impact of eight amino acid substitutions was quantitated at highly conserved sites in HIV-1 Gag and Env. PMID:23201292

  4. Improving T-cell assays for the diagnosis of latent TB infection: potential of a diagnostic test based on IP-10

    DEFF Research Database (Denmark)

    Ruhwald, Morten; Petersen, Janne; Kofoed, Kristian;

    2008-01-01

    or community controls). IP-10 and IL-2 was measured in plasma after stimulation of whole-blood with M.tuberculosis specific antigens and mitogen. Previously developed criteria for positive IP-10 and IL-2 tests were used and the diagnostic performances of the IP-10 and IL-2 tests were compared......BACKGROUND: There is a need for simple tools such as the M.tuberculosis specific IFN-gamma release assays (IGRA) to improve diagnosis of M.tuberculosis-infection in children. The aim of the study was to evaluate the performance of an IP-10 and IL-2 based tests for the diagnosis of M...... with the Quantiferon In-Tube (QFT-IT) and the Tuberculin Skin Tests (TST). In response to M.tuberculosis specific antigens, the high-risk children expressed significantly higher levels of IP-10 (1358 pg/ml[IQR 278-2535 pg/ml]) and IL-2 (164 pg/ml[11-590 pg/ml]) than low risk groups 149 pg/ml(25-497 pg/ml), and 0 pg...

  5. An improved assay for antibody dependent cellular cytotoxicity based on time resolved fluorometry.

    Science.gov (United States)

    Patel, A K; Boyd, P N

    1995-07-17

    A new and faster assay for antibody dependent cellular cytotoxicity based on release of europium from target cells is described. This has a number of important advantages over the traditional assays based on release of chromium-51 (51Cr). The new method involves labelling of Wein 133 target cells (B cell non-Hodgkin's lymphoma cells) which express the antigen, CDw52, with the chelate europium diethylenetriaminopentaacetic acid (EuDTPA) according to the method of Blomberg et al. (1986). Labelled cells are sensitised (coated) with the anti-lymphocytic monoclonal antibody, Campath-1H. Human peripheral blood mononuclear cells are added to mediate lysis of EuDTPA labelled Wein 133 cells by ADCC. Release of EuDTPA from lysed cells is determined by mixing supernatants with enhancement solution containing 2-naphthoyl trifluoroacetone, 2-NTA, to form a highly fluorescent chelate which is measured using time resolved fluorometry. Results obtained with the new EuDPTA release assays were comparable to traditional assays based on the release of the radioisotope 51Cr. It is anticipated that this assay will have a widespread application among laboratories performing ADCC assays. The method is non-hazardous and has been used routinely for over 2 years to monitor production and purification of Campath-1H. PMID:7622867

  6. Analysis of image-based phenotypic parameters for high throughput gene perturbation assays.

    Science.gov (United States)

    Song, Mee; Jeong, Euna; Lee, Tae-Kyu; Tsoy, Yury; Kwon, Yong-Jun; Yoon, Sukjoon

    2015-10-01

    Although image-based phenotypic assays are considered a powerful tool for siRNA library screening, the reproducibility and biological implications of various image-based assays are not well-characterized in a systematic manner. Here, we compared the resolution of high throughput assays of image-based cell count and typical cell viability measures for cancer samples. It was found that the optimal plating density of cells was important to obtain maximal resolution in both types of assays. In general, cell counting provided better resolution than the cell viability measure in diverse batches of siRNAs. In addition to cell count, diverse image-based measures were simultaneously collected from a single screening and showed good reproducibility in repetitions. They were classified into a few functional categories according to biological process, based on the differential patterns of hit (i.e., siRNAs) prioritization from the same screening data. The presented systematic analyses of image-based parameters provide new insight to a multitude of applications and better biological interpretation of high content cell-based assays. PMID:26256799

  7. A homogeneous nucleic acid hybridization assay based on strand displacement.

    OpenAIRE

    Vary, C P

    1987-01-01

    A homogeneous nucleic acid hybridization assay which is conducted in solution and requires no separation steps is described. The assay is based on the concept of strand displacement. In the strand displacement assay, an RNA "signal strand" is hybridized within a larger DNA strand termed the "probe strand", which is, in turn, complementary to the target nucleic acid of interest. Hybridization of the target nucleic acid with the probe strand ultimately results in displacement of the RNA signal ...

  8. Patterning cell using Si-stencil for high-throughput assay

    KAUST Repository

    Wu, Jinbo

    2011-01-01

    In this communication, we report a newly developed cell pattering methodology by a silicon-based stencil, which exhibited advantages such as easy handling, reusability, hydrophilic surface and mature fabrication technologies. Cell arrays obtained by this method were used to investigate cell growth under a temperature gradient, which demonstrated the possibility of studying cell behavior in a high-throughput assay. This journal is © The Royal Society of Chemistry 2011.

  9. Comparison of genotoxicity of textile dyestuffs in Salmonella mutagenicity assay, in vitro micronucleus assay, and single cell gel/comet assay.

    Science.gov (United States)

    Wollin, Klaus-M; Gorlitz, Bernd-D

    2004-01-01

    The mutagenicity of textile dyes is an important consideration for the assurance of consumer protection and work safety. The mutagenicity testing of textile dyestuffs is crucial for accurately predicting health risks for consumers and workers exposed to dyes. Unfortunately, these data are often lacking. We studied the genotoxic activity of ten selected commercial textile dyestuffs, which are made up of mixtures of azo dyes and azo metal complex dyes as well as two anthraquinone dyestuffs. We used the Salmonella mutagenicity assay and cultured human keratinocytes (HaCaT cell line). In the S. typhimurium strain TA98, with and without S9, eight often dyestuffs investigated, and in strain TA 100, with and without S9, six often dyes caused frameshift mutations and base-pair substitutions in the dose range of 1-5000 microg/plate in a dose-related manner. All dyes, including those negative in the Salmonella mutagenicity assay, induced clastogenic effects in the in vitro micronucleus (MN) test in HaCaT cells as direct-acting mutagens in the concentration range of 5-150 microg/mL and with maximum MN frequencies between 1.1 and 7.2%, compared to negative controls that showed 0.2-0.4% MN cells. In the single cell gel/comet assay, all ten dyestuffs investigated caused DNA damage in HaCaT keratinocytes. The alkaline (pH >13) version used is capable of detecting DNA single strand breaks, alkali-labile sites, and DNA-DNA/DNA-protein cross-linking. Under the conditions of these screening tests, the textile dyes investigated are direct-acting genotoxic substances. The HaCaT cells testing protocol proposed has been shown to be an appropriate test system for evaluating mutagenicity of textile dyes on a base level.

  10. Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) assay

    Science.gov (United States)

    The Embryonic Stem Cell Test (EST) is an assay which evaluates xenobiotic-induced effects using three endpoints: mouse embryonic stem cell (mESC) differentiation, mESC viability, and 3T3-cell viability. Our research goal was to develop an improved high-throughput assay by establi...

  11. Migratory properties of cultured olfactory ensheathing cells by single-cell migration assay

    Institute of Scientific and Technical Information of China (English)

    Zhi-hui Huang; Ying Wang; Li Cao; Zhi-da Su; Yan-ling Zhu; Yi-zhang Chen; Xiao-bing Yuan; Cheng He

    2008-01-01

    Olfactory ensheathing cells (OECs) are a unique type of glial cells that have axonal growth-promoting properties. OEC transplantation has emerged as a promising experimental therapy of axonal injuries and demyelinating diseases. However, some fundamental cellular properties of OECs remain unclear. In this study, we found that the distinct OEC subpopulations exhibited different migratory properties based on time-lapse imaging of single isolated cells, possibly due to their different cytoskeletal organizations. Moreover, OEC subpopulations displayed different attractive migratory responses to a gradient of lysophosphatidic acid (LPA) in single-cell migration assays. Finally, we found that OEC subpopulations transformed into each other spontaneously. Together, these results demonstrate, for the first time to our knowledge, that distinct OEC subpopulations display different migratory properties in vitro and provide new evidence to support the notion of OECs as a single cell type with malleable functional phenotypes.

  12. Computer-determined assay time based on preset precision

    Energy Technology Data Exchange (ETDEWEB)

    Foster, L.A.; Hagan, R.; Martin, E.R.; Wachter, J.R.; Bonner, C.A.; Malcom, J.E. [Los Alamos National Lab., NM (United States). Nuclear Materials Measurement and Accountability

    1994-08-01

    Most current assay systems for special nuclear materials (SNM) operate on the principle of a fixed assay time which provides acceptable measurement precision without sacrificing the required throughput of the instrument. Waste items to be assayed for SNM content can contain a wide range of nuclear material. Counting all items for the same preset assay time results in a wide range of measurement precision and wastes time at the upper end of the calibration range. A short time sample taken at the beginning of the assay could optimize the analysis time on the basis of the required measurement precision. To illustrate the technique of automatically determining the assay time, measurements were made with a segmented gamma scanner at the Plutonium Facility of Los Alamos National Laboratory with the assay time for each segment determined by counting statistics in that segment. Segments with very little SNM were quickly determined to be below the lower limit of the measurement range and the measurement was stopped. Segments with significant SNM were optimally assays to the preset precision. With this method the total assay time for each item is determined by the desired preset precision. This report describes the precision-based algorithm and presents the results of measurements made to test its validity.

  13. Cell-based assays in combination with ultra-high performance liquid chromatography-quadrupole time of flight tandem mass spectrometry for screening bioactive capilliposide C metabolites generated by rat intestinal microflora.

    Science.gov (United States)

    Cheng, Zhongzhe; Huang, Meilin; Chen, Guiying; Yang, Guangjie; Zhou, Xin; Chen, Chang; Zhang, Yang; Xu, Yong; Feng, Yulin; Zhang, Lin; Jiang, Hongliang

    2016-02-01

    Many plant-derived glycosides are used as medications. It is common that these glycosides show poor intestinal absorption but their metabolites generated by intestinal microflora demonstrate strong bioactivity. Hence, it is crucial to develop a method for the identification and characterization of the metabolites, and consequently reveal the pathway in which the glycosides are processed in gut. In this study, cell-based assays in combination with ultra-high performance liquid chromatography-quadrupole time of flight tandem mass spectrometry (UHPLC-QTOF-MS/MS) were developed for rapid discovery and evaluation of the metabolites of a glycoside compound, capilliposide C (LC-C). 92.7% of LC-C was biotransformed by rat intestinal microflora after 36-h incubation at 37°C. Human cancer cell lines HepG2, PC-3 and A549 was treated with metabolites pool, respectively, which was followed by cell viability assays and characterization of metabolites using UHPLC-QTOF-MS/MS. As a result, significant cytotoxicity was observed for the metabolites pool, from which six metabolites were identified. Based on the metabolites identified, deglycosylation and esterolysis were proposed as the major metabolic pathways of LC-C in rat intestinal microflora. In addition, M4, an esterolysis product of LC-C, was obtained and evaluated for its bioactivity in vitro. As a result, M4 exhibited a reduction in cell viability in HepG2 with an IC50 value of 17.46±1.55μg/mL.

  14. Comparison of clonogenic assay with premature chromosome condensation assay in prediction of human cell radiosensitivity

    Institute of Scientific and Technical Information of China (English)

    Zhuan-Zi Wang; Wen-Jian Li; Hong Zhang; Jian-She Yang; Rong Qiu; Xiao Wang

    2006-01-01

    AIM: To determine whether the number of non-rejoining G2-chromatid breaks can predict the radiosensitivity of human cell lines.METHODS: Cell lines of human ovary carcinoma cells (HO8910), human hepatoma cells (HepG2) and liver cells (L02) were irradiated with a range of doses and assessed both of cell survival and non-rejoining G2-chromatid breaks at 24 h after irradiation. Cell survival was documented by a colony assay. Non-rejoining G2-chromatid breaks were measured by counting the number of non-rejoining G2 chromatid breaks at 24 h after irradiation, detected by the prematurely chromosome condensed (PCC) technique.RESULTS: A linear-quadratic survival curve was observed in three cell lines, and HepG2 was the most sensitive to y-radiation. A dose-dependent linear increase was observed in radiation-induced non-rejoining G2-PCC breaks measured at 24 h after irradiation in all cell lines, and HepG2 was the most susceptible to induction of non-rejoining G2-PCC breaks. A close correlation was found between the clonogenic radiosensitivity and the radiation-induced non-rejoining G2-PCC breaks (r= 0.923). Furthermore, survival-aberration correlations for two or more than two doses lever were also significant.CONCLUSION: The number of non-rejoining G2 PCC breaks holds considerable promise for predicting the radiosensitivity of normal and tumor cells when two or more than two doses lever is tested.

  15. An image-based, dual fluorescence reporter assay to evaluate the efficacy of shRNA for gene silencing at the single-cell level [v1; ref status: indexed, http://f1000r.es/2tt

    Directory of Open Access Journals (Sweden)

    Shin-ichiro Kojima

    2014-02-01

    Full Text Available RNA interference (RNAi is widely used to suppress gene expression in a specific manner. The efficacy of RNAi is mainly dependent on the sequence of small interfering RNA (siRNA in relation to the target mRNA. Although several algorithms have been developed for the design of siRNA, it is still difficult to choose a really effective siRNA from among multiple candidates. In this article, we report the development of an image-based, quantitative, ratiometric fluorescence reporter assay to evaluate the efficacy of RNAi at the single-cell level. Two fluorescence reporter constructs are used. One expresses the candidate small hairpin RNA (shRNA together with an enhanced green fluorescent protein (EGFP; the other expresses a 19-nt target sequence inserted into a cassette expressing a red fluorescent protein (either DsRed or mCherry. Effectiveness of the candidate shRNA is evaluated as the extent to which it knocks down expression of the red fluorescent protein. Thus, the red-to-green fluorescence intensity ratio (appropriately normalized to controls is used as the read-out for quantifying the siRNA efficacy at the individual cell level. We tested this dual fluorescence assay and compared predictions to actual endogenous knockdown levels for three different genes (vimentin, lamin A/C and Arp3 and twenty different shRNAs. For each of the genes, our assay successfully predicted the target sequences for effective RNAi. To further facilitate testing of RNAi efficacy, we developed a negative selection marker (ccdB method for construction of shRNA and red fluorescent reporter plasmids that allowed us to purify these plasmids directly from transformed bacteria without the need for colony selection and DNA sequencing verification.

  16. An image-based, dual fluorescence reporter assay to evaluate the efficacy of shRNA for gene silencing at the single-cell level [v2; ref status: indexed, http://f1000r.es/39j

    Directory of Open Access Journals (Sweden)

    Shin-ichiro Kojima

    2014-05-01

    Full Text Available RNA interference (RNAi is widely used to suppress gene expression in a specific manner. The efficacy of RNAi is mainly dependent on the sequence of small interfering RNA (siRNA in relation to the target mRNA. Although several algorithms have been developed for the design of siRNA, it is still difficult to choose a really effective siRNA from among multiple candidates. In this article, we report the development of an image-based, quantitative, ratiometric fluorescence reporter assay to evaluate the efficacy of RNAi at the single-cell level. Two fluorescence reporter constructs are used. One expresses the candidate small hairpin RNA (shRNA together with an enhanced green fluorescent protein (EGFP; the other expresses a 19-nt target sequence inserted into a cassette expressing a red fluorescent protein (either DsRed or mCherry. Effectiveness of the candidate shRNA is evaluated as the extent to which it knocks down expression of the red fluorescent protein. Thus, the red-to-green fluorescence intensity ratio (appropriately normalized to controls is used as the read-out for quantifying the siRNA efficacy at the individual cell level. We tested this dual fluorescence assay and compared predictions to actual endogenous knockdown levels for three different genes (vimentin, lamin A/C and Arp3 and twenty different shRNAs. For each of the genes, our assay successfully predicted the target sequences for effective RNAi. To further facilitate testing of RNAi efficacy, we developed a negative selection marker (ccdB method for construction of shRNA and red fluorescent reporter plasmids that allowed us to purify these plasmids directly from transformed bacteria without the need for colony selection and DNA sequencing verification.

  17. Cell-Binding Assays for Determining the Affinity of Protein-Protein Interactions: Technologies and Considerations.

    Science.gov (United States)

    Hunter, S A; Cochran, J R

    2016-01-01

    Determining the equilibrium-binding affinity (Kd) of two interacting proteins is essential not only for the biochemical study of protein signaling and function but also for the engineering of improved protein and enzyme variants. One common technique for measuring protein-binding affinities uses flow cytometry to analyze ligand binding to proteins presented on the surface of a cell. However, cell-binding assays require specific considerations to accurately quantify the binding affinity of a protein-protein interaction. Here we will cover the basic assumptions in designing a cell-based binding assay, including the relevant equations and theory behind determining binding affinities. Further, two major considerations in measuring binding affinities-time to equilibrium and ligand depletion-will be discussed. As these conditions have the potential to greatly alter the Kd, methods through which to avoid or minimize them will be provided. We then outline detailed protocols for performing direct- and competitive-binding assays against proteins displayed on the surface of yeast or mammalian cells that can be used to derive accurate Kd values. Finally, a comparison of cell-based binding assays to other types of binding assays will be presented. PMID:27586327

  18. Measurement of separase proteolytic activity in single living cells by a fluorogenic flow cytometry assay.

    Directory of Open Access Journals (Sweden)

    Wiltrud Haaß

    Full Text Available ESPL1/Separase, an endopeptidase, is required for centrosome duplication and separation of sister-chromatides in anaphase of mitosis. Overexpression and deregulated proteolytic activity of Separase as frequently observed in human cancers is associated with the occurrence of supernumerary centrosomes, chromosomal missegregation and aneuploidy. Recently, we have hypothesized that increased Separase proteolytic activity in a small subpopulation of tumor cells may serve as driver of tumor heterogeneity and clonal evolution in chronic myeloid leukemia (CML. Currently, there is no quantitative assay to measure Separase activity levels in single cells. Therefore, we have designed a flow cytometry-based assay that utilizes a Cy5- and rhodamine 110 (Rh110-biconjugated Rad21 cleavage site peptide ([Cy5-D-R-E-I-M-R]2-Rh110 as smart probe and intracellular substrate for detection of Separase enzyme activity in living cells. As measured by Cy5 fluorescence the cellular uptake of the fluorogenic peptide was fast and reached saturation after 210 min of incubation in human histiocytic lymphoma U937 cells. Separase activity was recorded as the intensity of Rh110 fluorescence released after intracellular peptide cleavage providing a linear signal gain within a 90-180 min time slot. Compared to conventional cell extract-based methods the flow cytometric assay delivers equivalent results but is more reliable, bypasses the problem of vague loading controls and unspecific proteolysis associated with whole cell extracts. Especially suited for the investigaton of blood- and bone marrow-derived hematopoietic cells the flow cytometric Separase assay allows generation of Separase activity profiles that tell about the number of Separase positive cells within a sample i.e. cells that currently progress through mitosis and about the range of intercellular variation in Separase activity levels within a cell population. The assay was used to quantify Separase proteolytic

  19. Mixture models for single-cell assays with applications to vaccine studies.

    Science.gov (United States)

    Finak, Greg; McDavid, Andrew; Chattopadhyay, Pratip; Dominguez, Maria; De Rosa, Steve; Roederer, Mario; Gottardo, Raphael

    2014-01-01

    Blood and tissue are composed of many functionally distinct cell subsets. In immunological studies, these can be measured accurately only using single-cell assays. The characterization of these small cell subsets is crucial to decipher system-level biological changes. For this reason, an increasing number of studies rely on assays that provide single-cell measurements of multiple genes and proteins from bulk cell samples. A common problem in the analysis of such data is to identify biomarkers (or combinations of biomarkers) that are differentially expressed between two biological conditions (e.g. before/after stimulation), where expression is defined as the proportion of cells expressing that biomarker (or biomarker combination) in the cell subset(s) of interest. Here, we present a Bayesian hierarchical framework based on a beta-binomial mixture model for testing for differential biomarker expression using single-cell assays. Our model allows the inference to be subject specific, as is typically required when assessing vaccine responses, while borrowing strength across subjects through common prior distributions. We propose two approaches for parameter estimation: an empirical-Bayes approach using an Expectation-Maximization algorithm and a fully Bayesian one based on a Markov chain Monte Carlo algorithm. We compare our method against classical approaches for single-cell assays including Fisher's exact test, a likelihood ratio test, and basic log-fold changes. Using several experimental assays measuring proteins or genes at single-cell level and simulations, we show that our method has higher sensitivity and specificity than alternative methods. Additional simulations show that our framework is also robust to model misspecification. Finally, we demonstrate how our approach can be extended to testing multivariate differential expression across multiple biomarker combinations using a Dirichlet-multinomial model and illustrate this approach using single-cell gene

  20. Isolation of Treg cells and Treg cell suppression/death assay

    OpenAIRE

    sprotocols

    2015-01-01

    In vitro Treg suppression assays are performed to determine the functional effect of Treg cells on CD4 T cells. They are performed by co-culturing the responding population (Tresp) with the Treg cells or control CD4 cells (Tcon cells).

  1. Quality Assurance in the Polio Laboratory. Cell Sensitivity and Cell Authentication Assays.

    Science.gov (United States)

    Dunn, Glynis

    2016-01-01

    The accuracy of poliovirus surveillance is largely dependent on the quality of the cell lines used for virus isolation, which is the foundation of poliovirus diagnostic work. Many cell lines are available for the isolation of enteroviruses, whilst genetically modified L20B cells can be used as a diagnostic tool for the identification of polioviruses. To be confident that cells can consistently isolate the virus of interest, it is necessary to have a quality assurance system in place, which will ensure that the cells in use are not contaminated with other cell lines or microorganisms and that they remain sensitive to the viruses being studied.The sensitivity of cell lines can be assessed by the regular testing of a virus standard of known titer in the cell lines used for virus isolation. The titers obtained are compared to previously obtained titers in the same assay, so that any loss of sensitivity can be detected.However, the detection of cell line cross contamination is more difficult. DNA bar coding is a technique that uses a short DNA sequence from a standardized position in the genome as a molecular diagnostic assay for species-level identification. For almost all groups of higher animals, the cytochrome c oxidase subunit 1 of mitochondrial DNA (CO1) is emerging as the standard barcode region. This region is 648 nucleotide base pairs long in most phylogenetic groups and is flanked by regions of conserved sequences, making it relatively easy to isolate and analyze. DNA barcodes vary among individuals of the same species to a very minor degree (generally less than 1-2 %), and a growing number of studies have shown that the COI sequences of even closely related species differ by several per cent, making it possible to identify different species with high confidence. PMID:26983732

  2. Two novel nonradioactive polymerase chain reaction-based assays of dried blood spots, genomic DNA, or whole cells for fast, reliable detection of Z and S mutations in the alpha 1-antitrypsin gene

    DEFF Research Database (Denmark)

    Andresen, B S; Knudsen, I; Jensen, P K;

    1992-01-01

    Two new nonradioactive polymerase chain reaction (PCR)-based assays for the Z and S mutations in the alpha 1-antitrypsin gene are presented. The assays take advantage of PCR-mediated mutagenesis, creating new diagnostic restriction enzyme sites for unambiguous discrimination between test samples...... from individuals who are normal, heterozygous, or homozygous for the mutations. We show that the two assays can be performed with purified genomic DNA as well as with boiled blood spots. The new assays were validated by parallel testing with a technique in which PCR is combined with allele...

  3. Accuracy of the Fluorescence-Activated Cell Sorting Assay for the Aquaporin-4 Antibody (AQP4-Ab): Comparison with the Commercial AQP4-Ab Assay Kit

    Science.gov (United States)

    Kim, Yoo-Jin; Cheon, So Young; Kim, Boram; Jung, Kyeong Cheon; Park, Kyung Seok

    2016-01-01

    Background The aquaporin-4 antibody (AQP4-Ab) is a disease-specific autoantibody to neuromyelitis optica (NMO). We aimed to evaluate the accuracy of the FACS assay in detecting the AQP4-Ab compared with the commercial cell-based assay (C-CBA) kit. Methods Human embryonic kidney-293 cells were transfected with human aquaporin-4 (M23) cDNA. The optimal cut off values of FACS assay was tested using 1123 serum samples from patients with clinically definite NMO, those at high risk for NMO, patients with multiple sclerosis, patients with other idiopathic inflammatory demyelinating diseases, and negative controls. The accuracy of FACS assay and C-CBA were compared in consecutive 225 samples that were collected between January 2014 and June 2014. Results With a cut-off value of MFIi of 3.5 and MFIr of 2.0, the receiver operating characteristic curve for the FACS assay showed an area under the curve of 0.876. Among 225 consecutive sera, the FACS assay and C-CBA had a sensitivity of 77.3% and 69.7%, respectively, in differentiating the sera of definite NMO patients from sera of controls without IDD or of MS. Both assay had a good specificity of 100% in it. The overall positivity of the C-CBA among FACS-positive sera was 81.5%; moreover, its positivity was low as 50% among FACS-positive sera with relatively low MFIis. Conclusions Both the FACS assay and C-CBA are sensitive and highly specific assays in detecting AQP4-Ab. However, in some sera with relatively low antibody titer, FACS-assay can be a more sensitive assay option. In real practice, complementary use of FACS assay and C-CBA will benefit the diagnosis of NMO patients, because the former can be more sensitive among low titer sera and the latter are easier to use therefore can be widely used. PMID:27658059

  4. In-cell protease assay systems based on trans-localizing molecular beacon proteins using HCV protease as a model system.

    Directory of Open Access Journals (Sweden)

    Jeong Hee Kim

    Full Text Available This study describes a sensitive in-cell protease detection system that enables direct fluorescence detection of a target protease and its inhibition inside living cells. This live-cell imaging system provides a fluorescent molecular beacon protein comprised of an intracellular translocation signal sequence, a protease-specific cleavage sequence, and a fluorescent tag sequence(s. The molecular beacon protein is designed to change its intracellular localization upon cleavage by a target protease, i.e., from the cytosol to a subcellular organelle or from a subcellular organelle to the cytosol. Protease activity can be monitored at the single cell level, and accordingly the entire cell population expressing the protease can be accurately enumerated. The clear cellular change in fluorescence pattern makes this system an ideal tool for various life science and drug discovery research, including high throughput and high content screening applications.

  5. A Modified NK Cell Degranulation Assay Applicable for Routine Evaluation of NK Cell Function

    Science.gov (United States)

    Shabrish, Snehal; Gupta, Maya; Madkaikar, Manisha

    2016-01-01

    Natural killer (NK) cells play important role in innate immunity against tumors and viral infections. Studies show that lysosome-associated membrane protein-1 (LAMP-1, CD107a) is a marker for degranulation of NK and cytotoxic T cells and its expression is a sensitive marker for the cytotoxic activity determination. The conventional methods of determination of CD107a on NK cells involve use of peripheral blood mononuclear cells (PBMC) or pure NK cells and K562 cells as stimulants. Thus, it requires large volume of blood sample which is usually difficult to obtain in pediatric patients and patients with cytopenia and also requires specialized laboratory for maintaining cell line. We have designed a flow cytometric assay to determine CD107a on NK cells using whole blood, eliminating the need for isolation of PBMC or isolate NK cells. This assay uses phorbol-12-myristate-13-acetate (PMA) and calcium ionophore (Ca2+-ionophore) instead of K562 cells for stimulation and thus does not require specialized cell culture laboratory. CD107a expression on NK cells using modified NK cell degranulation assay compared to the conventional assay was significantly elevated (p < 0.0001). It was also validated by testing patients diagnosed with familial hemophagocytic lymphohistiocytosis (FHL) with defect in exocytosis. This assay is rapid, cost effective, and reproducible and requires significantly less volume of blood which is important for clinical evaluation of NK cells. PMID:27413758

  6. Development of a VHH-Based Erythropoietin Quantification Assay

    DEFF Research Database (Denmark)

    Kol, Stefan; Beuchert Kallehauge, Thomas; Adema, Simon;

    2015-01-01

    Erythropoietin (EPO) quantification during cell line selection and bioreactor cultivation has traditionally been performed with ELISA or HPLC. As these techniques suffer from several drawbacks, we developed a novel EPO quantification assay. A camelid single-domain antibody fragment directed against...

  7. Editor's Highlight: Analysis of the Effects of Cell Stress and Cytotoxicity on In Vitro Assay Activity Across a Diverse Chemical and Assay Space.

    Science.gov (United States)

    Judson, Richard; Houck, Keith; Martin, Matt; Richard, Ann M; Knudsen, Thomas B; Shah, Imran; Little, Stephen; Wambaugh, John; Woodrow Setzer, R; Kothya, Parth; Phuong, Jimmy; Filer, Dayne; Smith, Doris; Reif, David; Rotroff, Daniel; Kleinstreuer, Nicole; Sipes, Nisha; Xia, Menghang; Huang, Ruili; Crofton, Kevin; Thomas, Russell S

    2016-08-01

    Chemical toxicity can arise from disruption of specific biomolecular functions or through more generalized cell stress and cytotoxicity-mediated processes. Here, responses of 1060 chemicals including pharmaceuticals, natural products, pesticidals, consumer, and industrial chemicals across a battery of 815 in vitro assay endpoints from 7 high-throughput assay technology platforms were analyzed in order to distinguish between these types of activities. Both cell-based and cell-free assays showed a rapid increase in the frequency of responses at concentrations where cell stress/cytotoxicity responses were observed in cell-based assays. Chemicals that were positive on at least 2 viability/cytotoxicity assays within the concentration range tested (typically up to 100 μM) activated a median of 12% of assay endpoints whereas those that were not cytotoxic in this concentration range activated 1.3% of the assays endpoints. The results suggest that activity can be broadly divided into: (1) specific biomolecular interactions against one or more targets (eg, receptors or enzymes) at concentrations below which overt cytotoxicity-associated activity is observed; and (2) activity associated with cell stress or cytotoxicity, which may result from triggering specific cell stress pathways, chemical reactivity, physico-chemical disruption of proteins or membranes, or broad low-affinity non-covalent interactions. Chemicals showing a greater number of specific biomolecular interactions are generally designed to be bioactive (pharmaceuticals or pesticidal active ingredients), whereas intentional food-use chemicals tended to show the fewest specific interactions. The analyses presented here provide context for use of these data in ongoing studies to predict in vivo toxicity from chemicals lacking extensive hazard assessment. PMID:27208079

  8. Editor's Highlight: Analysis of the Effects of Cell Stress and Cytotoxicity on In Vitro Assay Activity Across a Diverse Chemical and Assay Space.

    Science.gov (United States)

    Judson, Richard; Houck, Keith; Martin, Matt; Richard, Ann M; Knudsen, Thomas B; Shah, Imran; Little, Stephen; Wambaugh, John; Woodrow Setzer, R; Kothya, Parth; Phuong, Jimmy; Filer, Dayne; Smith, Doris; Reif, David; Rotroff, Daniel; Kleinstreuer, Nicole; Sipes, Nisha; Xia, Menghang; Huang, Ruili; Crofton, Kevin; Thomas, Russell S

    2016-08-01

    Chemical toxicity can arise from disruption of specific biomolecular functions or through more generalized cell stress and cytotoxicity-mediated processes. Here, responses of 1060 chemicals including pharmaceuticals, natural products, pesticidals, consumer, and industrial chemicals across a battery of 815 in vitro assay endpoints from 7 high-throughput assay technology platforms were analyzed in order to distinguish between these types of activities. Both cell-based and cell-free assays showed a rapid increase in the frequency of responses at concentrations where cell stress/cytotoxicity responses were observed in cell-based assays. Chemicals that were positive on at least 2 viability/cytotoxicity assays within the concentration range tested (typically up to 100 μM) activated a median of 12% of assay endpoints whereas those that were not cytotoxic in this concentration range activated 1.3% of the assays endpoints. The results suggest that activity can be broadly divided into: (1) specific biomolecular interactions against one or more targets (eg, receptors or enzymes) at concentrations below which overt cytotoxicity-associated activity is observed; and (2) activity associated with cell stress or cytotoxicity, which may result from triggering specific cell stress pathways, chemical reactivity, physico-chemical disruption of proteins or membranes, or broad low-affinity non-covalent interactions. Chemicals showing a greater number of specific biomolecular interactions are generally designed to be bioactive (pharmaceuticals or pesticidal active ingredients), whereas intentional food-use chemicals tended to show the fewest specific interactions. The analyses presented here provide context for use of these data in ongoing studies to predict in vivo toxicity from chemicals lacking extensive hazard assessment.

  9. Comparison of two rapid assays for Clostridium difficile Common antigen and a C difficile toxin A/B assay with the cell culture neutralization assay.

    Science.gov (United States)

    Reller, Megan E; Alcabasa, Romina C; Lema, Clara A; Carroll, Karen C

    2010-01-01

    We compared 3 rapid assays for Clostridium difficile with a cell culture cytotoxicity neutralization assay (CCNA). Of 600 stool samples, 46 were positive for toxigenic C difficile. Both rapid common antigen assays were highly sensitive (91.3%-100%) and, therefore, were appropriate screening tests. The rapid toxin assay had poor sensitivity (61%) but excellent specificity (99.3%). Testing stools for glutamate dehydrogenase (step 1) and those positive with a rapid toxin assay (step 2) would correctly classify 81% of submitted specimens within 2 hours, including during periods of limited staffing (evenings, nights, and weekends). CCNA could then be used as a third step to test rapid toxin-negative samples, thereby providing a final result for the remaining 19% of samples by 48 to 72 hours. The use of rapid assays as outlined could enhance timely diagnosis of C difficile. PMID:20023265

  10. Comparison of two rapid assays for Clostridium difficile Common antigen and a C difficile toxin A/B assay with the cell culture neutralization assay.

    Science.gov (United States)

    Reller, Megan E; Alcabasa, Romina C; Lema, Clara A; Carroll, Karen C

    2010-01-01

    We compared 3 rapid assays for Clostridium difficile with a cell culture cytotoxicity neutralization assay (CCNA). Of 600 stool samples, 46 were positive for toxigenic C difficile. Both rapid common antigen assays were highly sensitive (91.3%-100%) and, therefore, were appropriate screening tests. The rapid toxin assay had poor sensitivity (61%) but excellent specificity (99.3%). Testing stools for glutamate dehydrogenase (step 1) and those positive with a rapid toxin assay (step 2) would correctly classify 81% of submitted specimens within 2 hours, including during periods of limited staffing (evenings, nights, and weekends). CCNA could then be used as a third step to test rapid toxin-negative samples, thereby providing a final result for the remaining 19% of samples by 48 to 72 hours. The use of rapid assays as outlined could enhance timely diagnosis of C difficile.

  11. Medically Relevant Assays with a Simple Smartphone and Tablet Based Fluorescence Detection System

    Directory of Open Access Journals (Sweden)

    Piotr Wargocki

    2015-05-01

    Full Text Available Cell phones and smart phones can be reconfigured as biomedical sensor devices but this requires specialized add-ons. In this paper we present a simple cell phone-based portable bioassay platform, which can be used with fluorescent assays in solution. The system consists of a tablet, a polarizer, a smart phone (camera and a box that provides dark readout conditions. The assay in a well plate is placed on the tablet screen acting as an excitation source. A polarizer on top of the well plate separates excitation light from assay fluorescence emission enabling assay readout with a smartphone camera. The assay result is obtained by analysing the intensity of image pixels in an appropriate colour channel. With this device we carried out two assays, for collagenase and trypsin using fluorescein as the detected fluorophore. The results of collagenase assay with the lowest measured concentration of 3.75 µg/mL and 0.938 µg in total in the sample were comparable to those obtained by a microplate reader. The lowest measured amount of trypsin was 930 pg, which is comparable to the low detection limit of 400 pg for this assay obtained in a microplate reader. The device is sensitive enough to be used in point-of-care medical diagnostics of clinically relevant conditions, including arthritis, cystic fibrosis and acute pancreatitis.

  12. Demonstration of the dynamic mass redistribution label-free technology as a useful cell-based pharmacological assay for endogenously expressed GABAA receptors

    DEFF Research Database (Denmark)

    Klein, Anders Bue; Nittegaard-Nielsen, Mia; Christensen, Julie T.;

    2015-01-01

    the immortalized IMR-32 neuroblastoma cell line, which expresses relatively high levels of several endogenous GABAA receptor subunits, we show that GABA produces concentration-dependent cellular responses that can be measured and quantified in real-time. With the aid of the GABAA receptor-specific agonist muscimol...

  13. Detection of hypoxic cells in murine tumors using the comet assay. Comparison with a conventional radiobiological assay

    International Nuclear Information System (INIS)

    The comet (single-cell electrophoresis) assay has been developed as a method for measuring DNA damage in single cells after irradiation. We have developed our own methods and image analysis system for the comet assay to identify hypoxic fractions. In vitro, we tested our system using a cultured tumor cell line (SCCVII). In vivo, we compared the hypoxic fractions detected by this assay with those determined by the in vivo-in vitro clonogenic assay using two rodent tumors (SCCVII/C3H, EMT6/KU/balb/c), which exhibit different types of hypoxia: acute and chronic. In vitro, our method could differentiate hypoxic cells from oxic cells, using the parameter of tail moment. In vivo, there were good correlations between the hypoxic fractions determined by the comet assay and by the clonogenic assay, in SCCVII/C3H (r=0.85) and in EMT6/KU/balb/c (r=0.75) tumors. By comparison of the two methods in chronically hypoxic and acutely hypoxic tumors, we further confirmed that the comet assay is clinically useful for estimating hypoxic fractions of solid tumors. (author)

  14. A High-Throughput Assay for Rho Guanine Nucleotide Exchange Factors Based on the Transcreener GDP Assay.

    Science.gov (United States)

    Reichman, Melvin; Schabdach, Amanda; Kumar, Meera; Zielinski, Tom; Donover, Preston S; Laury-Kleintop, Lisa D; Lowery, Robert G

    2015-12-01

    Ras homologous (Rho) family GTPases act as molecular switches controlling cell growth, movement, and gene expression by cycling between inactive guanosine diphosphate (GDP)- and active guanosine triphosphate (GTP)-bound conformations. Guanine nucleotide exchange factors (GEFs) positively regulate Rho GTPases by accelerating GDP dissociation to allow formation of the active, GTP-bound complex. Rho proteins are directly involved in cancer pathways, especially cell migration and invasion, and inhibiting GEFs holds potential as a therapeutic strategy to diminish Rho-dependent oncogenesis. Methods for measuring GEF activity suitable for high-throughput screening (HTS) are limited. We developed a simple, generic biochemical assay method for measuring GEF activity based on the fact that GDP dissociation is generally the rate-limiting step in the Rho GTPase catalytic cycle, and thus addition of a GEF causes an increase in steady-state GTPase activity. We used the Transcreener GDP Assay, which relies on selective immunodetection of GDP, to measure the GEF-dependent stimulation of steady-state GTP hydrolysis by small GTPases using Dbs (Dbl's big sister) as a GEF for Cdc42, RhoA, and RhoB. The assay is well suited for HTS, with a homogenous format and far red fluorescence polarization (FP) readout, and it should be broadly applicable to diverse Rho GEF/GTPase pairs.

  15. Identification of small molecule agonists of human relaxin family receptor 1 (RXFP1) by utilizing a homogenous cell-based cAMP assay

    OpenAIRE

    Chen, Catherine Z.; Southall, Noel; Xiao, Jingbo; Marugan, Juan J; Ferrer, Marc; Hu, Xin; Jones, Raisa E.; Feng, Shu; Agoulnik, Irina U; Zheng, Wei; Agoulnik, Alexander I.

    2012-01-01

    The relaxin hormone is involved in a variety of biological functions including female reproduction and parturition, regulation of cardiovascular, renal, pulmonary, and hepatic functions. It regulates extracellular matrix remodeling, cell invasiveness, proliferation, differentiation, and overall tissue homeostasis. The G protein-coupled receptor (GPCR) RXFP1, relaxin family receptor 1, is a cognate relaxin receptor that mainly signals through cyclic AMP second messenger. While agonists of the ...

  16. Towards a high throughput droplet-based agglutination assay

    KAUST Repository

    Kodzius, Rimantas

    2013-10-22

    This work demonstrates the detection method for a high throughput droplet based agglutination assay system. Using simple hydrodynamic forces to mix and aggregate functionalized microbeads we avoid the need to use magnetic assistance or mixing structures. The concentration of our target molecules was estimated by agglutination strength, obtained through optical image analysis. Agglutination in droplets was performed with flow rates of 150 µl/min and occurred in under a minute, with potential to perform high-throughput measurements. The lowest target concentration detected in droplet microfluidics was 0.17 nM, which is three orders of magnitude more sensitive than a conventional card based agglutination assay.

  17. A highly scalable peptide-based assay system for proteomics.

    Directory of Open Access Journals (Sweden)

    Igor A Kozlov

    Full Text Available We report a scalable and cost-effective technology for generating and screening high-complexity customizable peptide sets. The peptides are made as peptide-cDNA fusions by in vitro transcription/translation from pools of DNA templates generated by microarray-based synthesis. This approach enables large custom sets of peptides to be designed in silico, manufactured cost-effectively in parallel, and assayed efficiently in a multiplexed fashion. The utility of our peptide-cDNA fusion pools was demonstrated in two activity-based assays designed to discover protease and kinase substrates. In the protease assay, cleaved peptide substrates were separated from uncleaved and identified by digital sequencing of their cognate cDNAs. We screened the 3,011 amino acid HCV proteome for susceptibility to cleavage by the HCV NS3/4A protease and identified all 3 known trans cleavage sites with high specificity. In the kinase assay, peptide substrates phosphorylated by tyrosine kinases were captured and identified by sequencing of their cDNAs. We screened a pool of 3,243 peptides against Abl kinase and showed that phosphorylation events detected were specific and consistent with the known substrate preferences of Abl kinase. Our approach is scalable and adaptable to other protein-based assays.

  18. Identification of a new plant extract for androgenic alopecia treatment using a non-radioactive human hair dermal papilla cell-based assay

    OpenAIRE

    Jain, Ruchy; Monthakantirat, Orawan; Tengamnuay, Parkpoom; De-Eknamkul, Wanchai

    2016-01-01

    Background Androgenic alopecia (AGA) is a major type of human scalp hair loss, which is caused by two androgens: testosterone (T) and 5α-dihydrotestosterone (5α-DHT). Both androgens bind to the androgen receptor (AR) and induce androgen-sensitive genes within the human hair dermal papilla cells (HHDPCs), but 5α-DHT exhibits much higher binding affinity and potency than T does in inducing the involved androgen-sensitive genes. Changes in the induction of androgen-sensitive genes during AGA are...

  19. Micro-fluidic module for blood cell separation for gene expression radiobiological assays

    International Nuclear Information System (INIS)

    Advances in molecular techniques have improved discovery of biomarkers associated with radiation exposure. Gene expression techniques have been demonstrated as effective tools for biodosimetry, and different assay platforms with different chemistries are now available. One of the main challenges is to integrate the sample preparation processing of these assays into micro-fluidic platforms to be fully automated for point-of-care medical countermeasures in the case of a radiological event. Most of these assays follow the same workflow processing that comprises first the collection of blood samples followed by cellular and molecular sample preparation. The sample preparation is based on the specific reagents of the assay system and depends also on the different subsets of cells population and the type of biomarkers of interest. In this article, the authors present a module for isolation of white blood cells from peripheral blood as a prerequisite for automation of gene expression assays on a micro-fluidic cartridge. For each sample condition, the gene expression platform can be adapted to suit the requirements of the selected assay chemistry (authors)

  20. Evaluation of 1066 ToxCast Chemicals in a human stem cell assay for developmental toxicity (SOT)

    Science.gov (United States)

    To increase the diversity of assays used to assess potential developmental toxicity, the ToxCast chemical library was screened in the Stemina devTOX quickPREDICT assay using human embryonic stem (hES) cells. A model for predicting teratogenicity was based on a training set of 23 ...

  1. Sorting of cells of the same size, shape, and cell cycle stage for a single cell level assay without staining

    Directory of Open Access Journals (Sweden)

    Yomo Tetsuya

    2006-06-01

    Full Text Available Abstract Background Single-cell level studies are being used increasingly to measure cell properties not directly observable in a cell population. High-performance data acquisition systems for such studies have, by necessity, developed in synchrony. However, improvements in sample purification techniques are also required to reveal new phenomena. Here we assessed a cell sorter as a sample-pretreatment tool for a single-cell level assay. A cell sorter is routinely used for selecting one type of cells from a heterogeneous mixture of cells using specific fluorescence labels. In this case, we wanted to select cells of exactly the same size, shape, and cell-cycle stage from a population, without using a specific fluorescence label. Results We used four light scatter parameters: the peak height and area of the forward scatter (FSheight and FSarea and side scatter (SSheight and SSarea. The rat pheochromocytoma PC12 cell line, a neuronal cell line, was used for all experiments. The living cells concentrated in the high FSarea and middle SSheight/SSarea fractions. Single cells without cell clumps were concentrated in the low SS and middle FS fractions, and in the higher FSheight/FSarea and SSheight/SSarea fractions. The cell populations from these viable, single-cell-rich fractions were divided into twelve subfractions based on their FSarea-SSarea profiles, for more detailed analysis. We found that SSarea was proportional to the cell volume and the FSarea correlated with cell roundness and elongation, as well as with the level of DNA in the cell. To test the method and to characterize the basic properties of the isolated single cells, sorted cells were cultured in separate wells. The cells in all subfractions survived, proliferated and differentiated normally, suggesting that there was no serious damage. The smallest, roundest, and smoothest cells had the highest viability. There was no correlation between proliferation and differentiation. NGF increases

  2. An agonist sensitive, quick and simple cell-based signaling assay for determination of ligands mimicking prostaglandin E2 or E1 activity through subtype EP1 receptor: Suitable for high throughput screening

    Directory of Open Access Journals (Sweden)

    Ruan Ke-He

    2011-02-01

    Full Text Available Abstract Background Conventionally the active ingredients in herbal extracts are separated into individual components, by fractionation, desalting, and followed by high-performance liquid chromatography (HPLC. In this study we have tried to directly screen water-soluble fractions of herbs with potential active ingredients before purification or extraction. We propose that the herbal extracts mimicking prostaglandin E1 (PGE1 and E2 (PGE2 can be identified in the water-soluble non-purified fraction. PGE1 is a potent anti-inflammatory molecule used for treating peripheral vascular diseases while PGE2 is an inflammatory molecule. Methods We used cell-based assays (CytoFluor multi-well plate reader and fluorescence microscopy in which a calcium signal was generated by the recombinant EP1 receptor stably expressed in HEK293 cells (human embryonic kidney. PGE1 and PGE2 were tested for their ability to generate a calcium signal. Ninety-six water soluble fractions of Treasures of the east (single Chinese herb dietary supplements were screened. Results After screening, the top ten stimulators were identified. The identified herbs were then desalted and the calcium fluorescent signal reconfirmed using fluorescence microscopy. Among these top ten agonists identified, seven stimulated the calcium signaling (1-40 μM concentration using fluorescence microscopy. Conclusions Fluorescence microscopy and multi-well plate readers can be used as a target specific method for screening water soluble fractions with active ingredients at a very early stage, before purification. Our future work consists of purifying and separating the active ingredients and repeating fluorescence microscopy. Under ordinary circumstances we would have to purify the compounds first and then test all the extracts from 96 herbs. Conventionally, for screening natural product libraries, the procedure followed is the automated separation of all constituents into individual components using

  3. A homogeneous time-resolved fluorescence resonance energy transfer assay for phosphatidylserine exposure on apoptotic cells.

    Science.gov (United States)

    Gasser, Jean-Philippe; Hehl, Michaela; Millward, Thomas A

    2009-01-01

    A simple, "mix-and-measure" microplate assay for phosphatidylserine (PtdSer) exposure on the surface of apoptotic cells is described. The assay exploits the fact that annexin V, a protein with high affinity and specificity for PtdSer, forms trimers and higher order oligomers on binding to membranes containing PtdSer. The transition from soluble monomer to cell-bound oligomer is detected using time-resolved fluorescence resonance energy transfer from europium chelate-labeled annexin V to Cy5-labeled annexin V. PtdSer detection is achieved by a single addition of a reagent mix containing labeled annexins and calcium ions directly to cell cultures in a 96-well plate, followed by a brief incubation before fluorescence measurement. The assay can be used to quantify PtdSer exposure on both suspension cells and adherent cells in situ. This method is simpler and faster than existing annexin V binding assays based on flow cytometry or microscopy, and it yields precise data with Z' values of 0.6-0.7. PMID:18835236

  4. Single Cell Gel Electrophoresis Assay of Porcine Leydig Cell DNA Damage Induced by Zearalenone

    Institute of Scientific and Technical Information of China (English)

    Jianwei ZHEN; Qincl LIU; Jianhong GU; Yan YUAN; Xuezhong LIU; Handong WANG; Zongping LIU; Jianchun BIAN

    2012-01-01

    Abstract [Objective] This study aimed to investigate the effect of zearalenone (ZEN) on DNA damage of porcine leydig cells. [Method] Porcine leydig cells cultured in vitro were collected to determine the median lethal dose (LD~o) of ZEN with tetra- zolium-based colorimetric assay (MTT assay). Comet assay was carried out to de- tect the DNA damage of porcine leydig cells exposed to at 0 (negative group), 1, 5, 10, 20, 40 tJmol/L of ZEN. [Result] The percentage of cell tail was 16.67%, 34.00%, 40.67%. 52.00% and 64.67% under 0, 1, 5, 10 and 20 ~mo~/L o~ ZEN, respectively; the differences between the percentages of celt tail in various experimental groups had extremely significant statistical significance compared with the negative group (P〈0.01), showing a significant dose-effect relationship; Tail length in various groups was 57.60_+4.78, 57.75_+6.25, 78.97_+5.83, 100.50~6.94 and 146.83_+12.31 ~m, re- spectively; Tail DNA % in various groups was 21.29_+2.25%, 22.24_+2.43%, 31.21_+ 6.27%, 37.45_+4.33% and 60.68_+9.83%, respectively; Tail length and Tail DNA % in experimental groups with ZEN concentration above 5 ~mo~/L showed significant dif- ferences (P〈0.05) compared with the negative group, which showed an upward trend with the increase of ZEN concentration. [Conclusion] ZEN has genotoxic effect on porcine leydig cells, which can cause DNA damage, with a significant dose-effect relationship.

  5. Bioanalytical method transfer considerations of chromatographic-based assays.

    Science.gov (United States)

    Williard, Clark V

    2016-07-01

    Bioanalysis is an important part of the modern drug development process. The business practice of outsourcing and transferring bioanalytical methods from laboratory to laboratory has increasingly become a crucial strategy for successful and efficient delivery of therapies to the market. This chapter discusses important considerations when transferring various types of chromatographic-based assays in today's pharmaceutical research and development environment. PMID:27277876

  6. HIV-1 Reverse Transcriptase based assay to determine cellular dNTP concentrations

    Science.gov (United States)

    Hollenbaugh, Joseph A.; Kim, Baek

    2016-01-01

    Summary Deoxynucleoside triphosphates (dNTPs) are the building blocks of DNA and their biosynthesis are tightly regulated in the cell. HPLC-MS and enzyme-based methods are currently employed to determine dNTP concentrations from cellular extracts. Here, we describe a highly efficient, HIV-1 reverse transcriptase (RT)-based assay to quantitate dNTP concentrations. The assay is based on the ability of HIV-1 RT to function at very low dNTP concentrations, thus providing for the high sensitivity of detection. PMID:26714705

  7. A prospective longitudinal study evaluating a T-cell-based assay for latent tuberculosis infection in health-care workers in a general hospital in Beijing

    Institute of Scientific and Technical Information of China (English)

    ZHANG Li-fan; LIU Xiao-qing; ZHANG Yao; DENG Guo-hua; Manish Pareek; Ajit Lalvani

    2013-01-01

    Background The health-care workers (HCWs) are at high risk of acquiring infection with Mycobacterium tuberculosis.The objectives of this study were to compare the performance of the T-SPOT.TB and tuberculin skin test (TST) for latent tuberculosis infection (LTBI),evaluate diagnostic concordance and risk factors for LTBI,and observe the progression to active tuberculosis (TB) disease among HCWs in a general hospital in Beijing.Methods The prospective cohort study enrolled HCWs in a tertiary general hospital in Beijing,China,to evaluate LTBI with T-SPOT.TB and TST.The subjects were evaluated every 12 months during the 60-month follow-up.Results Of 101 participating HCWs,96 and 101 had valid TST and T-SPOT.TB results,respectively.Twenty-nine (28.7%,95% confidence interval (CI),19.9%-37.5%) were defined as positive by T-SPOT.TB and 53 (55.2%,95% CI,45.2%-64.9%) were defined as positive by TST (using a ≥10 mm cutoff).An agreement between the two tests was poor (57.3%,K=0.18,95% CI,0.01%-0.52%).In multivariate analysis,direct exposure to sputum smear-positive TB patients was a significant risk factor for a positive T-SPOT.TB (OR 5.76; 95% CI 1.38-24.00).Pooled frequency of antigenspecific IFN-γ secreting T-cells for subjects who reported direct contact with sputum smear-positive TB patients was significantly higher than that for participants without direct contact (P=-0.045).One of 20 participants with positive result of T-SPOT.TB and TST developed active TB at 24-month follow-up.Conclusion T-SPOT.TB is a more accurate,targeted method of diagnosing LTBI than TST.

  8. An automated cell-counting algorithm for fluorescently-stained cells in migration assays

    Directory of Open Access Journals (Sweden)

    Novielli Nicole M

    2011-10-01

    Full Text Available Abstract A cell-counting algorithm, developed in Matlab®, was created to efficiently count migrated fluorescently-stained cells on membranes from migration assays. At each concentration of cells used (10,000, and 100,000 cells, images were acquired at 2.5 ×, 5 ×, and 10 × objective magnifications. Automated cell counts strongly correlated to manual counts (r2 = 0.99, P

  9. Development of fluorescence-based high-throughput screening assays: choice of appropriate instrumentation

    Science.gov (United States)

    Burns, David J.; Alder, Elisabeth; Fan, Yi-Hong; McKeegan, Evelyn; Warrior, Usha; Beutel, Bruce

    1998-04-01

    Fluorescence-based assays have become increasingly popular in high throughput screening for a variety of reasons (e.g. sensitivity). However, new screening technologies are pushing the limits of conventional fluorescence plate readers. For example, instruments that have optical sensitivities beyond most of the commercially available plate readers are required to reproducibly measure the fluorescence generated by the green fluorescent protein (GFP)--a novel reporter gene. Also, miniaturization of screening formats (with densities higher than the conventional 96-well plate) requires high resolution instrumentation to measure fluorescence. Several assays based on optical fluorescence measurements have been developed and screened in our Biological Screening group. These assays include various fluorescence-based protease assays (standard end-point and kinetic modes) and a functional cell-based screen using the green fluorescent protein as a reporter gene. The choice of instrumentation was the critical factor in the performance and success of each of these arrays. Data will be presented for the cell- based reporter assay including the type of instrumentation (fluorescence plate readers; fluorescence imaging systems) used for detection of GFP fluorescence.

  10. An assay for secologanin in plant tissues based on enzymatic conversion into strictosidine

    DEFF Research Database (Denmark)

    Hallard, Didier; van der Heijden, Robert; Contin, Adriana;

    1998-01-01

    The secoiridoid glucoside secologanin is the terpenoid building block in the biosynthesis of terpenoid indole alkaloids. A method for its determination in plant tissues and cell suspension cultures has been developed. This assay is based on the condensation of secologanin with tryptamine, yieldin...

  11. A rapid and robust assay for detection of S-phase cell cycle progression in plant cells and tissues by using ethynyl deoxyuridine

    Directory of Open Access Journals (Sweden)

    Horváth Gábor V

    2010-01-01

    Full Text Available Abstract Background Progress in plant cell cycle research is highly dependent on reliable methods for detection of cells replicating DNA. Frequency of S-phase cells (cells in DNA synthesis phase is a basic parameter in studies on the control of cell division cycle and the developmental events of plant cells. Here we extend the microscopy and flow cytometry applications of the recently developed EdU (5-ethynyl-2'-deoxyuridine-based S-phase assay to various plant species and tissues. We demonstrate that the presented protocols insure the improved preservation of cell and tissue structure and allow significant reduction in assay duration. In comparison with the frequently used detection of bromodeoxyuridine (BrdU and tritiated-thymidine incorporation, this new methodology offers several advantages as we discuss here. Results Applications of EdU-based S-phase assay in microscopy and flow cytometry are presented by using cultured cells of alfalfa, Arabidopsis, grape, maize, rice and tobacco. We present the advantages of EdU assay as compared to BrdU-based replication assay and demonstrate that EdU assay -which does not require plant cell wall digestion or DNA denaturation steps, offers reduced assay duration and better preservation of cellular, nuclear and chromosomal morphologies. We have also shown that fast and efficient EdU assay can also be an efficient tool for dual parameter flow cytometry analysis and for quantitative assessment of replication in thick root samples of rice. Conclusions In plant cell cycle studies, EdU-based S-phase detection offers a superior alternative to the existing S-phase assays. EdU method is reliable, versatile, fast, simple and non-radioactive and it can be readily applied to many different plant systems.

  12. The quantitation of human growth hormone by a radioreceptor assay using an established human cell line

    International Nuclear Information System (INIS)

    Membrane receptors on cultured human lymphocytes (IM-9) have been shown to bind human growth hormone (hGH) in a specific manner. The aim of the present study was to develop an in vitro assay of hGH based on this binding. The binding of [125I]hGH was studied as a function of time, temperature, cell density, tracer concentration and the concentration of unlabelled hGH and other related hormones. Also, the dissociation of bound hGH and the chemical stability of hGH in the incubation medium were studied. From these studies, the conditions for an appropriate radioreceptor assay were determined. Briefly, 1.5-3.0 x 107 cells ml-1 were incubated with 5-20 x 10-12 M [125I]hGH and three different concentrations of unlabelled hGH chosen from the linear part of the [125I]hGH displacement curve. The results were analyzed according to general pharmacopoeial principles. The mean values for growth hormone activity tested by radioreceptor assay were within the fiducial limits (P = 0.05) of the corresponding activity determined by the hypophysectomized rat body-weight gain assay. The in vitro assay was found to be more precise and less resource demanding than the in vivo bioassay of hGH. It is concluded that the in vitro bioassay described here is well suited as a screening method for potency determination of hGH preparations. (author)

  13. An Enhanced ELISPOT Assay for Sensitive Detection of Antigen-Specific T Cell Responses to Borrelia burgdorferi

    OpenAIRE

    Kellermann, Gottfried H.; Lehmann, Paul V; Diana R. Roen; Chenggang Jin

    2013-01-01

    Lyme Borreliosis is an infectious disease caused by the spirochete Borrelia burgdorferi that is transmitted through the bite of infected ticks. Both B cell-mediated humoral immunity and T cell immunity develop during natural Borrelia infection. However, compared with humoral immunity, the T cell response to Borrelia infection has not been well elucidated. In this study, a novel T cell-based assay was developed and validated for the sensitive detection of antigen-specific T cell response to B....

  14. Quality Control Assays for Clinical-Grade Human Mesenchymal Stromal Cells: Methods for ATMP Release.

    Science.gov (United States)

    Radrizzani, Marina; Soncin, Sabrina; Lo Cicero, Viviana; Andriolo, Gabriella; Bolis, Sara; Turchetto, Lucia

    2016-01-01

    Mesenchymal stromal/stem cells (MSC) are promising candidates for the development of cell-based therapies for various diseases and are currently being evaluated in a number of clinical trials (Sharma et al., Transfusion 54:1418-1437, 2014; Ikebe and Suzuki, Biomed Res Int 2014:951512, 2014). MSC for therapeutic applications are classified as advanced therapy medicinal products (ATMP) (Regulation (EC) No 1394/2007 of the European Parliament and of the Council of 13 November 2007 on advanced therapy medicinal products and amending Directive 2001/83/EC and Regulation (EC) No 726/2004) and must be prepared according to good manufacturing practices ( http://ec.europa.eu/health/documents/eudralex/vol-4 ). They may be derived from different starting materials (mainly bone marrow (BM), adipose tissue, or cord blood) and applied as fresh or cryopreserved products, in the autologous as well as an allogeneic context (Sharma et al., Transfusion 54:1418-1437, 2014; Ikebe and Suzuki, Biomed Res Int 2014:951512, 2014; Sensebé and Bourin, Transplantation 87(9 Suppl):S49-S53, 2009). In any case, they require an approved and well-defined panel of assays in order to be released for clinical use.This chapter describes analytical methods implemented and performed in our cell factory as part of the release strategy for an ATMP consisting of frozen autologous BM-derived MSC. Such methods are designed to assess the safety (sterility, endotoxin, and mycoplasma assays) and identity/potency (cell count and viability, immunophenotype and clonogenic assay) of the final product. Some assays are also applied to the biological starting material (sterility) or carried out as in-process controls (sterility, cell count and viability, immunophenotype, clonogenic assay).The validation strategy for each analytical method is described in the accompanying Chapter 20 . PMID:27236681

  15. Quality Control Assays for Clinical-Grade Human Mesenchymal Stromal Cells: Methods for ATMP Release.

    Science.gov (United States)

    Radrizzani, Marina; Soncin, Sabrina; Lo Cicero, Viviana; Andriolo, Gabriella; Bolis, Sara; Turchetto, Lucia

    2016-01-01

    Mesenchymal stromal/stem cells (MSC) are promising candidates for the development of cell-based therapies for various diseases and are currently being evaluated in a number of clinical trials (Sharma et al., Transfusion 54:1418-1437, 2014; Ikebe and Suzuki, Biomed Res Int 2014:951512, 2014). MSC for therapeutic applications are classified as advanced therapy medicinal products (ATMP) (Regulation (EC) No 1394/2007 of the European Parliament and of the Council of 13 November 2007 on advanced therapy medicinal products and amending Directive 2001/83/EC and Regulation (EC) No 726/2004) and must be prepared according to good manufacturing practices ( http://ec.europa.eu/health/documents/eudralex/vol-4 ). They may be derived from different starting materials (mainly bone marrow (BM), adipose tissue, or cord blood) and applied as fresh or cryopreserved products, in the autologous as well as an allogeneic context (Sharma et al., Transfusion 54:1418-1437, 2014; Ikebe and Suzuki, Biomed Res Int 2014:951512, 2014; Sensebé and Bourin, Transplantation 87(9 Suppl):S49-S53, 2009). In any case, they require an approved and well-defined panel of assays in order to be released for clinical use.This chapter describes analytical methods implemented and performed in our cell factory as part of the release strategy for an ATMP consisting of frozen autologous BM-derived MSC. Such methods are designed to assess the safety (sterility, endotoxin, and mycoplasma assays) and identity/potency (cell count and viability, immunophenotype and clonogenic assay) of the final product. Some assays are also applied to the biological starting material (sterility) or carried out as in-process controls (sterility, cell count and viability, immunophenotype, clonogenic assay).The validation strategy for each analytical method is described in the accompanying Chapter 20 .

  16. Immunological assays for chemokine detection in in-vitro culture of CNS cells

    Directory of Open Access Journals (Sweden)

    Mahajan Supriya D.

    2003-01-01

    Full Text Available Herein we review the various methods currently in use for determining the expression of chemokines by CNS cells in vitro. Chemokine detection assays are used in conjuction with one another to provide a comprehensive, biologically relevant assessment of the chemokines which is necessary for correct data interpretation of a specific observed biological effect. The methods described include bioassays for soluble chemokine receptors, RNA extraction, RT-PCR, Real - time quantitative PCR, gene array analysis, northern blot analysis, Ribonuclease Protection assay, Flow cytometry, ELISPOT, western blot analysis, and ELISA. No single method of analysis meets the criteria for a comprehensive, biologically relevant assessment of the chemokines, therefore more than one assay might be necessary for correct data interpretation, a choice that is based on development of a scientific rationale for the method with emphasis on the reliability and relevance of the method.

  17. DNA damage in lung cells after radon exposure detected by comet assay

    International Nuclear Information System (INIS)

    The comet assay was applied to measure DNA breaks and oxidised bases in isolated alveolar macrophages and epithelial type II cells from the rat lung. The cells were exposed to radon for 60 min. Radon exposure was estimated at (1.25 - 2.45) MBq.h.m-3. Strand breaks were significantly elevated above the background level after irradiation of epithelial type II cells. In contrast, no strand breaks were induced in alveolar macrophages, but a high level of oxidised bases, mostly purines, was found. Alveolar macrophages and epithelial type II cells isolated from the rat lung provide and exceptionally suitable cell model for investigation of potential hazards of air-born environmental contaminants. (authors)

  18. Identifying Intracellular Sites of Eicosanoid Lipid Mediator Synthesis with EicosaCell Assays

    OpenAIRE

    Bandeira-Melo, Christianne; Weller, Peter Fahey; Bozza, Patricia T.

    2011-01-01

    Eicosanoids, arachidonic acid-derived signaling lipid mediators, are newly formed and nonstorable molecules that have important roles in physiological and pathological processes. EicosaCell is a microscopic assay that enables the intracellular detection and localization of eicosanoid lipid mediator-synthesizing compartments by means of a strategy to covalently cross-link and immobilize eicosanoids at their sites of synthesis followed by immunofluorescent-based localization of the targeted eic...

  19. Pseudotype-based neutralization assays for influenza: a systematic analysis

    Directory of Open Access Journals (Sweden)

    George William Carnell

    2015-04-01

    Full Text Available The use of vaccination against the influenza virus remains the most effective method of mitigating the significant morbidity and mortality caused by this virus. Antibodies elicited by currently licensed influenza vaccines are predominantly hemagglutination-inhibition (HI-competent antibodies that target the globular head of HA thus inhibiting influenza virus entry into target cells. These antibodies predominantly confer homosubtypic/strain specific protection and only rarely confer heterosubtypic protection. However, recent academia or pharma-led R&D towards the production of a universal vaccine has centered on the elicitation of antibodies directed against the stalk of the influenza HA that has been shown to confer broad protection across a range of different subtypes (H1 to H16. The accurate and sensitive measurement of antibody responses elicited by these next-generation influenza vaccines is however hampered by the lack of sensitivity of the traditional influenza serological assays hemagglutinin inhibition (HI, single radial hemolysis (SRH and microneutralization (MN. Assays utilizing pseudotypes, chimeric viruses bearing influenza glycoproteins, have been shown to be highly efficient for the measurement of homosubtypic and heterosubtypic broadly-neutralizing antibodies, making them ideal serological tools for the study of cross-protective responses against multiple influenza subtypes with pandemic potential. In this review, we will analyze and compare literature involving the production of influenza pseudotypes with particular emphasis on their use in serum antibody neutralization assays. This will enable us to establish the parameters required for optimization and propose a consensus protocol to be employed for the further deployment of these assays in influenza vaccine immunogenicity studies.

  20. Preliminary Validation of Tumor Cell Attachment Inhibition Assay for Developmental Toxicants With Mouse S180 Cells

    Institute of Scientific and Technical Information of China (English)

    LU RONG-ZHU; CHEN CHUAN-FEN; LIN HUI-FEN; HUANG LEI-MING; JIN Xl-PENG

    1999-01-01

    This study was designed to explore the possibility of using ascitic mouse sarcoma cell line(S180) to validate the mouse tumor cell attachment assay for developmental toxicants, and to test the inhibitory effects of various developmental toxicants. The results showed that 2 of 3 developmental toxicants under consideration, sodium pentobarbital and ethanol, significantly inhibited S180cells attachment to Concanavalin A-coated surfaces. Inhibition was dependent on concentration, and the IC5o(the concentration that reduced attachment by 50% ), of these 2 chemicals was 1.2 ×10-3 mol/L and 1.0 mol/L, respectively. Another developmental toxicant, hydrocortisone, did not show inhibitory activity. Two non-developmental toxicants, sodium chloride and glycine were also testedand these did not decrease attachment rates. The main results reported here were generally similar to those obtained with ascitic mouse ovarian tumor cells as a model. Therefore, this study added further evidence to the conclusion that cell specificity does not limit attachment inhibition to Con A-coated surfaces, so S180 cell may serve as an alternative cell model, especially when other cell lines are unavailable. Furthermore, after optimal validation, it can be suggested that an S180 cell attachment assay may be a candidate for a series of assays to detect developmental toxicants.

  1. Miniaturized and high-throughput assays for analysis of T-cell immunity specific for opportunistic pathogens and HIV.

    Science.gov (United States)

    Li Pira, Giuseppina; Ivaldi, Federico; Starc, Nadia; Landi, Fabiola; Locatelli, Franco; Rutella, Sergio; Tripodi, Gino; Manca, Fabrizio

    2014-04-01

    Monitoring of antigen-specific T-cell responses is valuable in numerous conditions that include infectious diseases, vaccinations, and opportunistic infections associated with acquired or congenital immune defects. A variety of assays that make use of peripheral lymphocytes to test activation markers, T-cell receptor expression, or functional responses are currently available. The last group of assays calls for large numbers of functional lymphocytes. The number of cells increases with the number of antigens to be tested. Consequently, cells may be the limiting factor, particularly in lymphopenic subjects and in children, the groups that more often require immune monitoring. We have developed immunochemical assays that measure secreted cytokines in the same wells in which peripheral blood mononuclear cells (PBMC) are cultured. This procedure lent itself to miniaturization and automation. Lymphoproliferation and the enzyme-linked immunosorbent spot (ELISPOT) assay have been adapted to a miniaturized format. Here we provide examples of immune profiles and describe a comparison between miniaturized assays based on cytokine secretion or proliferation. We also demonstrate that these assays are convenient for use in testing antigen specificity in established T-cell lines, in addition to analysis of PBMC. In summary, the applicabilities of miniaturization to save cells and reagents and of automation to save time and increase accuracy were demonstrated in this study using different methodological approaches valuable in the clinical immunology laboratory.

  2. Tissue-specific methylation of human insulin gene and PCR assay for monitoring beta cell death.

    Directory of Open Access Journals (Sweden)

    Mohamed I Husseiny

    Full Text Available The onset of metabolic dysregulation in type 1 diabetes (T1D occurs after autoimmune destruction of the majority of pancreatic insulin-producing beta cells. We previously demonstrated that the DNA encoding the insulin gene is uniquely unmethylated in these cells and then developed a methylation-specific PCR (MSP assay to identify circulating beta cell DNA in streptozotocin-treated mice prior to the rise in blood glucose. The current study extends to autoimmune non-obese diabetic (NOD mice and humans, showing in NOD mice that beta cell death occurs six weeks before the rise in blood sugar and coincides with the onset of islet infiltration by immune cells, demonstrating the utility of MSP for monitoring T1D. We previously reported unique patterns of methylation of the human insulin gene, and now extend this to other human tissues. The methylation patterns of the human insulin promoter, intron 1, exon 2, and intron 2 were determined in several normal human tissues. Similar to our previous report, the human insulin promoter was unmethylated in beta cells, but methylated in all other tissues tested. In contrast, intron 1, exon 2 and intron 2 did not exhibit any tissue-specific DNA methylation pattern. Subsequently, a human MSP assay was developed based on the methylation pattern of the insulin promoter and human islet DNA was successfully detected in circulation of T1D patients after islet transplantation therapy. Signal levels of normal controls and pre-transplant samples were shown to be similar, but increased dramatically after islet transplantation. In plasma the signal declines with time but in whole blood remains elevated for at least two weeks, indicating that association of beta cell DNA with blood cells prolongs the signal. This assay provides an effective method to monitor beta cell destruction in early T1D and in islet transplantation therapy.

  3. Tissue-Specific Methylation of Human Insulin Gene and PCR Assay for Monitoring Beta Cell Death

    Science.gov (United States)

    Husseiny, Mohamed I.; Kaye, Alexander; Zebadua, Emily; Kandeel, Fouad; Ferreri, Kevin

    2014-01-01

    The onset of metabolic dysregulation in type 1 diabetes (T1D) occurs after autoimmune destruction of the majority of pancreatic insulin-producing beta cells. We previously demonstrated that the DNA encoding the insulin gene is uniquely unmethylated in these cells and then developed a methylation-specific PCR (MSP) assay to identify circulating beta cell DNA in streptozotocin-treated mice prior to the rise in blood glucose. The current study extends to autoimmune non-obese diabetic (NOD) mice and humans, showing in NOD mice that beta cell death occurs six weeks before the rise in blood sugar and coincides with the onset of islet infiltration by immune cells, demonstrating the utility of MSP for monitoring T1D. We previously reported unique patterns of methylation of the human insulin gene, and now extend this to other human tissues. The methylation patterns of the human insulin promoter, intron 1, exon 2, and intron 2 were determined in several normal human tissues. Similar to our previous report, the human insulin promoter was unmethylated in beta cells, but methylated in all other tissues tested. In contrast, intron 1, exon 2 and intron 2 did not exhibit any tissue-specific DNA methylation pattern. Subsequently, a human MSP assay was developed based on the methylation pattern of the insulin promoter and human islet DNA was successfully detected in circulation of T1D patients after islet transplantation therapy. Signal levels of normal controls and pre-transplant samples were shown to be similar, but increased dramatically after islet transplantation. In plasma the signal declines with time but in whole blood remains elevated for at least two weeks, indicating that association of beta cell DNA with blood cells prolongs the signal. This assay provides an effective method to monitor beta cell destruction in early T1D and in islet transplantation therapy. PMID:24722187

  4. An assay to monitor HIV-1 protease activity for the identification of novel inhibitors in T-cells.

    Directory of Open Access Journals (Sweden)

    Brett J Hilton

    Full Text Available The emergence of resistant HIV strains, together with the severe side-effects of existing drugs and lack of development of effective anti-HIV vaccines highlight the need for novel antivirals, as well as innovative methods to facilitate their discovery. Here, we have developed an assay in T-cells to monitor the proteolytic activity of the HIV-1 protease (PR. The assay is based on the inducible expression of HIV-1 PR fused within the Gal4 DNA-binding and transactivation domains. The fusion protein binds to the Gal4 responsive element and activates the downstream reporter, enhanced green fluorescent protein (eGFP gene only in the presence of an effective PR Inhibitor (PI. Thus, in this assay, eGFP acts as a biosensor of PR activity, making it ideal for flow cytometry based screening. Furthermore, the assay was developed using retroviral technology in T-cells, thus providing an ideal environment for the screening of potential novel PIs in a cell-type that represents the natural milieu of HIV infection. Clones with the highest sensitivity, and robust, reliable and reproducible reporter activity, were selected. The assay is easily adaptable to other PR variants, a multiplex platform, as well as to high-throughput plate reader based assays and will greatly facilitate the search for novel peptide and chemical compound based PIs in T-cells.

  5. Cloning assay thresholds on cells exposed to ultrafast laser pulses

    Science.gov (United States)

    Koenig, Karsten; Riemann, Iris; Fischer, Peter; Becker, Thomas P.; Oehring, Hartmut; Halbhuber, Karl-Juergen

    1999-06-01

    The influence of the peak power, laser wavelength and the pulse duration of near infrared ultrashort laser pulses on the reproduction behavior of Chinese hamster ovary (CHO) cells has been studied. In particular, we determined the cloning efficiency of single cell pairs after exposure to ultrashort laser pulses with an intensity in the range of GW/cm2 and TW/cm2. A total of more than 3500 non- labeled cells were exposed to a highly focused scanning beam of a multiphoton laser microscope with 60 microsecond(s) pixel dwell time per scan. The beam was provided by a tunable argon ion laser pumped mode-locked 76 MHz Titanium:Sapphire laser as well as by a compact solid-state laser based system (Vitesse) at a fixed wavelength of 800 nm. Pulse duration (tau) was varied in the range of 100 fs to 4 ps by out-of- cavity pulse-stretching units consisting of SF14 prisms and blazed gratings. Within an optical (laser power) window CHO cells could be scanned for hours without severe impact on reproduction behavior, morphology and vitality. Ultrastructural studies reveal that mitochondria are the major targets of intense destructive laser pulses. Above certain laser power P thresholds, CHO cells started to delay or failed to undergo cell division and, in part, to develop uncontrolled cell growth (giant cell formation). The damage followed a P2/(tau) relation which is typical for a two- photon excitation process. Therefore, cell damage was found to be more pronounced at shorter pulses. Due to the same P2/(tau) relation for the efficiency of fluorescence excitation, two-photon microscopy of living cells does not require extremely short femtosecond laser pulses nor pulse compression units. Picosecond as well as femtosecond lasers can be used as efficient light sources in safe two photon fluorescence microscopy. Only in three photon fluorescence microscopy, femtosecond laser pulses are advantageous over picosecond pulses.

  6. Measurement of single-cell adhesion strength using a microfluidic assay.

    Science.gov (United States)

    Christ, Kevin V; Williamson, Kyle B; Masters, Kristyn S; Turner, Kevin T

    2010-06-01

    Despite the importance of cell adhesion in numerous physiological, pathological, and biomaterial-related responses, our understanding of adhesion strength at the cell-substrate interface and its relationship to cell function remains incomplete. One reason for this deficit is a lack of accessible experimental approaches that quantify adhesion strength at the single-cell level and facilitate large numbers of tests. The current work describes the design, fabrication, and use of a microfluidic-based method for single-cell adhesion strength measurements. By applying a monotonically increasing flow rate in a microfluidic channel in combination with video microscopy, the adhesion strength of individual NIH3T3 fibroblasts cultured for 24 h on various surfaces was measured. The small height of the channel allows high shear stresses to be generated under laminar conditions, allowing strength measurements on well-spread, strongly adhered cells that cannot be characterized in most conventional assays. This assay was used to quantify the relationship between morphological characteristics and adhesion strength for individual well-spread cells. Cell adhesion strength was found to be positively correlated with both cell area and circularity. Computational fluid dynamics (CFD) analysis was performed to examine the role of cell geometry in determining the actual stress applied to the cell. Use of this method to examine adhesion at the single-cell level allows the detachment of strongly-adhered cells under a highly-controllable, uniform loading to be directly observed and will enable the characterization of biological events and relationships that cannot currently be achieved using existing methods.

  7. Toxicity of South American snake venoms measured by an in vitro cell culture assay.

    Science.gov (United States)

    Oliveira, J C R; de Oca, H M; Duarte, M M; Diniz, C R; Fortes-Dias, C L

    2002-03-01

    Cytotoxicity of venoms from eight medically important South American Crotalidae snakes (Bothrops and Lachesis genera) was determined, based on a procedure originally described for the screening of cytotoxic agents in general. The assay, the conditions of which were adapted to snake venoms, determines the survival of viable cells in monolayer culture upon exposure to the toxic agent. Snake venom toxicity was expressed as the venom dose that killed 50% of the cells (CT(50)) under the assay conditions. Bothrops neuwieddi mattogrossensis (CT(50)=4.74+/-0.35 microg/ml) and Bothrops leucurus (CT(50)=4.95+/-0.51 microg/ml) were the most cytotoxic whereas Bothrops atrox (CT(50)=34.64+/-2.38 microg/ml) and Bothrops sp. (CT(50)=33.89+/-3.89 microg/ml) were the least cytotoxic venoms, respectively. The relationship between CT(50) and other biological activities of these snake venoms was evaluated. PMID:11711131

  8. A colorimetric sandwich-type assay for sensitive thrombin detection based on enzyme-linked aptamer assay.

    Science.gov (United States)

    Park, Jun Hee; Cho, Yea Seul; Kang, Sungmuk; Lee, Eun Jeong; Lee, Gwan-Ho; Hah, Sang Soo

    2014-10-01

    A colorimetric sandwich-type assay based on enzyme-linked aptamer assay has been developed for the fast and sensitive detection of as low as 25 fM of thrombin with high linearity. Aptamer-immobilized glass was used to capture the target analyte, whereas a second aptamer, functionalized with horseradish peroxidase (HRP), was employed for the conventional 3,5,3',5'-tetramethylbenzidine (TMB)-based colorimetric detection. Without the troublesome antibody requirement of the conventional enzyme-linked immunosorbent assay (ELISA), as low as 25 fM of thrombin could be rapidly and reproducibly detected. This assay has superior, or at least equal, recovery and accuracy to that of conventional antibody-based ELISA.

  9. Limitations of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay when compared to three commonly used cell enumeration assays

    OpenAIRE

    van Tonder, Alet; Joubert, Annie M; Cromarty, A Duncan

    2015-01-01

    Background The tetrazolium-based MTT assay has long been regarded as the gold standard of cytotoxicity assays as it is highly sensitive and has been miniaturised for use as a high-throughput screening assay. However, various reports refer to interference by different test compounds, including the glycolysis inhibitor 3-bromopyruvate, with the conversion of the dye to coloured formazan crystals. This study assessed the linear range and reproducibility of three commonly used cell enumeration as...

  10. Two-tiered keratinocyte assay: IL-18 production by NCTC2544 cells to determine the skin sensitizing capacity and an epidermal equivalent assay to determine sensitizer potency

    OpenAIRE

    Teunis, Marc; Corsini, Emanuela; Smits, Mieke; Madsen, Charlotte Bernhard; Eltze, Tobias; Ezendam, Janine; Galbiati, Valentina; Gremmer, Eric; Krul, Cyrille; Landin, Annette; Landsiedel, Robert; Pieters, Raymond; Rasmussen, Tina; Reinders, Judith; Roggen, Erwin

    2012-01-01

    At present, the identification of potentially sensitizing chemicals is carried out using animal models. However, it should be very important, both from ethical and economic point of view, to discriminate allergy and irritation events, and to classify sensitizers according to their potency, without the use of animals.The aim of the EU FP6 Integrated Project Sens-it-iv was to develop and optimize an integrated testing strategy consisting of in vitro, human cell based assays which will closely m...

  11. A new scintillation proximity assay-based approach for the detection of KRAS mutations

    International Nuclear Information System (INIS)

    KRAS is very commonly mutated resulting in a constitutively activated protein, which is independent of epidermal growth factor receptor (EGFR) ligand binding and resistant to anti-EGFR therapy. Although KRAS is frequently studied, there is still no uniform standard for detecting of KRAS mutations. In this report, a new scintillation proximity assay-based approach is described that determines the relative affinities of wild-type and mutated KRAS to the anti-KRAS antibody. We performed in vitro experiments using normal human colonic cells (CCD18Co), KRAS wild type (Caco-2) and KRAS mutant (HCT 116) cell lines to determine the relative affinities of wild type or mutated KRAS toward an anti-KRAS monoclonal antibody. The process consists of two primary steps: immunoprecipitation from cell lysate to enrich the KRAS protein and the scintillation proximity assay of the immunoprecipitant to determine the relative affinity against the antibody. A fixed concentration of cell lysates was purified by the immunoprecipitation method. The expressions of the KRAS protein in all cell lines was quantitatively confirmed by western blot analysis. For the scintillation proximity assay, the KRAS standard protein was radiolabeled with 125I by a simple mixing process in the iodogen tube immediately at room temperature immediately before use. The obtained CPM (count per minute) values of were used to calculate the KRAS concentration using purified KRAS as the standard. The calculated relative affinities of 7 μg of Caco-2 and HCT 116 immunoprecipitants for the anti-KRAS antibody were 77 and 0%, respectively. The newly developed scintillation proximity assay-based strategy determines the relative affinities of wild-type or mutated KRAS towards the anti-KRAS monoclonal antibody. This determination can help distinguish mutated KRAS from the wild type protein. The new SPA based approach for detecting KRAS mutations is applicable to many other cancer-related mutations.

  12. A new scintillation proximity assay-based approach for the detection of KRAS mutations

    Energy Technology Data Exchange (ETDEWEB)

    Lee, So-Young; Lim, Jae-Cheong; Cho, Eun-Ha; Jung, Sung-Hee [Korea Atomic Energy Research Institute (KAERI), Daejeon (Korea, Republic of). Radioisotope Research Div.

    2016-04-01

    KRAS is very commonly mutated resulting in a constitutively activated protein, which is independent of epidermal growth factor receptor (EGFR) ligand binding and resistant to anti-EGFR therapy. Although KRAS is frequently studied, there is still no uniform standard for detecting of KRAS mutations. In this report, a new scintillation proximity assay-based approach is described that determines the relative affinities of wild-type and mutated KRAS to the anti-KRAS antibody. We performed in vitro experiments using normal human colonic cells (CCD18Co), KRAS wild type (Caco-2) and KRAS mutant (HCT 116) cell lines to determine the relative affinities of wild type or mutated KRAS toward an anti-KRAS monoclonal antibody. The process consists of two primary steps: immunoprecipitation from cell lysate to enrich the KRAS protein and the scintillation proximity assay of the immunoprecipitant to determine the relative affinity against the antibody. A fixed concentration of cell lysates was purified by the immunoprecipitation method. The expressions of the KRAS protein in all cell lines was quantitatively confirmed by western blot analysis. For the scintillation proximity assay, the KRAS standard protein was radiolabeled with {sup 125}I by a simple mixing process in the iodogen tube immediately at room temperature immediately before use. The obtained CPM (count per minute) values of were used to calculate the KRAS concentration using purified KRAS as the standard. The calculated relative affinities of 7 μg of Caco-2 and HCT 116 immunoprecipitants for the anti-KRAS antibody were 77 and 0%, respectively. The newly developed scintillation proximity assay-based strategy determines the relative affinities of wild-type or mutated KRAS towards the anti-KRAS monoclonal antibody. This determination can help distinguish mutated KRAS from the wild type protein. The new SPA based approach for detecting KRAS mutations is applicable to many other cancer-related mutations.

  13. Dynamic monitoring of changes in endothelial cell-substrate adhesiveness during leukocyte adhesion by microelectrical impedance assay

    Institute of Scientific and Technical Information of China (English)

    Yakun Ge; Tongle Deng; Xiaoxiang Zheng

    2009-01-01

    Adhesion of leukocytes to endothelial cells in inflammation processes leads to changes of endothelial cell-substrate adhesiveness, and understanding of such changes will provide us with important information of inflammation processes. In this study, we used a non-invasive biosensor system referred to as real-time cell electronic sensor (RT-CES) system to monitor the changes in endothelial cell-substrate adhesiveness induced by human monoblastic cell line U937 cell adhesion in a dynamic and quantitative manner. This assay, which is based on cell-substrate impedance readout, is able to monitor transient changes in cell-substrate adhesiveness as a result of U937 cell adhesion. The U937 cell adhesion to endothelial cells was induced by lipopolysaccharide (LPS) in a dose-dependent manner. Although the number of adherent U937 cells to the endothelial cells was verified by a standard assay, the adhesiveness of endothelial cells after addition of U937 cells was monitored by the RT-CES system. Furthermore, focal adhesion kinase protein decrease and F-actin rearrangement in endothelial cells were observed after addition of U937 cells. Our results indicated that the adhesion of U937 cells to LPS-treated endothelial cells reduced the cell adhesiveness to the substrate, and such reduction might facilitate infiltration of leukocytes.

  14. A replication competent lentivirus (RCL) assay for equine infectious anaemia virus (EIAV)-based lentiviral vectors.

    Science.gov (United States)

    Miskin, J; Chipchase, D; Rohll, J; Beard, G; Wardell, T; Angell, D; Roehl, H; Jolly, D; Kingsman, S; Mitrophanous, K

    2006-02-01

    Lentiviral vectors are being developed to satisfy a wide range of currently unmet medical needs. Vectors destined for clinical evaluation have been rendered multiply defective by deletion of all viral coding sequences and nonessential cis-acting sequences from the transfer genome. The viral envelope and accessory proteins are excluded from the production system. The vectors are produced from separate expression plasmids that are designed to minimize the potential for homologous recombination. These features ensure that the regeneration of the starting virus is impossible. It is a regulatory requirement to confirm the absence of any replication competent virus, so we describe here the development and validation of a replication competent lentivirus (RCL) assay for equine infectious anaemia virus (EIAV)-based vectors. The assay is based on the guidelines developed for testing retroviral vectors, and uses the F-PERT (fluorescent-product enhanced reverse transcriptase) assay to test for the presence of a transmissible reverse transcriptase. We have empirically modelled the replication kinetics of an EIAV-like entity in human cells and devised an amplification protocol by comparison with a replication competent MLV. The RCL assay has been validated at the 20 litre manufacturing scale, during which no RCL was detected. The assay is theoretically applicable to any lentiviral vector and pseudotype combination. PMID:16208418

  15. Development of a competitive fluorescence-based synaptosome binding assay for brevetoxins.

    Science.gov (United States)

    McCall, Jennifer R; Jacocks, Henry M; Baden, Daniel G; Bourdelais, Andrea J

    2012-09-01

    Brevetoxins are a family of ladder-frame polyether toxins produced during blooms of the marine dinoflagellate Karenia brevis. Inhalation of brevetoxins aerosolized by wind and wave action can lead to asthma-like symptoms in beach goers. Consumption of either shellfish or finfish exposed to K. brevis blooms can lead to the development of neurotoxic shellfish poisoning. The toxic effects of brevetoxins are due to activation of voltage-sensitive sodium channels (VSSCs) in cell membranes. Binding of brevetoxin analogs and competitors to site 5 on these channels has historically been measured using a radioligand competition assay that is fraught with difficulty, including slow analysis time, production of radioactive waste, and cumbersome and expensive methods associated with the generation of radioactive labeled ligands. In this study, we describe the development of a novel fluorescent synaptosome binding assay for the brevetoxin receptor. BODIPY(®)-conjugated to PbTx-2 was used as the labeled ligand. The BODIPY(®)-PbTx-2 conjugate was found to displace [(3)H]-PbTx-3 from its binding site on VSSCs on rat brain synaptosomes with an equilibrium inhibition constant of 0.11 nM. We have shown that brevetoxin A and B analogs are all able to compete for binding with the fluorescent ligand. Most importantly, this assay was validated against the current site 5 receptor binding assay standard, the radioligand receptor assay for the brevetoxin receptor using [(3)H]-PbTx-3 as the labeled ligand. The fluorescence based assay yielded equilibrium inhibition constants comparable to the radioligand assay for all brevetoxin analogs. The fluorescence based assay was quicker, far less expensive, and did not generate radioactive waste or need radioactive facilities. As such, this fluorescence-based assay can be used to replace the current radioligand assay for site 5 on voltage-sensitive sodium channels and will be a vital tool for future experiments examining the binding affinity of

  16. Modified procedure for labelling target cells in a europium release assay of natural killer cell activity.

    Science.gov (United States)

    Pacifici, R; Di Carlo, S; Bacosi, A; Altieri, I; Pichini, S; Zuccaro, P

    1993-05-01

    Lanthanide europium chelated to diethylenetriaminopentaacetate (EuDTPA) can be used to label target cells such as tumor cells and lymphocytes (Blomberg et al., 1986a,b; Granberg et al., 1988). This procedure has permitted the development of new non-radioactive methods for the detection of target cell cytolysis by natural killer (NK) cells (Blomberg et al., 1986a,b), cytotoxic T lymphocytes (CTL) (Granberg et al., 1988) or complement-mediated cytolysis (Cui et al., 1992). However, we had no success with this method because of a lack of comparability between human NK cell activity simultaneously measured by a classical 51Cr release assay (Seaman et al., 1981) and EuDTPA release assay (Blomberg et al., 1986a). Furthermore, cell division and cell viability were significantly impaired by the suggested concentrations of EuCl3. In this paper, we present a modified non-cytotoxic method for target cell labelling with EuDTPA while cells are growing in culture medium. PMID:8486925

  17. Demonstration of DSI-semen--A novel DNA methylation-based forensic semen identification assay.

    Science.gov (United States)

    Wasserstrom, Adam; Frumkin, Dan; Davidson, Ariane; Shpitzen, Moshe; Herman, Yael; Gafny, Ron

    2013-01-01

    Determining whether the source tissue of biological material is semen is important in confirming sexual assaults, which account for a considerable percentage of crime cases. The gold standard for confirming the presence of semen is microscopic identification of sperm cells, however, this method is labor intensive and operator-dependent. Protein-based immunologic assays, such as PSA, are highly sensitive and relatively fast, but suffer from low specificity in some situations. In addition, proteins are less stable than DNA under most environmental insults. Recently, forensic tissue identification advanced with the development of several approaches based on mRNA and miRNA for identification of various body fluids. Herein is described DNA source identifier (DSI)-semen, a DNA-based assay that determines whether the source tissue of a sample is semen based on detection of semen-specific methylation patterns in five genomic loci. The assay is comprised of a simple single tube biochemical procedure, similar to DNA profiling, followed by automatic software analysis, yielding the identification (semen/non-semen) accompanied by a statistical confidence level. Three additional internal control loci are used to ascertain the reliability of the results. The assay, which aims to replace microscopic examination, can easily be integrated by forensic laboratories and is automatable. The kit was tested on 135 samples of semen, saliva, venous blood, menstrual blood, urine, and vaginal swabs and the identification of semen vs. non-semen was correct in all cases. In order to test the assay's applicability in "real-life" situations, 33 actual casework samples from the forensic biological lab of the Israeli police were analyzed, and the results were compared with microscopic examination performed by Israeli police personnel. There was complete concordance between both analyses except for one sample, in which the assay identified semen whereas no sperm was seen in the microscope. This

  18. Enzymatic assay for calmodulins based on plant NAD kinase activity

    Energy Technology Data Exchange (ETDEWEB)

    Harmon, A.C.; Jarrett, H.W.; Cormier, M.J.

    1984-01-01

    NAD kinase with increased sensitivity to calmodulin was purified from pea seedlings (Pisum sativum L., Willet Wonder). Assays for calmodulin based on the activities of NAD kinase, bovine brain cyclic nucleotide phosphodiesterase, and human erythrocyte Ca/sup 2 -/-ATPase were compared for their sensitivities to calmodulin and for their abilities to discriminate between calmodulins from different sources. The activities of the three enzymes were determined in the presence of various concentrations of calmodulins from human erythrocyte, bovine brain, sea pansy (Renilla reniformis), mung bean seed (Vigna radiata L. Wilczek), mushroom (Agaricus bisporus), and Tetrahymena pyriformis. The concentrations of calmodulin required for 50% activation of the NAD kinase (K/sub 0.5/) ranged from 0.520 ng/ml for Tetrahymena to 2.20 ng/ml for bovine brain. The A/sub 0.5/ s ranged from 19.6 ng/ml for bovine brain calmodulin to 73.5 ng/ml for mushroom calmodulin for phosphodiesterase activation. The K/sub 0.5/'s for the activation of Ca/sup 2 +/-ATPase ranged from 36.3 ng/mol for erythrocyte calmodulin to 61.7 ng/ml for mushroom calmodulin. NAD kinase was not stimulated by phosphatidylcholine, phosphatidylserine, cardiolipin, or palmitoleic acid in the absence or presence of Ca/sup 2 +/. Palmitic acid had a slightly stimulatory effect in the presence of Ca/sup 2 +/ (10% of maximum), but no effect in the absence of Ca/sup 2 +/. Palmitoleic acid inhibited the calmodulin-stimulated activity by 50%. Both the NAD kinase assay and radioimmunoassay were able to detect calmodulin in extracts containing low concentrations of calmodulin. Estimates of calmodulin contents of crude homogenates determined by the NAD kinase assay were consistent with amounts obtained by various purification procedures. 30 references, 1 figure, 4 tables.

  19. The application of single cell gel electrophoresis or comet assay to human monitoring studies

    Directory of Open Access Journals (Sweden)

    Valverde Mahara

    1999-01-01

    Full Text Available Objective. In the search of new human genotoxic biomarkers, the single cell gel electrophoresis assay has been proposed as a sensible alternative. Material and methods. This technique detects principally single strand breaks as well as alkali-labile and repair-retarded sites. Results. Herein we present our experience using the single cell gel electrophoresis assay in human population studies, both occupationally and environmentally exposed. Conclusions. We discuss the assay feasibility as a genotoxic biomarker.

  20. Functional characterisation of the human alpha1 glycine receptor in a fluorescence-based membrane potential assay

    DEFF Research Database (Denmark)

    Jensen, Anders A.; Kristiansen, Uffe

    2004-01-01

    In the present study, we have created a stable HEK293 cell line expressing the human homomeric alpha1 glycine receptor (GlyR) and characterised its functional pharmacology in a conventional patch-clamp assay and in the FLIPR Membrane Potential (FMP) assay, a fluorescence-based high throughput...... and RU 5135>strychnine>brucine>PMBA=picrotoxin>atropine for the antagonists. The actions of three allosteric modulators at the alpha1 GlyR cell line were also characterised in the FMP assay. Micromolar concentrations of Zn2+ inhibited alpha1 GlyR signalling but in contrast to previous reports the metal...... not be suited for sophisticated studies of GlyR pharmacology and kinetics. However, the assay offers several advantages in studies of ligand-receptor interactions. Furthermore, the assay could be highly useful in the search for structurally novel ligands acting at GlyRs....

  1. A droplet-to-digital (D2D) microfluidic device for single cell assays.

    Science.gov (United States)

    Shih, Steve C C; Gach, Philip C; Sustarich, Jess; Simmons, Blake A; Adams, Paul D; Singh, Seema; Singh, Anup K

    2015-01-01

    in other ILs/salts. The growth curve trends obtained by D2D matched conventional yeast culturing in microtiter wells, validating the D2D platform. We believe that our approach represents a generic platform for multi-step biochemical assays such as drug screening, digital PCR, enzyme assays, immunoassays and cell-based assays. PMID:25354549

  2. Measuring stem cell frequency in epidermis: A quantitative in vivo functional assay for long-term repopulating cells

    Science.gov (United States)

    Schneider, T. E.; Barland, C.; Alex, A. M.; Mancianti, M. L.; Lu, Y.; Cleaver, J. E.; Lawrence, H. J.; Ghadially, R.

    2003-09-01

    Epidermal stem cells play a central role in tissue homeostasis, wound repair, tumor initiation, and gene therapy. A major impediment to the purification and molecular characterization of epidermal stem cells is the lack of a quantitative assay for cells capable of long-term repopulation in vivo, such as exists for hematopoietic cells. The tremendous strides made in the characterization and purification of hematopoietic stem cells have been critically dependent on the availability of competitive transplantation assays, because these assays permit the accurate quantitation of long-term repopulating cells in vivo. We have developed an analogous functional assay for epidermal stem cells, and have measured the frequency of functional epidermal stem cells in interfollicular epidermis. These studies indicate that cells capable of long-term reconstitution of a squamous epithelium reside in the interfollicular epidermis. We find that the frequency of these long-term repopulating cells is 1 in 35,000 total epidermal cells, or in the order of 1 in 104 basal epidermal cells, similar to that of hematopoietic stem cells in the bone marrow, and much lower than previously estimated in epidermis. Furthermore, these studies establish a novel functional assay that can be used to validate immunophenotypic markers and enrichment strategies for epidermal stem cells, and to quantify epidermal stem cells in various keratinocyte populations. Thus further studies using this type of assay for epidermis should aid in the progress of cutaneous stem cell-targeted gene therapy, and in more basic studies of epidermal stem cell regulation and differentiation.

  3. A simple and novel modification of comet assay for determination of bacteriophage mediated bacterial cell lysis.

    Science.gov (United States)

    Khairnar, Krishna; Sanmukh, Swapnil; Chandekar, Rajshree; Paunikar, Waman

    2014-07-01

    The comet assay is the widely used method for in vitro toxicity testing which is also an alternative to the use of animal models for in vivo testing. Since, its inception in 1984 by Ostling and Johansson, it is being modified frequently for a wide range of application. In spite of its wide applicability, unfortunately there is no report of its application in bacteriophages research. In this study, a novel application of comet assay for the detection of bacteriophage mediated bacterial cell lysis was described. The conventional methods in bacteriophage research for studying bacterial lysis by bacteriophages are plaque assay method. It is time consuming, laborious and costly. The lytic activity of bacteriophage devours the bacterial cell which results in the release of bacterial genomic material that gets detected by ethidium bromide staining method by the comet assay protocol. The objective of this study was to compare efficacy of comet assay with different assay used to study phage mediated bacterial lysis. The assay was performed on culture isolates (N=80 studies), modified comet assay appear to have relatively higher sensitivity and specificity than other assay. The results of the study showed that the application of comet assay can be an economical, time saving and less laborious alternative to conventional plaque assay for the detection of bacteriophage mediated bacterial cell lysis.

  4. A simple and novel modification of comet assay for determination of bacteriophage mediated bacterial cell lysis.

    Science.gov (United States)

    Khairnar, Krishna; Sanmukh, Swapnil; Chandekar, Rajshree; Paunikar, Waman

    2014-07-01

    The comet assay is the widely used method for in vitro toxicity testing which is also an alternative to the use of animal models for in vivo testing. Since, its inception in 1984 by Ostling and Johansson, it is being modified frequently for a wide range of application. In spite of its wide applicability, unfortunately there is no report of its application in bacteriophages research. In this study, a novel application of comet assay for the detection of bacteriophage mediated bacterial cell lysis was described. The conventional methods in bacteriophage research for studying bacterial lysis by bacteriophages are plaque assay method. It is time consuming, laborious and costly. The lytic activity of bacteriophage devours the bacterial cell which results in the release of bacterial genomic material that gets detected by ethidium bromide staining method by the comet assay protocol. The objective of this study was to compare efficacy of comet assay with different assay used to study phage mediated bacterial lysis. The assay was performed on culture isolates (N=80 studies), modified comet assay appear to have relatively higher sensitivity and specificity than other assay. The results of the study showed that the application of comet assay can be an economical, time saving and less laborious alternative to conventional plaque assay for the detection of bacteriophage mediated bacterial cell lysis. PMID:24681053

  5. A high-throughput assay of NK cell activity in whole blood and its clinical application

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Saet-byul [Department of Microbiology and Brain Korea 21 Project for Medical Sciences, Yonsei University College of Medicine, Seoul (Korea, Republic of); Cha, Junhoe [ATGen Co. Ltd., Sungnam (Korea, Republic of); Kim, Im-kyung [Department of Surgery, Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul (Korea, Republic of); Yoon, Joo Chun [Department of Microbiology, Ewha Womans University School of Medicine, Seoul (Korea, Republic of); Lee, Hyo Joon [Department of Microbiology and Brain Korea 21 Project for Medical Sciences, Yonsei University College of Medicine, Seoul (Korea, Republic of); Park, Sang Woo; Cho, Sunjung; Youn, Dong-Ye; Lee, Heyja; Lee, Choong Hwan [ATGen Co. Ltd., Sungnam (Korea, Republic of); Lee, Jae Myun [Department of Microbiology and Brain Korea 21 Project for Medical Sciences, Yonsei University College of Medicine, Seoul (Korea, Republic of); Lee, Kang Young, E-mail: kylee117@yuhs.ac [Department of Surgery, Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul (Korea, Republic of); Kim, Jongsun, E-mail: jkim63@yuhs.ac [Department of Microbiology and Brain Korea 21 Project for Medical Sciences, Yonsei University College of Medicine, Seoul (Korea, Republic of)

    2014-03-14

    Graphical abstract: - Highlights: • We demonstrated a simple assay of NK cell activity from whole blood. • The measurement of secreted IFN-γ from NK cell enables high-throughput screening. • The NKA assay was validated by clinical results of colorectal cancer patients. - Abstract: Natural killer (NK) cells are lymphocytes of the innate immune system and have the ability to kill tumor cells and virus-infected cells without prior sensitization. Malignant tumors and viruses have developed, however, strategies to suppress NK cells to escape from their responses. Thus, the evaluation of NK cell activity (NKA) could be invaluable to estimate the status and the outcome of cancers, viral infections, and immune-mediated diseases. Established methods that measure NKA, such as {sup 51}Cr release assay and CD107a degranulation assay, may be used to determine NK cell function, but they are complicated and time-consuming because they require isolation of peripheral blood mononuclear cells (PBMC) or NK cells. In some cases these assays require hazardous material such as radioactive isotopes. To overcome these difficulties, we developed a simple assay that uses whole blood instead of PBMC or isolated NK cells. This novel assay is suitable for high-throughput screening and the monitoring of diseases, because it employs serum of ex vivo stimulated whole blood to detect interferon (IFN)-γ secreted from NK cells as an indicator of NKA. After the stimulation of NK cells, the determination of IFNγ concentration in serum samples by enzyme-linked immunosorbent assay (ELISA) provided a swift, uncomplicated, and high-throughput assay of NKA ex vivo. The NKA results microsatellite stable (MSS) colorectal cancer patients was showed significantly lower NKA, 263.6 ± 54.5 pg/mL compared with healthy subjects, 867.5 ± 50.2 pg/mL (p value <0.0001). Therefore, the NKA could be utilized as a supportive diagnostic marker for microsatellite stable (MSS) colorectal cancer.

  6. A high-throughput assay of NK cell activity in whole blood and its clinical application

    International Nuclear Information System (INIS)

    Graphical abstract: - Highlights: • We demonstrated a simple assay of NK cell activity from whole blood. • The measurement of secreted IFN-γ from NK cell enables high-throughput screening. • The NKA assay was validated by clinical results of colorectal cancer patients. - Abstract: Natural killer (NK) cells are lymphocytes of the innate immune system and have the ability to kill tumor cells and virus-infected cells without prior sensitization. Malignant tumors and viruses have developed, however, strategies to suppress NK cells to escape from their responses. Thus, the evaluation of NK cell activity (NKA) could be invaluable to estimate the status and the outcome of cancers, viral infections, and immune-mediated diseases. Established methods that measure NKA, such as 51Cr release assay and CD107a degranulation assay, may be used to determine NK cell function, but they are complicated and time-consuming because they require isolation of peripheral blood mononuclear cells (PBMC) or NK cells. In some cases these assays require hazardous material such as radioactive isotopes. To overcome these difficulties, we developed a simple assay that uses whole blood instead of PBMC or isolated NK cells. This novel assay is suitable for high-throughput screening and the monitoring of diseases, because it employs serum of ex vivo stimulated whole blood to detect interferon (IFN)-γ secreted from NK cells as an indicator of NKA. After the stimulation of NK cells, the determination of IFNγ concentration in serum samples by enzyme-linked immunosorbent assay (ELISA) provided a swift, uncomplicated, and high-throughput assay of NKA ex vivo. The NKA results microsatellite stable (MSS) colorectal cancer patients was showed significantly lower NKA, 263.6 ± 54.5 pg/mL compared with healthy subjects, 867.5 ± 50.2 pg/mL (p value <0.0001). Therefore, the NKA could be utilized as a supportive diagnostic marker for microsatellite stable (MSS) colorectal cancer

  7. A functional assay-based strategy for nanomaterial risk forecasting

    Energy Technology Data Exchange (ETDEWEB)

    Hendren, Christine Ogilvie, E-mail: christine.hendren@duke.edu [Center for the Environmental Implications of NanoTechnology, Duke University, Durham, NC 27708 (United States); Lowry, Gregory V., E-mail: glowry@andrew.cmu.edu [Center for the Environmental Implications of NanoTechnology, Duke University, Durham, NC 27708 (United States); Department of Civil and Environmental Engineering, Carnegie Mellon University, 119 Porter Hall, Pittsburgh, PA 15213 (United States); Unrine, Jason M., E-mail: jason.unrine@uky.edu [Center for the Environmental Implications of NanoTechnology, Duke University, Durham, NC 27708 (United States); Department of Plant and Soil Sciences, University of Kentucky, Agricultural Science Center, Lexington, KY 40546 (United States); Wiesner, Mark R., E-mail: wiesner@duke.edu [Center for the Environmental Implications of NanoTechnology, Duke University, Durham, NC 27708 (United States); Department of Civil and Environmental Engineering, Duke University, 121 Hudson Hall PO Box 90287, Durham, NC 27708 (United States)

    2015-12-01

    The study of nanomaterial impacts on environment, health and safety (nanoEHS) has been largely predicated on the assumption that exposure and hazard can be predicted from physical–chemical properties of nanomaterials. This approach is rooted in the view that nanoöbjects essentially resemble chemicals with additional particle-based attributes that must be included among their intrinsic physical–chemical descriptors. With the exception of the trivial case of nanomaterials made from toxic or highly reactive materials, this approach has yielded few actionable guidelines for predicting nanomaterial risk. This article addresses inherent problems in structuring a nanoEHS research strategy based on the goal of predicting outcomes directly from nanomaterial properties, and proposes a framework for organizing data and designing integrated experiments based on functional assays (FAs). FAs are intermediary, semi-empirical measures of processes or functions within a specified system that bridge the gap between nanomaterial properties and potential outcomes in complex systems. The three components of a functional assay are standardized protocols for parameter determination and reporting, a theoretical context for parameter application and reference systems. We propose the identification and adoption of reference systems where FAs may be applied to provide parameter estimates for environmental fate and effects models, as well as benchmarks for comparing the results of FAs and experiments conducted in more complex and varied systems. Surface affinity and dissolution rate are identified as two critical FAs for characterizing nanomaterial behavior in a variety of important systems. The use of these FAs to predict bioaccumulation and toxicity for initial and aged nanomaterials is illustrated for the case of silver nanoparticles and Caenorhabditis elegans. - Highlights: • Approaches to predict risk directly from nanomaterial (NM) properties are problematic. • We propose

  8. A functional assay-based strategy for nanomaterial risk forecasting

    International Nuclear Information System (INIS)

    The study of nanomaterial impacts on environment, health and safety (nanoEHS) has been largely predicated on the assumption that exposure and hazard can be predicted from physical–chemical properties of nanomaterials. This approach is rooted in the view that nanoöbjects essentially resemble chemicals with additional particle-based attributes that must be included among their intrinsic physical–chemical descriptors. With the exception of the trivial case of nanomaterials made from toxic or highly reactive materials, this approach has yielded few actionable guidelines for predicting nanomaterial risk. This article addresses inherent problems in structuring a nanoEHS research strategy based on the goal of predicting outcomes directly from nanomaterial properties, and proposes a framework for organizing data and designing integrated experiments based on functional assays (FAs). FAs are intermediary, semi-empirical measures of processes or functions within a specified system that bridge the gap between nanomaterial properties and potential outcomes in complex systems. The three components of a functional assay are standardized protocols for parameter determination and reporting, a theoretical context for parameter application and reference systems. We propose the identification and adoption of reference systems where FAs may be applied to provide parameter estimates for environmental fate and effects models, as well as benchmarks for comparing the results of FAs and experiments conducted in more complex and varied systems. Surface affinity and dissolution rate are identified as two critical FAs for characterizing nanomaterial behavior in a variety of important systems. The use of these FAs to predict bioaccumulation and toxicity for initial and aged nanomaterials is illustrated for the case of silver nanoparticles and Caenorhabditis elegans. - Highlights: • Approaches to predict risk directly from nanomaterial (NM) properties are problematic. • We propose

  9. γδ T Cell-Mediated Antibody-Dependent Cellular Cytotoxicity with CD19 Antibodies Assessed by an Impedance-Based Label-Free Real-Time Cytotoxicity Assay

    OpenAIRE

    Seidel, Ursula Jördis Eva; Vogt, Fabian; Grosse-Hovest, Ludger; Jung, Gundram; Handgretinger, Rupert; Lang, Peter

    2014-01-01

    γδ T cells are not MHC restricted, elicit cytotoxicity against various malignancies, are present in early post-transplant phases in novel stem cell transplantation strategies and have been shown to mediate antibody-dependent cellular cytotoxicity (ADCC) with monoclonal antibodies (mAbs). These features make γδ T cells promising effector cells for antibody-based immunotherapy in pediatric patients with B-lineage acute lymphoblastic leukemia (ALL). To evaluate combination of human γδ T cells wi...

  10. Differences in estimates of cisplatin-induced cell kill in vitro between colorimetric and cell count/colony assays.

    Science.gov (United States)

    Henriksson, Eva; Kjellén, Elisabeth; Wahlberg, Peter; Wennerberg, Johan; Kjellström, Johan H

    2006-01-01

    The aim of this study was to evaluate some bioassays that are different in principle: cell counting, colony forming assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), sulforhodamine B (SRB), crystal violet, and alamarBlue, with respect to their ability to measure cisplatin-induced cell death of in vitro-cultivated squamous cell carcinoma of the head and neck (SCCHN). Cisplatin was applied in concentrations of 1.0, 5.0, 10.0, 50.0, and 100 microM. The cells were incubated for 1 h, and the cell survival was measured 5 d after treatment. We found the colorimetric assays and cell counting to be comparable. The colony forming assay indicated a higher degree of cell kill compared with the other techniques. Measurement of cell survival after treatment with cisplatin can be done by use of any of the above tested assays. However, the majority of SCCHN cell lines available do not form colonies easily, or at all. Therefore, comparing the chemosensitivity between such cell lines is limited to alternative assays. In this respect, any of the tested colorimetric assays can be used. However, they seem to underestimate cell kill. Cell counting is also an alternative. This technique, however, is time consuming and operator dependent, as in the case of manual counting, or relatively expensive when counting is performed electronically, compared with the colorimetric assays. PMID:17316066

  11. An improved haemolytic plaque assay for the detection of cells secreting antibody to bacterial antigens

    DEFF Research Database (Denmark)

    Barington, T; Heilmann, C

    1992-01-01

    Recent advances in the development of conjugate polysaccharide vaccines for human use have stimulated interest in the use of assays detecting antibody-secreting cells (AbSC) with specificity for bacterial antigens. Here we present improved haemolytic plaque-forming cell (PFC) assays detecting Ab......SC with specificity for tetanus and diphtheria toxoid as well as for Haemophilus influenzae type b and pneumococcal capsular polysaccharides. These assays were found to be less time consuming, more economical and yielded 1.9-3.4-fold higher plaque numbers than traditional Jerne-type PFC assays. In the case of anti...

  12. The radiosensitivity of a murine fibrosarcoma as measured by three cell survival assays.

    Science.gov (United States)

    Rice, L; Urano, M; Suit, H D

    1980-04-01

    The radiation sensitivity of a weakly immunogenic spontaneous fibrosarcoma of the C3Hf/Sed mouse (designated FSa-II) was assessed by three in vivo cell survival methods: end-point dilution (TD50) assay, lung colony (LC) assay, and agar diffusion chamber (ADC) assay. The hypoxic fraction of this tumour was also determined by the ADC method. Although there was a good agreement of the cell survival data between the ADC and LC methods, the TD50 method yielded a considerably less steep cell survival curve. Beneficial aspects and limitations of each assay are discussed. In addition, the use of the ADC method for the growth of xenogeneic cell lines and a preliminary experiment with human tumour cells in non-immunosuppressed hosts suggest that this method may be a valuable adjunct for studying the growth and therapeutic responses of human tumour cells. PMID:6932931

  13. Immune Monitoring in Cancer Vaccine Clinical Trials: Critical Issues of Functional Flow Cytometry-Based Assays

    Directory of Open Access Journals (Sweden)

    Iole Macchia

    2013-01-01

    Full Text Available The development of immune monitoring assays is essential to determine the immune responses against tumor-specific antigens (TSAs and tumor-associated antigens (TAAs and their possible correlation with clinical outcome in cancer patients receiving immunotherapies. Despite the wide range of techniques used, to date these assays have not shown consistent results among clinical trials and failed to define surrogate markers of clinical efficacy to antitumor vaccines. Multiparameter flow cytometry- (FCM- based assays combining different phenotypic and functional markers have been developed in the past decade for informative and longitudinal analysis of polyfunctional T-cells. These technologies were designed to address the complexity and functional heterogeneity of cancer biology and cellular immunity and to define biomarkers predicting clinical response to anticancer treatment. So far, there is still a lack of standardization of some of these immunological tests. The aim of this review is to overview the latest technologies for immune monitoring and to highlight critical steps involved in some of the FCM-based cellular immune assays. In particular, our laboratory is focused on melanoma vaccine research and thus our main goal was the validation of a functional multiparameter test (FMT combining different functional and lineage markers to be applied in clinical trials involving patients with melanoma.

  14. A microsystem-based assay for studying pollen tube guidance in plant reproduction

    International Nuclear Information System (INIS)

    We present a novel microsystem-based assay to assess and quantify pollen tube behavior in response to pistil tissues. During plant reproduction, signals from female tissues (pistils) guide the sperm-carrying pollen tube to the egg cell to achieve fertilization and initiate seed development. Existing pollen tube guidance bioassays are performed in an isotropically diffusive environment (for example, a semi in vivo assay in petri dishes) instead of anisotropically diffusive conditions required to characterize guidance signal gradients. Lack of a sensitive pollen tube guidance bioassay has therefore compounded the difficulties of identifying and characterizing the guidance signals that are likely produced in minute quantities by the ovules. We therefore developed a novel microsystem-based assay that mimics the in vivo micro-environment of ovule fertilization by pollen tubes in the model research plant Arabidopsis thaliana. In this microdevice, the pollen tube growth rate, length and ovule targeting frequencies were similar to those obtained using a semi in vivo plate assay. As a direct measure of the microdevice's utility in monitoring pollen tube guidance, we demonstrated that in this device, pollen tubes preferentially enter chambers with unfertilized ovules, suggesting that the pollen tubes sense the concentration gradient and respond to the chemoattractants secreted by unfertilized ovules

  15. γδ T cell-mediated antibody-dependent cellular cytotoxicity with CD19 antibodies assessed by an impedance-based label-free real-time cytotoxicity assay

    OpenAIRE

    Ursula Jördis Eva Seidel; Fabian eVogt; Ludger eGrosse-Hovest; Gundram eJung; Rupert eHandgretinger; Peter eLang

    2014-01-01

    γδ T cells are not MHC restricted, elicit cytotoxicity against various malignancies, are present in early post-transplant phases in novel stem cell transplantation (SCT) strategies and have been shown to mediate antibody-dependent cellular cytotoxicity (ADCC) with monoclonal antibodies (mAbs). These features make γδ T cells promising effector cells for antibody-based immunotherapy in pediatric patients with B-lineage acute lymphoblastic leukemia (ALL). To evaluate combination of human γδ T ce...

  16. Human iPSC-Derived Endothelial Cell Sprouting Assay in Synthetic Hydrogel Arrays

    Science.gov (United States)

    Activation of vascular endothelial cells (ECs) by growth factors initiates a cascade of events in vivo consisting of EC tip cell selection, sprout formation, EC stalk cell proliferation, and ultimately vascular stabilization by support cells. Although EC functional assays can rec...

  17. Detection of Cronobacter sakazakii in powdered infant formula using an immunoliposome-based immunomagnetic concentration and separation assay

    Science.gov (United States)

    Shukla, Shruti; Lee, Gibaek; Song, Xinjie; Park, Jung Hyun; Cho, Hyunjeong; Lee, Eun Ju; Kim, Myunghee

    2016-01-01

    This study aimed to optimize the applicability of an immunoliposome-based immunomagnetic concentration and separation assay to facilitate rapid detection of Cronobacter sakazakii in powdered infant formula (PIF). To determine the detection limit, specificity, and pre-enrichment incubation time (0, 4, 6, and 8 h), assay tests were performed with different cell numbers of C. sakazakii (2 × 100 and 2 × 101 CFU/ml) inoculated in 10 g of PIF. The assay was able to detect as few as 2 cells of C. sakazakii/10 g of PIF sample after 6 h of pre-enrichment incubation with an assay time of 2 h 30 min. The assay was assessed for cross-reactivity with other bacterial strains and exhibited strong specificity to C. sakazakii. Moreover, the assay method was applied to the detection of C. sakazakii in PIF without pre-enrichment steps, and the results were compared with INC-ELISA and RT-PCR. The developed method was able to detect C. sakazakii in spiked PIF without pre-enrichment, whereas INC-ELISA failed to detect C. sakazakii. In addition, when compared with the results obtained with RT-PCR, our developed assay required lesser detection time. The developed assay was also not susceptible to any effect of the food matrix or background contaminant microflora. PMID:27721500

  18. Quantitative analysis of G-protein-coupled receptor internalization using DnaE intein-based assay.

    Science.gov (United States)

    Lu, Bin; Chen, Linjie; Zhang, Yaping; Shi, Ying; Zhou, Naiming

    2016-01-01

    G-protein-coupled receptors (GPCRs), the largest family of cell surface receptors, are involved in many physiological processes. They represent highly important therapeutic targets for drug discovery. Currently, there are numerous cell-based assays developed for the pharmacological profiling of GPCRs and the identification of novel agonists and antagonists. However, the development of new, faster, easier, and more cost-effective approaches to detect GPCR activity remains highly desirable. β-arrestin-dependent internalization has been demonstrated to be a common mechanism for most GPCRs. Here we describe a novel assay for quantitative analysis of GPCR internalization based on DnaE intein-mediated reconstitution of fragmented Renilla luciferase or Firefly luciferase when activated GPCRs interact with β-arrestin2 or Rab5. Further validation, using functionally divergent GPCRs, showed that EC50 values obtained for the known agonists and antagonists were in close agreement with the results of previous reports. This suggests that this assay is sensitive enough to permit quantification of GPCR internalization. Compared with conventional assays, this novel assay system is cost-effective, rapid, and easy to manipulate. These advantages may allow this assay to be used universally as a functional cell-based system for GPCR characterization and in the screening process of drug discovery. PMID:26928549

  19. Single Cell Adhesion Assay Using Computer Controlled Micropipette

    OpenAIRE

    Rita Salánki; Csaba Hős; Norbert Orgovan; Beatrix Péter; Noémi Sándor; Zsuzsa Bajtay; Anna Erdei; Robert Horvath; Bálint Szabó

    2014-01-01

    Cell adhesion is a fundamental phenomenon vital for all multicellular organisms. Recognition of and adhesion to specific macromolecules is a crucial task of leukocytes to initiate the immune response. To gain statistically reliable information of cell adhesion, large numbers of cells should be measured. However, direct measurement of the adhesion force of single cells is still challenging and today's techniques typically have an extremely low throughput (5-10 cells per day). Here, we introduc...

  20. Diversity in the applications of the single cell gel electrophoresis (comet) assay / Cristal Huysamen

    OpenAIRE

    Huysamen, Cristal

    2005-01-01

    The development of the single cell gel electrophoresis assay (Comet assay) as a powerful method for measuring DNA strand breakage and repair, has lead to a broader understanding of the impact of certain internal and external factors on DNA damage. This study describes the establishment of the Comet assay in our laboratory and its application in a diversity of studies. These studies include the monitoring of the effect of exercise on DNA damage and repair with the purpose of ...

  1. Quality Control Assays for Clinical-Grade Human Mesenchymal Stromal Cells: Validation Strategy.

    Science.gov (United States)

    Radrizzani, Marina; Soncin, Sabrina; Bolis, Sara; Lo Cicero, Viviana; Andriolo, Gabriella; Turchetto, Lucia

    2016-01-01

    The present chapter focuses on the validation of the following analytical methods for the control of mesenchymal stromal cells (MSC) for cell therapy clinical trials: Microbiological control for cellular product Endotoxin assay Mycoplasma assay Cell count and viability Immunophenotype Clonogenic potential (CFU-F assay) In our lab, these methods are in use for product release, process control or control of the biological starting materials. They are described in detail in the accompanying Chapter 19.For each method, validation goals and strategy are presented, and a detailed experimental scheme is proposed. PMID:27236682

  2. Induction and repair of DNA damage measured by the comet assay in human T lymphocytes separated by immunomagnetic cell sorting.

    Science.gov (United States)

    Bausinger, Julia; Speit, Günter

    2014-11-01

    The comet assay is widely used in human biomonitoring to measure DNA damage in whole blood or isolated peripheral blood mononuclear cells (PBMC) as a marker of exposure to genotoxic agents. Cytogenetic assays with phytohemagglutinin (PHA)-stimulated cultured T lymphocytes are also frequently performed in human biomonitoring. Cytogenetic effects (micronuclei, chromosome aberrations, sister chromatid exchanges) may be induced in vivo but also occur ex vivo during the cultivation of lymphocytes as a consequence of DNA damage present in lymphocytes at the time of sampling. To better understand whether DNA damage measured by the comet assay in PBMC is representative for DNA damage in T cells, we comparatively investigated DNA damage and its repair in PBMC and T cells obtained by immunomagnetic cell sorting. PBMC cultures and T cell cultures were exposed to mutagens with different modes of genotoxic action and DNA damage was measured by the comet assay after the end of a 2h exposure and after 18h post-incubation. The mutagens tested were methyl methanesulfonate (MMS), (±)-anti-B[a]P-7,8-dihydrodiol-9,10-epoxide (BPDE), 4-nitroquinoline-1-oxide (4NQO), styrene oxide and potassium bromate. MMS and potassium bromate were also tested by the modified comet assay with formamido pyrimidine glycosylase (FPG) protein. The results indicate that the mutagens tested induce DNA damage in PBMC and T cells in the same range of concentrations and removal of induced DNA lesions occurs to a comparable extent. Based on these results, we conclude that the comet assay with PBMC is suited to predict DNA damage and its removal in T cells.

  3. Flow cytometry-based invasion phenotyping assay for malaria

    OpenAIRE

    sprotocols

    2015-01-01

    Authors: Michel Theron, Richard Hesketh, Sathish Subramanian & Julian Rayner ### Abstract To facilitate the scale-up of erythrocyte invasion phenotyping for Plasmodium falciparum, we have developed a novel platform based on two-color flow cytometry that distinguishes parasite invasion from parasite growth. Target cells that had one or more receptors removed using enzymatic treatment were prelabeled with intracellular dyes CFDA-SE or DDAO-SE, incubated with P. falciparum parasites,...

  4. A dual immunocytochemical assay for oestrogen and epidermal growth factor receptors in tumour cell lines

    NARCIS (Netherlands)

    A.K. Sharma (Anisha K.); J.H. Horgan; R.L. McClelland (Robyn); A.G. Douglas-Jones (A.); T. van Agthoven (Ton); L.C.J. Dorssers (Lambert); R.I. Nicholson (R.)

    1994-01-01

    textabstractA new dual immunocytochemical assay for oestrogen receptor (ER) and epidermal growth factor receptor (EGFR) has been developed. It has been tested in a variety of conditions using cell culture lines and the results correlate well with those obtained from single immunocytochemical assays.

  5. Electrochemical chip-based genomagnetic assay for detection of high-risk human papillomavirus DNA.

    Science.gov (United States)

    Bartosik, Martin; Durikova, Helena; Vojtesek, Borivoj; Anton, Milan; Jandakova, Eva; Hrstka, Roman

    2016-09-15

    Cervical cancer, being the fourth leading cause of cancer death in women worldwide, predominantly originates from a persistent infection with a high-risk human papillomavirus (HPV). Detection of DNA sequences from these high-risk strains, mostly HPV-16 and HPV-18, represents promising strategy for early screening, which would help to identify women with higher risk of cervical cancer. In developing countries, inadequate screening options lead to disproportionately high mortality rates, making a fast and inexpensive detection schemes highly important. Electrochemical sensors and assays offer an alternative to current methods of detection. We developed an electrochemical-chip based assay, in which target HPV DNA is captured via magnetic bead-modified DNA probes, followed by an antidigoxigenin-peroxidase detection system at screen-printed carbon electrode chips, enabling parallel measurements of eight samples simultaneously. We show sensitive detection in attomoles of HPV DNA, selective discrimination between HPV-16 and HPV-18 and good reproducibility. Most importantly, we show application of the assay into both cancer cell lines and cervical smears from patients. The electrochemical results correlated well with standard methods, making this assay potentially applicable in clinical practice. PMID:27132004

  6. Electrochemical chip-based genomagnetic assay for detection of high-risk human papillomavirus DNA.

    Science.gov (United States)

    Bartosik, Martin; Durikova, Helena; Vojtesek, Borivoj; Anton, Milan; Jandakova, Eva; Hrstka, Roman

    2016-09-15

    Cervical cancer, being the fourth leading cause of cancer death in women worldwide, predominantly originates from a persistent infection with a high-risk human papillomavirus (HPV). Detection of DNA sequences from these high-risk strains, mostly HPV-16 and HPV-18, represents promising strategy for early screening, which would help to identify women with higher risk of cervical cancer. In developing countries, inadequate screening options lead to disproportionately high mortality rates, making a fast and inexpensive detection schemes highly important. Electrochemical sensors and assays offer an alternative to current methods of detection. We developed an electrochemical-chip based assay, in which target HPV DNA is captured via magnetic bead-modified DNA probes, followed by an antidigoxigenin-peroxidase detection system at screen-printed carbon electrode chips, enabling parallel measurements of eight samples simultaneously. We show sensitive detection in attomoles of HPV DNA, selective discrimination between HPV-16 and HPV-18 and good reproducibility. Most importantly, we show application of the assay into both cancer cell lines and cervical smears from patients. The electrochemical results correlated well with standard methods, making this assay potentially applicable in clinical practice.

  7. Exploitation of the Dose/Time-Response Relationship for a New Measure of DNA Repari in the Single-Cell Gel Electrophoresis (Comet) Assay

    International Nuclear Information System (INIS)

    The comet assay (also called the single-cell gel electrophoresis assay) has been widely used for detecting DNA damage and repair in individual cells. Since the conventional methods of evaluating comet assay data using frequency statistics are unsatisfactory we developed a new quantitative measure of DNA damage/repair that is based on all information residing in the dose/time-response curves of a comet experiment. Blood samples were taken from 25 breast cancer patients before undergoing radiotherapy. The comet assay was performed under alkaline conditions using isolated lymphocytes

  8. Single cell adhesion assay using computer controlled micropipette.

    Directory of Open Access Journals (Sweden)

    Rita Salánki

    Full Text Available Cell adhesion is a fundamental phenomenon vital for all multicellular organisms. Recognition of and adhesion to specific macromolecules is a crucial task of leukocytes to initiate the immune response. To gain statistically reliable information of cell adhesion, large numbers of cells should be measured. However, direct measurement of the adhesion force of single cells is still challenging and today's techniques typically have an extremely low throughput (5-10 cells per day. Here, we introduce a computer controlled micropipette mounted onto a normal inverted microscope for probing single cell interactions with specific macromolecules. We calculated the estimated hydrodynamic lifting force acting on target cells by the numerical simulation of the flow at the micropipette tip. The adhesion force of surface attached cells could be accurately probed by repeating the pick-up process with increasing vacuum applied in the pipette positioned above the cell under investigation. Using the introduced methodology hundreds of cells adhered to specific macromolecules were measured one by one in a relatively short period of time (∼30 min. We blocked nonspecific cell adhesion by the protein non-adhesive PLL-g-PEG polymer. We found that human primary monocytes are less adherent to fibrinogen than their in vitro differentiated descendants: macrophages and dendritic cells, the latter producing the highest average adhesion force. Validation of the here introduced method was achieved by the hydrostatic step-pressure micropipette manipulation technique. Additionally the result was reinforced in standard microfluidic shear stress channels. Nevertheless, automated micropipette gave higher sensitivity and less side-effect than the shear stress channel. Using our technique, the probed single cells can be easily picked up and further investigated by other techniques; a definite advantage of the computer controlled micropipette. Our experiments revealed the existence of a

  9. Psychoneuroimmunology and natural killer cells: the chromium release whole blood assay.

    Science.gov (United States)

    Fletcher, Mary Ann; Barnes, Zachary; Broderick, Gordon; Klimas, Nancy G

    2012-01-01

    Natural killer (NK) cells are an essential component of innate immunity. These lymphocytes are also sensitive barometers of the effects of endogenous and exogenous stressors on the immune system. This chapter will describe a chromium ((51)Cr) release bioassay designed to measure the target cell killing capacity of NK cells (NKCC). Key features of the cytotoxicity assay are that it is done with whole blood and that numbers of effector cells are determined for each sample by flow cytometry and lymphocyte count. Effector cells are defined as CD3-CD56+ lymphocytes. Target cells are the K562 eyrthroleukemia cell line. Killing capacity is defined as number of target cells killed per effector cell, at an effector cell/target cell ratio of 1:1 during a 4 h in vitro assay.

  10. Comet-electrophoresis assay as a method for determining radiosensitivities of tumor cells

    International Nuclear Information System (INIS)

    Objective: To explore the feasibility of applying comet-electrophoresis to determining the radiosensitivity of tumor cells. Methods: The residual rates of DNA damage at 30 minute after 2 Gy gamma irradiation in four human tumor cell lines (WM9839, KB, LS-T-117, PC3M) were determined with the comet assay. The cell survival fraction of tumor cell after 2 Gy gamma ray-irradiation was determined with clonogenic assay. Results: There were good correlations between cell survival fraction (SF2 ) and residual rate of DNA damage at 30 minute after 2 Gy gamma ray-irradiation in these four human tumor cell lines, separately. Conclusion: The comet-electrophoresis assay may be used as a repaid and sensitive method for determining inherent radiosensitivities of tumor cells

  11. Flow cytometric assay detecting cytotoxicity against human endogenous retrovirus antigens expressed on cultured multiple sclerosis cells

    DEFF Research Database (Denmark)

    Møller-Larsen, A; Brudek, T; Petersen, T;

    2013-01-01

    as control antibody. Without antibodies this system is suitable for analyses of natural killer cell activity. In optimization of the assay we have used effector lymphocytes from healthy donors. The most effective effector cells are CD56(+) cells. CD8(+) T cells also express CD107a in ADCC. Using the adapted......Damage of target cells by cytotoxicity, either mediated by specific lymphocytes or via antibody-dependent reactions, may play a decisive role in causing the central nervous system (CNS) lesions seen in multiple sclerosis (MS). Relevant epitopes, antibodies towards these epitopes and a reliable...... assay are all mandatory parts in detection and evaluation of the pertinence of such cytotoxicity reactions. We have adapted a flow cytometry assay detecting CD107a expression on the surface of cytotoxic effector cells to be applicable for analyses of the effect on target cells from MS patients...

  12. Nanobeads-based assays. The case of gluten detection

    Science.gov (United States)

    Venditti, Iole; Fratoddi, Ilaria; Vittoria Russo, Maria; Bellucci, Stefano; Crescenzo, Roberta; Iozzino, Luisa; Staiano, Maria; Aurilia, Vincenzo; Varriale, Antonio; Rossi, Mosè; D'Auria, Sabato

    2008-11-01

    In order to verify if the use of nanobeads of poly[phenylacetylene-(co-acrylic acid)] (PPA/AA) in the ELISA test would affect the immune-activity of the antibodies (Ab) and/or the activity of the enzymes used to label the Ab anti-rabbit IGg, in this work we immobilized the horse liver peroxidase labelled Ab anti-rabbit IGg onto PPA/AA nanobeads. The gluten test was chosen as the model to demonstrate the usefulness of these nanobeads in immunoassays. The synthesis of PPA/AA nanobeads was performed by a modified emulsion polymerization. Self-assembly of nanospheres with mean diameter equal to 200 nm was achieved by casting aqueous suspensions. The materials were characterized by traditional spectroscopic techniques, while the size and dispersion of the particles were analysed by scanning electron microscopy (SEM) measurements. The obtained results show that the immobilization process of the Abs onto PPA/AA did not affect either the immune-response of the Abs or the functional activity of the peroxidase suggesting the usefulness of PPA/AA for the design of advanced nanobeads-based assays for the simultaneous screening of several analytes in complex media.

  13. Gold nanoparticles-based colorimetric and visual creatinine assay

    International Nuclear Information System (INIS)

    We demonstrate a selective and sensitive method for determination of creatinine using citrate-stabilized gold nanoparticles (AuNPs) as a colorimetric probe. It is based on a direct cross-linking reaction that occurs between creatinine and AuNPs that causes aggregation of AuNPs and results in a color change from wine red to blue. The absorption peak is shifted from 520 to 670 nm. Under the optimized conditions, the shift in the absorption peak is related the logarithm of the creatinine concentration in the 0.1 to 20 mM range, and the instrumental detection limit (LOD) is 80 μM. This LOD is about one order of magnitude better than that that of the Jaffé method (720 μM). The assay displays good selectivity over interfering substances including various inorganic ions, organic small compounds, proteins, and biothiols. It was successfully employed to the determination of creatinine in spiked human urine. (author)

  14. GTP-specific fab fragment-based GTPase activity assay.

    Science.gov (United States)

    Kopra, Kari; Rozwandowicz-Jansen, Anita; Syrjänpää, Markku; Blaževitš, Olga; Ligabue, Alessio; Veltel, Stefan; Lamminmäki, Urpo; Abankwa, Daniel; Härmä, Harri

    2015-03-17

    GTPases are central cellular signaling proteins, which cycle between a GDP-bound inactive and a GTP-bound active conformation in a controlled manner. Ras GTPases are frequently mutated in cancer and so far only few experimental inhibitors exist. The most common methods for monitoring GTP hydrolysis rely on luminescent GDP- or GTP-analogs. In this study, the first GTP-specific Fab fragment and its application are described. We selected Fab fragments using the phage display technology. Six Fab fragments were found against 2'/3'-GTP-biotin and 8-GTP-biotin. Selected antibody fragments allowed specific detection of endogenous, free GTP. The most potent Fab fragment (2A4(GTP)) showed over 100-fold GTP-specificity over GDP, ATP, or CTP and was used to develop a heterogeneous time-resolved luminescence based assay for the monitoring of GTP concentration. The method allows studying the GEF dependent H-Ras activation (GTP binding) and GAP-catalyzed H-Ras deactivation (GTP hydrolysis) at nanomolar protein concentrations.

  15. COMBINED EXPERIMENTAL AND MATHEMATICAL APPROACH FOR DEVELOPMENT OF MICROFABRICATION-BASED CANCER MIGRATION ASSAY

    OpenAIRE

    Sarkar, Saheli; Bustard, Bethany L.; Welter, Jean F.; Baskaran, Harihara

    2011-01-01

    Migration of cancer cells is a key determinant of metastasis, which is correlated with poor prognosis in patients. Evidence shows that cancer cell motility is regulated by stromal cell interactions. To quantify the role of homotypic and heterotypic cell-cell interaction on migration, a two-dimensional migration assay has been developed by microfabrication techniques. Two breast cancer cell lines, MDA-MB-231 and MDA-MB-453, were used to develop micropatterns of cancer cells (cell islands) that...

  16. Optical scatter imaging: a microscopic modality for the rapid morphological assay of living cells

    Science.gov (United States)

    Boustany, Nada N.

    2007-02-01

    Tumors derived from epithelial cells comprise the majority of human tumors and their growth results from the accumulation of multiple mutations affecting cellular processes critical for tissue homeostasis, including cell proliferation and cell death. To understand these processes and address the complexity of cancer cell function, multiple cellular responses to different experimental conditions and specific genetic mutations must be analyzed. Fundamental to this endeavor is the development of rapid cellular assays in genetically defined cells, and in particular, the development of optical imaging methods that allow dynamic observation and real-time monitoring of cellular processes. In this context, we are developing an optical scatter imaging technology that is intended to bridge the gap between light and electron microscopy by rapidly providing morphometric information about the relative size and shape of non-spherical organelles, with sub-wavelength resolution. Our goal is to complement current microscopy techniques used to study cells in-vitro, especially in long-term time-lapse studies of living cells, where exogenous labels can be toxic, and electron microscopy will destroy the sample. The optical measurements are based on Fourier spatial filtering in a standard microscope, and could ultimately be incorporated into existing high-throughput diagnostic platforms for cancer cell research and histopathology of neoplastic tissue arrays. Using an engineered epithelial cell model of tumor formation, we are currently studying how organelle structure and function are altered by defined genetic mutations affecting the propensity for cell death and oncogenic potential, and by environmental conditions promoting tumor growth. This talk will describe our optical scatter imaging technology and present results from our studies on apoptosis, and the function of BCL-2 family proteins.

  17. Biotoxin Detection Using Cell-Based Sensors

    OpenAIRE

    Pratik Banerjee; Spyridon Kintzios; Balabhaskar Prabhakarpandian

    2013-01-01

    Cell-based biosensors (CBBs) utilize the principles of cell-based assays (CBAs) by employing living cells for detection of different analytes from environment, food, clinical, or other sources. For toxin detection, CBBs are emerging as unique alternatives to other analytical methods. The main advantage of using CBBs for probing biotoxins and toxic agents is that CBBs respond to the toxic exposures in the manner related to actual physiologic responses of the vulnerable subjects. The results ob...

  18. Towards in vitro DT/DNT testing: Assaying chemical susceptibility in early differentiating NT2 cells.

    Science.gov (United States)

    Menzner, Ann-Katrin; Abolpour Mofrad, Sepideh; Friedrich, Oliver; Gilbert, Daniel F

    2015-12-01

    Human pluripotent embryonal carcinoma (NT2) cells are increasingly considered as a suitable model for in vitro toxicity testing, e.g. developmental toxicity and neurotoxicity (DT/DNT) studies, as they undergo neuronal differentiation upon stimulation with retinoic acid (RA) and permit toxicity testing at different stages of maturation. NT2 cells have recently been reported to show specific changes in dielectric resistance profiles during differentiation which can be observed as early as 24h upon RA-stimulation. These observations suggest altered susceptibility to chemicals at an early stage of differentiation. However, chemical susceptibility of early differentiating NT cells has not yet been studied. To address this question, we have established a cell fitness screening assay based on the analysis of intracellular ATP levels and we applied the assay in a large-scale drug screening experiment in NT2 stem cells and early differentiating NT2 cells. Subsequent analysis of ranked fitness phenotypes revealed 19 chemicals with differential toxicity profile in early differentiating NT2 cells. To evaluate whether any of the identified drugs have previously been associated with DT/DNT, we conducted a literature search on the identified molecules and quantified the fraction of chemicals assigned to the FDA (Food and Drug Administration) pregnancy risk categories (PRC) N, A, B, C, D, and X in the hit list and the small molecule library. While the fractions of the categories N and B were decreased (0.81 and 0.35-fold), the classes C, D and X were increased (1.35, 1.47 and 3.27-fold) in the hit list compared to the chemical library. From these data as well as from the literature review, identifying large fractions of chemicals being directly (∼42%) and indirectly associated with DT/DNT (∼32%), we conclude that our method may be beneficial to systematic in vitro-based primary screening for developmental toxicants and neurotoxicants and we propose cell fitness screening in

  19. A novel assay of biofilm antifungal activity reveals that amphotericin B and caspofungin lyse Candida albicans cells in biofilms.

    Science.gov (United States)

    DiDone, Louis; Oga, Duana; Krysan, Damian J

    2011-08-01

    The ability of Candida albicans to form drug-resistant biofilms is an important factor in its contribution to human disease. Assays to identify and characterize molecules with activity against fungal biofilms are crucial for the development of drugs with improved anti-biofilm activity. Here we report the application of an adenylate kinase (AK)-based cytotoxicity assay of fungal cell lysis to the characterization of agents active against C. albicans biofilms. We have developed three protocols for the AK assay. The first measures AK activity in the supernatants of biofilms treated with antifungal drugs and can be performed in parallel with a standard 2,3-bis-(2-methoxy-4-nitro-5-sulphophenyl)-2H-tetrazolium-5-caboxanilide-based biofilm susceptibility assay; a second, more sensitive protocol measures the AK activity present within the biofilm matrix; and a third procedure allows the direct visualization of lytic activity toward biofilms formed on catheter material. Amphotericin B and caspofungin, the two most effective anti-biofilm drugs currently used to treat fungal infections, both directly lyse planktonic C. albicans cells in vitro, leading to the release of AK into the culture medium. These studies serve to validate the AK-based lysis assay as a useful addition to the methods for the characterization of antifungal agents active toward biofilms and provide insights into the mode of action of amphotericin B and caspofungin against C. albicans biofilms.

  20. A novel assay of biofilm antifungal activity reveals that amphotericin B and caspofungin lyse Candida albicans cells in biofilms.

    Science.gov (United States)

    DiDone, Louis; Oga, Duana; Krysan, Damian J

    2011-08-01

    The ability of Candida albicans to form drug-resistant biofilms is an important factor in its contribution to human disease. Assays to identify and characterize molecules with activity against fungal biofilms are crucial for the development of drugs with improved anti-biofilm activity. Here we report the application of an adenylate kinase (AK)-based cytotoxicity assay of fungal cell lysis to the characterization of agents active against C. albicans biofilms. We have developed three protocols for the AK assay. The first measures AK activity in the supernatants of biofilms treated with antifungal drugs and can be performed in parallel with a standard 2,3-bis-(2-methoxy-4-nitro-5-sulphophenyl)-2H-tetrazolium-5-caboxanilide-based biofilm susceptibility assay; a second, more sensitive protocol measures the AK activity present within the biofilm matrix; and a third procedure allows the direct visualization of lytic activity toward biofilms formed on catheter material. Amphotericin B and caspofungin, the two most effective anti-biofilm drugs currently used to treat fungal infections, both directly lyse planktonic C. albicans cells in vitro, leading to the release of AK into the culture medium. These studies serve to validate the AK-based lysis assay as a useful addition to the methods for the characterization of antifungal agents active toward biofilms and provide insights into the mode of action of amphotericin B and caspofungin against C. albicans biofilms. PMID:21674619

  1. Micro-arrayed human embryonic stem cells-derived cardiomyocytes for in vitro functional assay.

    Directory of Open Access Journals (Sweden)

    Elena Serena

    Full Text Available INTRODUCTION: The heart is one of the least regenerative organs in the body and any major insult can result in a significant loss of heart cells. The development of an in vitro-based cardiac tissue could be of paramount importance for many aspects of the cardiology research. In this context, we developed an in vitro assay based on human cardiomyocytes (hCMs and ad hoc micro-technologies, suitable for several applications: from pharmacological analysis to physio-phatological studies on transplantable hCMs. We focused on the development of an assay able to analyze not only hCMs viability, but also their functionality. METHODS: hCMs were cultured onto a poly-acrylamide hydrogel with tunable tissue-like mechanical properties and organized through micropatterning in a 20×20 array. Arrayed hCMs were characterized by immunofluorescence, GAP-FRAP analyses and live and dead assay. Their functionality was evaluated monitoring the excitation-contraction coupling. RESULTS: Micropatterned hCMs maintained the expression of the major cardiac markers (cTnT, cTnI, Cx43, Nkx2.5, α-actinin and functional properties. The spontaneous contraction frequency was (0.83±0.2 Hz, while exogenous electrical stimulation lead to an increase up to 2 Hz. As proof of concept that our device can be used for screening the effects of pathological conditions, hCMs were exposed to increasing levels of H(2O(2. Remarkably, hCMs viability was not compromised with exposure to 0.1 mM H(2O(2, but hCMs contractility was dramatically suppressed. As proof of concept, we also developed a microfluidic platform to selectively treat areas of the cell array, in the perspective of performing multi-parametric assay. CONCLUSIONS: Such system could be a useful tool for testing the effects of multiple conditions on an in vitro cell model representative of human heart physiology, thus potentially helping the processes of therapy and drug development.

  2. Adapted cytokinesis-block micronucleus assay (CBMn) for mouse embryonic stem cells

    OpenAIRE

    sprotocols

    2015-01-01

    Authors: Hamid Kalantari, Hamid Gourabi & Hossein Baharvand ### Abstract Our observation showed the addition of cytochalasin-B to mouse embryonic stem cells (mESC) culture for CBMn analysis led to the induction of apoptosis in these cells. On the other hand, addition of cyt-B is the most critical part of the cytokinesis-block micronucleus assay (CBMn) technique that cannot be omitted. Thus, modification of the traditional CBMn assay seems to be necessary. In this paper, we attempt...

  3. Convenient cell fusion assay for rapid screening for HIV entry inhibitors

    Science.gov (United States)

    Jiang, Shibo; Radigan, Lin; Zhang, Li

    2000-03-01

    Human immunodeficiency viruses (HIV)-induced cell fusion is a critical pathway of HIV spread from infected cells to uninfected cells. A rapid and simple assay was established to measure HIV-induce cell fusion. This study is particularly useful to rapid screen for HIV inhibitors that block HIV cell-to-cell transmission. Present study demonstrated that coculture of HIV-infected cells with uninfected cells at 37 degree(s)C for 2 hours resulted in the highest cell fusion rate. Using this cell fusion assay, we have identified several potent HIV inhibitors targeted to the HIV gp41 core. These antiviral agents can be potentially developed as antiviral drugs for chemotherapy and prophylaxis of HIV infection and AIDS.

  4. A NOVel ELISPOT assay to quantify HLA-specific B cells in HLA-immunized individuals

    NARCIS (Netherlands)

    Heidt, S.; Roelen, D.L.; Vaal, Y.J. de; Kester, M.G.; Eijsink, C.; Thomas, S.; Besouw, N.M. van; Volk, H.D.; Weimar, W.; Claas, F.H.; Mulder, A.

    2012-01-01

    Quantification of the humoral alloimmune response is generally achieved by measuring serum HLA antibodies, which provides no information about the cells involved in the humoral immune response. Therefore, we have developed an HLA-specific B-cell ELISPOT assay allowing for quantification of B cells p

  5. Single Cell Proteolytic Assays to Investigate Cancer Clonal Heterogeneity and Cell Dynamics Using an Efficient Cell Loading Scheme

    Science.gov (United States)

    Chen, Yu-Chih; Cheng, Yu-Heng; Ingram, Patrick; Yoon, Euisik

    2016-06-01

    Proteolytic degradation of the extracellular matrix (ECM) is critical in cancer invasion, and recent work suggests that heterogeneous cancer populations cooperate in this process. Despite the importance of cell heterogeneity, conventional proteolytic assays measure average activity, requiring thousands of cells and providing limited information about heterogeneity and dynamics. Here, we developed a microfluidic platform that provides high-efficiency cell loading and simple valveless isolation, so the proteolytic activity of a small sample (10–100 cells) can be easily characterized. Combined with a single cell derived (clonal) sphere formation platform, we have successfully demonstrated the importance of microenvironmental cues for proteolytic activity and also investigated the difference between clones. Furthermore, the platform allows monitoring single cells at multiple time points, unveiling different cancer cell line dynamics in proteolytic activity. The presented tool facilitates single cell proteolytic analysis using small samples, and our findings illuminate the heterogeneous and dynamic nature of proteolytic activity.

  6. The influence of the number of cells scored on the sensitivity in the comet assay

    DEFF Research Database (Denmark)

    Sharma, Anoop Kumar; Soussaline, Françoise; Sallette, Jerome;

    2012-01-01

    The impact on the sensitivity of the in vitro comet assay by increasing the number of cells scored has only been addressed in a few studies. The present study investigated whether the sensitivity of the assay could be improved by scoring more than 100 cells. Two cell lines and three different...... out by means of a fully automated scoring system and the results were analyzed by evaluating the % tail DNA of 100–700 randomly selected cells for each slide consisting of two gels. By increasing the number of cells scored, the coefficients of variance decreased, leading to an improved sensitivity...... of the assay. A two-way ANOVA analysis of variance showed that the contribution from the two variables “the number of cells scored” and “concentration” on the total variation in the coefficients of variance dataset was statistically significant (p...

  7. Analysis of three marine fish cell lines by rapd assay.

    Science.gov (United States)

    Guo, H R; Zhang, S C; Tong, S L; Xiang, J H

    2001-01-01

    We tested the applicability of the random amplified polymorphic deoxyribonucleic acid (RAPD) analysis for identification of three marine fish cell lines FG, SPH, and RSBF, and as a possible tool to detect cross-contamination. Sixty commercial 10-mer RAPD primers were tested on the cell lines and on samples collected from individual fish. The results obtained showed that the cell lines could be identified to the correspondent species on the basis of identical patterns produced by 35-48% of the primers tested; the total mean similarity indices for cell lines versus correspondent species of individual fish ranged from 0.825 to 0.851, indicating the existence of genetic variation in these cell lines in relation to the species of their origin. Also, four primers, which gave a monomorphic band pattern within species/line, but different among the species/line, were obtained. These primers can be useful for identification of these cell lines and for characterization of the genetic variation of these cell lines in relation to the species of their origin. This supported the use of RAPD analysis as an effective tool in species identification and cross-contamination test among different cell lines. PMID:11573817

  8. A versatile assay for the identification of RNA silencing suppressors based on complementation of viral movement.

    Science.gov (United States)

    Powers, Jason G; Sit, Tim L; Qu, Feng; Morris, T Jack; Kim, Kook-Hyung; Lommel, Steven A

    2008-07-01

    The cell-to-cell movement of Turnip crinkle virus (TCV) in Nicotiana benthamiana requires the presence of its coat protein (CP), a known suppressor of RNA silencing. RNA transcripts of a TCV construct containing a reporter gene (green fluorescent protein) (TCV-sGFP) in place of the CP open reading frame generated foci of three to five cells. TCV CP delivered in trans by Agrobacterium tumefaciens infiltration potentiated movement of TCV-sGFP and increased foci diameter, on average, by a factor of four. Deletion of the TCV movement proteins in TCV-sGFP (construct TCVDelta92-sGFP) abolished the movement complementation ability of TCV CP. Other known suppressors of RNA silencing from a wide spectrum of viruses also complemented the movement of TCV-sGFP when delivered in trans by Agrobacterium tumefaciens. These include suppressors from nonplant viruses with no known plant movement function, demonstrating that this assay is based solely on RNA silencing suppression. While the TCV-sGFP construct is primarily used as an infectious RNA transcript, it was also subcloned for direct expression from Agrobacterium tumefaciens for simple quantification of suppressor activity based on fluorescence levels in whole leaves. Thus, this system provides the flexibility to assay for suppressor activity in either the cytoplasm or nucleus, depending on the construct employed. PMID:18533829

  9. Smart polymer platforms for in vitro drug screening assays based on drug-loaded nanoparticles

    DEFF Research Database (Denmark)

    Faralli, Adele

    ). In this thesis we investigate the use of polymers for drug screening assays. The aim of this study is the development of a polymer platform that enables to overcome some of the limitations that characterize the existing screening methods, by requiring very small amounts of tissues and permitting a fast and low......-cost screening of individual drugs as well as combined drugs. Human colorectal adenocarcinoma cell line (HT-29) has been selected as cell culture model because easy to handle and phenotypically stable. The responsiveness of HT-29 cells to the individual and combined drug regimens normally selected for colorectal...... cancer therapy is finally evaluated using our technologies. Two main platforms are proposed: one is based on the use of poly(ethylene glycol) diacrylate (PEGDA) hydrogels for controlled drug release, and the second one consists on the use of poly(3,4-(1-azidomethylene)-dioxythiophene) (PEDOT-N3) micro...

  10. In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas.

    Science.gov (United States)

    Tremblay, Jacob R; LeBon, Jeanne M; Luo, Angela; Quijano, Janine C; Wedeken, Lena; Jou, Kevin; Riggs, Arthur D; Tirrell, David A; Ku, H Teresa

    2016-01-01

    Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. However, it is still unknown whether stem and progenitor cells exist in the adult pancreas. Research strategies using cre-lox lineage-tracing in adult mice have yielded results that either support or refute the idea that beta cells can be generated from the ducts, the presumed location where adult pancreatic progenitors may reside. These in vivo cre-lox lineage-tracing methods, however, cannot answer the questions of self-renewal and multi-lineage differentiation-two criteria necessary to define a stem cell. To begin addressing this technical gap, we devised 3-dimensional colony assays for pancreatic progenitors. Soon after our initial publication, other laboratories independently developed a similar, but not identical, method called the organoid assay. Compared to the organoid assay, our method employs methylcellulose, which forms viscous solutions that allow the inclusion of extracellular matrix proteins at low concentrations. The methylcellulose-containing assays permit easier detection and analyses of progenitor cells at the single-cell level, which are critical when progenitors constitute a small sub-population, as is the case for many adult organ stem cells. Together, results from several laboratories demonstrate in vitro self-renewal and multi-lineage differentiation of pancreatic progenitor-like cells from mice. The current protocols describe two methylcellulose-based colony assays to characterize mouse pancreatic progenitors; one contains a commercial preparation of murine extracellular matrix proteins and the other an artificial extracellular matrix protein known as a laminin hydrogel. The techniques shown here are 1) dissociation of the pancreas and sorting of CD133(+)Sox9/EGFP(+) ductal cells from adult mice, 2) single cell manipulation of the sorted cells, 3) single colony analyses using microfluidic q

  11. Radiotoxicity induced by auger electron emitters in human osteosarcoma cell line using comet assay

    International Nuclear Information System (INIS)

    The comet assay (single cell gel electrophoresis assay) was used to evaluate the radiotoxicity of Auger electron emitters in the human osteosarcoma cell line (HOS-8603). After internal exposure to 67Ga-EDTMP, the sarcoma cell has been injured severely. The comet length was longer along with the increase of dose, the appearance of comet tail was different from that with respect to the 60Co γ-ray irradiation. DNA damage of cell was mainly due to the radiation effect of Auger electrons. The 67Ga may be a therapeutic radionuclide with good prospect for tumor treatment and palliation of bone pain induced by metastasis

  12. Radiotoxicity induced by Auger electron emitters in human osteosarcoma cell line using comet assay

    Institute of Scientific and Technical Information of China (English)

    XU Yu-Jie; LI Qing-Nuan; ZHU Ran; ZHU Ben-Xing; ZHANG Yong-Ping; ZHANG Xiao-Dong; FAN Wo; HONG Cheng-Jiao; LI Wen-Xin

    2003-01-01

    The comet assay (single cell gel electrophoresis assay) was used to evaluate the radiotoxicity of Augerelectron emitters in the human osteosarcoma cell line (HOS-8603). After internal exposure to 67Ga-EDTMP, the sar-coma cell has been injured severely. The comet length was longer along with the increase of dose, the appearance ofcomet tail was different from that with respect to the 60Co γ-ray irradiation. DNA damage of cell was mainly due tothe radiation effect of Auger electrons. The 67Ga may be a therapeutic radionuclide with good prospect for tumortreatment and palliation of bone pain induced by metastasis.

  13. Antimetastatic function of concomitant antitumor immunity. I. Host Ly-1+2+ effector T cells prevent the enumeration of metastatic tumor cells in a biological assay

    International Nuclear Information System (INIS)

    A mouse survival assay was evaluated for its suitability to enumerate metastatic P815 tumor cells in the draining lymph node and spleen of a B6D2 F1 (H-2/sup b/ x H-2/sup d/) host bearing a primary intradermal P815 tumor. The mouse survival assay is based on the linear relationship between the log10 number of P815 tumor cells (H-2/sup d/) injected i.p. into mice and their mean survival time. It was found that the assay is capable of quantifying as few as 10 tumor cells in lymph node and spleen, but only if cell suspensions of these organs are treated with anti-H-2/sup b/ serum and complement, in order to selectively destroy H-2/sup bd/ host cells. This was necessary because host cells from the lymph node and spleen of a tumor-bearing host possessed antitumor functions, in that they were capable of destroying the H-2/sup d/ P815 tumor cells when admixed with the tumor cells and injected i.p. into 800-rad irradiated test recipients. The kinetics of acquisition and loss of host cells with antitumor function and the Ly phenotype of these host cells suggest that they are the same cells that give the tumor-bearing host the capacity to express concomitant immunity against a tumor implant

  14. Field-based multiplex and quantitative assay platforms for diagnostics

    Science.gov (United States)

    Venkatasubbarao, Srivatsa; Dixon, C. Edward; Chipman, Russell; Scherer, Axel; Beshay, Manal; Kempen, Lothar U.; Chandra Sekhar, Jai Ganesh; Yan, Hong; Puccio, Ava; Okonkwo, David; McClain, Stephen; Gilbert, Noah; Vyawahare, Saurabh

    2011-06-01

    The U.S. military has a continued interest in the development of handheld, field-usable sensors and test kits for a variety of diagnostic applications, such as traumatic brain injury (TBI) and infectious diseases. Field-use presents unique challenges for biosensor design, both for the readout unit and for the biological assay platform. We have developed robust biosensor devices that offer ultra-high sensitivity and also meet field-use needs. The systems under development include a multiplexed quantitative lateral flow test strip for TBI diagnostics, a field test kit for the diagnosis of pathogens endemic to the Middle East, and a microfluidic assay platform with a label-free reader for performing complex biological automated assays in the field.

  15. Limitations of MTT and MTS-based assays for measurement of antiproliferative activity of green tea polyphenols.

    Directory of Open Access Journals (Sweden)

    Piwen Wang

    Full Text Available BACKGROUND: The chemopreventive effect of green tea polyphenols, such as (--epigallocatechin-3-gallate (EGCG, has been well demonstrated in cell culture studies. However, a wide range of IC(50 concentrations has been observed in published studies of the anti-proliferative activity of EGCG from different laboratories. Although the susceptibility to EGCG treatment is largely dependent on cancer cell type, the particular cell viability and proliferation assays utilized may significantly influence quantitative results reported in the literature. METHODOLOGY/PRINCIPAL FINDINGS: We compared five widely used methods to measure cell proliferation and viability after EGCG treatment using LNCaP prostate cancer cells and MCF-7 breast cancer cells. Both methods using dyes to quantify adenosine triphosphate (ATP and deoxynucleic acid (DNA showed accuracy in the measurement of viable cells when compared to trypan blue assay and results showed good linear correlation (r = 0.95. However, the use of MTT (3-(4,5-Dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide and MTS (3-(4,5-dimethylthiazol-2-yl-5-(3-carboxymethoxyphenyl-2-(4-sulfophenyl-2H-tetrazolium as indicators of metabolically active mitochondria overestimated the number of viable cells by comparison with the ATP, DNA, or trypan blue determinations. As a result, the observed IC(50 concentration of EGCG was 2-fold higher using MTT and MTS compared to dyes quantifying ATP and DNA. In contrast, when cells were treated with apigenin MTT and MTS assays showed consistent results with ATP, DNA, or trypan blue assays. CONCLUSIONS/SIGNIFICANCE: These results demonstrate that MTT and MTS -based assays will provide an underestimation of the anti-proliferative effect of EGCG, and suggest the importance of careful evaluation of the method for in vitro assessment of cell viability and proliferation depending on the chemical nature of botanical supplements.

  16. An Enhanced ELISPOT Assay for Sensitive Detection of Antigen-Specific T Cell Responses to Borrelia burgdorferi

    Directory of Open Access Journals (Sweden)

    Gottfried H. Kellermann

    2013-09-01

    Full Text Available Lyme Borreliosis is an infectious disease caused by the spirochete Borrelia burgdorferi that is transmitted through the bite of infected ticks. Both B cell-mediated humoral immunity and T cell immunity develop during natural Borrelia infection. However, compared with humoral immunity, the T cell response to Borrelia infection has not been well elucidated. In this study, a novel T cell-based assay was developed and validated for the sensitive detection of antigen-specific T cell response to B. burgdorferi. Using interferon-g as a biomarker, we developed a new enzyme-linked immunospot method (iSpot Lyme™ to detect Borrelia antigen-specific effector/memory T cells that were activated in vivo by exposing them to recombinant Borrelia antigens ex vivo. To test this new method as a potential laboratory diagnostic tool, we performed a clinical study with a cohort of Borrelia positive patients and healthy controls. We demonstrated that the iSpot Lyme assay has a significantly higher specificity and sensitivity compared with the Western Blot assay that is currently used as a diagnostic measure. A comprehensive evaluation of the T cell response to Borrelia infection should, therefore, provide new insights into the pathogenesis, diagnosis, treatment and monitoring of Lyme disease.

  17. Cell invasion in the spheroid sprouting assay: a spatial organisation analysis adaptable to cell behaviour.

    Directory of Open Access Journals (Sweden)

    Silvia Blacher

    Full Text Available The endothelial cell spheroid assay provides a suitable in vitro model to study (lymph angiogenesis and test pro- and anti-(lymph angiogenic factors or drugs. Usually, the extent of cell invasion, observed through optical microscopy, is measured. The present study proposes the spatial distribution of migrated cells as a new descriptor of the (lymph angiogenic response. The utility of this novel method rests with its capacity to locally characterise spheroid structure, allowing not only the investigation of single and collective cell invasion but also the evolution of the spheroid core itself. Moreover, the proposed method can be applied to 2D-projected spheroid images obtained by optical microscopy, as well as to 3D images acquired by confocal microscopy. To validate the proposed methodology, endothelial cell invasion was evaluated under different experimental conditions. The results were compared with widely used global parameters. The comparison shows that our method prevents local spheroid modifications from being overlooked and leading to the possible misinterpretation of results.

  18. A novel in vitro survival assay of small intestinal stem cells after exposure to ionizing radiation

    International Nuclear Information System (INIS)

    The microcolony assay developed by Withers and Elkind has been a gold standard to assess the surviving fraction of small intestinal stem cells after exposure to high (≥8 Gy) doses of ionizing radiation (IR), but is not applicable in cases of exposure to lower doses. Here, we developed a novel in vitro assay that enables assessment of the surviving fraction of small intestinal stem cells after exposure to lower IR doses. The assay includes in vitro culture of small intestinal stem cells, which allows the stem cells to develop into epithelial organoids containing all four differentiated cell types of the small intestine. We used Lgr5-EGFP-IRES-CreERT2/ROSA26-tdTomato mice to identify Lgr5+ stem cells and their progeny. Enzymatically dissociated single crypt cells from the duodenum and jejunum of mice were irradiated with 7.25, 29, 101, 304, 1000, 2000 and 4000 mGy of X-rays immediately after plating, and the number of organoids was counted on Day 12. Organoid-forming efficiency of irradiated cells relative to that of unirradiated controls was defined as the surviving fraction of stem cells. We observed a significant decrease in the surviving fraction of stem cells at ≥1000 mGy. Moreover, fluorescence-activated cell sorting analyses and passage of the organoids revealed that proliferation of stem cells surviving IR is significantly potentiated. Together, the present study demonstrates that the in vitro assay is useful for quantitatively assessing the surviving fraction of small intestinal stem cells after exposure to lower doses of IR as compared with previous examinations using the microcolony assay. (author)

  19. Natural killer cell cytotoxicity assay with time-resolved fluorimetry

    Institute of Scientific and Technical Information of China (English)

    李建中; 章竹君; 金伯泉; 田方

    1996-01-01

    A new time-resolved fluorimetric method for the measurement of natural killer (NK) cell cytotoxicity has been developed by labelling the target cell K562 with a new synthesized fluorescence marker KLUK. The method has advantages of higher sensitivity, time-saving, good reproducibility and has no radioactivity problems. A satisfactory result is obtained by comparing it with 51Cr release method. It demonstrates that the new marker provides an alternative to currently used radioactive markers for the assessment of in vitro cellular cytotoxicity.

  20. Micronucleus assay prediction and application optimized by cytochalasin B-induced binucleated tumor cells

    International Nuclear Information System (INIS)

    Improvement in the predictive assertion of the micronucleus assay was achieved by treating human malignant melanoma cells (Mewo) with cytochalasin B (CB), generating binucleated cells (BNC) representing cells after a single karyokinesis. Optimal cell binucleation was determined by testing several cytochalasin B concentrations and different incubation times. On average, 56% binucleated cells were found after incubation with 2 to 3 μg/ml cytochalasin B for 48 h. Cells with at least one micronucleus (Mn) were defined as fraction of cells with micronuclei and describes the degree of damaged cells. We found in binucleated cells 2.2fold the fraction of cells with micronuclei than in mononucleated cells (MNC), as expected assuming that an induced micronucleus is associated with only one single daughter cell after mitosis. The mean of micronuclei per binucleated cells, however, was enhanced about 2.9fold in relation to that of micronuclei per mononucleated cells and is related to the nucelar damage per cell. The application of cytochalasin B did not enhance the fraction of damaged cells although the degree of the injury per cell is intensified. A micronuclei promoting or inhibiting effect of the experimental design due to changes in cell proliferation was excluded by cytofluorometric investigations of DNA content and synthesis after cytochalasin B application. A comparison of the modified with the conventional micronucleus assay shows the superiority of the former. (orig.)

  1. Ovine carotid artery-derived cells as an optimized supportive cell layer in 2-D capillary network assays.

    Directory of Open Access Journals (Sweden)

    Stefan Weinandy

    Full Text Available BACKGROUND: Endothelial cell co-culture assays are differentiation assays which simulate the formation of capillary-like tubules with the aid of a supportive cell layer. Different cell types have been employed as a supportive cell layer, including human pulmonary artery smooth muscle cells (PASMCs and human mammary fibroblasts. However, these sources of human tissue-derived cells are limited, and more readily accessible human or animal tissue-derived cell sources would simplify the endothelial cell co-culture assay. In the present study, we investigated the potential use of alternative, accessible supportive cells for endothelial cell co-culture assay, including human umbilical cord and ovine carotid artery. METHODS AND RESULTS: Human umbilical artery SMCs (HUASMCs and ovine carotid artery-derived cells were seeded into 96-well plates, followed by addition of human umbilical vein endothelial cells (HUVECs. Nine days after co-culture, cells were fixed, immunostained and analysed using an in vitro angiogenesis quantification tool. Capillary-like structures were detected on ovine carotid artery-derived supportive cell layers. The initial cell number, as well as pro- and anti-angiogenic factors (VEGF, PDGF-BB and Bevacizumab, had a positive or negative influence on the number of capillary-like structures. Furthermore, HUVECs from different donors showed distinct levels of VEGF receptor-2, which correlated with the amount of capillary-like structures. In the case of HUASMC supportive cell layers, HUVECs detached almost completely from the surface. CONCLUSIONS: Cells of different origin have a varying applicability regarding the endothelial cell co-culture assay: under the conditions described here, ovine carotid artery-derived cells seem to be more suitable than HUASMCs for an endothelial co-culture assay. Furthermore, the ovine carotid artery-derived cells are easier to obtain and are in more abundant supply than the currently used dermal or breast

  2. Discovery of a novel restriction endonuclease by genome comparison and application of a wheat-germ-based cell-free translation assay: PabI (5′-GTA/C) from the hyperthermophilic archaeon Pyrococcus abyssi

    OpenAIRE

    Ishikawa, Ken; Watanabe, Miki; Kuroita, Toshihiro; Uchiyama, Ikuo; Bujnicki, Janusz M.; Kawakami, Bunsei; Tanokura, Masaru; Kobayashi, Ichizo

    2005-01-01

    To search for restriction endonucleases, we used a novel plant-based cell-free translation procedure that bypasses the toxicity of these enzymes. To identify candidate genes, the related genomes of the hyperthermophilic archaea Pyrococcus abyssi and Pyrococcus horikoshii were compared. In line with the selfish mobile gene hypothesis for restriction–modification systems, apparent genome rearrangement around putative restriction genes served as a selecting criterion. Several candidate restricti...

  3. Development of an enzyme-linked immunosorbent assay (ELISA)-like fluorescence assay to investigate the interactions of glycosaminoglycans to cells

    Energy Technology Data Exchange (ETDEWEB)

    Boucas, Rodrigo Ippolito [Disciplina de Biologia Molecular, Departamento de Bioquimica, Universidade Federal de Sao Paulo, SP (Brazil); Trindade, Edvaldo S. [Disciplina de Biologia Molecular, Departamento de Bioquimica, Universidade Federal de Sao Paulo, SP (Brazil); Departamento de Biologia Celular, Universidade Federal do Parana, Curitiba, Parana (Brazil); Tersariol, Ivarne L.S. [Disciplina de Biologia Molecular, Departamento de Bioquimica, Universidade Federal de Sao Paulo, SP (Brazil); Centro Interdisciplinar de Investigacao Bioquimica, Universidade de Mogi das Cruzes, Mogi das Cruzes, SP (Brazil); Dietrich, Carl P. [Disciplina de Biologia Molecular, Departamento de Bioquimica, Universidade Federal de Sao Paulo, SP (Brazil); Nader, Helena B. [Disciplina de Biologia Molecular, Departamento de Bioquimica, Universidade Federal de Sao Paulo, SP (Brazil)], E-mail: hbnader.bioq@epm.br

    2008-06-23

    Sulfated glycosaminoglycans were labeled with biotin to study their interaction with cells in culture. Thus, heparin, heparan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate and dermatan sulfate were labeled using biotin-hydrazide, under different conditions. The structural characteristics of the biotinylated products were determined by chemical (molar ratios of hexosamine, uronic acid, sulfate and biotin) and enzymatic methods (susceptibility to degradation by chondroitinases and heparitinases). The binding of biotinylated glycosaminoglycans was investigated both in endothelial and smooth muscle cells in culture, using a novel time resolved fluorometric method based on interaction of europium-labeled streptavidin with the biotin covalently linked to the compounds. The interactions of glycosaminoglycans were saturable and number of binding sites could be obtained for each individual compound. The apparent dissociation constant varied among the different glycosaminoglycans and between the two cell lines. The interactions of the biotinylated glycosaminoglycans with the cells were also evaluated using confocal microscopy. We propose a convenient and reliable method for the preparation of biotinylated glycosaminoglycans, as well as a sensitive non-competitive fluorescence-based assay for studies of the interactions and binding of these compounds to cells in culture.

  4. Automated high-content assay for compounds selectively toxic to Trypanosoma cruzi in a myoblastic cell line.

    Directory of Open Access Journals (Sweden)

    Julio Alonso-Padilla

    2015-01-01

    Full Text Available Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, represents a very important public health problem in Latin America where it is endemic. Although mostly asymptomatic at its initial stage, after the disease becomes chronic, about a third of the infected patients progress to a potentially fatal outcome due to severe damage of heart and gut tissues. There is an urgent need for new drugs against Chagas disease since there are only two drugs available, benznidazole and nifurtimox, and both show toxic side effects and variable efficacy against the chronic stage of the disease.Genetically engineered parasitic strains are used for high throughput screening (HTS of large chemical collections in the search for new anti-parasitic compounds. These assays, although successful, are limited to reporter transgenic parasites and do not cover the wide T. cruzi genetic background. With the aim to contribute to the early drug discovery process against Chagas disease we have developed an automated image-based 384-well plate HTS assay for T. cruzi amastigote replication in a rat myoblast host cell line. An image analysis script was designed to inform on three outputs: total number of host cells, ratio of T. cruzi amastigotes per cell and percentage of infected cells, which respectively provides one host cell toxicity and two T. cruzi toxicity readouts. The assay was statistically robust (Z´ values >0.6 and was validated against a series of known anti-trypanosomatid drugs.We have established a highly reproducible, high content HTS assay for screening of chemical compounds against T. cruzi infection of myoblasts that is amenable for use with any T. cruzi strain capable of in vitro infection. Our visual assay informs on both anti-parasitic and host cell toxicity readouts in a single experiment, allowing the direct identification of compounds selectively targeted to the parasite.

  5. Automated High-Content Assay for Compounds Selectively Toxic to Trypanosoma cruzi in a Myoblastic Cell Line

    Science.gov (United States)

    Alonso-Padilla, Julio; Cotillo, Ignacio; Presa, Jesús L.; Cantizani, Juan; Peña, Imanol; Bardera, Ana I.; Martín, Jose J.; Rodriguez, Ana

    2015-01-01

    Background Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, represents a very important public health problem in Latin America where it is endemic. Although mostly asymptomatic at its initial stage, after the disease becomes chronic, about a third of the infected patients progress to a potentially fatal outcome due to severe damage of heart and gut tissues. There is an urgent need for new drugs against Chagas disease since there are only two drugs available, benznidazole and nifurtimox, and both show toxic side effects and variable efficacy against the chronic stage of the disease. Methodology/Principal Findings Genetically engineered parasitic strains are used for high throughput screening (HTS) of large chemical collections in the search for new anti-parasitic compounds. These assays, although successful, are limited to reporter transgenic parasites and do not cover the wide T. cruzi genetic background. With the aim to contribute to the early drug discovery process against Chagas disease we have developed an automated image-based 384-well plate HTS assay for T. cruzi amastigote replication in a rat myoblast host cell line. An image analysis script was designed to inform on three outputs: total number of host cells, ratio of T. cruzi amastigotes per cell and percentage of infected cells, which respectively provides one host cell toxicity and two T. cruzi toxicity readouts. The assay was statistically robust (Z´ values >0.6) and was validated against a series of known anti-trypanosomatid drugs. Conclusions/Significance We have established a highly reproducible, high content HTS assay for screening of chemical compounds against T. cruzi infection of myoblasts that is amenable for use with any T. cruzi strain capable of in vitro infection. Our visual assay informs on both anti-parasitic and host cell toxicity readouts in a single experiment, allowing the direct identification of compounds selectively targeted to the parasite. PMID:25615687

  6. Homogeneous time-resolved G protein-coupled receptor-ligand binding assay based on fluorescence cross-correlation spectroscopy.

    Science.gov (United States)

    Antoine, Thomas; Ott, David; Ebell, Katharina; Hansen, Kerrin; Henry, Luc; Becker, Frank; Hannus, Stefan

    2016-06-01

    G protein-coupled receptors (GPCRs) mediate many important physiological functions and are considered as one of the most successful therapeutic target classes for a wide spectrum of diseases. Drug discovery projects generally benefit from a broad range of experimental approaches for screening compound libraries and for the characterization of binding modes of drug candidates. Owing to the difficulties in solubilizing and purifying GPCRs, assay formats have been so far mainly limited to cell-based functional assays and radioligand binding assays. In this study, we used fluorescence cross-correlation spectroscopy (FCCS) to analyze the interaction of detergent-solubilized receptors to various types of GPCR ligands: endogenous peptides, small molecules, and a large surrogate antagonist represented by a blocking monoclonal antibody. Our work demonstrates the suitability of the homogeneous and time-resolved FCCS assay format for a robust, high-throughput determination of receptor-ligand binding affinities and kinetic rate constants for various therapeutically relevant GPCRs. PMID:26954998

  7. Prediction value of radiosensitivity of hepatocarcinoma cells for apoptosis and micronucleus assay

    Institute of Scientific and Technical Information of China (English)

    Zhi-Zhong Liu; Wen-Ying Huang; Xiao-Sheng Li; Ju-Sheng Lin; Xiao-Kun Cai; Kuo-Huang Lian; He-Jun Zhou

    2005-01-01

    AIM: To investigate the prediction value of radiosensitivity of hepatocarcinoma cells for apoptosis and micronucleus assay.METHODS: Clonogenic assay, flow cytometry, and CB micronuclei assay were used to survey the cell survival rate, radiation-induced apoptosis and micronucleus frequency of hepatocarcinoma cell lines SMMC-7721,HL-7702, and HepG2 after being irradiated by X-ray at the dosage ranging 0-8 Gy.RESULTS: After irradiation, there was a dose-effect relationship between micronucleus frequency and radiation dosage among the three cell lines (P<0.05). A positive relationship was observed between apoptosis and radiation dosage among the three cell lines. The HepG2 cells had a significant correlation (P<0.05) but apoptosis incidence had a negative relationship with micronucleus frequency. There was a positive relationship between apoptosis and radiation dosage and the correlation between SMMC-7721 and HL-7702 cell lines had a significant difference (P<0.01). After irradiation,a negative relationship between cell survival rate and radiation dosages was found among the three cell lines(P<0.01). There was a positive relationship between cell survival rate and micronucleus frequency (P<0.01). No correlation was observed between apoptosis and cell survival rate.CONCLUSION: The radiosensitivity of hepatocarcinoma cells can be reflected by apoptosis and micronuclei.Detection of apoptosis and micronuclei could enhance the accuracy for predicting radiosensitivity.

  8. Enumeration and Characterization of Human Memory T Cells by Enzyme-Linked Immunospot Assays

    Directory of Open Access Journals (Sweden)

    Sandra A. Calarota

    2013-01-01

    Full Text Available The enzyme-linked immunospot (ELISPOT assay has advanced into a useful and widely applicable tool for the evaluation of T-cell responses in both humans and animal models of diseases and/or vaccine candidates. Using synthetic peptides (either individually or as overlapping peptide mixtures or whole antigens, total lymphocyte or isolated T-cell subset responses can be assessed either after short-term stimulation (standard ELISPOT or after their expansion during a 10-day culture (cultured ELISPOT. Both assays detect different antigen-specific immune responses allowing the analysis of effector memory T cells and central memory T cells. This paper describes the principle of ELISPOT assays and discusses their application in the evaluation of immune correlates of clinical interest with a focus on the vaccine field.

  9. Improved assay for surface hydrophobic avidity of Candida albicans cells.

    OpenAIRE

    Hazen, K C; LeMelle, W G

    1990-01-01

    A simple method that distinguishes among hydrophobic avidity levels of highly hydrophobic isolates of the pathogenic fungus Candida albicans is described. This method involves mixing polystyrene microspheres at different concentrations with a constant concentration of yeast cells and plotting the data in accordance with the Langmuir isotherm equation. A 10-fold difference between the C. albicans isolates with the lowest and highest avidity (KH) values was found. This method may also demonstra...

  10. In Vitro Cell Culture Infectivity Assay for Human Noroviruses

    Energy Technology Data Exchange (ETDEWEB)

    Straub, Tim M.; Honer Zu Bentrup, Kerstin A.; Orosz Coghlan, Patricia A.; Dohnalkova, Alice; Mayer, Brooke K.; Bartholomew, Rachel A.; Valdez, Catherine O.; Bruckner-Lea, Cindy J.; Gerba, Charles P.; Abbaszadegan, Morteza; Nickerson, Cheryl A.

    2007-01-30

    Human noroviruses (NoV) cause severe, self-limiting gastroenteritis that typically lasts 24 - 48 hours. The true nature of NoV pathogenesis remains unknown due to the lack of suitable tissue culture or animal models. Here we show, for the first time, that NoV can infect and replicate in an organoid, three-dimensional (3-D) model of human small intestinal epithelium (INT-407). Cellular differentiation for this model was achieved by growing the cells in 3-D on porous collagen I-coated microcarrier beads under conditions of physiological fluid shear in rotating wall vessel bioreactors. Microscopy, PCR, and fluorescent in-situ hybridization were employed to provide evidence of NoV infection. CPE and norovirus RNA was detected at each of the five cell passages for both genogroup I and II viruses. Our results demonstrate that the highly differentiated 3-D cell culture model can support the natural growth of human noroviruses, whereas previous attempts using differentiated monolayer cultures failed.

  11. Development of a novel IGRA assay to test T cell responsiveness to HBV antigens in whole blood of chronic Hepatitis B patients

    OpenAIRE

    Dammermann, Werner; Bentzien, Frank; Stiel, Eva-Maria; Kühne, Claudia; Ullrich, Sebastian; zur Wiesch, Julian Schulze; Lüth, Stefan

    2015-01-01

    Background Interferon gamma release assays (IGRA) have been developed to support easy and fast diagnosis of diseases like tuberculosis, and CMV in transplant patients. IGRAs focus on cellular immunity especially memory T cells and thus also allow rapid screening prior to complex flow cytometric testing. Here, we describe a novel, sensitive whole blood based cytokine release assay capable of assessing T cell responsiveness to HBV antigens in Hepatitis B patients and assessing hepatitis B vacci...

  12. Identification of heme oxygenase-1 stimulators by a convenient ELISA-based bilirubin quantification assay.

    Science.gov (United States)

    Rücker, Hannelore; Amslinger, Sabine

    2015-01-01

    The upregulation of heme oxygenase-1 (HO-1) has proven to be a useful tool for fighting inflammation. In order to identify new HO-1 inducers, an efficient screening method was developed which can provide new lead structures for drug research. We designed a simple ELISA-based HO-1 enzyme activity assay, which allows for the screening of 12 compounds in parallel in the setting of a 96-well plate. The well-established murine macrophage cell line RAW264.7 is used and only about 26µg of protein from whole cell lysates is needed for the analysis of HO-1 activity. The quantification of HO-1 activity is based on an indirect ELISA using the specific anti-bilirubin antibody 24G7 to quantify directly bilirubin in the whole cell lysate, applying a horseradish peroxidase-tagged antibody together with ortho-phenylenediamine and H2O2 for detection. The bilirubin is produced on the action of HO enzymes by converting their substrate heme to biliverdin and additional recombinant biliverdin reductase together with NADPH at pH 7.4 in buffer. This sensitive assay allows for the detection of 0.57-82pmol bilirubin per sample in whole cell lysates. Twenty-three small molecules, mainly natural products with an α,β-unsaturated carbonyl unit such as polyphenols, including flavonoids and chalcones, terpenes, an isothiocyanate, and the drug oltipraz were tested at typically 6 or 24h incubation with RAW264.7 cells. The activity of known HO-1 inducers was confirmed, while the chalcones cardamonin, flavokawain A, calythropsin, 2',3,4'-trihydroxy-4-methoxychalcone (THMC), and 2',4'-dihydroxy-3,4-dimethoxychalcone (DHDMC) were identified as new potent HO-1 inducers. The highest inductive power after 6h incubation was found at 10µM for DHDMC (6.1-fold), carnosol (3.9-fold), butein (3.1-fold), THMC (2.9-fold), and zerumbone (2.5-fold). Moreover, the time dependence of HO-1 protein production for DHDMC was compared to its enzyme activity, which was further evaluated in the presence of

  13. Progress in high-throughput assays of MGMT and APE1 activities in cell extracts

    OpenAIRE

    Georgiadis, Panagiotis; Polychronaki, Nektaria; Kyrtopoulos, Soterios A.

    2012-01-01

    DNA repair activity is of interest as a potential biomarker of individual susceptibility to genotoxic agents. In view of the current trend for exploitation of large cohorts in molecular epidemiology projects, there is a pressing need for the development of phenotypic DNA repair assays that are high-throughput, very sensitive, inexpensive and reliable. Towards this goal we have developed and validated two phenotypic assays for the measurement of two DNA repair enzymes in cell extracts: (1) O(6...

  14. A molecular assay for sensitive detection of pathogen-specific T-cells.

    Directory of Open Access Journals (Sweden)

    Victoria O Kasprowicz

    Full Text Available Here we describe the development and validation of a highly sensitive assay of antigen-specific IFN-γ production using real time quantitative PCR (qPCR for two reporters--monokine-induced by IFN-γ (MIG and the IFN-γ inducible protein-10 (IP10. We developed and validated the assay and applied it to the detection of CMV, HIV and Mycobacterium tuberculosis (MTB specific responses, in a cohort of HIV co-infected patients. We compared the sensitivity of this assay to that of the ex vivo RD1 (ESAT-6 and CFP-10-specific IFN-γ Elispot assay. We observed a clear quantitative correlation between the two assays (P<0.001. Our assay proved to be a sensitive assay for the detection of MTB-specific T cells, could be performed on whole blood samples of fingerprick (50 uL volumes, and was not affected by HIV-mediated immunosuppression. This assay platform is potentially of utility in diagnosis of infection in this and other clinical settings.

  15. Production of Genetically Engineered Biotinylated Interleukin-2 and Its Application in a Rapid Nonradioactive Assay for T-Cell Activation

    OpenAIRE

    Jordan, Robert A.; Preissler, Mark T.; Banas, Jeffrey A.; Gosselin, Edmund J.

    2003-01-01

    The development of reliable assay systems that can measure lymphocyte activation in vitro has been a major goal of immunodiagnostics. Traditionally, tritiated thymidine incorporation has been used to monitor T-cell activation. Other methods include enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunospot assay, and colorimetric assays. We have established a lymphocyte activation assay that utilizes fluorescein isothiocyanate (FITC)-streptavidin bound to recombinant biotinylated hum...

  16. A simple non-perturbing cell migration assay insensitive to proliferation effects.

    Science.gov (United States)

    Glenn, Honor L; Messner, Jacob; Meldrum, Deirdre R

    2016-01-01

    Migration is a fundamental cellular behavior that plays an indispensable role in development and homeostasis, but can also contribute to pathology such as cancer metastasis. Due to its relevance to many aspects of human health, the ability to accurately measure cell migration is of broad interest, and numerous approaches have been developed. One of the most commonly employed approaches, because of its simplicity and throughput, is the exclusion zone assay in which cells are allowed to migrate into an initially cell-free region. A major drawback of this assay is that it relies on simply counting cells in the exclusion zone and therefore cannot distinguish the effects of proliferation from migration. We report here a simple modification to the exclusion zone migration assay that exclusively measures cell migration and is not affected by proliferation. This approach makes use of a lineage-tracing vital stain that is retained through cell generations and effectively reads out migration relative to the original, parental cell population. This modification is simple, robust, non-perturbing, and inexpensive. We validate the method in a panel of cell lines under conditions that inhibit or promote migration and demonstrate its use in normal and cancer cell lines as well as primary cells. PMID:27535324

  17. 12C6+ ion beam induced DNA damage in human hepatocyte L02 cells detected by comet assay

    International Nuclear Information System (INIS)

    Human hepatocyte L02 cells were irradiated by the carbon ion beam with LET of 30 keV/μm and DNA strand breaks were detected immediately after the irradiation using comet assay. Based on the comet images, all the indexes of comet assay including head DNA%, tail DNA%, comet length, tail length, tail moment and olive tail moment were analyzed with CASP and SPSS 11.5 code. Statistically significant dose-effect relationships could be observed in all the indexes of comet assay and TM increased with increasing the radiation dose. These experimental results suggest that carbon ion beam with intermediate LET value would cause remarkable DNA strand breaks immediately and the damage increases in a dose-dependent manner. This work provides basic data and evidence for the risk assessment of heavy ion radiation to healthy tissue. (authors)

  18. Application of the comet assay in studies of programmed cell death (PCD in plants

    Directory of Open Access Journals (Sweden)

    Maria Charzyńska

    2014-02-01

    Full Text Available Programmed cell death (PCD in plants is an intensively investigated process. One of the main characteristics of PCD in both animal and plant organisms is the non-random, internucleosomal fragmentation of nuclear DNA, usually analysed using total DNA gel electrophoresis or TUNEL method. In this paper we present application of the "comet assay" (Single Cell Gel Electrophoresis for detection of nDNA degradation in studies of PCD during plant life cycle. We analyzed three types of tissue: anther tapetum, endosperm and mesophyll which were prepared in different ways to obtain a suspension of viable cells (without cell walls. The comet assay gives a possibility of examination of the nDNA degradation in individual cell. This method is significant for studies of the plant tissue differentiation and senescence especially in the cases when it is not possible to isolate large number of cells at the same developmental stage.

  19. Observation of DNA damage of human hepatoma cells irradiated by heavy ions using comet assay

    Institute of Scientific and Technical Information of China (English)

    Li-Mei Qiu; Wen-Jian Li; Xin-Yue Pang; Qing-Xiang Gao; Yan Feng; Li-Bin Zhou; Gao-Hua Zhang

    2003-01-01

    AIM: Now many countries have developed cancer therapy with heavy ions, especially in GSI (Gesellschaft fur Schwerionenforschung mbH, Darmstadt, Germany),remarkable results have obtained, but due to the complexity of particle track structure, the basic theory still needs further researching. In this paper, the genotoxic effects of heavy ions irradiation on SMMC-7721 cells were measured using the single cell gel electrophoresis (comet assay). The information about the DNA damage made by other radiations such as X-ray, γ-ray, UV and fast neutron irradiation is very plentiful, while little work have been done on the heavy ions so far. Hereby we tried to detect the reaction of liver cancer cells to heavy ion using comet assay, meanwhile to establish a database for clinic therapy of cancer with the heavy ions.METHODS: The human hepatoma cells were chosen as the test cell line irradiated by 80Mev/u 20Ne10+ on HIRFL (China), the radiation-doses were 0, 0.5, 1, 2, 4 and 8 Gy,and then comet assay was used immediately to detect the DNA damages, 100-150 cells per dose-sample (30-50 cells were randomly observed at constant depth of the gel). The tail length and the quantity of the cells with the tail were put down. EXCEL was used for statistical analysis.RESULTS: We obtained clear images by comet assay and found that SMMC-7721 cells were all damaged apparently from the dose 0.5Gy to 8Gy (t-test: P<0.001, vs control).The tail length and tail moment increased as the doses increased, and the number of cells with tails increased with increasing doses. When doses were higher than 2Gy, nearly 100 % cells were damaged. Furthermore, both tail length and tail moment, showed linear equation.CONCLUSION: From the clear comet assay images, our experiment proves comet assay can be used to measure DNA damages by heavy ions. Meanwhile DNA damages have a positive correlation with the dose changes of heavy ions and SMMC-7721 cells have a great radiosensitivity to 20Ne10+.Different reactions

  20. An Evaluation of Dose Equivalence between Synchrotron Microbeam Radiation Therapy and Conventional Broadbeam Radiation Using Clonogenic and Cell Impedance Assays

    OpenAIRE

    Mohammad Johari Ibahim; Crosbie, Jeffrey C.; Yuqing Yang; Marina Zaitseva; Andrew W Stevenson; Rogers, Peter A. W.; Premila Paiva

    2014-01-01

    BACKGROUND: High-dose synchrotron microbeam radiation therapy (MRT) has shown the potential to deliver improved outcomes over conventional broadbeam (BB) radiation therapy. To implement synchrotron MRT clinically for cancer treatment, it is necessary to undertake dose equivalence studies to identify MRT doses that give similar outcomes to BB treatments. AIM: To develop an in vitro approach to determine biological dose equivalence between MRT and BB using two different cell-based assays. METHO...

  1. Objective scoring of transformed foci in BALB/c 3T3 cell transformation assay by statistical image descriptors

    OpenAIRE

    Urani, Chiara; Corvi, Raffaella; CALLEGARO G.; Stefanini, Federico Mattia

    2013-01-01

    In vitro cell transformation assays (CTAs) have been shown to model important stages of in vivo carcinogenesis and have the potential to predict carcinogenicity in humans. Advantages of CTAs are their ability of revealing both genotoxic and non-genotoxic carcinogens while reducing both experimental costs and the number of animals used. The endpoint of the CTA is foci formation, and requires classification under light microscopy based on morphology. Thus current limitations for the wide ado...

  2. An assay for transient gene expression in transfected Drosophila cells, using [3H]guanine incorporation.

    OpenAIRE

    Burke, J F; Sinclair, J H; Sang, J. H.; Ish-Horowicz, D.

    1984-01-01

    We have developed an assay for transient gene expression using a dominant-selectable marker previously employed to transform Drosophila cultured cells. Drosophila hydei cells transfected with a functional Escherichia coli xanthine guanine phosphoribosyl transferase gene (gpt), under the control of the long terminal repeats (LTRs) of the copia transposable element, rapidly incorporate guanine into acid-precipitable counts. Autoradiographic analysis in situ shows that approximately 20% of cells...

  3. Properties of kojic acid and curcumin: Assay on cell B16-F1

    Science.gov (United States)

    Sugiharto, Ariff, Arbakariya; Ahmad, Syahida; Hamid, Muhajir

    2016-03-01

    Ultra violet (UV) exposure and oxidative stress are casually linked to skin disorders. They can increase melanin synthesis, proliferation of melanocytes, and hyperpigmentation. It is possible that antioxidants or inhibitors may have a beneficial effect on skin health to reduce hyperpigmentation. In the last few years, a huge number of natural herbal extracts have been tested to reduce hyperpigmentation. The objective of this study was to determine and to compare of kojic acid and curcumin properties to viability cell B16-F1. In this study, our data showed that the viability of cell B16-F1 was 63.91% for kojic acid and 64.12% for curcumin at concentration 100 µg/ml. Further investigation assay of antioxidant activities, indicated that IC50 for kojic acid is 63.8 µg/ml and curcumin is 16.05 µg/ml. Based on the data, kojic acid and curcumin have potential antioxidant properties to reduce hyperpigmentation with low toxicity effect in cell B16-F1.

  4. p53 Promoter-based Reporter Gene in vitro Assays for Quick Assessment of Agents with Genotoxic Potential

    Institute of Scientific and Technical Information of China (English)

    Huaixing LI; Ke SHI; Ruiwen CHEN; Yan HE; Dan WU; Shuhan SUN

    2007-01-01

    The p53 promoter-based green fluorescent protein (GFP) and luciferase reporter gene assays have been established for detecting DNA damage induced by genotoxic agents.To evaluate the system,NIH3T3 cells transfected with either pHP53-GFP or pMP53-GFP construct were treated with mitomycin or 5-fluorouracil.Expression of the GFP reporter gene was significantly and specifically induced in the cells exposed to mitomycin or 5-fluorouracil.Then we treated NIH3T3 cells harboring pHP53-Luc or pMP53-Luc vector with mitomycin,5-fluorouracil or cisplatin at various concentrations.Similarly,exposure of the cells to these agents with genotoxic potentials resulted in a dose-dependent induction in luciferase reporter gene expression.Thus,these in vitro reporter gene assays could provide an ideal system for quick assessment or screening of agents with genotoxic potential.

  5. Identification of ataxia telangiectasia heterozygotes, a cancer-prone population, using the single-cell gel electrophoresis (Comet) assay.

    Science.gov (United States)

    Djuzenova, C S; Schindler, D; Stopper, H; Hoehn, H; Flentje, M; Oppitz, U

    1999-06-01

    Heterozygotes of ataxia telangiectasia (AT) may comprise up to 1% of the general population. Because these individuals have no clinical expression of AT but may be highly radiosensitive and strongly predisposed for several forms of cancer, identification of AT carriers represents a considerable interest in cancer epidemiology and radiotherapy. We report a new approach for the in vitro identification of AT-heterozygotes based on the evaluation of the radiosensitivity and DNA damage repair ability of peripheral blood mononuclear cells using the single-cell gel electrophoresis (Comet) assay. The assay was performed on cells isolated from four different groups of individuals: (1) apparently healthy donors (n = 10); (2) patients with breast cancer showing a normal reaction to radiotherapy (n = 10); (3) a group of obligate AT carriers (parents of AT-homozygotes, n = 20); and (4) AT-homozygotes (n = 4). Cells irradiated with 3 Gy of x-rays were assayed for three parameters: (1) the initial and (2) residual DNA damage and (3) the kinetics of DNA damage repair. Both AT-heterozygotes' and AT-homozygotes' cells were found to be highly sensitive to x-irradiation. Quantitative evaluation of the single-cell electrophoregrams revealed that the average initial DNA damage in AT-heterozygous and AT-homozygous cells was almost three times higher than that in control non-AT cells. In addition, the DNA repair process in irradiated AT carrier cells was almost three times slower, and the extent of irreparable DNA damage in these cells was three times greater than in controls. Simultaneous assessment of the three parameters enabled correct identification of all tested AT carriers. This method seems to be a sensitive and useful tool for populational studies as a rapid prescreening test for a mutated AT status. The approach can also be extended for prediction of the in vivo radiosensitivity, which would enable optimization of individual radiotherapy schedules. PMID:10378512

  6. In vitro permeation models for healthy and compromised skin: The Phospholipid Vesicle-based Permeation Assay (PVPA) for skin applications

    OpenAIRE

    Engesland, André

    2015-01-01

    In vitro models with the ability to estimate drug penetration through healthy and compromised skin may reduce animal testing of drugs and cosmetics to a minimum. The phospholipid vesicle based permeation assay (PVPA) is based on a tight barrier composed of liposomes mimicking cells. It was originally made to mimic the intestinal epithelial barrier and in this project further developed to mimic the stratum corneum barrier of the skin. The lipid composition was changed to better mimic the lipid...

  7. The potential value of the neutral comet assay and γH2AX foci assay in assessing the radiosensitivity of carbon beam in human tumor cell lines

    International Nuclear Information System (INIS)

    Carbon ions (12C6+) are high linear energy transfer (LET) radiation characterized by higher relative biological effectiveness than low LET radiation. The assessment of tumour radiosensitivity would be particularly useful in optimizing the radiation dose during radiotherapy. The aim of the current study was to evaluate the potential value of the neutral comet assay and γH2AX foci assay in assessing 12C6+ radiosensitivity of tumour cells. The doses of 12C6+ and X-rays used in the present study were 2 and 4 Gy. The survival fraction, DNA double-strand breaks (DSB) and repair kinetics of DSB were assayed with clonogenic survival, neutral comet assay and γH2AX foci assay in human cervical carcinoma HeLa cells, hepatoma HepG2 cells, and mucoepidermoid carcinoma MEC-1 cells at the time points of 0.5, 4, 16 and 24 h after 12C6+ and X-rays irradiation. The survival fraction for 12C6+ irradiation was much more inhibited than for X-rays (p < 0.05) in all three tumour cell lines tested. Substantial amounts of residual damage, assessed by the neutral comet assay, were present after irradiation (p < 0.05). The highest residual damage was observed at 0.5 or 4 h, both for 12C6+ and X-ray irradiation. However, the residual damage in HeLa and MEC-1 cells was higher for 12C6+ than X-rays (p < 0.05). The strongest induction of γH2AX foci was observed after 30 min, for all three tumour cell lines (p < 0.01). The franction of γH2AX foci persisted for at least 24 h after 12C6+ irradiation; in HeLa cells and MEC-1 was higher than after X-ray irradiation (p < 0.05). The correlation coefficients between the clonogenic survival, neutral comet assay and γH2AX foci assay were not statistically significant, except for some tumour cells at individual irradiation doses and types. Our study demonstrated that the neutral comet assay and γ-H2AX foci assay could be used to assess the radiosensitivity of 12C6+ in human tumour cells

  8. Src homology 2 domain-based high throughput assays for profiling downstream molecules in receptor tyrosine kinase pathways.

    Science.gov (United States)

    Yaoi, Takuro; Chamnongpol, Sangpen; Jiang, Xin; Li, Xianqiang

    2006-05-01

    Src homology 2 (SH2) domains are evolutionary conserved small protein modules that bind specifically to tyrosine-phosphorylated peptides. More than 100 SH2 domains have been identified in proteins encoded by the human genome. The binding specificity of these domains plays a critical role in signaling within the cell, mediating the relocalization and interaction of proteins in response to changes in tyrosine phosphorylation states. Here we developed an SH2 domain profiling method based on a multiplexed fluorescent microsphere assay in which various SH2 domains are used to probe the global state of tyrosine phosphorylation within a cell and to screen synthetic peptides that specifically bind to each SH2 domain. The multiplexed, fluorescent microsphere-based assay is a recently developed technology that can potentially detect a wide variety of interactions between biological molecules. We constructed 25-plex SH2 domain-GST fusion protein-conjugated fluorescent microsphere sets to investigate phosphorylation-mediated cell signaling through the specific binding of SH2 domains to activated target proteins. The response of HeLa, COS-1, A431, and 293 cells and four breast cancer cell lines to epidermal growth factor and insulin were quantitatively profiled using this novel microsphere-based, multiplexed, high throughput assay system. PMID:16477079

  9. Molecular-Based Assay for Simultaneous Detection of Four Plasmodium spp. and Wuchereria bancrofti Infections

    OpenAIRE

    MEHLOTRA, RAJEEV K.; Gray, Laurie R; Blood-Zikursh, Melinda J.; Kloos, Zachary; Henry-Halldin, Cara N.; Tisch, Daniel J.; Thomsen, Edward; Reimer, Lisa; Kastens, Will; Baea, Manasseh; Baea, Kaye; Baisor, Moses; Tarongka, Nandao; Kazura, James W; Zimmerman, Peter A

    2010-01-01

    Four major malaria-causing Plasmodium spp. and lymphatic filariasis-causing Wuchereria bancrofti are co-endemic in many tropical and sub-tropical regions. Among molecular diagnostic assays, multiplex polymerase chain reaction (PCR)–based assays for the simultaneous detection of DNAs from these parasite species are currently available only for P. falciparum and W. bancrofti or P. vivax and W. bancrofti. Using a post-PCR oligonucleotide ligation detection reaction–fluorescent microsphere assay ...

  10. Novel biosensor-based microarray assay for detecting rs8099917 and rs12979860 genotypes

    Institute of Scientific and Technical Information of China (English)

    Pei-Yuan Li; Xiao-Jun Zhou; Lan Yao; Xin-Hua Fang; Jiang-Nan Ren; Jia-Wu Song

    2012-01-01

    AIM:To evaluate a novel biosensor-based microarray (BBM) assay for detecting rs12979860 and rs8099917genotypes.METHODS:Four probes specific for rs8099917C/T or rs12979860G/T detection and three sets of quality control probes were designed,constructed and arrayed on an optical biosensor to develop a microarray assay.Two sets of primers were used in a one tube polymerase chain reaction (PCR) system to amplify two target fragments simultaneously.The biosensor microarray contained probes that had been sequenced to confirm that they included the rsS099917C/T or rs12979860G/T alleles of interest and could serve as the specific assay standards.In addition to rehybridization of four probes of known sequence,a total of 40 clinical samples collected from hepatitis C seropositive patients were also tested.The target fragments of all 40 samples were amplified in a 50 μL PCR system.Ten μL of each amplicon was tested by BBM assay,and another 40 μL was used for sequencing.The agreement of the results obtained by the two methods was tested statistically using the kappa coefficient.The sensitivity of the BBM assay was evaluated using serial dilutions of ten clinical blood samples containing 103-104 white cells/lμL.RESULTS:As shown by polyacrylamide gel electrophoresis,two target segments of the interleukin 28B-associated polymorphisms (SNPs) were successfully amplified in the one-tube PCR system.The lengths of the two amplified fragments were consistent with the known length of the target sequences,137 and 159bps.After hybridization of the PCR amplicons with the probes located on the BBM array,the signals of each allele of both the rs8099917 SNPs and rs12979860 SNPs were observed simultaneously and were clearly visible by the unaided eye.The signals were distinct from each other,could be interpreted visually,and accurately recorded using an ordinary digital camera.To evaluate the specificity of the assay,both the plasmids and clinical samples were applied to the microarray

  11. High performance magnesium anode in paper-based microfluidic battery, powering on-chip fluorescence assay.

    Science.gov (United States)

    Koo, Youngmi; Sankar, Jagannathan; Yun, Yeoheung

    2014-09-01

    A high power density and long-lasting stable/disposable magnesium battery anode was explored for a paper-based fluidic battery to power on-chip functions of various Point of Care (POC) devices. The single galvanic cell with magnesium foil anode and silver foil cathode in Origami cellulose chip provided open circuit potential, 2.2 V, and power density, 3.0 mW/cm(2). A paper-based fluidic galvanic cell was operated with one drop of water (80 μl) and continued to run until it was dry. To prove the concept about powering on-chip POC devices, two-serial galvanic cells are developed and incorporated with a UV-light emitting diode (λ = 365 nm) and fluorescence assay for alkaline phosphatase reaction. Further, detection using smart phones was performed for quantitative measurement of fluorescent density. To conclude, a magnesium-based fluidic battery paper chip was extremely low-cost, required minute sample volumes, was easy to dispose of, light weight, easy to stack, store and transport, easy to fabricate, scalable, and has faster analysis times.

  12. Two-tiered keratinocyte assay: IL-18 production by NCTC2544 cells to determine the skin sensitizing capacity and an epidermal equivalent assay to determine sensitizer potency

    DEFF Research Database (Denmark)

    Teunis, Marc; Corsini, Emanuela; Smits, Mieke;

    2012-01-01

    method to the LLNA. Both assays are based on the use of human keratinocytes, which have been shown, over the last two decades, to play a key role in all phases of skin sensitization. First, 4 known chemicals were tested during a transferability study in which 6 laboratories participated. Three......At present, the identification of potentially sensitizing chemicals is carried out using animal models. However, it should be very important, both from ethical and economic point of view, to discriminate allergy and irritation events, and to classify sensitizers according to their potency, without......2544 IL-18 assay can be used to identify the sensitizing capacity of a chemical (NCTC assay, tier 1) while the Epidermal Equivalent potency assay is used to quantify the potency of the sensitizing agent (EE assay, tier 2). These assays combined, may offer an unique opportunity to provide an alternative...

  13. Dissecting functions of the conserved oligomeric Golgi tethering complex using a cell-free assay.

    Science.gov (United States)

    Cottam, Nathanael P; Wilson, Katherine M; Ng, Bobby G; Körner, Christian; Freeze, Hudson H; Ungar, Daniel

    2014-01-01

    Vesicle transport sorts proteins between compartments and is thereby responsible for generating the non-uniform protein distribution along the eukaryotic secretory and endocytic pathways. The mechanistic details of specific vesicle targeting are not yet well characterized at the molecular level. We have developed a cell-free assay that reconstitutes vesicle targeting utilizing the recycling of resident enzymes within the Golgi apparatus. The assay has physiological properties, and could be used to show that the two lobes of the conserved oligomeric Golgi tethering complex play antagonistic roles in trans-Golgi vesicle targeting. Moreover, we can show that the assay is sensitive to several different congenital defects that disrupt Golgi function and therefore cause glycosylation disorders. Consequently, this assay will allow mechanistic insight into the targeting step of vesicle transport at the Golgi, and could also be useful for characterizing some novel cases of congenital glycosylation disorders.

  14. Cytotoxicity and genotoxicity assessment of Euphorbia hirta in MCF-7 cell line model using comet assay

    Institute of Scientific and Technical Information of China (English)

    Kwan Yuet Ping; Ibrahim Darah; Yeng Chen; Sreenivasan Sasidharan

    2013-01-01

    Objective:To evaluate the cytotoxicity and genotoxicity activity of Euphorbia hirta (E. hirta) in MCF-7 cell line model using comet assay. Methods: The cytotoxicity of E. hirta extract was investigated by employing brine shrimp lethality assay and the genotoxicity of E. hirta was assessed by using Comet assay. Results: Both toxicity tests exhibited significant toxicity result. In the comet assay, the E. hirta extract exhibited genotoxicity effects against MCF-7 DNA in a time-dependent manner by increasing mean percentage of DNA damage. The extract of E. hirta showed significant toxicity against brine shrimp with an LC50 value of 620.382 μg/mL (24 h). Comparison with positive control potassium dichromate signifies that cytotoxicity exhibited by the methanol extract might have moderate activity. Conclusion:The present work confirmed the cytotoxicity and genotoxicity of E. hirta. However, the observed toxicity of E. hirta extracts needs to be confirmed in additional studies.

  15. A microfluidic digital single-cell assay for the evaluation of anticancer drugs.

    Science.gov (United States)

    Wang, Yao; Tang, Xiaolong; Feng, Xiaojun; Liu, Chao; Chen, Peng; Chen, Dongjuan; Liu, Bi-Feng

    2015-02-01

    Digital single-cell assays hold high potentials for the analysis of cell apoptosis and the evaluation of chemotherapeutic reagents for cancer therapy. In this paper, a microfluidic hydrodynamic trapping system was developed for digital single-cell assays with the capability of monitoring cellular dynamics over time. The microfluidic chip was designed with arrays of bypass structures for trapping individual cells without the need for surface modification, external electric force, or robotic equipment. After optimization of the bypass structure by both numerical simulations and experiments, a single-cell trapping efficiency of ∼90 % was achieved. We demonstrated the method as a digital single-cell assay for the evaluation of five clinically established chemotherapeutic reagents. As a result, the half maximal inhibitory concentration (IC50) values of these compounds could be conveniently determined. We further modeled the gradual decrease of active drugs over time which was often observed in vivo after an injection to investigate cell apoptosis against chemotherapeutic reagents. The developed method provided a valuable means for cell apoptotic analysis and evaluation of anticancer drugs. PMID:25433683

  16. Optimization and standardization of the ''comet assay'' for analyzing the repair of DNA damage in cells

    International Nuclear Information System (INIS)

    Human tumor cells or isolated human peripheral blood lymphocytes were analyzed in the experiments. The amount of DNA damage and the effectiveness of DNA repair was measured after X-irradiation using the 'comet assay' technique. Results: In this presentation the influences of different methodological factors like agarose concentration, buffer pH, electrophoresis time, electric field strength on the applicability of the 'comet assay' are described in detail and optimum conditions for 'comet assay' experiments have been evaluated. Additionally the authors will show a comparison of different fluorescent DNA dyes pointing out their advantages or disadvantages for 'comet' analysis. The usefulness of this technique and its capabilities are exemplified by showing DNA repair kinetics of human lymphocytes of different healthy or radiosensitive donors after in-vitro irradiation with 2 Gy X-rays. Conclusions: This paper presents data on the optimization and standardization of the original 'comet assay' leading to an extremely fast and practicable protocol in the field of single cell gel electrophoresis. After irradiation with 0.1 Gy an increase in the amount of DNA damage can be measured with high statistical significance and the DNA repair capacity of individual cells after X-ray doses of 2 Gy can be analyzed with high reproducibility. The results comparing DNA repair capacities of different donors point out that the 'comet assay' may have the potential for the estimation of individual radiosensitivity. (orig./MG)

  17. Plaque assay for human coronavirus NL63 using human colon carcinoma cells

    Directory of Open Access Journals (Sweden)

    Drosten Christian

    2008-11-01

    Full Text Available Abstract Background Coronaviruses cause a broad range of diseases in animals and humans. Human coronavirus (hCoV NL63 is associated with up to 10% of common colds. Viral plaque assays enable the characterization of virus infectivity and allow for purifying virus stock solutions. They are essential for drug screening. Hitherto used cell cultures for hCoV-NL63 show low levels of virus replication and weak and diffuse cytopathogenic effects. It has not yet been possible to establish practicable plaque assays for this important human pathogen. Results 12 different cell cultures were tested for susceptibility to hCoV-NL63 infection. Human colon carcinoma cells (CaCo-2 replicated virus more than 100 fold more efficiently than commonly used African green monkey kidney cells (LLC-MK2. CaCo-2 cells showed cytopathogenic effects 4 days post infection. Avicel, agarose and carboxymethyl-cellulose overlays proved suitable for plaque assays. Best results were achieved with Avicel, which produced large and clear plaques from the 4th day of infection. The utility of plaque assays with agrose overlay was demonstrated for purifying virus, thereby increasing viral infectivity by 1 log 10 PFU/mL. Conclusion CaCo-2 cells support hCoV-NL63 better than LLC-MK2 cells and enable cytopathogenic plaque assays. Avicel overlay is favourable for plaque quantification, and agarose overlay is preferred for plaque purification. HCoV-NL63 virus stock of increased infectivity will be beneficial in antiviral screening, animal modelling of disease, and other experimental tasks.

  18. Activity-based assay for ricin-like toxins

    Science.gov (United States)

    Keener, William K.; Ward, Thomas E.

    2007-02-06

    A method of detecting N-glycosylase activity in a sample involves incubating an oligodeoxyribonucleotide substrate containing a deoxyadenosine or deoxyuridine residue with the sample to be tested such that the N-glycosylase, if present, hydrolyzes the deoxyadenosine or deoxyuridine residue to result in an N-glycosylase product having an abasic site. A primer is annealed to the N-glycosylase product, and the primer is extended with a DNA polymerase, such as Taq DNA polymerase, that pauses at abasic sites. The resulting extension products are melted from the N-glycosylase product, allowed to form hairpins due to self-complementarity, and further extended in the presence of labeled precursors to result in labeled products. Extension products synthesized from undigested substrate as template do not result in labeled products. Thus, detection of labeled products results in detection of N-glycosylase activity. Oligodeoxyribonucleotide substrates, primer, and positive controls and a kit for N-glycosylase assay are also disclosed.

  19. Development of a lipase-based optical assay for detection of DNA

    DEFF Research Database (Denmark)

    Pinijsuwan, Suttiporn; Shipovskov, Stepan; Surareungchai, Werasak;

    2011-01-01

    A lipase-based assay for detection of specific DNA sequences has been developed. Lipase from Candida antarctica was conjugated to DNA and captured on magnetic beads in a sandwich assay, in which the binding was dependent on the presence of a specific target DNA. For amplification and to generate...... a detectable readout the captured lipase was applied to an optical assay that takes advantage of the enzymatic activity of lipase. The assay applies p-nitrophenol octanoate (NPO) as the substrate and in the presence of lipase the ester is hydrolyzed to p-nitrophenolate which has a strong absorbance at 405 nm...

  20. A competitive and reversible deactivation approach to catalysis-based quantitative assays.

    Science.gov (United States)

    Koide, Kazunori; Tracey, Matthew P; Bu, Xiaodong; Jo, Junyong; Williams, Michael J; Welch, Christopher J

    2016-01-01

    Catalysis-based signal amplification makes optical assays highly sensitive and widely useful in chemical and biochemical research. However, assays must be fine-tuned to avoid signal saturation, substrate depletion and nonlinear performance. Furthermore, once stopped, such assays cannot be restarted, limiting the dynamic range to two orders of magnitude with respect to analyte concentrations. In addition, abundant analytes are difficult to quantify under catalytic conditions due to rapid signal saturation. Herein, we report an approach in which a catalytic reaction competes with a concomitant inactivation of the catalyst or consumption of a reagent required for signal generation. As such, signal generation proceeds for a limited time, then autonomously and reversibly stalls. In two catalysis-based assays, we demonstrate restarting autonomously stalled reactions, enabling accurate measurement over five orders of magnitude, including analyte levels above substrate concentration. This indicates that the dynamic range of catalysis-based assays can be significantly broadened through competitive and reversible deactivation. PMID:26891765

  1. Development of a Coxsackievirus A16 neutralization assay based on pseudoviruses for measurement of neutralizing antibody titer in human serum.

    Science.gov (United States)

    Jin, Jun; Ma, Hongxia; Xu, Lin; An, Dong; Sun, Shiyang; Huang, Xueyong; Kong, Wei; Jiang, Chunlai

    2013-02-01

    Serum neutralizing antibody titers are indicative of protective immunity against Coxsackievirus A16 (CV-A16) and Enterovirus 71 (EV71), the two main etiological agents of hand, foot and mouth disease (HFMD), and provide the basis for evaluating vaccine efficacy. The current CV-A16 neutralization assay based on inhibition of cytopathic effects requires manual microscopic examination, which is time-consuming and labor-intensive. In this study, a high-throughput neutralization assay was developed by employing CV-A16 pseudoviruses expressing luciferase for detecting infectivity in rhabdomyosarcoma (RD) cells and measuring serum viral neutralizing antibodies. Without the need to use infectious CV-A16 strains, the neutralizing antibody titer against CV-A16 could be determined within 15h by measuring luciferase signals by this assay. The pseudovirus CV-A16 neutralization assay (pCNA) was validated by comparison with a conventional CV-A16 neutralization assay (cCNA) in testing 174 human serum samples collected from children (age <5 years). The neutralizing antibody titers determined by these two assays were well correlated (R(2)=0.7689). These results suggest that the pCNA can serve as a rapid and objective procedure for the measurement of neutralizing antibodies against CV-A16. PMID:23178532

  2. Development of a Coxsackievirus A16 neutralization assay based on pseudoviruses for measurement of neutralizing antibody titer in human serum.

    Science.gov (United States)

    Jin, Jun; Ma, Hongxia; Xu, Lin; An, Dong; Sun, Shiyang; Huang, Xueyong; Kong, Wei; Jiang, Chunlai

    2013-02-01

    Serum neutralizing antibody titers are indicative of protective immunity against Coxsackievirus A16 (CV-A16) and Enterovirus 71 (EV71), the two main etiological agents of hand, foot and mouth disease (HFMD), and provide the basis for evaluating vaccine efficacy. The current CV-A16 neutralization assay based on inhibition of cytopathic effects requires manual microscopic examination, which is time-consuming and labor-intensive. In this study, a high-throughput neutralization assay was developed by employing CV-A16 pseudoviruses expressing luciferase for detecting infectivity in rhabdomyosarcoma (RD) cells and measuring serum viral neutralizing antibodies. Without the need to use infectious CV-A16 strains, the neutralizing antibody titer against CV-A16 could be determined within 15h by measuring luciferase signals by this assay. The pseudovirus CV-A16 neutralization assay (pCNA) was validated by comparison with a conventional CV-A16 neutralization assay (cCNA) in testing 174 human serum samples collected from children (age <5 years). The neutralizing antibody titers determined by these two assays were well correlated (R(2)=0.7689). These results suggest that the pCNA can serve as a rapid and objective procedure for the measurement of neutralizing antibodies against CV-A16.

  3. Using a medium-throughput comet assay to evaluate the global DNA methylation status of single cells

    OpenAIRE

    Lewies, Angélique; Van Dyk, Etresia; Johannes F. Wentzel; Pieter J. Pretorius

    2014-01-01

    The comet assay is a simple and cost effective technique, commonly used to analyze and quantify DNA damage in individual cells. The versatility of the comet assay allows introduction of various modifications to the basic technique. The difference in the methylation sensitivity of the isoschizomeric restriction enzymes HpaII and MspI are used to demonstrate the ability of the comet assay to measure the global DNA methylation level of individual cells when using cell cultures. In the experiment...

  4. DNA Microarray Assay Helps to Identify Functional Genes Specific for Leukemia Stem Cells

    Directory of Open Access Journals (Sweden)

    Haojian Zhang

    2013-01-01

    Full Text Available Chronic myeloid leukemia (CML is a myeloproliferative disease derived from an abnormal hematopoietic stem cell (HSC and is consistently associated with the formation of Philadelphia (Ph chromosome. Tyrosine kinase inhibitors (TKIs are highly effective in treating chronic phase CML but do not eliminate leukemia stem cells (LSCs, which are believed to be related to disease relapse. Therefore, one major challenge in the current CML research is to understand the biology of LSCs and to identify the molecular difference between LSCs and its normal stem cell counterparts. Comparing the gene expression profiles between LSCs and normal HSCs by DNA microarray assay is a systematic and unbiased approach to address this issue. In this paper, we present a DNA microarray dataset for CML LSCs and normal HSCs to show that the microarray assay will benefit the current and future studies of the biology of CML stem cells.

  5. A Rapid and Efficient Luminescence-based Method for Assaying Phosphoglycosyltransferase Enzymes.

    Science.gov (United States)

    Das, Debasis; Walvoort, Marthe T C; Lukose, Vinita; Imperiali, Barbara

    2016-01-01

    Phosphoglycosyltransferases (PGTs) are families of integral membrane proteins with intriguingly diverse architectures. These enzymes function to initiate many important biosynthetic pathways including those leading to peptidoglycan, N-linked glycoproteins and lipopolysaccharide O-antigen. In spite of tremendous efforts, characterization of these enzymes remains a challenge not only due to the inherent difficulties associated with the purification of integral membrane proteins but also due to the limited availability of convenient assays. Current PGT assays include radioactivity-based methods, which rely on liquid-liquid or solid-liquid extractions, multienzyme systems linked to lactate dehydrogenase and NAD(+) generation, and HPLC-based approaches, all of which may suffer from low sensitivity and low throughput. Herein, we present the validation of a new luminescence-based assay (UMP-Glo) for measuring activities of PGT enzymes. This assay measures UMP, the by-product of PGT reactions, in a sensitive and quantitative manner by measuring the luminescence output in a discontinuous coupled assay system. The assay is rapid and robust in nature, and also compatible with microtiter plate formats. Activity and kinetic parameters of PglC, a PGT from Campylobacter jejuni, were quickly established using this assay. The efficacy of the assay was further corroborated using two different PGTs; PglC from Helicobacter pullorum and WecA from Thermatoga maritima. PMID:27624811

  6. Immunological-based assays for specific detection of shrimp viruses.

    Science.gov (United States)

    Chaivisuthangkura, Parin; Longyant, Siwaporn; Sithigorngul, Paisarn

    2014-02-12

    Among shrimp viral pathogens, white spot syndrome virus (WSSV) and yellow head virus (YHV) are the most lethal agents, causing serious problems for both the whiteleg shrimp, Penaeus (Litopenaeus) vannamei, and the black tiger shrimp, Penaeus (Penaeus) monodon. Another important virus that infects P. vannamei is infectious myonecrosis virus (IMNV), which induces the white discoloration of affected muscle. In the cases of taura syndrome virus and Penaeus stylirostris densovirus (PstDNV; formerly known as infectious hypodermal and hematopoietic necrosis virus), their impacts were greatly diminished after the introduction of tolerant stocks of P. vannamei. Less important viruses are Penaeus monodon densovirus (PmDNV; formerly called hepatopancreatic parvovirus), and Penaeus monodon nucleopolyhedrovirus (PemoNPV; previously called monodon baculovirus). For freshwater prawn, Macrobrachium rosenbergii nodavirus and extra small virus are considered important viral pathogens. Monoclonal antibodies (MAbs) specific to the shrimp viruses described above have been generated and used as an alternative tool in various immunoassays such as enzyme-linked immunosorbent assay, dot blotting, Western blotting and immunohistochemistry. Some of these MAbs were further developed into immunochromatographic strip tests for the detection of WSSV, YHV, IMNV and PemoNPV and into a dual strip test for the simultaneous detection of WSSV/YHV. The strip test has the advantages of speed, as the result can be obtained within 15 min, and simplicity, as laboratory equipment and specialized skills are not required. Therefore, strip tests can be used by shrimp farmers for the pond-side monitoring of viral infection. PMID:24567913

  7. Filter-based assay for Escherichia coli in aqueous samples using bacteriophage-based amplification.

    Science.gov (United States)

    Derda, Ratmir; Lockett, Matthew R; Tang, Sindy K Y; Fuller, Renee C; Maxwell, E Jane; Breiten, Benjamin; Cuddemi, Christine A; Ozdogan, Aysegul; Whitesides, George M

    2013-08-01

    This paper describes a method to detect the presence of bacteria in aqueous samples, based on the capture of bacteria on a syringe filter, and the infection of targeted bacterial species with a bacteriophage (phage). The use of phage as a reagent provides two opportunities for signal amplification: (i) the replication of phage inside a live bacterial host and (ii) the delivery and expression of the complementing gene that turns on enzymatic activity and produces a colored or fluorescent product. Here we demonstrate a phage-based amplification scheme with an M13KE phage that delivers a small peptide motif to an F(+), α-complementing strain of Escherichia coli K12, which expresses the ω-domain of β-galactosidase (β-gal). The result of this complementation-an active form of β-gal-was detected colorimetrically, and the high level of expression of the ω-domain of β-gal in the model K12 strains allowed us to detect, on average, five colony-forming units (CFUs) of this strain in 1 L of water with an overnight culture-based assay. We also detected 50 CFUs of the model K12 strain in 1 L of water (or 10 mL of orange juice, or 10 mL of skim milk) in less than 4 h with a solution-based assay with visual readout. The solution-based assay does not require specialized equipment or access to a laboratory, and is more rapid than existing tests that are suitable for use at the point of access. This method could potentially be extended to detect many different bacteria with bacteriophages that deliver genes encoding a full-length enzyme that is not natively expressed in the target bacteria.

  8. Acute shear stress direction dictates adherent cell remodeling and verifies shear profile of spinning disk assays

    International Nuclear Information System (INIS)

    Several methods have been developed to quantify population level changes in cell attachment strength given its large heterogeneity. One such method is the rotating disk chamber or ‘spinning disk’ in which a range of shear forces are applied to attached cells to quantify detachment force, i.e. attachment strength, which can be heterogeneous within cell populations. However, computing the exact force vectors that act upon cells is complicated by complex flow fields and variable cell morphologies. Recent observations suggest that cells may remodel their morphology and align during acute shear exposure, but contrary to intuition, shear is not orthogonal to the radial direction. Here we theoretically derive the magnitude and direction of applied shear and demonstrate that cells, under certain physiological conditions, align in this direction within minutes. Shear force magnitude is also experimentally verified which validates that for spread cells shear forces and not torque or drag dominate in this assay, and demonstrates that the applied force per cell area is largely independent of initial morphology. These findings suggest that direct quantified comparison of the effects of shear on a wide array of cell types and conditions can be made with confidence using this assay without the need for computational or numerical modeling. (paper)

  9. Application of the Single Cell Gel Electrophoresis (SCGE) Assay to Genotoxicity Evaluation in Plants and Animals

    International Nuclear Information System (INIS)

    Application of the Single Cell Gel Electrophoresis (SCGE) Assay to Genotoxicity Evaluation in Plants and Animals. Recently, the importance of ionizing radiation and chemicals has been recognized since radio- and chemical therapy is directly related to the control of various diseases such as cancer. Radiation and the chemicals can cause biological damages while they have great applicability. It is of necessity to analyze rapidly, easily and accurately the biological effects, especially DNA damage due to those factors. Recently SCGE (single cell gel electrophoresis assay, alias comet assay) has been developed for the efficient evaluation of DNA damage. In this report, the comprehensive review will be given on the rationale, the technical applications and the advantages and shortcomings of SCGE assay. This method can be directly applied to study on toxicity, cancer, and aging in terms of the evaluation of DNA damages due to radiation and chemicals on human cellular level. It is also suggested that comet assay be used for testing genotoxicity of suspected substances, detecting irradiated foods, screening radioprotective candidates, and studying DNA repair process in various biological systems

  10. Application of the Single Cell Gel Electrophoresis (SCGE) Assay to Genotoxicity Evaluation in Plants and Animals

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jin Kyu

    2007-10-15

    Application of the Single Cell Gel Electrophoresis (SCGE) Assay to Genotoxicity Evaluation in Plants and Animals. Recently, the importance of ionizing radiation and chemicals has been recognized since radio- and chemical therapy is directly related to the control of various diseases such as cancer. Radiation and the chemicals can cause biological damages while they have great applicability. It is of necessity to analyze rapidly, easily and accurately the biological effects, especially DNA damage due to those factors. Recently SCGE (single cell gel electrophoresis assay, alias comet assay) has been developed for the efficient evaluation of DNA damage. In this report, the comprehensive review will be given on the rationale, the technical applications and the advantages and shortcomings of SCGE assay. This method can be directly applied to study on toxicity, cancer, and aging in terms of the evaluation of DNA damages due to radiation and chemicals on human cellular level. It is also suggested that comet assay be used for testing genotoxicity of suspected substances, detecting irradiated foods, screening radioprotective candidates, and studying DNA repair process in various biological systems.

  11. Sensitive hepatocyte-mediated assay for the metabolism of nitrosamines to mutagens for mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Jones, C.A.; Huberman, E.

    1980-02-01

    A sensitive cell-mediated assay has been developed for testing mutagenesis in Chinese hamster V79 cells by carcinogenic nitrosamines. Mutations were characterized by resistance to ouabian and 6-thioguanine. Since V79 cells do not metabolize nitrosamines, mutagenesis in the V79 cells was tested in the presence of primary hepatocytes capable of metabolizing nitrosamines. The hepatocytes were isolated after collagenase and hyaluronidase digestion of liver slices. All seven liver carcinogens of the nine tested nitrosamines exhibited a mutagenic response in this cell-mediated assay. The potent liver carcinogens nitrosodimethylamine, nitrosodiethylamine, nitrosoethylmethylamine, and nitrosodipropylamine could be detected with doses as low as 1 ..mu..m. The noncarcinogenic nitrosodiphenylamine was not mutagenic. Nitrosomethoxymethylamine was the only nitrosamine that exhibited mutagenic activity in the absence of hepatocytes, and this activity was diminished in the presence of hepatocytes. It is suggested that the use of hepatocytes prepared by the slicing method for carcinogen metabolism and mutable V79 cells offers a highly sensitive assay for determining the mutagenic potential of carcinogenic nitrosamines and probably of other classes of hazardous chemicals occurring in the environment.

  12. Sensitivity and specificity of tritiated thymidine incorporation and ELISPOT assays in identifying antigen specific T cell immune responses

    Directory of Open Access Journals (Sweden)

    MacLeod Beth

    2007-09-01

    Full Text Available Abstract Background Standardization of cell-based immunologic monitoring is becoming increasingly important as methods for measuring cellular immunity become more complex. We assessed the ability of two commonly used cell-based assays, tritiated thymidine incorporation (proliferation and IFN-gamma ELISPOT, to predict T cell responses to HER-2/neu, tetanus toxoid (tt, and cytomegalovirus (CMV antigens. These antigens were determined to be low (HER-2/neu, moderate (tt, and robustly (CMV immunogenic proteins. Samples from 27 Stage II, III, and IV HER-2/neu positive breast cancer patients, vaccinated against the HER-2/neu protein and tt, were analyzed by tritiated thymidine incorporation and IFN-gamma ELISPOT for T cell response. Results Linear regression analysis indicates that both stimulation index (SI (p = 0.011 and IFN-gamma secreting precursor frequency (p Conclusion These data underscore the importance of taking into consideration the performance characteristics of assays used to measure T cell immunity. This consideration is particularly necessary when determining which method to utilize for assessing responses to immunotherapeutic manipulations in cancer patients.

  13. Real-time fluorescent quantitative PCR assay for measuring cytomegalovirus DNA load in patients after haematopoietic stem cell transplantation

    Institute of Scientific and Technical Information of China (English)

    FAN Jun; MA Wei-hang; YANG Mei-fang; XUE Han; GAO Hai-nü; LI Lan-juan

    2006-01-01

    @@ Cytomegalovirus (CMV) infection is a major and often deadly complication of haematopoietic stem cell (HSC) transplantation.1 Successful preemptive CMV therapy in transplant patients depends on the availability of sensitive, specific, and timely diagnostic tests for CMV infections.2 The pp65antigenemia assay has been used for this purposewith considerable success but has disadvantages of being time-consuming and labor-intensive.3 Recently,commercial quantitative polymerase chain reaction (PCR) methods based on TaqMan technique have become available and proven to be useful in the diagnosis of microbial infection as well as the determination of viral load.4 In this study, we developed a fluorescent-based quantitative real-time PCR (RT-FQ PCR) assay using TaqMan chemistry for rapid and quantitative detection of CMV DNA and assessed its clinic value for monitoring the reinfection of CMV in patients after HSC transplantion.

  14. An aptamer-based dipstick assay for the rapid and simple detection of aflatoxin B1.

    Science.gov (United States)

    Shim, Won-Bo; Kim, Min Jin; Mun, Hyoyoung; Kim, Min-Gon

    2014-12-15

    A rapid and simple dipstick assay based on an aptamer has been developed for the determination of aflatoxin B1 (AFB1). The dipstick assay format was based on a competitive reaction of the biotin-modified aptamer specific to AFB1 between target and cy5-modified DNA probes. Streptavidin and anti-cy5 antibody as capture reagents were immobilized at test and control lines on a membrane of the dipstick assay. After optimization, the limit of detection for the dipstick assay was 0.1 ng/ml AFB1 in buffer. The method was confirmed to be specific to AFB1, and the entire process of the assay can be completed within 30 min. Aqueous methanol (20%) provided a good extraction efficiency, and the matrix influence from corn extracts was successfully reduced through 2-fold dilution. The results of AFB1 analysis for corn samples spiked with known concentration of AFB1 by the dipstick assay and ELISA showed good agreement. The cut-off value of the dipstick assay for corn samples was 0.3 ng/g AFB1. Therefore, the dipstick assay is first reported and considered as a rapid, simple, on-site and inexpensive screening tool for AFB1 determination in grains as well as a corn. PMID:25032679

  15. Detection of hypoxic cells in a C3H mouse mammary carcinoma using the comet assay.

    OpenAIRE

    Olive, P. L.; Horsman, M R; C. Grau; Overgaard, J.

    1997-01-01

    The comet assay was used to estimate radiobiological hypoxic fraction across a full range of tumour oxygenations in C3H mammary tumours implanted into the feet of female CDF1 mice. Tumours were either clamped before irradiation or mice were allowed to breath air, 100% oxygen, carbogen or carbon monoxide for 5-35 min before and during exposure to 15 Gy. For the alkaline comet assay, tumours were excised after irradiation and individual tumour cells were analysed for DNA single-strand breaks. H...

  16. Homogeneous plate based antibody internalization assay using pH sensor fluorescent dye.

    Science.gov (United States)

    Nath, Nidhi; Godat, Becky; Zimprich, Chad; Dwight, Stephen J; Corona, Cesear; McDougall, Mark; Urh, Marjeta

    2016-04-01

    Receptor-mediated antibody internalization is a key mechanism underlying several anti-cancer antibody therapeutics. Delivering highly toxic drugs to cancer cells, as in the case of antibody drug conjugates (ADCs), efficient removal of surface receptors from cancer cells and changing the pharmacokinetics profile of the antibody drugs are some of key ways that internalization impacts the therapeutic efficacy of the antibodies. Over the years, several techniques have been used to study antibody internalization including radiolabels, fluorescent microscopy, flow cytometry and cellular toxicity assays. While these methods allow analysis of internalization, they have limitations including a multistep process and limited throughput and are generally endpoint assays. Here, we present a new homogeneous method that enables time and concentration dependent measurements of antibody internalization. The method uses a new hydrophilic and bright pH sensor dye (pHAb dye), which is not fluorescent at neutral pH but becomes highly fluorescent at acidic pH. For receptor mediated antibody internalization studies, antibodies against receptors are conjugated with the pHAb dye and incubated with the cells expressing the receptors. Upon binding to the receptor, the dyes conjugated to the antibody are not fluorescent because of the neutral pH of the media, but upon internalization and trafficking into endosomal and lysosomal vesicles the pH drops and dyes become fluorescent. The enabling attributes of the pHAb dyes are the hydrophilic nature to minimize antibody aggregation and bright fluorescence at acidic pH which allows development of simple plate based assays using a fluorescent reader. Using two different therapeutic antibodies--Trastuzumab (anti-HER2) and Cetuximab (anti-EGFR)--we show labeling with pHAb dye using amine and thiol chemistries and impact of chemistry and dye to antibody ration on internalization. We finally present two new approaches using the pHAb dye, which will be

  17. Pharmacological characterization of human excitatory amino acid transporters EAAT1, EAAT2 and EAAT3 in a fluorescence-based membrane potential assay

    DEFF Research Database (Denmark)

    Jensen, Anders A.; Bräuner-Osborne, Hans

    2004-01-01

    (FMP) assay. The K(m) and K(i) values obtained for 12 standard EAAT ligands at EAAT1, EAAT2 and EAAT3 in the FMP assay correlated well with the K(i) values obtained in the [(3) H]-d-aspartate assay (r(2) values of 0.92, 0.92, and 0.95, respectively). Furthermore, the pharmacological characteristics......We have expressed the human excitatory amino acid transporters EAAT1, EAAT2 and EAAT3 stably in HEK293 cells and characterized the transporters pharmacologically in a conventional [(3) H]-d-aspartate uptake assay and in a fluorescence-based membrane potential assay, the FLIPR Membrane Potential...... conventional electrophysiology set-ups might be superior in terms of studying sophisticated kinetic aspects of the uptake process, the FMP assay enables the collection of considerable amounts of highly reproducible data with relatively little labor. Furthermore, considering that the number of EAAT ligands...

  18. Biocompatibility of various ferrite nanoparticles evaluated by in vitro cytotoxicity assays using HeLa cells

    Energy Technology Data Exchange (ETDEWEB)

    Tomitaka, Asahi [Department of Electrical and Computer Engineering, Yokohama National University, Tokiwadai 79-5, Yokohama, Kanagawa 240-8501 (Japan)], E-mail: d07gd158@ynu.ac.jp; Hirukawa, Atsuo; Yamada, Tsutomu [Department of Electrical and Computer Engineering, Yokohama National University, Tokiwadai 79-5, Yokohama, Kanagawa 240-8501 (Japan); Morishita, Shin [Department of Mechanical Engineering and Materials Science, Yokohama National University, Tokiwadai 79-5, Yokohama, Kanagawa 240-8501 (Japan); Takemura, Yasushi [Department of Electrical and Computer Engineering, Yokohama National University, Tokiwadai 79-5, Yokohama, Kanagawa 240-8501 (Japan)

    2009-05-15

    Magnetic nanoparticles for thermotherapy must be biocompatible and possess high thermal efficiency as heating elements. The biocompatibility of Fe{sub 3}O{sub 4} (20-30 nm), ZnFe{sub 2}O{sub 4} (15-30 nm) and NiFe{sub 2}O{sub 4} (20-30 nm) nanoparticles was studied using a cytotoxicity colony formation assay and a cell viability assay. The Fe{sub 3}O{sub 4} sample was found to be biocompatible on HeLa cells. While ZnFe{sub 2}O{sub 4} and NiFe{sub 2}O{sub 4} were non-toxic at low concentrations, HeLa cells exhibited cytotoxic effects when exposed to concentrations of 100 {mu}g/ml nanoparticles.

  19. Scaling and automation of a high-throughput single-cell-derived tumor sphere assay chip.

    Science.gov (United States)

    Cheng, Yu-Heng; Chen, Yu-Chih; Brien, Riley; Yoon, Euisik

    2016-10-01

    Recent research suggests that cancer stem-like cells (CSCs) are the key subpopulation for tumor relapse and metastasis. Due to cancer plasticity in surface antigen and enzymatic activity markers, functional tumorsphere assays are promising alternatives for CSC identification. To reliably quantify rare CSCs (1-5%), thousands of single-cell suspension cultures are required. While microfluidics is a powerful tool in handling single cells, previous works provide limited throughput and lack automatic data analysis capability required for high-throughput studies. In this study, we present the scaling and automation of high-throughput single-cell-derived tumor sphere assay chips, facilitating the tracking of up to ∼10 000 cells on a chip with ∼76.5% capture rate. The presented cell capture scheme guarantees sampling a representative population from the bulk cells. To analyze thousands of single-cells with a variety of fluorescent intensities, a highly adaptable analysis program was developed for cell/sphere counting and size measurement. Using a Pluronic® F108 (poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol)) coating on polydimethylsiloxane (PDMS), a suspension culture environment was created to test a controversial hypothesis: whether larger or smaller cells are more stem-like defined by the capability to form single-cell-derived spheres. Different cell lines showed different correlations between sphere formation rate and initial cell size, suggesting heterogeneity in pathway regulation among breast cancer cell lines. More interestingly, by monitoring hundreds of spheres, we identified heterogeneity in sphere growth dynamics, indicating the cellular heterogeneity even within CSCs. These preliminary results highlight the power of unprecedented high-throughput and automation in CSC studies.

  20. A modified fluorimetric host cell reactivation assay to determine the repair capacity of primary keratinocytes, melanocytes and fibroblasts

    Directory of Open Access Journals (Sweden)

    Gebhard Daniel

    2010-06-01

    Full Text Available Abstract Background The Host Cell Reactivation Assay (HCRA is widely used to identify circumstances and substances affecting the repair capacity of cells, however, it is restricted by the transfection procedure used and the sensitivity of the detection method. Primary skin cells are particularly difficult to transfect, and therefore sensitive methods are needed to detect any variations due to the cell-type or inter-individual differences or changes induced by diverse substances. A sensitive and repeatable method to detect the repair capacity of skin cells would be useful in two different aspects: On the one hand, to identify substances influencing the repair capacity in a positive manner (these substances could be promising ingredients for cosmetic products and on the other hand, to exclude the negative effects of substances on the repair capacity (this could serve as one step further towards replacing or at least reducing animal testing. Results In this paper, we present a rapid and sensitive assay to determine the repair capacity of primary keratinocytes, melanocytes and fibroblasts based on two wave-length Green Fluorescent Protein (GFP and DsRed reporter technology in order to test different substances and their potential to influence the DNA repair capacity. For the detection of plasmid restoration, we used FACS technology, which, in comparison to luminometer technology, is highly sensitive and allows single cell based analysis. The usefulness of this assay and studying the repair capacity is demonstrated by the evidence that DNA repair is repressed by Cyclosporin A in fibroblasts. Conclusions The methodology described in this paper determines the DNA repair capacity in different types of human skin cells. The described transfection protocol is suitable for the transfection of melanocytes, keratinocytes and fibroblasts, reaching efficacies suitable for the detection of the restored plasmids by FACS technology. Therefore the repair capacity

  1. Extraction, amplification and detection of DNA in microfluidic chip-based assays

    KAUST Repository

    Wu, Jinbo

    2013-12-20

    This review covers three aspects of PCR-based microfluidic chip assays: sample preparation, target amplification, and product detection. We also discuss the challenges related to the miniaturization and integration of each assay and make a comparison between conventional and microfluidic schemes. In order to accomplish these essential assays without human intervention between individual steps, the micro-components for fluid manipulation become critical. We therefore summarize and discuss components such as microvalves (for fluid regulation), pumps (for fluid driving) and mixers (for blending fluids). By combining the above assays and microcomponents, DNA testing of multi-step bio-reactions in microfluidic chips may be achieved with minimal external control. The combination of assay schemes with the use of micro-components also leads to rapid methods for DNA testing via multi-step bioreactions. Contains 259 references.

  2. Extraction, amplification and detection of DNA in microfluidic chip-based assays

    International Nuclear Information System (INIS)

    This review covers three aspects of PCR-based microfluidic chip assays: sample preparation, target amplification, and product detection. We also discuss the challenges related to the miniaturization and integration of each assay and make a comparison between conventional and microfluidic schemes. In order to accomplish these essential assays without human intervention between individual steps, the micro-components for fluid manipulation become critical. We therefore summarize and discuss components such as microvalves (for fluid regulation), pumps (for fluid driving) and mixers (for blending fluids). By combining the above assays and microcomponents, DNA testing of multi-step bio-reactions in microfluidic chips may be achieved with minimal external control. The combination of assay schemes with the use of micro-components also leads to rapid methods for DNA testing via multi-step bioreactions. (author)

  3. Cell culture-Taqman PCR assay for evaluation of Cryptosporidium parvum disinfection.

    Science.gov (United States)

    Keegan, Alexandra R; Fanok, Stella; Monis, Paul T; Saint, Christopher P

    2003-05-01

    Cryptosporidium parvum represents a challenge to the water industry and a threat to public health. In this study, we developed a cell culture-quantitative PCR assay to evaluate the inactivation of C. parvum with disinfectants. The assay was validated by using a range of disinfectants in common use in the water industry, including low-pressure UV light (LP-UV), ozone, mixed oxidants (MIOX), and chlorine. The assay was demonstrated to be reliable and sensitive, with a lower detection limit of a single infectious oocyst. Effective oocyst inactivation was achieved (>2 log(10) units) with LP-UV (20 mJ/cm(2)) or 2 mg of ozone/liter (for 10 min). MIOX and chlorine treatments of oocysts resulted in minimal effective disinfection, with disinfection systems for drinking water and recycled water.

  4. Stem cell-like differentiation potentials of endometrial side population cells as revealed by a newly developed in vivo endometrial stem cell assay.

    Directory of Open Access Journals (Sweden)

    Kaoru Miyazaki

    Full Text Available BACKGROUND: Endometrial stem/progenitor cells contribute to the cyclical regeneration of human endometrium throughout a woman's reproductive life. Although the candidate cell populations have been extensively studied, no consensus exists regarding which endometrial population represents the stem/progenitor cell fraction in terms of in vivo stem cell activity. We have previously reported that human endometrial side population cells (ESP, but not endometrial main population cells (EMP, exhibit stem cell-like properties, including in vivo reconstitution of endometrium-like tissues when xenotransplanted into immunodeficient mice. The reconstitution efficiency, however, was low presumably because ESP cells alone could not provide a sufficient microenvironment (niche to support their stem cell activity. The objective of this study was to establish a novel in vivo endometrial stem cell assay employing cell tracking and tissue reconstitution systems and to examine the stem cell properties of ESP through use of this assay. METHODOLOGY/PRINCIPAL FINDINGS: ESP and EMP cells isolated from whole endometrial cells were infected with lentivirus to express tandem Tomato (TdTom, a red fluorescent protein. They were mixed with unlabeled whole endometrial cells and then transplanted under the kidney capsule of ovariectomized immunodeficient mice. These mice were treated with estradiol and progesterone for eight weeks and nephrectomized. All of the grafts reconstituted endometrium-like tissues under the kidney capsules. Immunofluorescence revealed that TdTom-positive cells were significantly more abundant in the glandular, stromal, and endothelial cells of the reconstituted endometrium in mice transplanted with TdTom-labeled ESP cells than those with TdTom-labeled EMP cells. CONCLUSIONS/SIGNIFICANCE: We have established a novel in vivo endometrial stem cell assay in which multi-potential differentiation can be identified through cell tracking during in vivo

  5. Heterogeneity in radiation-induced DNA damage and repair in tumor and normal cells measured using the comet assay

    International Nuclear Information System (INIS)

    A method for measuring DNA damage to individual cells, based on the technique of microelectrophoresis, was described by Ostling and Johanson in 1984. Cells embedded in agarose are lysed, subjected briefly to an electric field, stained with a fluorescent DNA-binding stain, and viewed using a fluorescence microscope. Broken DNA migrates farther in the electric field, and the cell then resembles a comet with a brightly fluorescent head and a tail region which increases as damage increases. We have used video image analysis to define appropriate features of the comet as a measure of DNA damage, and have quantified damage and repair by ionizing radiation. The assay was optimized for lysing solution, lysing time, electrophoresis time, and propidium iodide concentration using Chinese hamster V79 cells. To assess heterogeneity of response of normal versus malignant cells, damage to both tumor cells and normal cells within mouse SCC-VII tumors was assessed. Tumor cells were separated from macrophages using a cell-sorting method based on differential binding of FITC-conjugated goat anti-mouse IgG. The tail moment, the product of the amount of DNA in the tail and the mean distance of migration in the tail, was the most informative feature of the comet image. Tumor and normal cells showed significant heterogeneity in damage produced by ionizing radiation, although the average amount of damage increased linearly with dose (0-15 Gy) and suggested similar net radiosensitivities for the two cell types. Similarly, DNA repair rate was not significantly different for tumor and normal cells, and most of the cells had repaired the damage by 30 min following exposure to 15 Gy. The heterogeneity in response did not appear to be a result of differences in response through the cell cycle

  6. In vitro comet and micronucleus assays do not predict morphological transforming effects of silica particles in Syrian Hamster Embryo cells.

    Science.gov (United States)

    Darne, Christian; Coulais, Catherine; Terzetti, Francine; Fontana, Caroline; Binet, Stéphane; Gaté, Laurent; Guichard, Yves

    2016-01-15

    Crystalline silica particles and asbestos have both been classified as carcinogenic by the International Agency for Research on Cancer (IARC). However, because of the limited data available, amorphous silica was not classifiable. In vitro, the carcinogenic potential of natural crystalline and amorphous silica particles has been revealed by the Syrian Hamster Embryo (SHE) cell transformation assay. On the other hand, the genotoxic potential of those substances has not been investigated in SHE cells. And yet, genotoxicity assays are commonly used for hazard evaluation and they are often used as in vitro assays of reference to predict a possible carcinogenic potential. The main objective of this study was to compare the genotoxic potential and the carcinogenic potential of different crystalline and amorphous silica particles in SHE cells. Three silica samples of different crystallinity were used: natural amorphous silica, partially crystallized silica and quartz silica particles. Their genotoxicity were tested through the in vitro micronucleus assay and the comet assay in SHE, and their carcinogenic potential through the SHE transformation assay. In addition, silica samples were also tested with the same genotoxicity assays in V79 hamster-lung cells, a common in vitro model for particle exposure. Results obtained in the micronucleus and the comet assays show that none of the silica was capable of inducing genotoxic effects in SHE cells and only the amorphous silica induced genotoxic effects in V79 cells. However in the SHE cell transformation assays, the partially crystallized and quartz silica were able to induce morphological cell transformation. Together, these data suggest that, in vitro, the short-term genotoxic assays alone are not sufficient to predict the hazard and the carcinogenic potential of this type of particles; SHE transformation assay appears a more reliable tool for this purpose and should be included in the "in vitro battery assays" for hazard

  7. A cost effective base-matching assay with low backgrounds.

    OpenAIRE

    SU, X.; Mushinsky, G; Comeau, A.M.

    1996-01-01

    Base-matching or so-called mini-sequencing is a powerful technique for genotyping and mutation identification. However, its application is often hampered by high background and high cost. We have decreased the background by approximately 5-fold by incorporating an end-blocking step and using only 1/10 of the usual nucleotide concentrations.

  8. Microchip-based ultrafast serodiagnostic assay for tuberculosis

    Science.gov (United States)

    Mani, Vigneshwaran; Paleja, Bhairav; Larbi, Karima; Kumar, Pavanish; Tay, Jo Ann; Siew, Jie Yee; Inci, Fatih; Wang, ShuQi; Chee, Cynthia; Wang, Yee Tang; Demirci, Utkan; De Libero, Gennaro; Singhal, Amit

    2016-01-01

    Access to point-of-care (POC), rapid, inexpensive, sensitive, and instrument-free tests for the diagnosis of tuberculosis (TB) remains a major challenge. Here, we report a simple and low-cost microchip-based TB ELISA (MTBE) platform for the detection of anti-mycobacterial IgG in plasma samples in less than 15 minutes. The MTBE employs a flow-less, magnet-actuated, bead-based ELISA for simultaneous detection of IgG responses against multiple mycobacterial antigens. Anti-trehalose 6,6′-dimycolate (TDM) IgG responses were the strongest predictor for differentiating active tuberculosis (ATB) from healthy controls (HC) and latent tuberculosis infections (LTBI). The TDM-based MTBE demonstrated superior sensitivity compared to sputum microscopy (72% vs. 56%) with 80% and 63% positivity among smear-positive and smear-negative confirmed ATB samples, respectively. Receiver operating characteristic analysis indicated good accuracy for differentiating ATB from HC (AUC = 0.77). Thus, TDM-based MTBE can be potentially used as a screening device for rapid diagnosis of active TB at the POC. PMID:27775039

  9. Quantitative Analysis of NF-κB Transactivation Specificity Using a Yeast-Based Functional Assay.

    Directory of Open Access Journals (Sweden)

    Vasundhara Sharma

    Full Text Available The NF-κB transcription factor family plays a central role in innate immunity and inflammation processes and is frequently dysregulated in cancer. We developed an NF-κB functional assay in yeast to investigate the following issues: transactivation specificity of NF-κB proteins acting as homodimers or heterodimers; correlation between transactivation capacity and in vitro DNA binding measurements; impact of co-expressed interacting proteins or of small molecule inhibitors on NF-κB-dependent transactivation. Full-length p65 and p50 cDNAs were cloned into centromeric expression vectors under inducible GAL1 promoter in order to vary their expression levels. Since p50 lacks a transactivation domain (TAD, a chimeric construct containing the TAD derived from p65 was also generated (p50TAD to address its binding and transactivation potential. The p50TAD and p65 had distinct transactivation specificities towards seventeen different κB response elements (κB-REs where single nucleotide changes could greatly impact transactivation. For four κB-REs, results in yeast were predictive of transactivation potential measured in the human MCF7 cell lines treated with the NF-κB activator TNFα. Transactivation results in yeast correlated only partially with in vitro measured DNA binding affinities, suggesting that features other than strength of interaction with naked DNA affect transactivation, although factors such as chromatin context are kept constant in our isogenic yeast assay. The small molecules BAY11-7082 and ethyl-pyruvate as well as expressed IkBα protein acted as NF-κB inhibitors in yeast, more strongly towards p65. Thus, the yeast-based system can recapitulate NF-κB features found in human cells, thereby providing opportunities to address various NF-κB functions, interactions and chemical modulators.

  10. Development of an in-house enzyme-linked immunosorbent assay based on surface whole cell antigen for diagnosis of Helicobacter pylori infection in patients with gastroduodenal ulcer disease.

    Science.gov (United States)

    Aziz, Faisal; Sherwani, Sikander Khan; Akhtar, Syed Shakeel; Kazmi, Shahana Urooj

    2014-01-01

    Helicobacter pylori (H. pylori) is a causative agent of gastritis, gastroduodenal ulcers and gastric adenocarcinoma. More than 50% world population is colonized by H. pylori, which is closely related to the chronic gastritis and gastric ulcer infection. In this study, a total of 214 gastritis patient's serum samples were screened for anti-H. pylori IgG antibody. A 96-well plate coated with 20 μg/ml antigen and hundred-fold diluted patient's serum was allowed to react. After extensive washing with buffer, 1:2,500 diluted conjugated secondary antibody was added. Later substrate was added to observe positivity by measuring the intensity of color. Statistical analyses were performed, and p value of <0.01 was taken as significant; 84% male patients and 89% female patients, respectively, tested positive for H. pylori, while agewise distribution was 35-45 years males (40%) and 35-55 years females (52%) were found highest number of H. pylori infected patients. In-house ELISA based on surface whole cell antigen (wELISA) showed a sensitivity of 93%, specificity of 100%, accuracy 94% and κ value 0.86 with significant correlation R-0.77020; p < 0.0001. We conclude that H. pylori local isolates surface antigen was satisfactory for diagnosis as different parameters were adjusted according to the local H. pylori isolates. Fluctuations in serum antibody titer predict the variation in an individual's response of the host against H. pylori. In-house wELISA could provide a reliable and a clinically useful method for the diagnosis of H. pylori infection in patients of Karachi, Pakistan.

  11. Development of a heavy metals enzymatic-based assay using papain

    Energy Technology Data Exchange (ETDEWEB)

    Shukor, Yunus [Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, University Putra Malaysia, 43400 Serdang, Selangor (Malaysia)]. E-mail: yunus@biotech.upm.edu.my; Baharom, Nor Azlan [Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, University Putra Malaysia, 43400 Serdang, Selangor (Malaysia); Rahman, Fadhil Abd. [Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, University Putra Malaysia, 43400 Serdang, Selangor (Malaysia); Abdullah, Mohd. Puad [Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, University Putra Malaysia, 43400 Serdang, Selangor (Malaysia); Shamaan, Nor Aripin [Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, University Putra Malaysia, 43400 Serdang, Selangor (Malaysia); Syed, Mohd. Arif [Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, University Putra Malaysia, 43400 Serdang, Selangor (Malaysia)

    2006-05-04

    A heavy metals enzymatic-based assay using papain was developed. Papain was assayed using the Casein-coomassie-dye-binding assay. The assay is sensitive to several heavy metals. The IC{sub 50} (concentration of toxicant giving 50% inhibition) of Hg{sup 2+}, Ag{sup 2+}, Pb{sup 2}, Zn{sup 2+} is 0.39, 0.40, 2.16, 2.11 mg l{sup -1}, respectively. For Cu{sup 2+} and Cd{sup 2+} the LOQ (limits of quantitation) is 0.004 and 0.1 mg l{sup -1}, respectively. The IC{sub 50} and LOQ values were found to be generally comparable to several other enzymatic and bioassays tests such as: immobilized urease, 15-min Microtox{sup TM}, 48 h Daphnia magna, and 96 h Rainbow trout. The papain assay is xenobiotics tolerant, has a wide pH for optimum activity, is temperature stable, and has a relatively quick assay time. The papain assay was used to identify polluted water samples from industrial sources in Penang, Malaysia. We found one site where the assay gave a positive toxic response. The toxicity of the site was confirmed using Atomic Emission Spectrometry analysis.

  12. Using Exclusion-Based Sample Preparation (ESP to Reduce Viral Load Assay Cost.

    Directory of Open Access Journals (Sweden)

    Scott M Berry

    Full Text Available Viral load (VL measurements are critical to the proper management of HIV in developing countries. However, access to VL assays is limited by the high cost and complexity of existing assays. While there is a need for low cost VL assays, performance must not be compromised. Thus, new assays must be validated on metrics of limit of detection (LOD, accuracy, and dynamic range. Patient plasma samples from the Joint Clinical Research Centre in Uganda were de-identified and measured using both an existing VL assay (Abbott RealTime HIV-1 and our assay, which combines low cost reagents with a simplified method of RNA isolation termed Exclusion-Based Sample Preparation (ESP.71 patient samples with VLs ranging from 3,000,000 copies/mL were used to compare the two methods. We demonstrated equivalent LOD (~50 copies/mL and high accuracy (average difference between methods of 0.08 log, R2 = 0.97. Using expenditures from this trial, we estimate that the cost of the reagents and consumables for this assay to be approximately $5 USD. As cost is a significant barrier to implementation of VL testing, we anticipate that our assay will enhance access to this critical monitoring test in developing countries.

  13. DETECTION OF B LYMPHOMA CELLS UNDERGOING APOPTOSIS BY ANNEXIN V ASSAY

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Objective.To quantitatively analyze apoptotic and secondary necrotic cells under apoptosis conditions. Methods.The cells of Burkitt lymphoma (BL) cell line Raji were incubated with 1.0 μ mol/L dexamethasone (DEX) for 2,4 and 8 h respectively,then stained with Annexin V FITC (fluorescein isothiocyanate conjugated) which was used to detect the exposed phosphatidylserine (PS) on the epimembrane resulting from a loss of phospholipid asymmetry in the early stage of apoptosis,and also stained with propidium iodide (PI) which allows analysis of secondary necrotic cells related with cell membrane and DNA damage that probably represent late stage of apoptosis,then apoptotic cells were quantified by flow cytometry (FCM).Furthermore,Annexin+ /PI and Annexin+ /PI+ cells were sorted by fluoresence activated cell sorter (FACS),and identified by electron microscopy (EM) and DNA gel electrophoresis. Results.The percentage of apoptotic cells was found to increase with the incubation time (r=0.97).This method was sensitive with low detection limit (0.02% ),and was reproducible with low coefficient variance (CV)(4.2% ).Meanwhile,the Annexin+ /PI and Annexin+ /PI+ cells were identified as apoptotic and necrotic cells under EM,and DNA extracted from the Annexin+ /PI cells was characterized by " ladder pattern" . Conclusions.Annexin V assay is a specific,sensitive,accurate,reproductive and quantitative method for analyzing apoptotic cells.

  14. Real-time PCR assay is superior to other methods for the detection of mycoplasma contamination in the cell lines of the National Cell Bank of Iran.

    Science.gov (United States)

    Molla Kazemiha, Vahid; Bonakdar, Shahin; Amanzadeh, Amir; Azari, Shahram; Memarnejadian, Arash; Shahbazi, Shirin; Shokrgozar, Mohammad Ali; Mahdian, Reza

    2016-08-01

    Mycoplasmas are the most important contaminants of cell cultures throughout the world. They are considered as a major problem in biological studies and biopharmaceutical economic issues. In this study, our aim was to find the best standard technique as a rapid method with high sensitivity, specificity and accuracy for the detection of mycoplasma contamination in the cell lines of the National Cell Bank of Iran. Thirty cell lines suspected to mycoplasma contamination were evaluated by five different techniques including microbial culture, indirect DNA DAPI staining, enzymatic mycoalert(®) assay, conventional PCR and real-time PCR. Five mycoplasma-contaminated cell lines were assigned as positive controls and five mycoplasma-free cell lines as negative controls. The enzymatic method was performed using the mycoalert(®) mycoplasma detection kit. Real-time PCR technique was conducted by PromoKine diagnostic kits. In the conventional PCR method, mycoplasma genus-specific primers were designed to analyze the sequences based on a fixed and common region on 16S ribosomal RNA with PCR product size of 425 bp. Mycoplasma contamination was observed in 60, 56.66, 53.33, 46.66 and 33.33 % of 30 different cell cultures by real-time PCR, PCR, enzymatic mycoalert(®), indirect DNA DAPI staining and microbial culture methods, respectively. The analysis of the results of the different methods showed that the real-time PCR assay was superior the other methods with the sensitivity, specificity, accuracy, predictive value of positive and negative results of 100 %. These values were 94.44, 100, 96.77, 100 and 92.85 % for the conventional PCR method, respectively. Therefore, this study showed that real-time PCR and PCR assays based on the common sequences in the 16S ribosomal RNA are reliable methods with high sensitivity, specificity and accuracy for detection of mycoplasma contamination in cell cultures and other biological products.

  15. Real-time PCR assay is superior to other methods for the detection of mycoplasma contamination in the cell lines of the National Cell Bank of Iran.

    Science.gov (United States)

    Molla Kazemiha, Vahid; Bonakdar, Shahin; Amanzadeh, Amir; Azari, Shahram; Memarnejadian, Arash; Shahbazi, Shirin; Shokrgozar, Mohammad Ali; Mahdian, Reza

    2016-08-01

    Mycoplasmas are the most important contaminants of cell cultures throughout the world. They are considered as a major problem in biological studies and biopharmaceutical economic issues. In this study, our aim was to find the best standard technique as a rapid method with high sensitivity, specificity and accuracy for the detection of mycoplasma contamination in the cell lines of the National Cell Bank of Iran. Thirty cell lines suspected to mycoplasma contamination were evaluated by five different techniques including microbial culture, indirect DNA DAPI staining, enzymatic mycoalert(®) assay, conventional PCR and real-time PCR. Five mycoplasma-contaminated cell lines were assigned as positive controls and five mycoplasma-free cell lines as negative controls. The enzymatic method was performed using the mycoalert(®) mycoplasma detection kit. Real-time PCR technique was conducted by PromoKine diagnostic kits. In the conventional PCR method, mycoplasma genus-specific primers were designed to analyze the sequences based on a fixed and common region on 16S ribosomal RNA with PCR product size of 425 bp. Mycoplasma contamination was observed in 60, 56.66, 53.33, 46.66 and 33.33 % of 30 different cell cultures by real-time PCR, PCR, enzymatic mycoalert(®), indirect DNA DAPI staining and microbial culture methods, respectively. The analysis of the results of the different methods showed that the real-time PCR assay was superior the other methods with the sensitivity, specificity, accuracy, predictive value of positive and negative results of 100 %. These values were 94.44, 100, 96.77, 100 and 92.85 % for the conventional PCR method, respectively. Therefore, this study showed that real-time PCR and PCR assays based on the common sequences in the 16S ribosomal RNA are reliable methods with high sensitivity, specificity and accuracy for detection of mycoplasma contamination in cell cultures and other biological products. PMID:25742733

  16. In vitro assay for HCV serine proteinase expressed in insect cells

    Institute of Scientific and Technical Information of China (English)

    Li-Hua Hou; Gui-Xin Du; Rong-Bin Guan; Yi-Gang Tong; Hai-Tao Wang

    2003-01-01

    AIM: To produce the recombinant NS3 protease of hepatitis C virus with enzymatic activity in insect cells.METHODS: The gene of HCV serine proteinase domain which encodes 181 amino acids was inserted into pFastBacHTc and the recombinant plasmid pFBCNS3N was transformed into DH10Bac competent cells for transposition.After the recombinant bacmids had been determined to be correct by both blue-white colonies and PCR analysis, the isolated bacmid DNAs were transfected into Sf9 insect cells.The bacmids DNA was verified to replicate in insect cells and packaged into baculovirus particles via PCR and electronic microscopic analysis. The insect cells infected with recombinant baculovirus were determined by SDS-PAGE and Western-blot assays. The recombinant protein was soluted in N-lauryl sarcosine sodium (NLS) and purifed by metalchelated-affinity chromatography, then the antigenicity of recombinant protease was determined by enzyme-linked immunoabsorbant assay and its enzymatic activity was detected.RESULTS: The HCV NS3 protease domain was expressed in insect cells at high level and it was partially solved in NLS.Totally 0.2 mg recombinant serine proteinase domain with high purity was obtained by metal-chelated-affinity chromatography from 5×107 cells, and both antigenicity and specificity of the protein were evaluated to be high when used as antigen to detect hepatitis C patients′ sera in indirect ELISA format. In vitro cleavage assay corroborated its enzymatic activity.CONCLUSION: The recombinant HCV NS3 proteinase expressed by insect cells is a membrane-binding protein with good antigenicity and enzymatic activity.

  17. A comparison of cell-collecting methods for the Comet assay in urinary bladders of rats.

    Science.gov (United States)

    Wada, Kunio; Ohnuma, Aya; Kojima, Sayuri; Yoshida, Toshinori; Matsumoto, Kyomu

    2012-02-18

    Conducting the single-cell gel electrophoresis (Comet) assay in the urinary bladders of rodents is technically problematic because the bladder is small and thin, which makes it difficult to collect its mucosal cells by scraping. We performed the Comet assay using a simple mincing method in which tissues are minced with scissors. We then compared data obtained with this method with data obtained using the scraping method. Sprague-Dawley rats of both sexes were orally given twice the known carcinogens N-methyl-N-nitrosourea (MNU), ethyl methanesulfonate (EMS), or o-anisidine (OA). Three hours after the second administration, the bladder of each rat was divided into two parts and each part was processed by either the mincing or the scraping method. Both mincing and scraping methods detected DNA damage in MNU-, EMS-, but not OA-treated rats, and thus the mincing method had a sufficient capability to detect DNA damaging agents. The morphological analysis of the prepared cell suspensions revealed that more than 80% of the cells collected by the mincing method were from the epithelium. Because the mincing method requires only one-half of a bladder, the other half remains intact and can be used for histopathological examination. We conclude that the mincing method is easier and more appropriate for the Comet assay in urinary bladder tissue than the scraping method. PMID:22155339

  18. Crucial ignored parameters on nanotoxicology: the importance of toxicity assay modifications and "cell vision".

    Directory of Open Access Journals (Sweden)

    Sophie Laurent

    Full Text Available Until now, the results of nanotoxicology research have shown that the interactions between nanoparticles (NPs and cells are remarkably complex. In order to get a deep understanding of the NP-cell interactions, scientists have focused on the physicochemical effects. However, there are still considerable debates about the regulation of nanomaterials and the reported results are usually in contradictions. Here, we are going to introduce the potential key reasons for these conflicts. In this case, modification of conventional in vitro toxicity assays, is one of the crucial ignored matter in nanotoxicological sciences. More specifically, the conventional methods neglect important factors such as the sedimentation of NPs and absorption of proteins and other essential biomolecules onto the surface of NPs. Another ignored matter in nanotoxicological sciences is the effect of cell "vision" (i.e., cell type. In order to show the effects of these ignored subjects, we probed the effect of superparamagnetic iron oxide NPs (SPIONs, with various surface chemistries, on various cell lines. We found thatthe modification of conventional toxicity assays and the consideration of the "cell vision" concept are crucial matters to obtain reliable, and reproducible nanotoxicology data. These new concepts offer a suitable way to obtain a deep understanding on the cell-NP interactions. In addition, by consideration of these ignored factors, the conflict of future toxicological reports would be significantly decreased.

  19. Late radiation response of kidney assayed by tubule-cell survival

    International Nuclear Information System (INIS)

    An assay for the survival of renal tubule cells was developed using mice, analogous to other in-situ clonogenic cell survival assays. One kidney was irradiated using a 137Cs irradiator and removed 60-68 weeks later for histological examination. In unirradiated animals there were about 370 tubules in contact with the capsule in a coronal cross section at the middle of the kidney. After irradiation, extensive tubular damage was the dominant lesion. The number of epithelialised tubules in contact with the capsule showed a dose-dependent logarithmic decline. The dose-survival relationship for the clonogenic cells responsible for the regeneration of tubule epithelium was described by a D0 value of 1.5 Gy over the dose range 11-16 Gy. This radiosensitivity resembles that of stem cells in acutely responding tissues. The lack of histological evidence of damage to the arterial vasculature at the time the tubules are initially denuded of epithelium, and the similarity of renal tubule cell radiosensitivity to that of other mammalian cells, support the hypothesis that ''late'' radiation injury results primarily from depletion of parenchymal cells, not indirectly from injury to blood vessels, as has been the prevailing belief. (author)

  20. Responses of the L5178Y mouse Lymphoma cell forward mutation assay. V: 27 coded chemicals.

    Science.gov (United States)

    McGregor, D B; Brown, A G; Howgate, S; McBride, D; Riach, C; Caspary, W J

    1991-01-01

    Twenty-seven chemicals were tested for their mutagenic potential in the L5178Y tk+/tk- mouse lymphoma cell forward mutation assay using procedures based upon those described by McGregor et al. (McGregor DB, Martin R, Cattanach P, Edwards I, McBride D, Caspary WJ (1987): Environ Mol Mutagen 9:143-160). Cultures were exposed to the chemicals for 4 hr, then cultured for 2 days before plating in soft agar with or without trifluorothymidine (TFT), 3 micrograms/ml. The chemicals were tested at least twice. Statistically significant responses were obtained with acid orange 10, aniline, benzaldehyde, o-chloroaniline, chlorodibromomethane, cytembena, 1,2-dibromo-4-(1,2-dibromomethyl) cyclohexane, dieldrin, lithocholic acid, oxytetracycline, phenazopyridine HCl, 1-phenyl-3-methyl-5-pyrazolone, sodium diethyldithiocarbamate, solvent yellow 14, tetraethylthiuram disulfide (disulfiram), 2,4-toluene diisocyanate, and 2,6-toluene diisocyanate. Apart from phenazopyridine HCl, acid orange 10, and solvent yellow 14, rat liver S9 mix was not a requirement for the mutagenic activity of these compounds. Chemical not identified as mutagens were N-4-acetylaminofluorene, chlorpheniramine maleate, chloropropamide, 1,4-dioxane, endrin, ethylene glycol, iron dextran, methapyrilene, sodium(2-ethylhexyl)alcohol PMID:1902415

  1. Redox-Reaction Based Spectrophotometric Assay of Isoniazid in Pharmaceuticals

    OpenAIRE

    N Swamy; K. N. Prashanth; K. Basavaiah

    2014-01-01

    Two spectrophotometric methods are described for the determination of isoniazid (INH) in pharmaceuticals. In the first method (FCR method), INH is reacted with Folin-Ciocalteu reagent in Na2CO3 medium and the resulting blue colored chromogen measured at 760 nm. Iron(II), formed as a result of reaction between INH and iron(III), is made to react with ferricyanide, and the resulting Prussian blue is measured at 760 nm, basing the second method (FFC method). The conditions for better performance...

  2. Smartphone based visual and quantitative assays on upconversional paper sensor.

    Science.gov (United States)

    Mei, Qingsong; Jing, Huarong; Li, You; Yisibashaer, Wuerzha; Chen, Jian; Nan Li, Bing; Zhang, Yong

    2016-01-15

    The integration of smartphone with paper sensors recently has been gain increasing attentions because of the achievement of quantitative and rapid analysis. However, smartphone based upconversional paper sensors have been restricted by the lack of effective methods to acquire luminescence signals on test paper. Herein, by the virtue of 3D printing technology, we exploited an auxiliary reusable device, which orderly assembled a 980nm mini-laser, optical filter and mini-cavity together, for digitally imaging the luminescence variations on test paper and quantitative analyzing pesticide thiram by smartphone. In detail, copper ions decorated NaYF4:Yb/Tm upconversion nanoparticles were fixed onto filter paper to form test paper, and the blue luminescence on it would be quenched after additions of thiram through luminescence resonance energy transfer mechanism. These variations could be monitored by the smartphone camera, and then the blue channel intensities of obtained colored images were calculated to quantify amounts of thiram through a self-written Android program installed on the smartphone, offering a reliable and accurate detection limit of 0.1μM for the system. This work provides an initial demonstration of integrating upconversion nanosensors with smartphone digital imaging for point-of-care analysis on a paper-based platform.

  3. Smartphone based visual and quantitative assays on upconversional paper sensor.

    Science.gov (United States)

    Mei, Qingsong; Jing, Huarong; Li, You; Yisibashaer, Wuerzha; Chen, Jian; Nan Li, Bing; Zhang, Yong

    2016-01-15

    The integration of smartphone with paper sensors recently has been gain increasing attentions because of the achievement of quantitative and rapid analysis. However, smartphone based upconversional paper sensors have been restricted by the lack of effective methods to acquire luminescence signals on test paper. Herein, by the virtue of 3D printing technology, we exploited an auxiliary reusable device, which orderly assembled a 980nm mini-laser, optical filter and mini-cavity together, for digitally imaging the luminescence variations on test paper and quantitative analyzing pesticide thiram by smartphone. In detail, copper ions decorated NaYF4:Yb/Tm upconversion nanoparticles were fixed onto filter paper to form test paper, and the blue luminescence on it would be quenched after additions of thiram through luminescence resonance energy transfer mechanism. These variations could be monitored by the smartphone camera, and then the blue channel intensities of obtained colored images were calculated to quantify amounts of thiram through a self-written Android program installed on the smartphone, offering a reliable and accurate detection limit of 0.1μM for the system. This work provides an initial demonstration of integrating upconversion nanosensors with smartphone digital imaging for point-of-care analysis on a paper-based platform. PMID:26356763

  4. Cp*Rh-based indicator-displacement assays for the identification of amino sugars and aminoglycosides.

    Science.gov (United States)

    Zaubitzer, Friederike; Buryak, Andrey; Severin, Kay

    2006-05-01

    Indicator-displacement assays based on the organometallic complex [{Cp*RhCl2}2] (Cp*=pentamethylcyclopentadienyl) and the dye gallocyanine were used to sense amino sugars and aminoglycosides in buffered aqueous solution by conducting UV-visible spectroscopy. The data of three assays at pH 7.0, 8.0, and 9.0 were sufficient to distinguish between the amino sugars galactosamine, glucosamine, mannosamine and the aminoglycosides kanamycin A, kanamycin B, amikacin, apramycin, paromomycin, and streptomycin. Furthermore, the assays were used to characterize mixtures of aminoglycosides and obtain quantitative information about the respective analytes. PMID:16521137

  5. Plasmid-Based Materials as Multiplex Quality Controls and Calibrators for Clinical Next-Generation Sequencing Assays.

    Science.gov (United States)

    Sims, David J; Harrington, Robin D; Polley, Eric C; Forbes, Thomas D; Mehaffey, Michele G; McGregor, Paul M; Camalier, Corinne E; Harper, Kneshay N; Bouk, Courtney H; Das, Biswajit; Conley, Barbara A; Doroshow, James H; Williams, P Mickey; Lih, Chih-Jian

    2016-05-01

    Although next-generation sequencing technologies have been widely adapted for clinical diagnostic applications, an urgent need exists for multianalyte calibrator materials and controls to evaluate the performance of these assays. Control materials will also play a major role in the assessment, development, and selection of appropriate alignment and variant calling pipelines. We report an approach to provide effective multianalyte controls for next-generation sequencing assays, referred to as the control plasmid spiked-in genome (CPSG). Control plasmids that contain approximately 1000 bases of human genomic sequence with a specific mutation of interest positioned near the middle of the insert and a nearby 6-bp molecular barcode were synthesized, linearized, quantitated, and spiked into genomic DNA derived from formalin-fixed, paraffin-embedded-prepared hapmap cell lines at defined copy number ratios. Serial titration experiments demonstrated the CPSGs performed with similar efficiency of variant detection as formalin-fixed, paraffin-embedded cell line genomic DNA. Repetitive analyses of one lot of CPSGs 90 times during 18 months revealed that the reagents were stable with consistent detection of each of the plasmids at similar variant allele frequencies. CPSGs are designed to work across most next-generation sequencing methods, platforms, and data analysis pipelines. CPSGs are robust controls and can be used to evaluate the performance of different next-generation sequencing diagnostic assays, assess data analysis pipelines, and ensure robust assay performance metrics. PMID:27105923

  6. A protocol for the systematic and quantitative measurement of protein-lipid interactions using the liposome-microarray-based assay.

    Science.gov (United States)

    Saliba, Antoine-Emmanuel; Vonkova, Ivana; Deghou, Samy; Ceschia, Stefano; Tischer, Christian; Kugler, Karl G; Bork, Peer; Ellenberg, Jan; Gavin, Anne-Claude

    2016-06-01

    Lipids organize the activity of the cell's proteome through a complex network of interactions. The assembly of comprehensive atlases embracing all protein-lipid interactions is an important challenge that requires innovative methods. We recently developed a liposome-microarray-based assay (LiMA) that integrates liposomes, microfluidics and fluorescence microscopy and which is capable of measuring protein recruitment to membranes in a quantitative and high-throughput manner. Compared with previous assays that are labor-intensive and difficult to scale up, LiMA improves the protein-lipid interaction assay throughput by at least three orders of magnitude. Here we provide a step-by-step LiMA protocol that includes the following: (i) the serial and generic production of the liposome microarray; (ii) its integration into a microfluidic format; (iii) the measurement of fluorescently labeled protein (either purified proteins or from cell lysate) recruitment to liposomal membranes using high-throughput microscopy; (iv) automated image analysis pipelines to quantify protein-lipid interactions; and (v) data quality analysis. In addition, we discuss the experimental design, including the relevant quality controls. Overall, the protocol-including device preparation, assay and data analysis-takes 6-8 d. This protocol paves the way for protein-lipid interaction screens to be performed on the proteome and lipidome scales. PMID:27149326

  7. Neutral Comet Assay

    OpenAIRE

    2013-01-01

    The Comet assay (or Single Cell Gel Electrophoresis assay) is a sensitive technique to detect DNA damage at the level of an individual cell. This technique is based on micro-electrophoresis of cells DNA content. Briefly, cells are embedded in agarose, lysed and submitted to an electric field, before the staining step with a fluorescent DNA binding dye. Damaged DNA (charged DNA) migrates in this field, forming the tail of a “comet”, while undamaged DNA remained in the head of the “comet”. The ...

  8. A fluorescence-based quantitative real-time PCR assay for accurate Pocillopora damicornis species identification

    Science.gov (United States)

    Thomas, Luke; Stat, Michael; Evans, Richard D.; Kennington, W. Jason

    2016-09-01

    Pocillopora damicornis is one of the most extensively studied coral species globally, but high levels of phenotypic plasticity within the genus make species identification based on morphology alone unreliable. As a result, there is a compelling need to develop cheap and time-effective molecular techniques capable of accurately distinguishing P. damicornis from other congeneric species. Here, we develop a fluorescence-based quantitative real-time PCR (qPCR) assay to genotype a single nucleotide polymorphism that accurately distinguishes P. damicornis from other morphologically similar Pocillopora species. We trial the assay across colonies representing multiple Pocillopora species and then apply the assay to screen samples of Pocillopora spp. collected at regional scales along the coastline of Western Australia. This assay offers a cheap and time-effective alternative to Sanger sequencing and has broad applications including studies on gene flow, dispersal, recruitment and physiological thresholds of P. damicornis.

  9. Spectrophotometric total reducing sugars assay based on cupric reduction.

    Science.gov (United States)

    Başkan, Kevser Sözgen; Tütem, Esma; Akyüz, Esin; Özen, Seda; Apak, Reşat

    2016-01-15

    As the concentration of reducing sugars (RS) is controlled by European legislation for certain specific food and beverages, a simple and sensitive spectrophotometric method for the determination of RS in various food products is proposed. The method is based on the reduction of Cu(II) to Cu(I) with reducing sugars in alkaline medium in the presence of 2,9-dimethyl-1,10-phenanthroline (neocuproine: Nc), followed by the formation of a colored Cu(I)-Nc charge-transfer complex. All simple sugars tested had the linear regression equations with almost equal slope values. The proposed method was successfully applied to fresh apple juice, commercial fruit juices, milk, honey and onion juice. Interference effect of phenolic compounds in plant samples was eliminated by a solid phase extraction (SPE) clean-up process. The method was proven to have higher sensitivity and precision than the widely used dinitrosalicylic acid (DNS) colorimetric method.

  10. Spectrophotometric total reducing sugars assay based on cupric reduction.

    Science.gov (United States)

    Başkan, Kevser Sözgen; Tütem, Esma; Akyüz, Esin; Özen, Seda; Apak, Reşat

    2016-01-15

    As the concentration of reducing sugars (RS) is controlled by European legislation for certain specific food and beverages, a simple and sensitive spectrophotometric method for the determination of RS in various food products is proposed. The method is based on the reduction of Cu(II) to Cu(I) with reducing sugars in alkaline medium in the presence of 2,9-dimethyl-1,10-phenanthroline (neocuproine: Nc), followed by the formation of a colored Cu(I)-Nc charge-transfer complex. All simple sugars tested had the linear regression equations with almost equal slope values. The proposed method was successfully applied to fresh apple juice, commercial fruit juices, milk, honey and onion juice. Interference effect of phenolic compounds in plant samples was eliminated by a solid phase extraction (SPE) clean-up process. The method was proven to have higher sensitivity and precision than the widely used dinitrosalicylic acid (DNS) colorimetric method. PMID:26592591

  11. High-throughput pseudovirion-based neutralization assay for analysis of natural and vaccine-induced antibodies against human papillomaviruses.

    Directory of Open Access Journals (Sweden)

    Peter Sehr

    Full Text Available A highly sensitive, automated, purely add-on, high-throughput pseudovirion-based neutralization assay (HT-PBNA with excellent repeatability and run-to-run reproducibility was developed for human papillomavirus types (HPV 16, 18, 31, 45, 52, 58 and bovine papillomavirus type 1. Preparation of 384 well assay plates with serially diluted sera and the actual cell-based assay are separated in time, therefore batches of up to one hundred assay plates can be processed sequentially. A mean coefficient of variation (CV of 13% was obtained for anti-HPV 16 and HPV 18 titers for a standard serum tested in a total of 58 repeats on individual plates in seven independent runs. Natural antibody response was analyzed in 35 sera from patients with HPV 16 DNA positive cervical intraepithelial neoplasia grade 2+ lesions. The new HT-PBNA is based on Gaussia luciferase with increased sensitivity compared to the previously described manual PBNA (manPBNA based on secreted alkaline phosphatase as reporter. Titers obtained with HT-PBNA were generally higher than titers obtained with the manPBNA. A good linear correlation (R(2 = 0.7 was found between HT-PBNA titers and anti-HPV 16 L1 antibody-levels determined by a Luminex bead-based GST-capture assay for these 35 sera and a Kappa-value of 0.72, with only 3 discordant sera in the low titer range. In addition to natural low titer antibody responses the high sensitivity of the HT-PBNA also allows detection of cross-neutralizing antibodies induced by commercial HPV L1-vaccines and experimental L2-vaccines. When analyzing the WHO international standards for HPV 16 and 18 we determined an analytical sensitivity of 0.864 and 1.105 mIU, respectively.

  12. A flow cytometric in vivo chalone assay using retransplanted old murine JB-1 ascites tumour cells.

    Science.gov (United States)

    Barfod, N M

    1981-07-01

    A flow cytometric in vivo chalone assay is described. Transplantation of old JB-1 ascites tumour cells to new hosts induced an influx of tumour cells, with G1 DNA content, to the S phase. This induction could be reversibly and specifically blocked by injections of an ultrafiltrate of old JB-1 ascites fluid. The method described is superior to a previously published in vivo chalone assay using regenerating ascites tumours. Owing to a reduced variability in time of onset of DNA synthesis, a smaller scatter of observations is achieved and thus the number of mice per group may be reduced using the new method. In contrast to the older technique, the present one does not necessitate killing of mice during the observation period.

  13. Comparison of multiple assays for detecting human antibodies directed against antigens on normal and malignant tissue culture cells

    International Nuclear Information System (INIS)

    Four separate assays of human antibody reactivity to four separate normal and malignant human tissue culture cells lines from two patients have been evaluated using a single highly-reactive allogeneic serum. The visual end-point cytolysis assay and the chromium-51 release assay were equally sensitive in measuring complement mediated antibody cytotoxicity and both were far more sensitive than a trypan blue dye exclusion assay. The assay of antibody reactivity by hemadsorption technique was about 10 times more sensitive than any of the cytotoxicity assays. This latter assay measures only IgG antibody however. These assays showed that cell lines from different patients may differ greatly in 'reactivity' to an allogeneic serum and emphasized the importance of utilizing tumor and normal cells from the same patient when using tissue culture cells to search for tumor specific reactivity. These observations emphasize the importance of utilizing multiple assays against paired normal and malignant cells from the same patient to be certain of the specificity and magnitude of the measured antibody

  14. Microtiter plate based colorimetric assay for characterization of dehalogenation activity of GAC/Fe0 composite

    DEFF Research Database (Denmark)

    Hwang, Yuhoon; Salatas, Apostolos; Mines, Paul D.;

    2015-01-01

    Even though nanoscale zero valent iron (nZVI) has been intensively studied for the treatment of a plethora of pollutants through reductive reaction, a quantification of nZVI reactivity has not been standardized. Here, we developed series of colorimetric assays for determining reductive activity o...... based color assay will be useful and simple tool in various nZVI related research topics, e.g. different stabilization, immobilization, etc....

  15. MOBE-ChIP: a large-scale chromatin immunoprecipitation assay for cell type-specific studies.

    Science.gov (United States)

    Lau, On Sun; Bergmann, Dominique C

    2015-10-01

    Cell type-specific transcriptional regulators play critical roles in the generation and maintenance of multicellularity. As they are often expressed at low levels, in vivo DNA-binding studies of these regulators by standard chromatin immunoprecipitation (ChIP) assays are technically challenging. We describe here an optimized ChIP protocol termed Maximized Objects for Better Enrichment (MOBE)-ChIP, which enhances the sensitivity of ChIP assays for detecting cell type-specific signals. The protocol, which is based on the disproportional increase of target signals over background at higher scales, uses substantially greater volume of starting materials than conventional ChIPs to achieve high signal enrichment. This technique can capture weak binding events that are ambiguous in standard ChIP assays, and is useful both in gene-specific and whole-genome analysis. This protocol has been optimized for Arabidopsis, but should be applicable to other model systems with minor modifications. The full procedure can be completed within 3 days.

  16. Immunological assays for chemokine detection in in-vitro culture of CNS cells

    OpenAIRE

    Mahajan Supriya D.; Schwartz Stanley A; Nair Madhavan P.N.

    2003-01-01

    Herein we review the various methods currently in use for determining the expression of chemokines by CNS cells in vitro. Chemokine detection assays are used in conjuction with one another to provide a comprehensive, biologically relevant assessment of the chemokines which is necessary for correct data interpretation of a specific observed biological effect. The methods described include bioassays for soluble chemokine receptors, RNA extraction, RT-PCR, Real - time quantitative PCR, gene arra...

  17. CYTOKINESIS-BLOCK MICRONUCLEUS ASSAY IN HUMAN GLIOMA CELLS EXPOSED TO RADIATION

    Directory of Open Access Journals (Sweden)

    Jerzy Slowinski

    2011-05-01

    Full Text Available Biological tests are efficient in reflecting the biological influences of several types of generally harmful exposures. The micronucleus assay is widely used in genotoxicity studies or studies on genomic damage in general. We present methodological aspects of cytokinesis-block micronucleus assay performed in human gliomas irradiated in vitro. Eight human glioblastoma cell lines obtained from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Germany were gamma-irradiated (60Co over a dose range of 0-10 Gy. Cytokinesis-block micronucleus assay was performed to quantitate cytogenetic damage. The cells were fixed directly on dishes, stained with fluorochrome DAPI and evaluated under fluorescent and phase contrast microscope. The micronucleus frequency was expressed as a micronuclei (MN per binucleated cell (BNC ratio, calculated after scoring at least 100 BNC per dish. The frequency of spontaneous MN ranged from 0.17 to 0.613 (mean: 0.29 ± 0.14. After irradiation increase of MN frequency in the range of 0.312 - 2.241 (mean: 0.98 ± 0.68 was found at 10 Gy. Gliomas are extremely heterogenous in regard to cytogenetic effects of irradiation, as shown in this study by cytokinesis-block micronucleus assay. This test is easily performed on irradiated glioma cell lines and can assist in determining their radiosensitivity. However, in order to obtain reliable and reproducible results, precise criteria for MN scoring must be strictly followed. Simultaneous use of fluorescent and phase contrast equipment improves imaging of morphological details and can further optimize MN scoring.

  18. Nanoparticle-based assays in automated flow systems: A review

    International Nuclear Information System (INIS)

    Nanoparticles (NPs) exhibit a number of distinctive and entrancing properties that explain their ever increasing application in analytical chemistry, mainly as chemosensors, signaling tags, catalysts, analytical signal enhancers, reactive species generators, analyte recognition and scavenging/separation entities. The prospect of associating NPs with automated flow-based analytical is undoubtedly a challenging perspective as it would permit confined, cost-effective and reliable analysis, within a shorter timeframe, while exploiting the features of NPs. This article aims at examining state-of-the-art on continuous flow analysis and microfluidic approaches involving NPs such as noble metals (gold and silver), magnetic materials, carbon, silica or quantum dots. Emphasis is devoted to NP format, main practical achievements and fields of application. In this context, the functionalization of NPs with distinct chemical species and ligands is debated in what concerns the motivations and strengths of developed approaches. The utilization of NPs to improve detector's performance in electrochemical application is out of the scope of this review. The works discussed in this review were published in the period of time comprised between the years 2000 and 2013. - Highlights: • The state of the art of flowing stream systems comprising NPs was reviewed. • The use of different types of nanoparticles in each flow technique is discussed. • The most expressive and profitable applications are summarized. • The main conclusions and future perspectives were compiled in the final section

  19. Detection of DNA damage induced by heavy ion irradiation in the individual cells with comet assay

    Science.gov (United States)

    Wada, S.; Natsuhori, M.; Ito, N.; Funayama, T.; Kobayashi, Y.

    2003-05-01

    Investigating the biological effects of high-LET heavy ion irradiation at low fluence is important to evaluate the risk of charged particles. Especially it is important to detect radiation damage induced by the precise number of heavy ions in the individual cells. Thus we studied the relationship between the number of ions traversing the cell and DNA damage produced by the ion irradiation. We applied comet assay to measure the DNA damage in the individual cells. Cells attached on the ion track detector CR-39 were irradiated with ion beams at TIARA, JAERI-Takasaki. After irradiation, the cells were stained with ethidium bromide and the opposite side of the CR-39 was etched. We observed that the heavy ions with higher LET values induced the heavier DNA damage. The result indicated that the amount of DNA damage induced by one particle increased with the LET values of the heavy ions.

  20. Assay of the multiple energy-producing pathways of mammalian cells.

    Directory of Open Access Journals (Sweden)

    Barry R Bochner

    Full Text Available BACKGROUND: To elucidate metabolic changes that occur in diabetes, obesity, and cancer, it is important to understand cellular energy metabolism pathways and their alterations in various cells. METHODOLOGY AND PRINCIPAL FINDINGS: Here we describe a technology for simultaneous assessment of cellular energy metabolism pathways. The technology employs a redox dye chemistry specifically coupled to catabolic energy-producing pathways. Using this colorimetric assay, we show that human cancer cell lines from different organ tissues produce distinct profiles of metabolic activity. Further, we show that murine white and brown adipocyte cell lines produce profiles that are distinct from each other as well as from precursor cells undergoing differentiation. CONCLUSIONS: This technology can be employed as a fundamental tool in genotype-phenotype studies to determine changes in cells from shared lineages due to differentiation or mutation.

  1. Cell-free assay measuring repair DNA synthesis in human fibroblasts

    International Nuclear Information System (INIS)

    Osmotic disruption of confluent cultured human fibroblasts that have been irradiated or exposed to chemical carcinogens allows the specific measurement of repair DNA synthesis using dTTP as a precursor. Fibroblasts similarly prepared from various xeroderma pigmentosum cell lines show the deficiencies of uv-induced DNA synthesis predicted from in vivo studies, while giving normal responses to methylmethanesulfonate. A pyrimidine-dimer-specific enzyme, T4 endonuclease V, stimulated the rate of uv-induced repair synthesis with normal and xeroderma pigmentosum cell lines. This system should prove useful for identifying agents that induce DNA repair, and cells that respond abnormally to such induction. It should also be applicable to an in vitro complementation assay with repair-defective cells and proteins obtained from repair-proficient cells. Finally, by using actively growing fibroblasts and thymidine in the system, DNA replication can be measured and studied in vitro

  2. Identifying tumor cell growth inhibitors by combinatorial chemistry and zebrafish assays.

    Directory of Open Access Journals (Sweden)

    Jing Xiang

    Full Text Available Cyclin-dependent kinases (CDKs play important roles in regulating cell cycle progression, and altered cell cycles resulting from over-expression or abnormal activation of CDKs observed in many human cancers. As a result, CDKs have become extensive studied targets for developing chemical inhibitors for cancer therapies; however, protein kinases share a highly conserved ATP binding pocket at which most chemical inhibitors bind, therefore, a major challenge in developing kinase inhibitors is achieving target selectivity. To identify cell growth inhibitors with potential applications in cancer therapy, we used an integrated approach that combines one-pot chemical synthesis in a combinatorial manner to generate diversified small molecules with new chemical scaffolds coupled with growth inhibition assay using developing zebrafish embryos. We report the successful identification of a novel lead compound that displays selective inhibitory effects on CDK2 activity, cancer cell proliferation, and tumor progression in vivo. Our approaches should have general applications in developing cell proliferation inhibitors using an efficient combinatorial chemical genetic method and integrated biological assays. The novel cell growth inhibitor we identified should have potential as a cancer therapeutic agent.

  3. Cytotoxic Effect of Iron Oxide Nanoparticles on Mouse Embryonic Stem Cells by MTT Assay

    Directory of Open Access Journals (Sweden)

    Homa Mohseni Kouchesfehani

    2016-07-01

    Full Text Available Background: Despite the wide range of applications, there is a serious lack of information on the impact of the nanoparticles on human health and the environment. The present study was done to determine the range of dangerous concentrations of iron oxide nanoparticle and their effects on mouse embryonic stem cells. Methods: Iron oxide nanoparticles with less than 20 nanometers diameter were encapsulated by a PEG-phospholipid. The suspension of iron oxide nanoparticles was prepared using the culture media and cell viability was determined by MTT assay. Results: MTT assay was used to examine the cytotoxicity of iron oxide nanoparticle s. Royan B1 cells were treated with medium containing different concentrations (10, 20, 30, 40, 50, and 60µg/ml of the iron oxide nanoparticle. Cell viability was determined at 12 and 24 hours after treatment which showed significant decreases when concentration and time period increased. Conclusion: The main mechanism of nanoparticles action is still unknown, but in vivo and in vitro studies in different environments suggest that they are capable of producing reactive oxygen species (ROS. Therefore, they may have an effect on the concentration of intracellular calcium, activation of transcription factors, and changes in cytokine. The results of this study show that the higher concentration and duration of treatment of cells with iron oxide nanoparticles increase the rate of cell death.

  4. Automated low dose assay system for survival measurements of mammalian cells in vitro

    International Nuclear Information System (INIS)

    It has been suggested that at low doses of radiation both surviving cells (S) and inactivated cells (K) should be identified to obtain accurate data. One way to achieve this is to microscopically examine individual cells attached to a culture vessel, record their positions and observe and classify subsequent cell growth. In this way most systematic errors (counting, pipetting, diluting, etc.) are eliminated and since both S and K cells are scored, statistical accuracy (binomial) is also improved. For this purpose the authors have developed a semi automatic low dose assay system (ALDAS) whereby a microscope state was modified and equipped with two stepping motors under computer control. The computer automatically scans tissue culture flasks in which cells were plated after irradiation. When a cell is observed, the operator assumes command of the stage monitor, centres the cell in the field of view using a ''joystick'' control and signals the computer to record the cell's X-Y coordinates. After one week of incubation each cell location is revisited automatically and the operator scores the cells as S or K

  5. Development of a Direct and Continuous Phospholipase D Assay Based on the Chelation-Enhanced Fluorescence Property of 8-Hydroxyquinoline.

    Science.gov (United States)

    Rahier, Renaud; Noiriel, Alexandre; Abousalham, Abdelkarim

    2016-01-01

    Through its production of phosphatidic acid (PA), phospholipase D (PLD) is strongly involved in vesicular trafficking and cell signaling, making this enzyme an important therapeutic target. However, most PLD assays developed so far are either discontinuous or based on the indirect determination of choline released during PLD-catalyzed phosphatidylcholine hydrolysis, making its kinetic characterization difficult. We present here the development of a direct, specific, and continuous PLD assay that is based on the chelation-enhanced fluorescence property of 8-hydroxyquinoline (8HQ) following Ca(2+) complexation with PLD-generated PA. The real-time fluorescence intensity from 8HQ/Ca(2+)/PA complexes can be converted to concentrations of product using a calibration curve, with a detection limit of 1.2 μM of PA on a microplate scale, thus allowing measurement of the PLD-catalyzed reaction rate parameters. Hence, this assay is well adapted for studying the substrate specificity of PLD, together with its kinetic parameters, using natural phospholipids with various headgroups. In addition, the assay was found to be effective in monitoring the competitive inhibition of PA formation in the production of phosphatidylalcohols following the addition of primary alcohols, such as ethanol, propan-1-ol, or butan-1-ol. Finally, this assay was validated using the purified recombinant Vigna unguiculata PLD, as well as the PLD from Streptomyces chromofuscus, cabbage, or peanuts, and no PA production could be detected using phospholipase A1, phospholipase A2, or phospholipase C, allowing for a reliable determination of PLD activity in crude protein extract samples. This easy to handle PLD assay constitutes, to our knowledge, the first direct and continuous PA determination method on a microplate scale. PMID:26636829

  6. Development of a recombinant DNA assay system for the detection of genetic change in astronauts' cells

    International Nuclear Information System (INIS)

    We are developing a new recombinant DNA system for the detection and measurement of genetic change in humans caused by exposure to low level ionizing radiation. A unique feature of the method is the use of cloned repetitive DNA probes to assay human DNA for structural changes during or after irradiation. Repetitive sequences exist in different families. Collectively they constitute over 25% of the DNA in a human cell. Repeat families have between 10 and 500,000 members. We have constructed repetitive DNA sequence libraries using recombinant DNA techniques. From these libraries we have isolated and characterized individual repeats comprising 75 to 90% of the mass of human repetitive DNA. Repeats used in our assay system exist in tandem arrays in the genome. Perturbation of these sequences in a cell, followed by detection with a repeat probe, produces a new, multimeric ''ladder'' pattern on an autoradiogram. The repeat probe used in our initial study is complementary to 1% of human DNA. Therefore, the sensitivity of this method is several orders of magnitude better than existing assays. Preliminary evidence from human skin cells exposed to acute, low-dose x-ray treatments indicates that DNA is affected at a dose as low as 5R. The radiation doses used in this system are well within the range of doses received by astronauts during spaceflight missions. Due to its small material requirements, this technique could easily be adapted for use in space. 16 refs., 1 fig

  7. Functional characterisation of human glycine receptors in a fluorescence-based high throughput screening assay

    DEFF Research Database (Denmark)

    Jensen, Anders A.

    2005-01-01

    The human glycine receptor subtypes alpha1beta and alpha2 have been expressed stably in HEK293 cells, and the functional characteristics of the receptors have been characterised in the FLIPR Membrane Potential Assay. The pharmacological properties obtained for nine standard ligands at the two rec...

  8. Assay of Peripheral Regulatory Vδ1 T Cells in Ankylosing Spondylitis and its Significance

    Science.gov (United States)

    Wang, Hongliang; Sun, Na; Li, Ka; Tian, Jiguang; Li, Jianmin

    2016-01-01

    Background Ankylosing spondylitis (AS) involves inflammation at the sacroiliac joint and spine attachment site. This study aimed to observe the ratio and function of peripheral regulatory Vδ1 T cells in AS patients to investigate their roles in AS pathogenesis. Material/Methods Peripheral blood mononuclear cells (PBMC) were separated by density-gradient centrifugation from AS patients and healthy controls. Flow cytometry was used to determine the ratio between Vδ1 and CD4 T cells of PBMC in AS patients and controls. Flow cytometry sorting (FCS) was used to obtain Vδ1 and naïve CD4 T cells with purity higher than 90%. CFSE staining method was used to detect the effect of Vδ1 T cells on proliferation of naïve CD4 T cells. The effect of Vδ1 T cells on secretion of IFN-γ from naïve CD4 T cells and the ability to secrete IL-10 from Vδ1 T cells were determined by flow cytometry. Results AS patients had significantly lower Vδ1 T cell ratio in PBMC compared to controls (p<0.05), but their CD4 T cell ratio was significantly elevated (p<0.05). Functional assay showed suppression of naïve CD4 T cell proliferation and IFN-γ secretion by peripheral Vδ1 T cells in AS patients (p<0.01). AS patients also had lower IL-10 secreting level from peripheral derived Vδ1 T cells (p<0.01). Conclusions The immune suppression of peripheral Vδ1 T cell in AS patient increases the ratio of peripheral CD4 T cells and IFN-γ level, leading to AS pathogenesis. This immune suppression is mainly due to suppressed IL-10 secretion. PMID:27598263

  9. Enzyme activity assay of glycoprotein enzymes based on a boronate affinity molecularly imprinted 96-well microplate.

    Science.gov (United States)

    Bi, Xiaodong; Liu, Zhen

    2014-12-16

    Enzyme activity assay is an important method in clinical diagnostics. However, conventional enzyme activity assay suffers from apparent interference from the sample matrix. Herein, we present a new format of enzyme activity assay that can effectively eliminate the effects of the sample matrix. The key is a 96-well microplate modified with molecularly imprinted polymer (MIP) prepared according to a newly proposed method called boronate affinity-based oriented surface imprinting. Alkaline phosphatase (ALP), a glycoprotein enzyme that has been routinely used as an indicator for several diseases in clinical tests, was taken as a representative target enzyme. The prepared MIP exhibited strong affinity toward the template enzyme (with a dissociation constant of 10(-10) M) as well as superb tolerance for interference. Thus, the enzyme molecules in a complicated sample matrix could be specifically captured and cleaned up for enzyme activity assay, which eliminated the interference from the sample matrix. On the other hand, because the boronate affinity MIP could well retain the enzymatic activity of glycoprotein enzymes, the enzyme captured by the MIP was directly used for activity assay. Thus, additional assay time and possible enzyme or activity loss due to an enzyme release step required by other methods were avoided. Assay of ALP in human serum was successfully demonstrated, suggesting a promising prospect of the proposed method in real-world applications.

  10. Evaluation of the sensitivity of three sublethal cytotoxicity assays in human HepG2 cell line using water contaminants

    International Nuclear Information System (INIS)

    The in vitro toxicological index IC50 (the millimolar concentration of compound which inhibits response assay by 50% compared to the solvent control) of 11 water contaminants (acrylamide, atrazine, B[a]P, BPA, 2,4-DAT, 17-αEE, H2O2, 4-OP, sodium bromate, sodium chlorate, sodium nitrate) was evaluated on the human hepatoma (HepG2) cells using three short-term bioassays related to their morbidity status [radiometric RNA synthesis assay (RNA), luminometric ATP assay (ATP), fluorometric Alamar blue assay (AB)]. Among all substances, we were not able to determine atrazine IC50 value whatever the test used. Furthermore, B[a]P was not cytotoxic in the ATP and AB assays. Statistical analysis revealed a correlation between the IC50 values obtained in the three assays. Except with 4-OP, RNA assay was always inhibited at lower concentrations than those required in the other assays, suggesting that this assay is a very sensitive indicator of the presence of toxic compounds. ATP and AB assays responded to a similar pattern. Due to its higher sensitivity and its reliability, RNA synthesis assay using HepG2 cell line provides the most suitable tool for the screening of water contaminants

  11. Bimolecular Fluorescence Complementation to Assay the Interactions of Ubiquitylation Enzymes in Living Yeast Cells.

    Science.gov (United States)

    Blaszczak, Ewa; Prigent, Claude; Rabut, Gwenaël

    2016-01-01

    Ubiquitylation is a versatile posttranslational protein modification catalyzed through the concerted action of ubiquitin-conjugating enzymes (E2s) and ubiquitin ligases (E3s). These enzymes form transient complexes with each other and their modification substrates and determine the nature of the ubiquitin signals attached to their substrates. One challenge in the field of protein ubiquitylation is thus to identify the E2-E3 pairs that function in the cell. In this chapter, we describe the use of bimolecular fluorescence complementation to assay E2-E3 interactions in living cells, using budding yeast as a model organism. PMID:27613039

  12. Dissecting the T Cell Response: Proliferation Assays vs. Cytokine Signatures by ELISPOT

    Directory of Open Access Journals (Sweden)

    Magdalena Tary-Lehmann

    2012-05-01

    Full Text Available Chronic allograft rejection is in part mediated by host T cells that recognize allogeneic antigens on transplanted tissue. One factor that determines the outcome of a T cell response is clonal size, while another is the effector quality. Studies of alloimmune predictors of transplant graft survival have most commonly focused on only one measure of the alloimmune response. Because differing qualities and frequencies of the allospecific T cell response may provide distinctly different information we analyzed the relationship between frequency of soluble antigen and allo-antigen specific memory IFN-g secreting CD4 and CD8 T cells, their ability to secrete IL-2, and their proliferative capacity, while accounting for cognate and bystander proliferation. The results show proliferative responses primarily reflect on IL-2 production by antigen-specific T cells, and that proliferating cells in such assays entail a considerable fraction of bystander cells. On the other hand, proliferation (and IL-2 production did not reflect on the frequency of IFN-γ producing memory cells, a finding particularly accentuated in the CD8 T cell compartment. These data provide rationale for considering both frequency and effector function of pre-transplant T cell reactivity when analyzing immune predictors of graft rejection.

  13. Comparative analysis of dynamic cell viability, migration and invasion assessments by novel real-time technology and classic endpoint assays.

    Directory of Open Access Journals (Sweden)

    Ridha Limame

    Full Text Available BACKGROUND: Cell viability and motility comprise ubiquitous mechanisms involved in a variety of (pathobiological processes including cancer. We report a technical comparative analysis of the novel impedance-based xCELLigence Real-Time Cell Analysis detection platform, with conventional label-based endpoint methods, hereby indicating performance characteristics and correlating dynamic observations of cell proliferation, cytotoxicity, migration and invasion on cancer cells in highly standardized experimental conditions. METHODOLOGY/PRINCIPAL FINDINGS: Dynamic high-resolution assessments of proliferation, cytotoxicity and migration were performed using xCELLigence technology on the MDA-MB-231 (breast cancer and A549 (lung cancer cell lines. Proliferation kinetics were compared with the Sulforhodamine B (SRB assay in a series of four cell concentrations, yielding fair to good correlations (Spearman's Rho 0.688 to 0.964. Cytotoxic action by paclitaxel (0-100 nM correlated well with SRB (Rho>0.95 with similar IC(50 values. Reference cell migration experiments were performed using Transwell plates and correlated by pixel area calculation of crystal violet-stained membranes (Rho 0.90 and optical density (OD measurement of extracted dye (Rho>0.95. Invasion was observed on MDA-MB-231 cells alone using Matrigel-coated Transwells as standard reference method and correlated by OD reading for two Matrigel densities (Rho>0.95. Variance component analysis revealed increased variances associated with impedance-based detection of migration and invasion, potentially caused by the sensitive nature of this method. CONCLUSIONS/SIGNIFICANCE: The xCELLigence RTCA technology provides an accurate platform for non-invasive detection of cell viability and motility. The strong correlations with conventional methods imply a similar observation of cell behavior and interchangeability with other systems, illustrated by the highly correlating kinetic invasion profiles on

  14. A versatile electrowetting-based digital microfluidic platform for quantitative homogeneous and heterogeneous bio-assays

    International Nuclear Information System (INIS)

    Electrowetting-on-dielectric (EWOD) lab-on-a-chip systems have already proven their potential within a broad range of bio-assays. Nevertheless, research on the analytical performance of those systems is limited, yet crucial for a further breakthrough in the diagnostic field. Therefore, this paper presents the intrinsic possibilities of an EWOD lab-on-a-chip as a versatile platform for homogeneous and heterogeneous bio-assays with high analytical performance. Both droplet dispensing and splitting cause variations in droplet size, thereby directly influencing the assay's performance. The extent to which they influence the performance is assessed by a theoretical sensitivity analysis, which allows the definition of a basic framework for the reduction of droplet size variability. Taking advantage of the optimized droplet manipulations, both homogeneous and heterogeneous bio-assays are implemented in the EWOD lab-on-a-chip to demonstrate the analytical capabilities and versatility of the device. A fully on-chip enzymatic assay is realized with high analytical performance. It demonstrates the promising capabilities of an EWOD lab-on-a-chip in food-related and medical applications, such as nutritional and blood analyses. Further, a magnetic bio-assay for IgE detection using superparamagnetic nanoparticles is presented whereby the nanoparticles are used as solid carriers during the bio-assay. Crucial elements are the precise manipulation of the superparamagnetic nanoparticles with respect to dispensing and separation. Although the principle of using nano-carriers is demonstrated for protein detection, it can be easily extended to a broader range of bio-related applications like DNA sensing. In heterogeneous bio-assays the chip surface is actively involved during the execution of the bio-assay. Through immobilization of specific biological compounds like DNA, proteins and cells a reactive chip surface is realized, which enhances the bio-assay performance. To

  15. A versatile electrowetting-based digital microfluidic platform for quantitative homogeneous and heterogeneous bio-assays

    Science.gov (United States)

    Vergauwe, Nicolas; Witters, Daan; Ceyssens, Frederik; Vermeir, Steven; Verbruggen, Bert; Puers, Robert; Lammertyn, Jeroen

    2011-05-01

    Electrowetting-on-dielectric (EWOD) lab-on-a-chip systems have already proven their potential within a broad range of bio-assays. Nevertheless, research on the analytical performance of those systems is limited, yet crucial for a further breakthrough in the diagnostic field. Therefore, this paper presents the intrinsic possibilities of an EWOD lab-on-a-chip as a versatile platform for homogeneous and heterogeneous bio-assays with high analytical performance. Both droplet dispensing and splitting cause variations in droplet size, thereby directly influencing the assay's performance. The extent to which they influence the performance is assessed by a theoretical sensitivity analysis, which allows the definition of a basic framework for the reduction of droplet size variability. Taking advantage of the optimized droplet manipulations, both homogeneous and heterogeneous bio-assays are implemented in the EWOD lab-on-a-chip to demonstrate the analytical capabilities and versatility of the device. A fully on-chip enzymatic assay is realized with high analytical performance. It demonstrates the promising capabilities of an EWOD lab-on-a-chip in food-related and medical applications, such as nutritional and blood analyses. Further, a magnetic bio-assay for IgE detection using superparamagnetic nanoparticles is presented whereby the nanoparticles are used as solid carriers during the bio-assay. Crucial elements are the precise manipulation of the superparamagnetic nanoparticles with respect to dispensing and separation. Although the principle of using nano-carriers is demonstrated for protein detection, it can be easily extended to a broader range of bio-related applications like DNA sensing. In heterogeneous bio-assays the chip surface is actively involved during the execution of the bio-assay. Through immobilization of specific biological compounds like DNA, proteins and cells a reactive chip surface is realized, which enhances the bio-assay performance. To demonstrate

  16. Miniaturized and High-Throughput Assays for Analysis of T-Cell Immunity Specific for Opportunistic Pathogens and HIV

    OpenAIRE

    Li Pira, G.; Ivaldi, F.; Starc, N.; Landi, F.; Locatelli, F; Rutella, S; Tripodi, G; Manca, F

    2014-01-01

    Monitoring of antigen-specific T-cell responses is valuable in numerous conditions that include infectious diseases, vaccinations, and opportunistic infections associated with acquired or congenital immune defects. A variety of assays that make use of peripheral lymphocytes to test activation markers, T-cell receptor expression, or functional responses are currently available. The last group of assays calls for large numbers of functional lymphocytes. The number of cells increases with the nu...

  17. Sensitivity of the Erythrocyte Micronucleus Assay: Dependence on Number of Cells Scored and Inter-animal Variability

    OpenAIRE

    Kissling, Grace E.; Dertinger, Stephen; Hayashi, Makoto; MacGregor, James T.

    2007-01-01

    Until recently, the in vivo erythrocyte micronucleus assay has been scored using microscopy. Because the frequency of micronucleated cells is typically low, cell counts are subject to substantial binomial counting error. Counting error, along with inter-animal variability, limit the sensitivity of this assay. Recently, flow cytometric methods have been developed for scoring micronucleated erythrocytes and these methods enable many more cells to be evaluated than is possible with microscopic s...

  18. Cy5 labeled single-stranded DNA-polydopamine nanoparticle conjugate-based FRET assay for reactive oxygen species detection

    Directory of Open Access Journals (Sweden)

    Lina Ma

    2015-03-01

    Full Text Available This work reports on a simple and feasible fluorescence resonance energy transfer (FRET assay for detecting reactive oxygen species (ROS both in solution and living cell using polydopamine nanoparticle (PDA NP as energy acceptor and Cy5 labeled single-stranded DNA (Cy5-ssDNA as energy donor. The Cy5-ssDNA and PDA NPs form self-assembled conjugates (Cy5-ssDNA-PDA NP conjugates via π-stacking interactions. In the presence of ROS, the PDA NP adsorbed Cy5-ssDNAs can be effectively cleaved, resulting in the release of Cy5 molecules into solution and recovery of fluorescence emission of Cy5. In order to obtain ROS solution, the glucose oxidase-catalyzed oxidation reaction of glucose with O2 is employed to generate hydrogen peroxide for Fenton-like reaction. The formation of ROS in Fenton-like reaction can be detected as low as glucose oxidase-catalyzed oxidation of 100 pM glucose by the Cy5-ssDNA-PDA NP conjugate-based FRET assay. The recovery ratio of Cy5 fluorescence intensity is increased linearly with logarithm of glucose concentration from 100 pM to 1 μM, demonstrating that the FRET assay has wide dynamic range. In particular, intracellular ROS has been successfully detected in chemical stimulated HepG-2 cells by the Cy5-ssDNA-PDA NP conjugate-based FRET assay with a fluorescence microscopy, indicating that this approach has great potential to monitor ROS in living cells.

  19. Limit-dilution assay and clonal expansion of all T cells capable of proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Chen, W.F.; Wilson, A.; Scollay, R.; Shortman, K. (Walter and Eliza Hall Inst. of Medical Research, Parkville (Australia))

    1982-08-13

    A limit-dilution microculture system is presented in which almost all mature T cells, cultured at a level of about 1 cell/well, grow and expand to clones averaging 60,000 cells over an 8-9 day period. Cloning efficiency is 70-100%, so the set of expanded clones is representative of the starting T-cell population. T cells of all Lyt phenotypes form clones of progeny cells. The system involves culture in flat-bottom microtitre trays, in the presence of concanavalin A as the initiating stimulus, together with appropriately irradiated spleen filler cells and a supplementary source of soluble T cell growth factors. The resultant clones may be screened for cytolytic function, as described in the accompanying paper. The system may be used to assay the level of T cells capable of expansion or precursor function (PTL-p) by using (/sup 3/H)TdR uptake as a readout for the presence or absence of proliferating clones. Analysis of the frequency of positive cultures shows a good fit to the expected Poisson distribution, with no evidence of complicating suppressor or helper effects.

  20. Scalable DNA-Based Magnetic Nanoparticle Agglutination Assay for Bacterial Detection in Patient Samples

    DEFF Research Database (Denmark)

    Mezger, Anja; Fock, Jeppe; Antunes, Paula Soares Martins;

    2015-01-01

    We demonstrate a nanoparticle-based assay for the detection of bacteria causing urinary tract infections in patient samples with a total assay time of 4 h. This time is significantly shorter than the current gold standard, plate culture, which can take several days depending on the pathogen....... The assay is based on padlock probe recognition followed by two cycles of rolling circle amplification (RCA) to form DNA coils corresponding to the target bacterial DNA. The readout of the RCA products is based on optomagnetic measurements of the specific agglutination of DNA-bound magnetic nanoparticles...... (MNPs) using low-cost optoelectronic components from Blu-ray drives. We implement a detection approach, which relies on the monomerization of the RCA products, the use of the monomers to link and agglutinate two populations of MNPs functionalized with universal nontarget specific detection probes...

  1. Protein-carbohydrate complex reveals circulating metastatic cells in a microfluidic assay

    KAUST Repository

    Simone, Giuseppina

    2013-02-11

    Advances in carbohydrate sequencing technologies reveal the tremendous complexity of the glycome and the role that glycomics might have to bring insight into the biological functions. Carbohydrate-protein interactions, in particular, are known to be crucial to most mammalian physiological processes as mediators of cell adhesion and metastasis, signal transducers, and organizers of protein interactions. An assay is developed here to mimic the multivalency of biological complexes that selectively and sensitively detect carbohydrate-protein interactions. The binding of β-galactosides and galectin-3 - a protein that is correlated to the progress of tumor and metastasis - is examined. The efficiency of the assay is related to the expression of the receptor while anchoring to the interaction\\'s strength. Comparative binding experiments reveal molecular binding preferences. This study establishes that the assay is robust to isolate metastatic cells from colon affected patients and paves the way to personalized medicine. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. A flow cytometry-optimized assay using an SOS-green fluorescent protein (SOS-GFP) whole-cell biosensor for the detection of genotoxins in complex environments

    DEFF Research Database (Denmark)

    Norman, Anders; Hansen, Lars H.; Sørensen, Søren Johannes

    2006-01-01

    /mL, and proved far more sensitive than a previously published assay using the same biosensor strain. By applying the SOS-green fluorescent protein (GFP) whole-cell biosensor directly to soil microcosms we were also able to evaluate both the applicability and sensitivity of a biosensor based on SOS...

  3. Development of a quantal assay in primary shrimp cell culture for yellow head baculovirus (YBV) of penaeid shrimp.

    Science.gov (United States)

    Lu, Y; Tapay, L M; Loh, P C; Brock, J A; Gose, R

    1995-03-01

    A 50% tissue culture infectious dose assay (TCID50) using primary culture of shrimp lymphoid organ (Oka) cells was developed for the quantitative titration of yellow-head baculovirus (YBV), a newly isolated virus of penaeid shrimp. The assay protocol includes the use of Primaria-grade 96-well tissue culture plates to grow the primary lymphoid organ cells of penaeid shrimp. A 15% gill suspension from YBV-infected shrimp was determined to have an infectious virus titer of 5 x 10(5.75) TCID50/ml. This report represents the first convenient assay protocol using cell culture derived from penaeid shrimp to titer a shrimp virus.

  4. Interactive effects of metals as measured in cytotoxicity assays with established fish cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Segner, H.; Schuurmann, G. [Centre for Environmental Research, Leipzig (Germany). Dept. of Chemical Ecotoxicology

    1995-12-31

    The environmental toxicity of chemicals is often judged on the basis of toxicity tests with single compounds. One major drawback of this approach is the fact, that mixture effects occurring in aquatic ecosystems with a multitude of different chemicals are not accounted for. The present work explores the use of cytotoxicity assays with established fish cell lines as a rapid and economic approach to derive basic data on joint toxicity effects of heavy metals. For the assessment of mixture toxicity, concentration addition is taken as the reference model of no interaction, and both isobolographic analysis and calculation of mixture toxicity indices are used to analyze the effect profile of various equitoxic compound mixtures. Cytotoxic endpoints used include neutral red uptake inhibition assay as a measure of cell viability, proliferation measurements to estimate toxic effects on cell growth, and analysis of glutathion contents to estimate metabolic stress effects. The single toxicity of the metals silver, mercury, cadmium, copper, zinc, lead and nickel towards the cell lines RI from rainbow trout liver and RTG-2 from rainbow trout gonads was found to depend on the chemical softness parameter of the cations. The joint effect profile will be discussed in terms of the single effects and softness domain of the heavy metals.

  5. Inverse dose rate effect in tumour cells measured by the comet assay

    International Nuclear Information System (INIS)

    Reduction of the dose rate of low LET radiation from high (Gy/min) to low (Gy/h) usually leads to a reduced effect as measured by the survival methods. If the dose rate is reduced, cells are able to repair sublethal damage even during irradiation. During the last few years a comet assay has been widely used to measure DNA damage induction and repair in single cells. In our study we used the alkaline version of the comet assay for comparison of high (0.833 Gy/min) and low dose rate (0.0707 Gy/min) effects on DNA damage and repair in R1 rat rhabdomyosarcoma and Me45 human malignant melanoma cells. Cells gathered from exponential culture by trypsynization were suspended in a growth medium and irradiated at room temperature, with 5 Gy of photons X generated by linear accelerator at both dose rates. Comets were analysed automatically using self-made software for measurement of percentage DNA in the tail, and tail moment and inertia. Our results show that tail inertia is the best parameter expressing DNA damage and repair. Although the level of DNA damage induced by low dose rate was comparable with that induced by a high dose rate, the damage induced by the low dose rate are repair more slowly than after high dose rate irradiation. This inverse dose rate effect suggest that nature of damage can differ in both groups. (author)

  6. A buccal cell model comet assay: Development and evaluation for human biomonitoring and nutritional studies

    International Nuclear Information System (INIS)

    The comet assay is a widely used biomonitoring tool for DNA damage. The most commonly used cells in human studies are lymphocytes. There is an urgent need to find an alternative target human cell that can be collected from normal subjects with minimal invasion. There are some reports of buccal cells, collected easily from the inside of the mouth, being used in studies of DNA damage and repair, and these were of interest. However, our preliminary studies following the published protocol showed that buccal cells sustained massive damage and disintegrated at the high pH [O. Ostling, K.J. Johanson. Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Biochem. Biophys. Res. Commun. 123 (1984) 291-298] used, but that at lower pH were extremely resistant to lysis, an essential step in the comet assay. Therefore, the aims of this study were to develop a protocol than enabled buccal cell lysis and DNA damage testing in the comet assay, and to use the model to evaluate the potential use of the buccal cell model in human biomonitoring and nutritional study. Specifically, we aimed to investigate intra- and inter-individual differences in buccal cell DNA damage (as strand breaks), the effect of in vitro exposure to both a standard oxidant challenge and antioxidant treatment, as well as in situ exposure to an antioxidant-rich beverage and supplementation-related effects using a carotenoid-rich food. Successful lysis was achieved using 0.25% trypsin for 30 min followed by proteinase K (1 mg/ml) treatment for 60 min. When this procedure was performed on cells pre-embedded in agarose on a microscope slide, followed by electrophoresis (in 0.01 M NaOH, 1 mM EDTA, pH 9.1, 18 min at 12 V), a satisfactory comet image was obtained, though inter-individual variation was quite wide. Pre-lysis exposure of cells to a standard oxidant challenge (induced by H2O2) increased DNA strand breaks in a dose related manner, and incubation of cells in Trolox

  7. Evaluation of an in vitro cell culture assay for the potency assessment of recombinant human erythropoietin.

    Science.gov (United States)

    Machado, Francine T; Maldaner, Fernanda P S; Perobelli, Rafaela F; Xavier, Bruna; da Silva, Francielle S; de Freitas, Guilherme W; Bartolini, Paolo; Ribela, M Tereza C P; Dalmora, Sérgio L

    2016-05-01

    Recombinant human erythropoietin is a sialoglycoprotein that stimulates erythropoiesis. To assess potency of human erythropoietin produced by recombinant technology, we investigated an in vitro TF-1 cell proliferation assay, which was applied in conjunction with a reversed-phase liquid chromatography method for the determination of the content of sialic acids. The results obtained, which were higher than 126.8ng/μg, were compared with those obtained with the in vivo normocythaemic mouse bioassay. The in vitro assay resulted in a non-significant lower mean difference of the estimated potencies (0.61% ± 0.026, p > 0.05). The use of this combination of methods represents an advance toward the establishment of alternative in vitro approaches, in the context of the Three Rs, for the potency assessment of biotechnology-derived medicines. PMID:27256453

  8. Evaluation of a Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) assay (SOT)

    Science.gov (United States)

    The Embryonic Stem Cell Test (EST) has been used to evaluate the effects of xenobiotics using three endpoints, stem cell differentiation, stem cell viability and 3T3-cell viability. Our research goal is to establish amodel system that would evaluate chemical effects using a singl...

  9. Rapid alternative to the clonogenic assay for measuring antibody and complement-mediated killing of tumor cells

    International Nuclear Information System (INIS)

    A study of the methods used to quantitate killing of tumor cells by antibody and complement has highlighted a number of problems. Using leukemia as a model, the authors have found that the release of 51Cr from labeled tumor cells treated with antibody and complement can be an equivocal measure of cell viability. Combined with its restricted sensitivity (less than a 2 log range of cell killing) this makes this widely used assay of questionable value for detecting small numbers of viable cells, or for identifying subpopulations of complement-resistant cells. As an alternative a [125I]iododeoxyuridine uptake assay has been developed, that combines the simplicity and rapidity of the 51Cr release technique with the sensitivity of a clonogenic assay. This method eliminates the problem of spontaneous isotope release, inherent in prelabeling assays, and variability from experiment to experiment can be avoided by including a viable cell standard curve within each assay. The sensitivity of the 125IUdR uptake method, which can be completed within a day, is similar to that of a 10 day methylcellulose cloning assay and was capable of detecting the presence of a minor subpopulation of complement-resistant tumor cells

  10. Simultaneous Detection of Antigen-Specific IgG- and IgM-Secreting Cells with a B Cell Fluorospot Assay

    OpenAIRE

    M Anthony Moody; Mattia Bonsignori

    2012-01-01

    The traditional enzyme-linked immunospot (ELISpot) assay is the gold standard for the enumeration of antigen-specific B cells. Since B cell availability from biological samples is often limited, either because of sample size/volume or the need of performing multiple analyses on the same sample, the implementation of ELISpot assay formats that allow the simultaneous detection of multiple antibody types is desirable. While dual-color ELISpot assays have been described, technical complexities ha...

  11. Mammalian Cell-Based Sensor System

    Science.gov (United States)

    Banerjee, Pratik; Franz, Briana; Bhunia, Arun K.

    Use of living cells or cellular components in biosensors is receiving increased attention and opens a whole new area of functional diagnostics. The term "mammalian cell-based biosensor" is designated to biosensors utilizing mammalian cells as the biorecognition element. Cell-based assays, such as high-throughput screening (HTS) or cytotoxicity testing, have already emerged as dependable and promising approaches to measure the functionality or toxicity of a compound (in case of HTS); or to probe the presence of pathogenic or toxigenic entities in clinical, environmental, or food samples. External stimuli or changes in cellular microenvironment sometimes perturb the "normal" physiological activities of mammalian cells, thus allowing CBBs to screen, monitor, and measure the analyte-induced changes. The advantage of CBBs is that they can report the presence or absence of active components, such as live pathogens or active toxins. In some cases, mammalian cells or plasma membranes are used as electrical capacitors and cell-cell and cell-substrate contact is measured via conductivity or electrical impedance. In addition, cytopathogenicity or cytotoxicity induced by pathogens or toxins resulting in apoptosis or necrosis could be measured via optical devices using fluorescence or luminescence. This chapter focuses mainly on the type and applications of different mammalian cell-based sensor systems.

  12. An improved enzyme-linked immunosorbent assay for whole-cell determination of methanogens in samples from anaerobic reactors

    DEFF Research Database (Denmark)

    Sørensen, A.H.; Ahring, B.K.

    1997-01-01

    An enzyme-linked immunosorbent assay was developed for the detection of whole cells of methanogens in samples from anaerobic continuously stirred tank digesters treating slurries of solid waste. The assay was found to allow for quantitative analysis of the most important groups of methanogens in ...

  13. Comparison of in vitro cell culture and a mouse assay for measuring infectivity of Cryptosporidium parvum.

    Science.gov (United States)

    Rochelle, Paul A; Marshall, Marilyn M; Mead, Jan R; Johnson, Anne M; Korich, Dick G; Rosen, Jeffrey S; De Leon, Ricardo

    2002-08-01

    In vitro cell cultures were compared to neonatal mice for measuring the infectivity of five genotype 2 isolates of Cryptosporidium parvum. Oocyst doses were enumerated by flow cytometry and delivered to animals and cell monolayers by using standardized procedures. Each dose of oocysts was inoculated into up to nine replicates of 9 to 12 mice or 6 to 10 cell culture wells. Infections were detected by hematoxylin and eosin staining in CD-1 mice, by reverse transcriptase PCR in HCT-8 and Caco-2 cells, and by immunofluorescence microscopy in Madin-Darby canine kidney (MDCK) cells. Infectivity was expressed as a logistic transformation of the proportion of animals or cell culture wells that developed infection at each dose. In most instances, the slopes of the dose-response curves were not significantly different when we compared the infectivity models for each isolate. The 50% infective doses for the different isolates varied depending on the method of calculation but were in the range from 16 to 347 oocysts for CD-1 mice and in the ranges from 27 to 106, 31 to 629, and 13 to 18 oocysts for HCT-8, Caco-2, and MDCK cells, respectively. The average standard deviations for the percentages of infectivity for all replicates of all isolates were 13.9, 11.5, 13.2, and 10.7% for CD-1 mice, HCT-8 cells, Caco-2 cells, and MDCK cells, respectively, demonstrating that the levels of variability were similar in all assays. There was a good correlation between the average infectivity for HCT-8 cells and the results for CD-1 mice across all isolates for untreated oocysts (r = 0.85, n = 25) and for oocysts exposed to ozone and UV light (r = 0.89, n = 29). This study demonstrated that in vitro cell culture was equivalent to the "gold standard," mouse infectivity, for measuring the infectivity of C. parvum and should therefore be considered a practical and accurate alternative for assessing oocyst infectivity and inactivation. However, the high levels of variability displayed by all

  14. Cultivar origin and admixture detection in Turkish olive oils by SNP-based CAPS assays.

    Science.gov (United States)

    Uncu, Ali Tevfik; Frary, Anne; Doganlar, Sami

    2015-03-01

    The aim of this study was to establish a DNA-based identification key to ascertain the cultivar origin of Turkish monovarietal olive oils. To reach this aim, we sequenced short fragments from five olive genes for SNP (single nucleotide polymorphism) identification and developed CAPS (cleaved amplified polymorphic DNA) assays for SNPs that alter restriction enzyme recognition motifs. When applied on the oils of 17 olive cultivars, a maximum of five CAPS assays were necessary to discriminate the varietal origin of the samples. We also tested the efficiency and limit of our approach for detecting olive oil admixtures. As a result of the analysis, we were able to detect admixing down to a limit of 20%. The SNP-based CAPS assays developed in this work can be used for testing and verification of the authenticity of Turkish monovarietal olive oils, for olive tree certification, and in germplasm characterization and preservation studies.

  15. Use of PCR-based assays for the detection of the adventitious agent porcine circovirus type 1 (PCV1) in vaccines, and for confirming the identity of cell substrates and viruses used in vaccine production.

    Science.gov (United States)

    Kumar, Deepak; Beach, Nathan M; Meng, Xiang-Jin; Hegde, Nagendra R

    2012-01-01

    Safety and quality are important issues for vaccines. Whereas reversion to virulence poses a safety risk with live attenuated vaccines, the potential for the presence of adventitious agents is also an issue of vaccine quality. The recent detection or porcine circovirus type 1 (PCV1) in human vaccines has further highlighted the importance of quality control in vaccine production. The purpose of this study was to use a novel conventional PCR to detect PCV1, and subsequently screen materials used in the manufacture of vaccines at Bharat Biotech International Limited, India. The genome or gene fragments of PCV1 were not detected in any of the vaccines and materials tested, including the live attenuated rotavirus vaccine candidate ROTAVAC(®). Further, the identity of the cells and the viruses used as starting materials in the manufacture of these vaccines was confirmed by species-specific PCR or virus-specific RT-PCR, and no cross-contamination was detected in any case. The methods can be applied for regular in-house quality control screening of raw materials and seeds/banks, as well as formulated vaccines.

  16. Comparison of the colony formation and crystal violet cell proliferation assays to determine cellular radiosensitivity in a repair-deficient MCF10A cell line

    Energy Technology Data Exchange (ETDEWEB)

    Vandersickel, Veerle [Department of Basic Medical Sciences, Ghent University, Campus Heymans, De Pintelaan 185 (6B3), 9000 Gent (Belgium); Slabbert, Jacobus [NRF iThemba LABS (Laboratory for Accelerated Based Sciences), PO box 722, 7129 Somerset West (South Africa); Thierens, Hubert [Department of Basic Medical Sciences, Ghent University, Campus Heymans, De Pintelaan 185 (6B3), 9000 Gent (Belgium); Vral, Anne, E-mail: anne.Vral@UGent.b [Department of Basic Medical Sciences, Ghent University, Campus Heymans, De Pintelaan 185 (6B3), 9000 Gent (Belgium)

    2011-01-15

    Colony formation as measured by the in vitro clonogenic assay is a very important endpoint to determine cellular radiosensitivity and tumor response to radiotherapy. In the framework of assessing in vitro cellular radiosensitivity, proliferation assays could represent an attractive alternative to the clonogenic assay for cell lines that do not form proper colonies. In the present study, we compared cellular radiosensitivity measurements obtained by the crystal violet (CV) cell proliferation assay and the standard colony formation assay in repair-deficient and-proficient human MCF10A cell lines. Compared to the clonogenic assay, the CV cell proliferation assay yielded higher surviving fractions for the same radiation dose. This is reflected in larger mean inactivation dose values - a parameter that reflects the area under the survival curve. However, as the dose modifying factors obtained by both assays are comparable, the CV cell proliferation assay can be used to compare the in vitro cellular radiosensitivity of cell lines that lack the ability to form well-defined colonies.

  17. The Fibrin slide assay for detecting urokinase activity in human fetal kidney cells

    Science.gov (United States)

    Sedor, K.

    1985-01-01

    The Fibrin Slide Technique of Hau C. Kwaan and Tage Astrup is discussed. This relatively simple assay involves two steps: the formation of an artificial clot and then the addition of an enzyme (UKOKINASE) to dissolve the clot. The actual dissolving away of the clot is detected by the appearance of holes (lysis zones) in the stained clot. The procedure of Kwaan and Astrup is repeated, along with modifications and suggestions for improvements based on experience with the technique.

  18. An image-based high-content screening assay for compounds targeting intracellular Leishmania donovani amastigotes in human macrophages.

    Science.gov (United States)

    Siqueira-Neto, Jair L; Moon, Seunghyun; Jang, Jiyeon; Yang, Gyongseon; Lee, Changbok; Moon, Hong Kee; Chatelain, Eric; Genovesio, Auguste; Cechetto, Jonathan; Freitas-Junior, Lucio H

    2012-01-01

    Leishmaniasis is a tropical disease threatening 350 million people from endemic regions. The available drugs for treatment are inadequate, with limitations such as serious side effects, parasite resistance or high cost. Driven by this need for new drugs, we developed a high-content, high-throughput image-based screening assay targeting the intracellular amastigote stage of different species of Leishmania in infected human macrophages. The in vitro infection protocol was adapted to a 384-well-plate format, enabling acquisition of a large amount of readouts by automated confocal microscopy. The reading method was based on DNA staining and required the development of a customized algorithm to analyze the images, which enabled the use of non-modified parasites. The automated analysis generated parameters used to quantify compound activity, including infection ratio as well as the number of intracellular amastigote parasites and yielded cytotoxicity information based on the number of host cells. Comparison of this assay with one that used the promastigote form to screen 26,500 compounds showed that 50% of the hits selected against the intracellular amastigote were not selected in the promastigote screening. These data corroborate the idea that the intracellular amastigote form of the parasite is the most appropriate to be used in primary screening assay for Leishmania. PMID:22720099

  19. An image-based high-content screening assay for compounds targeting intracellular Leishmania donovani amastigotes in human macrophages.

    Directory of Open Access Journals (Sweden)

    Jair L Siqueira-Neto

    Full Text Available Leishmaniasis is a tropical disease threatening 350 million people from endemic regions. The available drugs for treatment are inadequate, with limitations such as serious side effects, parasite resistance or high cost. Driven by this need for new drugs, we developed a high-content, high-throughput image-based screening assay targeting the intracellular amastigote stage of different species of Leishmania in infected human macrophages. The in vitro infection protocol was adapted to a 384-well-plate format, enabling acquisition of a large amount of readouts by automated confocal microscopy. The reading method was based on DNA staining and required the development of a customized algorithm to analyze the images, which enabled the use of non-modified parasites. The automated analysis generated parameters used to quantify compound activity, including infection ratio as well as the number of intracellular amastigote parasites and yielded cytotoxicity information based on the number of host cells. Comparison of this assay with one that used the promastigote form to screen 26,500 compounds showed that 50% of the hits selected against the intracellular amastigote were not selected in the promastigote screening. These data corroborate the idea that the intracellular amastigote form of the parasite is the most appropriate to be used in primary screening assay for Leishmania.

  20. Phenotypic characterization of bovine memory cells responding to mycobacteria in IFNγ enzyme linked immunospot assays.

    Science.gov (United States)

    Blunt, Laura; Hogarth, Philip J; Kaveh, Daryan A; Webb, Paul; Villarreal-Ramos, Bernardo; Vordermeier, Hans Martin

    2015-12-16

    Bovine tuberculosis (bTB) remains a globally significant veterinary health problem. Defining correlates of protection can accelerate the development of novel vaccines against TB. As the cultured IFNγ ELISPOT (cELISPOT) assay has been shown to predict protection and duration of immunity in vaccinated cattle, we sought to characterize the phenotype of the responding T-cells. Using expression of CD45RO and CD62L we purified by cytometric cell sorting four distinct CD4(+) populations: CD45RO(+)CD62L(hi), CD45RO(+)CD62L(lo), CD45RO(-)CD62L(hi) and CD45RO(-)CD62L(lo) (although due to low and inconsistent cell recovery, this population was not considered further in this study), in BCG vaccinated and Mycobacterium bovis infected cattle. These populations were then tested in the cELISPOT assay. The main populations contributing to production of IFNγ in the cELISPOT were of the CD45RO(+)CD62L(hi) and CD45RO(+)CD62L(lo) phenotypes. These cell populations have been described in other species as central and effector memory cells, respectively. Following in vitro culture and flow cytometry we observed plasticity within the bovine CD4(+) T-cell phenotype. Populations switched phenotype, increasing or decreasing expression of CD45RO and CD62L within 24h of in vitro stimulation. After 14 days all IFNγ producing CD4(+) T cells expressed CD45RO regardless of the original phenotype of the sorted population. No differences were detected in behavior of cells derived from BCG-vaccinated animals compared to cells derived from naturally infected animals. In conclusion, although multiple populations of CD4(+) T memory cells from both BCG vaccinated and M. bovis infected animals contributed to cELISPOT responses, the dominant contributing population consists of central-memory-like T cells (CD45RO(+)CD62L(hi)).

  1. A facile, branched DNA assay to quantitatively measure glucocorticoid receptor auto-regulation in T-cell acute lymphoblastic leukemia

    Institute of Scientific and Technical Information of China (English)

    Jason R. Schwartz; Purvaba J. Sarvaiya; Lily E. Leiva; Maria C. Velez; Tammuella C. Singleton; Lolie C. Yu; Wayne V. Vedeckis

    2012-01-01

    Glucocorticoid (GC) steroid hormones are used to treat acute lymphoblastic leukemia (ALL) because of their pro-apoptotic effects in hematopoietic cells.However,not all leukemia cells are sensitive to GC,and no assay to stratify patients is available.In the GC-sensitive T-cell ALL cell line CEM-C7,auto-up-regulation of RNA transcripts for the glucocorticoid receptor (GR) correlates with increased apoptotic response.This study aimed to determine if a facile assay of GR transcript levels might be promising for stratifying ALL patients into hormone-sensitive and hormone-resistant populations.The GR transcript profiles of various lymphoid cell lines and 4 bone marrow samples from patients with T-cell ALL were analyzed using both an optimized branched DNA (bDNA) assay and a real-time quantitative reverse transcription-polymerase chain reaction assay.There were significant correlations between both assay platforms when measuring total GR (exon 5/6) transcripts in various cell lines and patient samples,but not for a probe set that detects a specific,low abundance GR transcript (exon 1A3).Our results suggest that the bDNA platform is reproducible and precise when measuring total GR transcripts and,with further development,may ultimately offer a simple clinical assay to aid in the prediction of GC-sensitivity in ALL patients.

  2. 14C-glucose binding assay of the glucose transporter binding sites in muscular cell membrane

    International Nuclear Information System (INIS)

    A method of determining the binding sites of glucose transporter in rat muscular cell membrane was introduced. The crude products of cell membrane form the skeletal muscle of control and insulin treated rats were prepared, and then fractionated in sucrose gradient. Both plasma membrane and microsome membrane were incubated with D-[U-14C] glucose respectively for the measurement of radioactivity and Scatchard plot analysis. It was found that the binding sites of glucose transporter in plasma membrane and intracellular membrane were 5.6 nmol 14C-glucose/mg protein and 8.7 nmol 14C-glucose-mg protein respectively at basic state. Insulin treatment in experimental groups caused approximately 146% increase in plasma membrane fraction and 88% decrease in intracellular membrane fraction. Moreover, the kinetic data of Scatchard plot curve were similar to those of the [3H]-cytochalasin B binding assay. D-[U-14C] glucose binding assay of glucose transporter binding sites in muscular cell membrane is simple, easy and practicable. The D-[U-14C] glucose is commercially available

  3. A MULTIPLEXED ASSAY FOR DETERMINATION OF NEUROTOXICANT EFFECTS ON SPONTANEOUS NETWORK ACTIVITY AND CELL VIABILITY FROM MICROELECTRODE ARRAYS

    Science.gov (United States)

    AbstractTITLE: A MULTIPLEXED ASSAY FOR DETERMINATION OF NEUROTOXICANT EFFECTS ON SPONTANEOUS NETWORK ACTIVITY AND CELL VIABILITY FROM MICROELECTRODE ARRAYSABSTRACT BODY: Microelectrode array (MEA) recordings are increasingly being used as an in vitro method to detect and characte...

  4. High-Throughput Assay Development for Cystine-Glutamate Antiporter (xc-) Highlights Faster Cystine Uptake than Glutamate Release in Glioma Cells.

    Science.gov (United States)

    Thomas, Ajit G; Sattler, Rita; Tendyke, Karen; Loiacono, Kara A; Hansen, Hans; Sahni, Vishal; Hashizume, Yutaka; Rojas, Camilo; Slusher, Barbara S

    2015-01-01

    The cystine-glutamate antiporter (system xc-) is a Na+-independent amino acid transporter that exchanges extracellular cystine for intracellular glutamate. It is thought to play a critical role in cellular redox processes through regulation of intracellular glutathione synthesis via cystine uptake. In gliomas, system xc- expression is universally up-regulated while that of glutamate transporters down-regulated, leading to a progressive accumulation of extracellular glutamate and excitotoxic cell death of the surrounding non-tumorous tissue. Additionally, up-regulation of system xc- in activated microglia has been implicated in the pathogenesis of several neurodegenerative disorders mediated by excess glutamate. Consequently, system xc- is a new drug target for brain cancer and neuroinflammatory diseases associated with excess extracellular glutamate. Unfortunately no potent and selective small molecule system xc- inhibitors exist and to our knowledge, no high throughput screening (HTS) assay has been developed to identify new scaffolds for inhibitor design. To develop such an assay, various neuronal and non-neuronal human cells were evaluated as sources of system xc-. Human glioma cells were chosen based on their high system xc- activity. Using these cells, [14C]-cystine uptake and cystine-induced glutamate release assays were characterized and optimized with respect to cystine and protein concentrations and time of incubation. A pilot screen of the LOPAC/NINDS libraries using glutamate release demonstrated that the logistics of the assay were in place but unfortunately, did not yield meaningful pharmacophores. A larger, HTS campaign using the 384-well cystine-induced glutamate release as primary assay and the 96-well 14C-cystine uptake as confirmatory assay is currently underway. Unexpectedly, we observed that the rate of cystine uptake was significantly faster than the rate of glutamate release in human glioma cells. This was in contrast to the same rates of

  5. MICROSPHERE-BASED FLOW CYTOMETRY PROTEASE ASSAYS FOR USE IN PROTEASE ACTIVITY DETECTION AND HIGH-THROUGHPUT SCREENING

    OpenAIRE

    Saunders, Matthew J.; Edwards, Bruce S.; Zhu, Jingshu; Sklar, Larry A.; Graves, Steven W.

    2010-01-01

    This protocol describes microsphere-based protease assays for use in flow cytometry and high-throughput screening. This platform measures a loss of fluorescence from the surface of a microsphere due to the cleavage of an attached fluorescent protease substrate by a suitable protease enzyme. The assay format can be adapted to any site or protein specific protease of interest and results can be measured in both real time and as end point fluorescence assays on a flow cytometer. End point assays...

  6. Innovative mode of action based in vitro assays for detection of marine neurotoxins

    NARCIS (Netherlands)

    Nicolas, J.A.Y.

    2015-01-01

    Innovative mode of action based in vitro assays for detection of marine neurotoxins J. Nicolas, P.J.M. Hendriksen, T.F.H. Bovee, I.M.C.M. Rietjens Marine biotoxins are naturally occurring compounds produced by particular phytoplankton species. These toxins often accumulate in seafood and thereby rep

  7. Lipopolysaccharide Specific Immunochromatography Based Lateral Flow Assay for Serogroup Specific Diagnosis of Leptospirosis in India.

    Directory of Open Access Journals (Sweden)

    Shanmugam Vanithamani

    Full Text Available Leptospirosis is a re-emerging infectious disease that is under-recognized due to low-sensitivity and cumbersome serological tests. MAT is the gold standard test and it is the only serogroup specific test used till date. Rapid reliable alternative serogroup specific tests are needed for surveillance studies to identify locally circulating serogroups in the study area.In the present investigation the serological specificity of leptospiral lipopolysaccharides (LPS was evaluated by enzyme linked immunosorbent assay (ELISA, dot blot assay and rapid immunochromatography based lateral flow assay (ICG-LFA. Sera samples from 120 MAT positive cases, 174 cases with febrile illness other than leptospirosis, and 121 seronegative healthy controls were evaluated for the diagnostic sensitivity and specificity of the developed assays. LPS was extracted from five locally predominant circulating serogroups including: Australis (27.5%, Autumnalis (11.7%, Ballum (25.8%, Grippotyphosa (12.5%, Pomona (10% and were used as antigens in the diagnostics to detect IgM antibodies in patients' sera. The sensitivity observed by IgM ELISA and dot blot assay using various leptospiral LPS was >90% for homologous sera. Except for Ballum LPS, no other LPS showed cross-reactivity to heterologous sera. An attempt was made to develop LPS based ICG-LFA for rapid and sensitive serogroup specific diagnostics of leptospirosis. The developed ICG-LFA showed sensitivity in the range between 93 and 100% for homologous sera. The Wilcoxon analysis showed LPS based ICG-LFA did not differ significantly from the gold standard MAT (P>0.05.The application of single array of LPS for serogroup specific diagnosis is first of its kind. The developed assay could potentially be evaluated and employed for as MAT alternative.

  8. Serological Assays Based on Recombinant Viral Proteins for the Diagnosis of Arenavirus Hemorrhagic Fevers

    Directory of Open Access Journals (Sweden)

    Masayuki Saijo

    2012-10-01

    Full Text Available The family Arenaviridae, genus Arenavirus, consists of two phylogenetically independent groups: Old World (OW and New World (NW complexes. The Lassa and Lujo viruses in the OW complex and the Guanarito, Junin, Machupo, Sabia, and Chapare viruses in the NW complex cause viral hemorrhagic fever (VHF in humans, leading to serious public health concerns. These viruses are also considered potential bioterrorism agents. Therefore, it is of great importance to detect these pathogens rapidly and specifically in order to minimize the risk and scale of arenavirus outbreaks. However, these arenaviruses are classified as BSL-4 pathogens, thus making it difficult to develop diagnostic techniques for these virus infections in institutes without BSL-4 facilities. To overcome these difficulties, antibody detection systems in the form of an enzyme-linked immunosorbent assay (ELISA and an indirect immunofluorescence assay were developed using recombinant nucleoproteins (rNPs derived from these viruses. Furthermore, several antigen-detection assays were developed. For example, novel monoclonal antibodies (mAbs to the rNPs of Lassa and Junin viruses were generated. Sandwich antigen-capture (Ag-capture ELISAs using these mAbs as capture antibodies were developed and confirmed to be sensitive and specific for detecting the respective arenavirus NPs. These rNP-based assays were proposed to be useful not only for an etiological diagnosis of VHFs, but also for seroepidemiological studies on VHFs. We recently developed arenavirus neutralization assays using vesicular stomatitis virus (VSV-based pseudotypes bearing arenavirus recombinant glycoproteins. The goal of this article is to review the recent advances in developing laboratory diagnostic assays based on recombinant viral proteins for the diagnosis of VHFs and epidemiological studies on the VHFs caused by arenaviruses.

  9. Miniaturization of cytotoxicity tests for concentration range-finding studies prior to conducting the pH 6.7 Syrian hamster embryo cell-transformation assay.

    Science.gov (United States)

    Plöttner, Sabine; Käfferlein, Heiko U; Brüning, Thomas

    2013-08-15

    The Syrian hamster embryo (SHE) cell-transformation assay (SHE assay) is a promising alternative method to animal testing for the identification of potential carcinogens in vitro. Prior to conducting the SHE assay the appropriate concentration range for each test chemical must be established, with a maximum concentration causing approximately 50% cytotoxicity. Concentration range-finding is done in separate experiments, which are similar to the final SHE assay but with less replicates and more concentrations. Here we present an alternative for the cytotoxicity testing by miniaturization of the test procedure by use of 24-well plates and surpluses from feeder-cell preparations as target cells. In addition, we integrated the photometry-based neutral red (NR) assay. For validation of the assay, incubations with dimethyl sulf-oxide, p-phenylenediamine-2HCl, aniline, o-toluidine-HCl, 2,4-diaminotoluene, and 2-naphthylamine were carried out in the miniaturized approach and compared with the standard procedure in terms of calculating the relative plating efficiencies (RPEs). To directly compare both methods, concentrations that produced 50% cytotoxicity (IC50) were calculated. Excellent associations were observed between the number of colonies and NR uptake. For all test substances a concentration-dependent, concomitant decrease of NR uptake in the miniaturized approach and RPEs in the standard test was observed after a 7-day incubation. The results from both test setups showed a comparable order of magnitude and the IC50 values differed by a factor <2 (1.4-1.9), depending on the substance in question. Overall, the miniaturized approach should be considered an improved alternative for cytotoxicity testing in the SHE assay, as it saves valuable SHE cells and speeds-up the time, to obtain test results more rapidly. PMID:23830925

  10. HLA-B27 detection – comparison of genetic sequence-based method and flow cytometry assay

    OpenAIRE

    Skalska, Urszula; Kozakiewicz, Anna; Maśliński, Włodzimierz; Jurkowska, Monika

    2015-01-01

    Objectives The presence of human leukocyte antigen B27 (HLA-B27) is strongly associated with ankylosing spondylitis. HLA-B27 testing is routinely applied in the diagnosis of this disease. The aim of the present study was to compare two methods of HLA-B27 detection – a genetic sequence-based method and a flow cytometry assay. Material and methods Peripheral blood was obtained from 300 individuals with suspected spondyloarthropathy. Expression of HLA-B27 on the T cell surface was analysed by fl...

  11. Toxicity evaluation of ZnO nanostructures on L929 fibroblast cell line using MTS assay

    Energy Technology Data Exchange (ETDEWEB)

    Bakhori, Siti Khadijah Mohd; Mahmud, Shahrom; Ann, Ling Chuo [Nano-optoelectronics Research and Technology Laboratory (NOR.), School of Physics, Universiti Sains Malaysia, 11800, USM, Pulau Pinang (Malaysia); Mohamed, Azman Seeni; Saifuddin, Siti Nazmin [Integrative Medicine Cluster, Advanced Medical and Dental Institute, Universiti Sains Malaysia, Bandar Putra Bertam, 13200 Kepala Batas, Pulau Pinang (Malaysia); Masudi, Sam’an Malik; Mohamad, Dasmawati [Craniofacial Science Laboratory, School of Dentistry, Health Campus, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan (Malaysia)

    2015-04-24

    ZnO has wide applications in medical and dentistry apart from being used as optoelectronic devices such as solar cells, photodetectors, sensors and light emitting diodes (LEDs). Therefore, the toxicity evaluation is important to know the toxicity level on normal cell line. The toxicity of two grades ZnO nanostructures, ZnO-4 and ZnO-8 have been carried out using cytotoxicity test of MTS assay on L929 rat fibroblast cell line. Prior to that, ZnO-4 and ZnO-8 were characterized for its morphology, structure and optical properties using FESEM, X-ray diffraction, and Photoluminescence respectively. The two groups revealed difference in morphology and exhibit slightly shifted of near band edge emission of Photoluminescence other than having a similar calculated crystallite size of nanostructures. The viability of cells after 72h were obtained and the statistical significance value was calculated using SPSS v20. The p value is more than 0.05 between untreated and treated cell with ZnO. This insignificant value of p>0.05 can be summarized as a non-toxic level of ZnO-4 and ZnO-8 on the L929 cell line.

  12. Toxicity evaluation of ZnO nanostructures on L929 fibroblast cell line using MTS assay

    Science.gov (United States)

    Bakhori, Siti Khadijah Mohd; Mahmud, Shahrom; Ann, Ling Chuo; Mohamed, Azman Seeni; Saifuddin, Siti Nazmin; Masudi, Sam'an Malik; Mohamad, Dasmawati

    2015-04-01

    ZnO has wide applications in medical and dentistry apart from being used as optoelectronic devices such as solar cells, photodetectors, sensors and light emitting diodes (LEDs). Therefore, the toxicity evaluation is important to know the toxicity level on normal cell line. The toxicity of two grades ZnO nanostructures, ZnO-4 and ZnO-8 have been carried out using cytotoxicity test of MTS assay on L929 rat fibroblast cell line. Prior to that, ZnO-4 and ZnO-8 were characterized for its morphology, structure and optical properties using FESEM, X-ray diffraction, and Photoluminescence respectively. The two groups revealed difference in morphology and exhibit slightly shifted of near band edge emission of Photoluminescence other than having a similar calculated crystallite size of nanostructures. The viability of cells after 72h were obtained and the statistical significance value was calculated using SPSS v20. The p value is more than 0.05 between untreated and treated cell with ZnO. This insignificant value of p>0.05 can be summarized as a non-toxic level of ZnO-4 and ZnO-8 on the L929 cell line.

  13. Detection of Hepatitis B Virus DNA by Duplex Scorpion Primer-based PCR Assay

    Institute of Scientific and Technical Information of China (English)

    KONG De-Ming孔德明; SHEN Han-Xi沈含熙; MI Huai-Feng宓怀风

    2004-01-01

    The application of a new fiuorogenic probe-based PCR assay (PCR duplex scorpion primer assay) to the detection of Hepatitis B virus (HBV) DNA in human sera was described. Duplex scorpion primer is a modified variant of duplex Amplifluor, and the incorporation of a PCR stopper between probe and primer sequences improve the detection specificity and sensitivity. Combined with PCR amplification, this probe can give unambiguous positive results for the reactions initiated with more than 20 HBV molecules. In addition, the particular unimolecular probing mechanism of this probe makes the use of short target-specific probe sequence possible, which will render this probe applicable in some specific systems.

  14. Microbial based assay for specific detection of β-lactam group of antibiotics in milk

    OpenAIRE

    Das, Sougata; Kumar, Naresh; Vishweswaraiah, Raghu Hirikyathanahalli; Haldar, Lopamudra; Gaare, Manju; Singh, Vinai Kumar; Puniya, Anil Kumar

    2011-01-01

    The spore forming Bacillus cereus (66) was screened for the induction of β-lactamase in presence of an inducer using iodometric assay. A significant induction in marker enzyme was observed in B. cereus 66 at maximum residual limit (MRL) of penicillin, ampicillin, cloxacillin, amoxicillin, cefalexin, and cephazolin belonging to β-lactam group of antibiotics. A microbial based assay, where enzyme induction was optimized at pH 7.0, temperature 30°C, and whey powder (0.25%) after 4 h of incubatio...

  15. Development of an upconverting chelate assay

    Science.gov (United States)

    Xiao, Xudong; Haushalter, Jeanne P.; Kotz, Kenneth T.; Faris, Gregory W.

    2005-04-01

    We report progress on performing a cell-based assay for the detection of EGFR on cell surfaces by using upconverting chelates. An upconversion microscope has been developed for performing assays and testing optical response. A431 cells are labeled with europium DOTA and imaged using this upconverting microscope.

  16. Measurement of X-ray-induced DNA double-strand breaks at various stages of the cell cycle using the total fluorescence as a comet assay parameter

    International Nuclear Information System (INIS)

    The aim of the study was to develop a protocol for both estimating cell cycle position and the level of ionizing radiation-induced DNA dsb using the neutral comet assay. Using DNA histograms, cell cycle positions were determined for human dermal fibroblasts. The tail intensity was used to estimate the level of DNA damage induced by X-rays, at different positions of the cell cycle. The results of tail intensity versus DNA content bivariate analysis of exponentially growing cells showed a remarkable decrease in tail intensity with transition of cells from G1 to S-phase and increases slightly with transition to G2/M phase. This effect is observed at all doses including unirradiated cells, indicating that the effect is not caused by X-rays and the comet assay based on the current tail parameters is not relevant to measure DNA damage at various stages of the cell cycle. The results of dose response curves showed a linear decrease in the comet fluorescence with the X-ray dose. This observation provides a basis for estimating the fraction of damaged DNA, based on the fluorescence decrement induced by ionizing radiation. The results of this new approach showed a linear increase in DNA damage with dose, at various stages of the cell cycle, with rates, which vary in the following order G0>G2/M>S/G1 cells. These results suggest that G0 and G2/M cells are the most sensitive to X-rays among all phases of the cell cycle and suggest synchronization of cells at these phases to increase the cellular radiosensitivity during radiotherapy. - Display Omitted Highlights: → Increase in DNA damage with dose. → Introduction of a new technique for measuring DNA damage using a new approach of the neutral comet assay. → Estimation of DNA damage in mammalian cells.

  17. Measurement of X-ray-induced DNA double-strand breaks at various stages of the cell cycle using the total fluorescence as a comet assay parameter

    Energy Technology Data Exchange (ETDEWEB)

    Attia, Atef M.M. [Department of Biochemistry, Biophysical laboratory, National Research Center, Dokki, Cairo (Egypt); Nabil, Ghada M., E-mail: gmnabilnooh@hotmail.com [Department of Biochemistry, Biophysical laboratory, National Research Center, Dokki, Cairo (Egypt); Frankenberg, Dieter; Frankenberg-Schwager, M. [Abteilung Klinische Strahlenbiologie und Klinische Strahlenphysik, Zentrum, Radiologie, Georg-August-Universitaet Goettingen, Von-Siebold-Str.3 (Germany)

    2011-11-15

    The aim of the study was to develop a protocol for both estimating cell cycle position and the level of ionizing radiation-induced DNA dsb using the neutral comet assay. Using DNA histograms, cell cycle positions were determined for human dermal fibroblasts. The tail intensity was used to estimate the level of DNA damage induced by X-rays, at different positions of the cell cycle. The results of tail intensity versus DNA content bivariate analysis of exponentially growing cells showed a remarkable decrease in tail intensity with transition of cells from G1 to S-phase and increases slightly with transition to G2/M phase. This effect is observed at all doses including unirradiated cells, indicating that the effect is not caused by X-rays and the comet assay based on the current tail parameters is not relevant to measure DNA damage at various stages of the cell cycle. The results of dose response curves showed a linear decrease in the comet fluorescence with the X-ray dose. This observation provides a basis for estimating the fraction of damaged DNA, based on the fluorescence decrement induced by ionizing radiation. The results of this new approach showed a linear increase in DNA damage with dose, at various stages of the cell cycle, with rates, which vary in the following order G0>G2/M>S/G1 cells. These results suggest that G0 and G2/M cells are the most sensitive to X-rays among all phases of the cell cycle and suggest synchronization of cells at these phases to increase the cellular radiosensitivity during radiotherapy. - Display Omitted Highlights: > Increase in DNA damage with dose. > Introduction of a new technique for measuring DNA damage using a new approach of the neutral comet assay. > Estimation of DNA damage in mammalian cells.

  18. Microalgae on display: a microfluidic pixel-based irradiance assay for photosynthetic growth.

    Science.gov (United States)

    Graham, Percival J; Riordon, Jason; Sinton, David

    2015-08-01

    Microalgal biofuel is an emerging sustainable energy resource. Photosynthetic growth is heavily dependent on irradiance, therefore photobioreactor design optimization requires comprehensive screening of irradiance variables, such as intensity, time variance and spectral composition. Here we present a microfluidic irradiance assay which leverages liquid crystal display technology to provide multiplexed screening of irradiance conditions on growth. An array of 238 microreactors are operated in parallel with identical chemical environments. The approach is demonstrated by performing three irradiance assays. The first assay evaluates the effect of intensity on growth, quantifying saturating intensity. The second assay quantifies the influence of time-varied intensity and the threshold frequency for growth. Lastly, the coupled influence of red-blue spectral composition and intensity is assessed. Each multiplexed assay is completed within three days. In contrast, completing the same number of experiments using conventional incubation flasks would require several years. Not only does our approach enable more rapid screening, but the short optical path avoids self-shading issues inherent to flask based systems. PMID:26085371

  19. An accurate assay for HCV based on real-time fluorescence detection of isothermal RNA amplification.

    Science.gov (United States)

    Wu, Xuping; Wang, Jianfang; Song, Jinyun; Li, Jiayan; Yang, Yongfeng

    2016-09-01

    Hepatitis C virus (HCV) is one of the common reasons of liver fibrosis and hepatocellular carcinoma (HCC). Early, rapid and accurate HCV RNA detection is important to prevent and control liver disease. A simultaneous amplification and testing (SAT) assay, which is based on isothermal amplification of RNA and real-time fluorescence detection, was designed to optimize routine HCV RNA detection. In this study, HCV RNA and an internal control (IC) were amplified and analyzed simultaneously by SAT assay and detection of fluorescence using routine real-time PCR equipment. The assay detected as few as 10 copies of HCV RNA transcripts. We tested 705 serum samples with SAT, among which 96.4% (680/705) showed consistent results compared with routine real-time PCR. About 92% (23/25) discordant samples were confirmed to be same results as SAT-HCV by using a second real-time PCR. The sensitivity and specificity of SAT-HCV assay were 99.6% (461/463) and 100% (242/242), respectively. In conclusion, the SAT assay is an accurate test with a high specificity and sensitivity which may increase the detection rate of HCV. It is therefore a promising tool to diagnose HCV infection. PMID:27283884

  20. An accurate assay for HCV based on real-time fluorescence detection of isothermal RNA amplification.

    Science.gov (United States)

    Wu, Xuping; Wang, Jianfang; Song, Jinyun; Li, Jiayan; Yang, Yongfeng

    2016-09-01

    Hepatitis C virus (HCV) is one of the common reasons of liver fibrosis and hepatocellular carcinoma (HCC). Early, rapid and accurate HCV RNA detection is important to prevent and control liver disease. A simultaneous amplification and testing (SAT) assay, which is based on isothermal amplification of RNA and real-time fluorescence detection, was designed to optimize routine HCV RNA detection. In this study, HCV RNA and an internal control (IC) were amplified and analyzed simultaneously by SAT assay and detection of fluorescence using routine real-time PCR equipment. The assay detected as few as 10 copies of HCV RNA transcripts. We tested 705 serum samples with SAT, among which 96.4% (680/705) showed consistent results compared with routine real-time PCR. About 92% (23/25) discordant samples were confirmed to be same results as SAT-HCV by using a second real-time PCR. The sensitivity and specificity of SAT-HCV assay were 99.6% (461/463) and 100% (242/242), respectively. In conclusion, the SAT assay is an accurate test with a high specificity and sensitivity which may increase the detection rate of HCV. It is therefore a promising tool to diagnose HCV infection.

  1. Improved Activity Assay Method for Arginine Kinase Based on a Ternary Heteropolyacid System

    Institute of Scientific and Technical Information of China (English)

    陈宝玉; 郭勤; 郭智; 王希成

    2003-01-01

    This paper presents a new system for the activity assay of arginine kinase (AK), based on the spectrophotometric determination of an ascorbic acid-reduced blue ternary heteropolyacid composed of bismuth, molybdate and the released phosphate from N-phospho-L-arginine (PArg) formed in the forward catalysis reaction.The assay conditions, including the formulation of the phosphate determination reagent (PDR), the assay timing, and the linear activity range of the enzyme concentration, have been tested and optimized.For these conditions, the ternary heteropolyacid color is completely developed within 1 min and is stable for at least 15 min, with an absorbance maximum at 700 nm and a molar extinction coefficient of 15.97 (mmol/L)-1 · cm-1 for the phosphate.Standard curves for phosphate show a good linearity of 0.999.Compared with previous activity assay methods for AK, this system exhibits superior sensitivity, reproducibility, and adaptability to various conditions in enzymological studies.This method also reduces the assay time and avoids the use of some expensive instruments and reagents.

  2. Multiple detection of single nucleotide polymorphism by microarray-based resonance light scattering assay with enlarged gold nanoparticle probes.

    Science.gov (United States)

    Gao, Jiaxue; Ma, Lan; Lei, Zhen; Wang, Zhenxin

    2016-03-01

    The mapping of specific single nucleotide polymorphisms (SNPs) in patients' genome is a critical process for the development of personalized therapy. In this work, a DNA microarray-based resonance light scattering (RLS) assay has been developed for multiplexed detection of breast cancer related SNPs with high sensitivity and selectivity. After hybridization of the desired target single-stranded DNAs (ssDNAs) with the ssDNA probes on a microarray, the polyvalent ssDNA modified 13 nm gold nanoparticles (GNPs) are employed to label the hybridization reaction through the formation of a three-stranded DNA system. The H2O2-mediated enlargement of GNPs is then used to enhance the RLS signal. The microarray-based RLS assay provides a detection limit of 10 pM (S/N = 3) for the target ssDNA and determines an allele frequency as low as 1.0% in the target ssDNA cocktail. Combined with an asymmetric PCR technique, the proposed assay shows good accuracy and sensitivity in profiling 4 SNPs related to breast cancer of three selected cell lines.

  3. Interlaboratory studies with the Chinese hamster V79 cell metabolic cooperation assay to detect tumor-promoting agents

    Energy Technology Data Exchange (ETDEWEB)

    Bohrman, J.S.; Burg, J.R.; Elmore, E.; Gulati, D.K.; Barfknecht, T.R.; Niemeier, R.W.; Dames, B.L.; Toraason, M.; Langenbach, R.

    1988-01-01

    Three laboratories participated in an interlaboratory study to evaluate the usefulness of the Chinese hamster V79 cell metabolic cooperation assay to predict the tumor-promoting activity of selected chemical. Twenty-three chemicals of different chemical structures (phorbol esters, barbiturates, phenols, artificial sweeteners, alkanes, and peroxides) were chosen for testing based on in vivo promotion activities, as reported in the literature. Assay protocols and materials were standardized, and the chemicals were coded to facilitate unbiased evaluation. A chemical was tested only once in each laboratory, with one of the three laboratories testing only 15 out of 23 chemicals. Dunnett's test was used for statistical analysis. Chemicals were scored as positive (at least two concentration levels statistically different than control), equivocal (only one concentration statistically different), or negative. For 15 chemicals tested in all three laboratories, there was complete agreement among the laboratories for nine chemicals. For the 23 chemicals tested in only two laboratories, there was agreement on 16 chemicals. With the exception of the peroxides and alkanes, the metabolic cooperation data were in general agreement with in vivo data. However, an overall evaluation of the V79 cell system for predicting in vivo promotion activity was difficult because of the organ specificity of certain chemicals and/or the limited number of adequately tested nonpromoting chemicals.

  4. Comparison of chemical binding to recombinant fathead minnow and human estrogen receptors alpha in whole cell and cell-free binding assays.

    Science.gov (United States)

    Rider, Cynthia V; Hartig, Phillip C; Cardon, Mary C; Wilson, Vickie S

    2009-10-01

    Mammalian receptors and assay systems are generally used for in vitro screening of endocrine-disrupting chemicals with the assumption that minor differences in amino acid sequences among species do not translate into significant differences in receptor function. Objectives of the present study were to evaluate the performance of two different in vitro assay systems (a whole cell and a cell-free competitive binding assay) in assessing whether binding of chemicals differs significantly between full-length recombinant estrogen receptors from fathead minnows (fhERalpha) and those from humans (hERalpha). It was confirmed that 17beta-estradiol displays a reduction in binding to fhERalpha at an elevated temperature (37 degrees C), as has been reported with other piscine estrogen receptors. Several of the chemicals (17beta-estradiol, ethinylestradiol, alpha-zearalanol, fulvestrant, dibutyl phthalate, benzyl butyl phthalate, and cadmium chloride) displayed higher affinity for fhERalpha than for hERalpha in the whole cell assay, while only dibutyl phthalate had a higher affinity for fhERalpha than for hERalpha in the cell-free assay. Both assays were effective in identifying strong binders, weak binders, and nonbinders to the two receptors. However, the cell-free assay provided a less complicated and more efficient binding platform and is, therefore, recommended over the whole cell binding assay. In conclusion, no strong evidence showed species-specific binding among the chemicals tested. PMID:19453209

  5. Simultaneous Detection of Antigen-Specific IgG- and IgM-Secreting Cells with a B Cell Fluorospot Assay

    Directory of Open Access Journals (Sweden)

    M. Anthony Moody

    2012-03-01

    Full Text Available The traditional enzyme-linked immunospot (ELISpot assay is the gold standard for the enumeration of antigen-specific B cells. Since B cell availability from biological samples is often limited, either because of sample size/volume or the need of performing multiple analyses on the same sample, the implementation of ELISpot assay formats that allow the simultaneous detection of multiple antibody types is desirable. While dual-color ELISpot assays have been described, technical complexities have so far prevented their wide utilization as well as further expansion of their multicolor capability. An attractive solution is to replace the chromogenic reaction of the traditional ELISpot assay with a fluorescent detection system (fluorospot assay. Fluorospot assays using fluorophore-conjugated secondary antibodies in conjunction with fluorescence enhancers, FITC/anti-FITC and biotin/avidin amplification systems and dedicated equipment for spot detection have been developed to enumerate T-cells secreting two or three specific cytokines and, more recently, IgG and IgA antibody-secreting cells (ASCs. We hereby report a method for a multiplex B cell fluorospot assay that utilizes quantum-dot nanocrystals as reporters without further amplification systems or need of dedicated equipment. With this method we simultaneously enumerated HIV-1 gp41 envelope glycoprotein-specific IgG and IgM antibody-secreting cells with sensitivity comparable to that of the traditional ELISpot assay.

  6. Yeast-based assay identifies novel Shh/Gli target genes in vertebrate development

    Directory of Open Access Journals (Sweden)

    Milla Luis A

    2012-01-01

    Full Text Available Abstract Background The increasing number of developmental events and molecular mechanisms associated with the Hedgehog (Hh pathway from Drosophila to vertebrates, suggest that gene regulation is crucial for diverse cellular responses, including target genes not yet described. Although several high-throughput, genome-wide approaches have yielded information at the genomic, transcriptional and proteomic levels, the specificity of Gli binding sites related to direct target gene activation still remain elusive. This study aims to identify novel putative targets of Gli transcription factors through a protein-DNA binding assay using yeast, and validating a subset of targets both in-vitro and in-vivo. Testing in different Hh/Gli gain- and loss-of-function scenarios we here identified known (e.g., ptc1 and novel Hh-regulated genes in zebrafish embryos. Results The combined yeast-based screening and MEME/MAST analysis were able to predict Gli transcription factor binding sites, and position mapping of these sequences upstream or in the first intron of promoters served to identify new putative target genes of Gli regulation. These candidates were validated by qPCR in combination with either the pharmacological Hh/Gli antagonist cyc or the agonist pur in Hh-responsive C3H10T1/2 cells. We also used small-hairpin RNAs against Gli proteins to evaluate targets and confirm specific Gli regulation their expression. Taking advantage of mutants that have been identified affecting different components of the Hh/Gli signaling system in the zebrafish model, we further analyzed specific novel candidates. Studying Hh function with pharmacological inhibition or activation complemented these genetic loss-of-function approaches. We provide evidence that in zebrafish embryos, Hh signaling regulates sfrp2, neo1, and c-myc expression in-vivo. Conclusion A recently described yeast-based screening allowed us to identify new Hh/Gli target genes, functionally important in

  7. Biofilm vivacity and destruction on antimicrobial nanosurfaces assayed within a microbial fuel cell.

    Science.gov (United States)

    Sugnaux, Marc; Fischer, Fabian

    2016-08-01

    A novel method was developed to assay the antimicrobial capacity of nanostructured surfaces for medical implants in a bicathodic microbial fuel cell. Nano-structured gold surfaces with protruding nanopillars and nanorings were investigated. Escherichia coli K12 were used as a model microbe to record electronic effects caused by the interaction with nanosurfaces. The nanostructured gold surfaces enabled power density maxima up to 1910mW/m(2), indicating fair vivacity, while flat surfaces on the nanoscale provided almost no power 0.35mW/m(2). The biofilm presence on antimicrobial nanosurfaces was confirmed by the addition of ampicillin and its bactericidal effect resulted in oscillating and declining potentiometric signals. Current density experiments showed that biofilms on antimicrobial nanostructured electrodes caused low currents, indicating that E.coli biofilm remained functional before destruction. The bicathodic microbial fuel cell sensor is a novel tool for evaluating antimicrobial effects caused by nanosurfaces and antibiotics. PMID:27071334

  8. Development and validation of a novel Taqman-based real-time RT-PCR assay suitable for demonstrating freedom from viral haemorrhagic septicaemia virus

    DEFF Research Database (Denmark)

    Jonstrup, Søren Peter; Kahns, Søren; Skall, Helle Frank;

    2013-01-01

    shortened and the need for maintaining expensive cell culture facilities reduced. Here we present the validation, according to OIE guidelines, of a sensitive and specific Taqman-based real-time RT-PCR. The assay detects all isolates in a panel of 79 VHSV isolates covering all known genotypes and subtypes...

  9. Assessment of DNA integrity (COMET assay) in sperm cells of boron-exposed workers.

    Science.gov (United States)

    Duydu, Yalçin; Başaran, Nurşen; Ustündağ, Aylin; Aydin, Sevtap; Undeğer, Ulkü; Ataman, Osman Yavuz; Aydos, Kaan; Düker, Yalçin; Ickstadt, Katja; Waltrup, Britta Schulze; Golka, Klaus; Bolt, Hermann M

    2012-01-01

    An extension of a male reproductive study conducted in a boric acid/borate production zone at Bandırma, Turkey, is presented. The relation between DNA-strand breaks (COMET assay, neutral and alkaline version) in sperm cells and previously described sperm quality parameters was investigated in boron-exposed males. A correlation between blood boron levels and mean DNA-strand breaks in sperm was weak, and DNA-strand breaks in sperm were statistically not different between control and exposed groups. Therefore, increasing boron exposures had no additional contribution in addition to already pre-existing DNA-strand breaks in the sperm cells. Weak but statistically significant correlations between DNA-strand breaks and motility/morphology parameters of sperm samples were observed in the neutral version of the COMET assay, while correlations between the same variables were statistically not significant in the alkaline version. A likely reason for these negative results, even in highly exposed humans, is that experimental exposures that had led to reproductive toxicity in animals were significantly higher than any boron exposures, which may be reached under realistic human conditions.

  10. Cytogenetic dosimetry by micronucleus assay using peripheral blood cells is modified by thyroid hormones

    International Nuclear Information System (INIS)

    Cytokinesis-block micronucleus (CBMN) assay is a convenient and easy method of radiation biodosimetry that uses peripheral blood (PB) cells. However, for micronuclei (MN) frequency induced by ionising radiation, a dose-response relationship in abnormal condition, such as in cancer patients, has not been assessed. To clarify the difference between the dose-response curve generated by the CBMN assay in conditions when thyroid hormone levels were normal and during thyroid hormone withdrawal (THW) prior to 131I treatment, 12 thyroid cancer patients who underwent thyroidectomy were studied. The collected PB mononuclear cells were exposed to 0.5-3.0 Gy X-ray irradiation. Under normal conditions, dose dependency and independence of MN frequency were observed in 92 % and 8 %, respectively. In contrast, during THW, the number of patients who showed dose independence significantly increased to 42 % in comparison with control. Furthermore, a higher concentration of serum thyroglobulin in dose-independent patients was observed. These results suggest that MN frequency in cytogenetic dosimetry is affected by thyroid hormones. (authors)