Mkit: A cell migration assay based on microfluidic device and smartphone.

Science.gov (United States)

Yang, Ke; Wu, Jiandong; Peretz-Soroka, Hagit; Zhu, Ling; Li, Zhigang; Sang, Yaoshuo; Hipolito, Jolly; Zhang, Michael; Santos, Susy; Hillier, Craig; de Faria, Ricardo Lobato; Liu, Yong; Lin, Francis

2018-01-15

Mobile sensing based on the integration of microfluidic device and smartphone, so-called MS 2 technology, has enabled many applications over recent years, and continues to stimulate growing interest in both research communities and industries. In particular, it has been envisioned that MS 2 technology can be developed for various cell functional assays to enable basic research and clinical applications. Toward this direction, in this paper, we describe the development of a MS 2 -based cell functional assay for testing cell migration (the M kit ). The system is constructed as an integrated test kit, which includes microfluidic chips, a smartphone-based imaging platform, the phone apps for image capturing and data analysis, and a set of reagent and accessories for performing the cell migration assay. We demonstrated that the M kit can effectively measure purified neutrophil and cancer cell chemotaxis. Furthermore, neutrophil chemotaxis can be tested from a drop of whole blood using the M kit with red blood cell (RBC) lysis. The effects of chemoattractant dose and gradient profile on neutrophil chemotaxis were also tested using the M kit . In addition to research applications, we demonstrated the effective use of the M kit for on-site test at the hospital and for testing clinical samples from chronic obstructive pulmonary disease patient. Thus, this developed M kit provides an easy and integrated experimental platform for cell migration related research and potential medical diagnostic applications. Copyright © 2017 Elsevier B.V. All rights reserved.

Challenges in the Development of Functional Assays of Membrane Proteins

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Sophie Demarche

2012-11-01

Full Text Available Lipid bilayers are natural barriers of biological cells and cellular compartments. Membrane proteins integrated in biological membranes enable vital cell functions such as signal transduction and the transport of ions or small molecules. In order to determine the activity of a protein of interest at defined conditions, the membrane protein has to be integrated into artificial lipid bilayers immobilized on a surface. For the fabrication of such biosensors expertise is required in material science, surface and analytical chemistry, molecular biology and biotechnology. Specifically, techniques are needed for structuring surfaces in the micro- and nanometer scale, chemical modification and analysis, lipid bilayer formation, protein expression, purification and solubilization, and most importantly, protein integration into engineered lipid bilayers. Electrochemical and optical methods are suitable to detect membrane activity-related signals. The importance of structural knowledge to understand membrane protein function is obvious. Presently only a few structures of membrane proteins are solved at atomic resolution. Functional assays together with known structures of individual membrane proteins will contribute to a better understanding of vital biological processes occurring at biological membranes. Such assays will be utilized in the discovery of drugs, since membrane proteins are major drug targets.

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.

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

G protein-coupled receptors (GPCRs) have been one of the most productive classes of drug targets for several decades, and new technologies for GPCR-based discovery promise to keep this field active for years to come. While molecular screens for GPCR receptor agonist- and antagonist-based drugs...... 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...

A robust and scalable TCR-based reporter cell assay to measure HIV-1 Nef-mediated T cell immune evasion.

Science.gov (United States)

Anmole, Gursev; Kuang, Xiaomei T; Toyoda, Mako; Martin, Eric; Shahid, Aniqa; Le, Anh Q; Markle, Tristan; Baraki, Bemuluyigza; Jones, R Brad; Ostrowski, Mario A; Ueno, Takamasa; Brumme, Zabrina L; Brockman, Mark A

2015-11-01

HIV-1 evades cytotoxic T cell responses through Nef-mediated downregulation of HLA class I molecules from the infected cell surface. Methods to quantify the impact of Nef on T cell recognition typically employ patient-derived T cell clones; however, these assays are limited by the cost and effort required to isolate and maintain primary cell lines. The variable activity of different T cell clones and the limited number of cells generated by re-stimulation can also hinder assay reproducibility and scalability. Here, we describe a heterologous T cell receptor reporter assay and use it to study immune evasion by Nef. Induction of NFAT-driven luciferase following co-culture with peptide-pulsed or virus-infected target cells serves as a rapid, quantitative and antigen-specific measure of T cell recognition of its cognate peptide/HLA complex. We demonstrate that Nef-mediated downregulation of HLA on target cells correlates inversely with T cell receptor-dependent luminescent signal generated by effector cells. This method provides a robust, flexible and scalable platform that is suitable for studies to measure Nef function in the context of different viral peptide/HLA antigens, to assess the function of patient-derived Nef alleles, or to screen small molecule libraries to identify novel Nef inhibitors. Copyright © 2015 Elsevier B.V. All rights reserved.

Isotopic rubidium ion efflux assay for the functional characterization of nicotinic acetylcholine receptors on clonal cell lines

International Nuclear Information System (INIS)

Lukas, R.J.; Cullen, M.J.

1988-01-01

An isotopic rubidium ion efflux assay has been developed for the functional characterization of nicotinic acetylcholine receptors on cultured neurons. This assay first involves the intracellular sequestration of isotopic potassium ion analog by the ouabain-sensitive action of a sodium-potassium ATPase. Subsequently, the release of isotopic rubidium ion through nicotinic acetylcholine receptor-coupled monovalent cation channels is activated by application of nicotinic agonists. Specificity of receptor-mediated efflux is demonstrated by its sensitivity to blockade by nicotinic, but not muscarinic, antagonists. The time course of agonist-mediated efflux, within the temporal limitations of the assay, indicates a slow inactivation of receptor function on prolonged exposure to agonist. Dose-response profiles (i) have characteristic shapes for different nicotinic agonists, (ii) are described by three operationally defined parameters, and (iii) reflect different affinities of agonists for binding sites that control receptor activation and functional inhibition. The rubidium ion efflux assay provides fewer hazards but greater sensitivity and resolution than isotopic sodium or rubidium ion influx assays for functional nicotinic receptors

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...

Whole blood microculture assay of human lymphocyte function.

Science.gov (United States)

Pauly, J L; Han, T

1976-11-01

A whole blood microculture assay is described for measuring lymphocyte reactivity to mitogenic and antigenic stimulants. This assay employs heparinized whole blood, serum-free culture medium, microtiter plates, and a Multiple Automated Sample Harvester (MASH). When this assay is compared to other leukocyte assays, its major advantages include (1) the utilization of fewer lymphocytes per microculture, thuus reducing the amount of blood required per test while increasing the number of test agents and replicate cultures which can be employed in any given experiment; (2) the conservation of mitogens, antigens, drugs, enzymes, hormones, lymphokines, and other test agents, some of which are either expensive of difficult to prepare in large quantities; (3) the elimination of lymphocyte isolation and purification procedures which may disrupt the relative proportion of T cells, B cells and antigen-processing cells; and (4) the application of an automated harvester which simplifies and expedites procedures required for processing cells for liquid scintillation counting.

Reference cells and ploidy in the comet assay

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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.

Critical elements in the development of cell therapy potency assays for ischemic conditions.

Science.gov (United States)

Porat, Yael; Abraham, Eytan; Karnieli, Ohad; Nahum, Sagi; Woda, Juliana; Zylberberg, Claudia

2015-07-01

A successful potency assay for a cell therapy product (CTP) used in the treatment of ischemic conditions should quantitatively measure relevant biological properties that predict therapeutic activity. This is especially challenging because of numerous degrees of complexity stemming from factors that include a multifactorial complex mechanism of action, cell source, inherent cell characteristics, culture method, administration mode and the in vivo conditions to which the cells are exposed. The expected biological function of a CTP encompasses complex interactions that range from a biochemical, metabolic or immunological activity to structural replacement of damaged tissue or organ. Therefore, the requirements for full characterization of the active substance with respect to biological function could be taxing. Moreover, the specific mechanism of action is often difficult to pinpoint to a specific molecular entity; rather, it is more dependent on the functionality of the cellular components acting in a in a multifactorial fashion. In the case of ischemic conditions, the cell therapy mechanism of action can vary from angiogenesis, vasculogenesis and arteriogenesis that may activate different pathways and clinical outcomes. The CTP cellular attributes with relation to the suggested mechanism of action can be used for the development of quantitative and reproducible analytical potency assays. CTPs selected and released on the basis of such potency assays should have the highest probability of providing meaningful clinical benefit for patients. This White Paper will discuss and give examples for key elements in the development of a potency assay for treatment of ischemic disorders treated by the use of CTPs. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

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

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

Epithelial cells as alternative human biomatrices for comet assay.

Science.gov (United States)

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 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 30 year period, the comet assay in epithelial cells has been little 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.

Functional assay of the alternative complement pathway of rat serum

International Nuclear Information System (INIS)

Coonrod, J.D.; Jenkins, S.D.

1979-01-01

Two functional assays of the alternative pathway of complement activation in rat serum were developed. In the first assay, conditions were established for titration of alternative pathway activity by use of the 50% hemolytic end-point of rabbit red blood cells (RaRBC) in serum treated with ethyleneglycol-bis-(beta-aminoethyl ether)-N, N'-tetraacetic acid (EGTA). The second assay of alternative pathway activity was based on the opsonization of heat-killed radiolabeled pneumococci of serotype 25 (Pn25). Opsonization of Pn25 was shown to proceed entirely via the alternative pathway in rat serum. There was excellent correlation between the results obtained with the RaRBC lysis test and those obtained with the opsonization test. Because of its technical simplicity, the RaRBC lysis test appeared to be the single most useful test of alternative pathway activity in rat serum. (Auth.)

A critical assessment of the use of microculture tetrazolium assays to measure cell growth and function.

Science.gov (United States)

Marshall, N J; Goodwin, C J; Holt, S J

1995-06-01

Microculture tetrazolium assays (MTAs) are being widely applied to probe the relationships between cell survival, growth, and differentiation and also to investigate associations between compromised cell metabolism, oxidative stress, and programmed cell death as occurs in apoptosis. MTAs rely upon the cellular reduction of tetrazolium salts to their intensely coloured formazans. The resulting colorimetric assays form the basis of exceptionally precise systems which are technically amenable and capable of a high throughput of samples. As a consequence, MTAs are being used to monitor responses to both extracellular activators and toxic agents in disciplines as diverse as radiobiology and endocrinology. We review the chemistry and histochemical applications of tetrazolium salts and subsequently discuss the criteria for their use in MTAs. These assays are one of the latest examples of the application of the tetrazolium/formazan system to cell biology. We outline current views on the mechanisms of the bioreduction of tetrazolium salts. These probably combine to reflect the integrated pyridine nucleotide dependent redox state of the cell. We try to illustrate how an understanding of these mechanisms helps to avoid some of the pitfalls of the MTA systems. There is now for example, extensive evidence that changes in cell culture environments, such as glucose supply or pH of the medium, influence the reduction of tetrazolium salts and thereby introduce artefacts into MTAs. Finally, we provide examples of situations in which MTAs can be used to complement other more established experimental systems. They then act as unique probes with which to investigate changes in the redox state of the cell. These changes are associated with regulation of cell growth, proliferation and differentiation and conversely, the different pathways leading to cell death.

Functional cell mediated lympholysis I. Description of the assay

International Nuclear Information System (INIS)

Goeken, N.E.; Thompson, J.S.

1981-01-01

The anamnestic response by human bi-directional (BD) mixed lymphocyte cultures (MLC) to restimulation by cells of the original stimulating type is generally strikingly reduced as compared to that of standard one-way cultures. This difference was shown not to be related to a change in kinetics nor was it due to exhaustion of the media or soluble factors since fresh media did not ameliorate the effect nor were supernatants from BD cultures found to be suppressive. The relative inhibition was also not reversed by removal of the allogeneic cells by phenotype specific antiserum. Cytotoxic tests with donor and responder specific antisera revealed that the cells bearing that phenotype were dramatically reduced in BD as compared to one-way cultures. Thus, the diminished secondary response appears to be due to cytotoxic elimination of the responder cells. This allogeneic cytotoxicity is dependent on non-T, phagocytic, adherent cells. The phenomenon is called Functional Cell Mediated Lympholysis (F-CML). (author)

Development and characterization of a pre-treatment procedure to eliminate human monoclonal antibody therapeutic drug and matrix interference in cell-based functional neutralizing antibody assays.

Science.gov (United States)

Xu, Weifeng; Jiang, Hao; Titsch, Craig; Haulenbeek, Jonathan R; Pillutla, Renuka C; Aubry, Anne-Françoise; DeSilva, Binodh S; Arnold, Mark E; Zeng, Jianing; Dodge, Robert W

2015-01-01

Biological therapeutics can induce an undesirable immune response resulting in the formation of anti-drug antibodies (ADA), including neutralizing antibodies (NAbs). Functional (usually cell-based) NAb assays are preferred to determine NAb presence in patient serum, but are often subject to interferences from numerous serum factors, such as growth factors and disease-related cytokines. Many functional cell-based NAb assays are essentially drug concentration assays that imply the presence of NAbs by the detection of small changes in functional drug concentration. Any drug contained in the test sample will increase the total amount of drug in the assay, thus reducing the sensitivity of NAb detection. Biotin-drug Extraction with Acid Dissociation (BEAD) has been successfully applied to extract ADA, thereby removing drug and other interfering factors from human serum samples. However, to date there has been no report to estimate the residual drug level after BEAD treatment when the drug itself is a human monoclonal antibody; mainly due to the limitation of traditional ligand-binding assays. Here we describe a universal BEAD optimization procedure for human monoclonal antibody (mAb) drugs by using a LC-MS/MS method to simultaneously measure drug (a mutant human IgG4), NAb positive control (a mouse IgG), and endogenous human IgGs as an indicator of nonspecific carry-over in the BEAD eluate. This is the first report demonstrating that residual human mAb drug level in clinical sample can be measured after BEAD pre-treatment, which is critical for further BEAD procedure optimization and downstream immunogenicity testing. Copyright © 2014 Elsevier B.V. All rights reserved.

Frequent sgRNA-barcode recombination in single-cell perturbation assays.

Directory of Open Access Journals (Sweden)

Shiqi Xie

Full Text Available Simultaneously detecting CRISPR-based perturbations and induced transcriptional changes in the same cell is a powerful approach to unraveling genome function. Several lentiviral approaches have been developed, some of which rely on the detection of distally located genetic barcodes as an indirect proxy of sgRNA identity. Since barcodes are often several kilobases from their corresponding sgRNAs, viral recombination-mediated swapping of barcodes and sgRNAs is feasible. Using a self-circularization-based sgRNA-barcode library preparation protocol, we estimate the recombination rate to be ~50% and we trace this phenomenon to the pooled viral packaging step. Recombination is random, and decreases the signal-to-noise ratio of the assay. Our results suggest that alternative approaches can increase the throughput and sensitivity of single-cell perturbation assays.

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

International Nuclear Information System (INIS)

Lee, Saet-byul; Cha, Junhoe; Kim, Im-kyung; Yoon, Joo Chun; Lee, Hyo Joon; Park, Sang Woo; Cho, Sunjung; Youn, Dong-Ye; Lee, Heyja; Lee, Choong Hwan; Lee, Jae Myun; Lee, Kang Young; Kim, Jongsun

2014-01-01

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 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

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.

Rapid assay for cell age response to radiation by electronic volume flow cell sorting

International Nuclear Information System (INIS)

Freyer, J.P.; Wilder, M.E.; Raju, M.R.

1987-01-01

A new technique is described for measuring cell survival as a function of cell cycle position using flow cytometric cell sorting on the basis of electronic volume signals. Sorting of cells into different cell age compartments is demonstrated for three different cell lines commonly used in radiobiological research. Using flow cytometric DNA content analysis and [ 3 H]thymidine autoradiography of the sorted cell populations, it is demonstrated that resolution of the age compartment separation is as good as or better than that reported for other cell synchronizing techniques. Variation in cell survival as a function of position in the cell cycle after a single dose of radiation as measured by volume cell sorting is similar to that determined by other cell synchrony techniques. Advantages of this method include: (1) no treatment of the cells is required, thus, this method is noncytotoxic; (2) no cell cycle progression is needed to obtain different cell age compartments; (3) the cell population can be held in complete growth medium at any desired temperature during sorting; (4) a complete radiation age - response assay can be plated in 2 h. Applications of this method are discussed, along with some technical limitations. (author)

Self-Checking Cell-Based Assays for GPCR Desensitization and Resensitization.

Science.gov (United States)

Fisher, Gregory W; Fuhrman, Margaret H; Adler, Sally A; Szent-Gyorgyi, Christopher; Waggoner, Alan S; Jarvik, Jonathan W

2014-09-01

G protein-coupled receptors (GPCRs) play stimulatory or modulatory roles in numerous physiological states and processes, including growth and development, vision, taste and olfaction, behavior and learning, emotion and mood, inflammation, and autonomic functions such as blood pressure, heart rate, and digestion. GPCRs constitute the largest protein superfamily in the human and are the largest target class for prescription drugs, yet most are poorly characterized, and of the more than 350 nonolfactory human GPCRs, over 100 are orphans for which no endogenous ligand has yet been convincingly identified. We here describe new live-cell assays that use recombinant GPCRs to quantify two general features of GPCR cell biology-receptor desensitization and resensitization. The assays employ a fluorogen-activating protein (FAP) reporter that reversibly complexes with either of two soluble organic molecules (fluorogens) whose fluorescence is strongly enhanced when complexed with the FAP. Both assays require no wash or cleanup steps and are readily performed in microwell plates, making them adaptable to high-throughput drug discovery applications. © 2014 Society for Laboratory Automation and Screening.

Xenografts in zebrafish embryos as a rapid functional assay for breast cancer stem-like cell identification.

Science.gov (United States)

Eguiara, Arrate; Holgado, Olaia; Beloqui, Izaskun; Abalde, Leire; Sanchez, Yolanda; Callol, Carles; Martin, Angel G

2011-11-01

The cancer stem cell is defined by its capacity to self-renew, the potential to differentiate into all cells of the tumor and the ability to proliferate and drive the expansion of the tumor. Thus, targeting these cells may provide novel anti-cancer treatment strategies. Breast cancer stem cells have been isolated according to surface marker expression, ability to efflux fluorescent dyes, increased activity of aldehyde dehydrogenase or the capacity to form spheres in non-adherent culture conditions. In order to test novel drugs directed towards modulating self-renewal of cancer stem cells, rapid, easy and inexpensive assays must be developed. Using 2 days-post-fertilization (dpf) zebrafish embryos as transplant recipients, we show that cells grown in mammospheres from breast carcinoma cell lines migrate to the tail of the embryo and form masses with a significantly higher frequency than parental monolayer populations. When stem-like self-renewal was targeted in the parental population by the use of the dietary supplement curcumin, cell migration and mass formation were reduced, indicating that these effects were associated with stem-like cell content. This is a proof of principle report that proposes a rapid and inexpensive assay to target in vivo cancer stem-like cells, which may be used to unravel basic cancer stem cell biology and for drug screening.

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.

Protein-carbohydrate complex reveals circulating metastatic cells in a microfluidic assay

KAUST Repository

Simone, Giuseppina; Malara, Natalia Maria; Trunzo, Valentina; Perozziello, Gerardo; Neužil, Pavel; Francardi, Marco; Roveda, Laura; Renne, Maria; Prati, Ubaldo; Mollace, Vincenzo; Manz, Andreas; Di Fabrizio, Enzo M.

2013-01-01

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.

Interspecific in vitro assay for the chimera-forming ability of human pluripotent stem cells.

Science.gov (United States)

Masaki, Hideki; Kato-Itoh, Megumi; Umino, Ayumi; Sato, Hideyuki; Hamanaka, Sanae; Kobayashi, Toshihiro; Yamaguchi, Tomoyuki; Nishimura, Ken; Ohtaka, Manami; Nakanishi, Mahito; Nakauchi, Hiromitsu

2015-09-15

Functional assay limitations are an emerging issue in characterizing human pluripotent stem cells (PSCs). With rodent PSCs, chimera formation using pre-implantation embryos is the gold-standard assay of pluripotency (competence of progeny to differentiate into all three germ layers). In human PSCs (hPSCs), however, this can only be monitored via teratoma formation or in vitro differentiation, as ethical concerns preclude generation of human-human or human-animal chimeras. To circumvent this issue, we developed a functional assay utilizing interspecific blastocyst injection and in vitro culture (interspecies in vitro chimera assay) that enables the development and observation of embryos up to headfold stage. The assay uses mouse pre-implantation embryos and rat, monkey and human PSCs to create interspecies chimeras cultured in vitro to the early egg-cylinder stage. Intra- and interspecific chimera assays with rodent PSC lines were performed to confirm the consistency of results in vitro and in vivo. The behavior of chimeras developed in vitro appeared to recapitulate that of chimeras developed in vivo; that is, PSC-derived cells survived and were integrated into the epiblast of egg-cylinder-stage embryos. This indicates that the interspecific in vitro chimera assay is useful in evaluating the chimera-forming ability of rodent PSCs. However, when human induced PSCs (both conventional and naïve-like types) were injected into mouse embryos and cultured, some human cells survived but were segregated; unlike epiblast-stage rodent PSCs, they never integrated into the epiblast of egg-cylinder-stage embryos. These data suggest that the mouse-human interspecies in vitro chimera assay does not accurately reflect the early developmental potential/process of hPSCs. The use of evolutionarily more closely related species as host embryos might be necessary to evaluate the developmental potency of hPSCs. © 2015. Published by The Company of Biologists Ltd.

A new sensitive and quantitative HTLV-I-mediated cell fusion assay in T cells

International Nuclear Information System (INIS)

Pare, Marie-Eve; Gauthier, Sonia; Landry, Sebastien; Sun Jiangfeng; Legault, Eric; Leclerc, Denis; Tanaka, Yuetsu; Marriott, Susan J.; Tremblay, Michel J.; Barbeau, Benoit

2005-01-01

Similar to several other viruses, human T cell leukemia virus type I (HTLV-I) induces the formation of multinucleated giant cells (also known as syncytium) when amplified in tissue culture. These syncytia result from the fusion of infected cells with uninfected cells. Due to the intrinsic difficulty of infecting cells with cell-free HTLV-I virions, syncytium formation has become an important tool in the study of HTLV-I infection and transmission. Since most HTLV-I-based cell fusion assays rely on the use of non-T cells, the aim of this study was to optimize a new HTLV-I-induced cell fusion assay in which HTLV-I-infected T cell lines are co-cultured with T cells that have been transfected with an HTLV-I long terminal repeat (LTR) luciferase reporter construct. We demonstrate that co-culture of various HTLV-I-infected T cells with different transfected T cell lines resulted in induction of luciferase activity. Cell-to-cell contact and expression of the viral gp46 envelope protein was crucial for this induction while other cell surface proteins (including HSC70) did not have a significant effect. This quantitative assay was shown to be very sensitive. In this assay, the cell fusion-mediated activation of NF-κB and the HTLV-I LTR occurred through previously described Tax-dependent signaling pathways. This assay also showed that cell fusion could activate Tax-inducible cellular promoters. These results thus demonstrate that this new quantitative HTLV-I-dependent cell fusion assay is versatile, highly sensitive, and can provide an important tool to investigate cellular promoter activation and intrinsic signaling cascades that modulate cellular gene expression

Alternative Methods for the Detection of Emerging Marine Toxins: Biosensors, Biochemical Assays and Cell-Based Assays

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Laia Reverté

2014-11-01

Full Text Available The emergence of marine toxins in water and seafood may have a considerable impact on public health. Although the tendency in Europe is to consolidate, when possible, official reference methods based on instrumental analysis, the development of alternative or complementary methods providing functional or toxicological information may provide advantages in terms of risk identification, but also low cost, simplicity, ease of use and high-throughput analysis. This article gives an overview of the immunoassays, cell-based assays, receptor-binding assays and biosensors that have been developed for the screening and quantification of emerging marine toxins: palytoxins, ciguatoxins, cyclic imines and tetrodotoxins. Their advantages and limitations are discussed, as well as their possible integration in research and monitoring programs.

Alternative Methods for the Detection of Emerging Marine Toxins: Biosensors, Biochemical Assays and Cell-Based Assays

Science.gov (United States)

Reverté, Laia; Soliño, Lucía; Carnicer, Olga; Diogène, Jorge; Campàs, Mònica

2014-01-01

The emergence of marine toxins in water and seafood may have a considerable impact on public health. Although the tendency in Europe is to consolidate, when possible, official reference methods based on instrumental analysis, the development of alternative or complementary methods providing functional or toxicological information may provide advantages in terms of risk identification, but also low cost, simplicity, ease of use and high-throughput analysis. This article gives an overview of the immunoassays, cell-based assays, receptor-binding assays and biosensors that have been developed for the screening and quantification of emerging marine toxins: palytoxins, ciguatoxins, cyclic imines and tetrodotoxins. Their advantages and limitations are discussed, as well as their possible integration in research and monitoring programs. PMID:25431968

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

International Nuclear Information System (INIS)

Hodzic, Jasmina; Dingjan, Ilse; Maas, Mariëlle JP; Meulen-Muileman, Ida H van der; Menezes, Renee X de; Heukelom, Stan; Verheij, Marcel; Gerritsen, Winald R; Geldof, Albert A; Triest, Baukelien van; Beusechem, Victor W van

2015-01-01

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

A novel microculture kinetic assay (MiCK assay) for malignant cell growth and chemosensitivity.

Science.gov (United States)

Kravtsov, V D

1994-01-01

The THERMOmax microplate reader was adapted for monitoring the growth kinetics of human leukaemic OCI/AML-2 and mouse tumour J-774.1 cell lines in continuous culture. Fluid evaporation from wells, CO2 escape and contamination were prevented by hermetic sealing of the microcultures in wells of a 96-well microplate, thus enabling the cells to grow exponentially for 72 h under the conditions of the incubated microplate reader. For both OCI/AML-2 cells, which grow in suspension, and adherent J-774.1 cells, a linear correlation was demonstrated between the number of unstained cells seeded in a given microplate well and the optical density (OD) of that well. Therefore, the OD/time curve of the culture could be deemed to be its growth curve. By the use of the linear fit equation, the actual number of the cells in the wells was computable at any time point of the assay. In the chemosensitivity test, an inhibitory effect of ARA-C on the growth of the cells could be estimated by viewing of the growth curves plotted on the screen. The maximum kinetic rates (Vmax) of the curves in the control and the ARA-C-treated wells were compared, yielding a growth inhibition index (GII). Comparison of results of the kinetic chemosensitivity assay with those of a [3H]thymidine incorporation assay revealed that the novel assay is suitable for precise quantitation of the cell chemosensitivity, is more informative and has the added technical advantage of performance without recourse to radioactive or chemically hazardous substances.

A novel flow cytometric HTS assay reveals functional modulators of ATP binding cassette transporter ABCB6.

Science.gov (United States)

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

2012-01-01

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

Single-cell nanotoxicity assays of superparamagnetic iron oxide nanoparticles.

Science.gov (United States)

Eustaquio, Trisha; Leary, James F

2012-01-01

Properly evaluating the nanotoxicity of nanoparticles involves much more than bulk-cell assays of cell death by necrosis. Cells exposed to nanoparticles may undergo repairable oxidative stress and DNA damage or be induced into apoptosis. Exposure to nanoparticles may cause the cells to alter their proliferation or differentiation or their cell-cell signaling with neighboring cells in a tissue. Nanoparticles are usually more toxic to some cell subpopulations than others, and toxicity often varies with cell cycle. All of these facts dictate that any nanotoxicity assay must be at the single-cell level and must try whenever feasible and reasonable to include many of these other factors. Focusing on one type of quantitative measure of nanotoxicity, we describe flow and scanning image cytometry approaches to measuring nanotoxicity at the single-cell level by using a commonly used assay for distinguishing between necrotic and apoptotic causes of cell death by one type of nanoparticle. Flow cytometry is fast and quantitative, provided that the cells can be prepared into a single-cell suspension for analysis. But when cells cannot be put into suspension without altering nanotoxicity results, or if morphology, attachment, and stain location are important, a scanning image cytometry approach must be used. Both methods are described with application to a particular type of nanoparticle, a superparamagnetic iron oxide nanoparticle (SPION), as an example of how these assays may be applied to the more general problem of determining the effects of nanomaterial exposure to living cells.

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

A novel flow cytometric HTS assay reveals functional modulators of ATP binding cassette transporter ABCB6.

Directory of Open Access Journals (Sweden)

Kishore Polireddy

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

Clinical and Functional Assays of Radiosensitivity and Radiation-Induced Second Cancer

Directory of Open Access Journals (Sweden)

Mohammad Habash

2017-10-01

Full Text Available Whilst the near instantaneous physical interaction of radiation energy with living cells leaves little opportunity for inter-individual variation in the initial yield of DNA damage, all the downstream processes in how damage is recognized, repaired or resolved and therefore the ultimate fate of cells can vary across the population. In the clinic, this variability is observed most readily as rare extreme sensitivity to radiotherapy with acute and late tissue toxic reactions. Though some radiosensitivity can be anticipated in individuals with known genetic predispositions manifest through recognizable phenotypes and clinical presentations, others exhibit unexpected radiosensitivity which nevertheless has an underlying genetic cause. Currently, functional assays for cellular radiosensitivity represent a strategy to identify patients with potential radiosensitivity before radiotherapy begins, without needing to discover or evaluate the impact of the precise genetic determinants. Yet, some of the genes responsible for extreme radiosensitivity would also be expected to confer susceptibility to radiation-induced cancer, which can be considered another late adverse event associated with radiotherapy. Here, the utility of functional assays of radiosensitivity for identifying individuals susceptible to radiotherapy-induced second cancer is discussed, considering both the common mechanisms and important differences between stochastic radiation carcinogenesis and the range of deterministic acute and late toxic effects of radiotherapy.

In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells.

Science.gov (United States)

Fang, Mingzhu; Kang, Hwan-Goo; Park, Youngil; Estrella, Brian; Zarbl, Helmut

2017-09-28

The circadian rhythm is a fundamental physiological process present in all organisms that regulates biological processes ranging from gene expression to sleep behavior. In vertebrates, circadian rhythm is controlled by a molecular oscillator that functions in both the suprachiasmatic nucleus (SCN; central pacemaker) and individual cells comprising most peripheral tissues. More importantly, disruption of circadian rhythm by exposure to light-at-night, environmental stressors and/or toxicants is associated with increased risk of chronic diseases and aging. The ability to identify agents that can disrupt central and/or peripheral biological clocks, and agents that can prevent or mitigate the effects of circadian disruption, has significant implications for prevention of chronic diseases. Although rodent models can be used to identify exposures and agents that induce or prevent/mitigate circadian disruption, these experiments require large numbers of animals. In vivo studies also require significant resources and infrastructure, and require researchers to work all night. Thus, there is an urgent need for a cell-type appropriate in vitro system to screen for environmental circadian disruptors and enhancers in cell types from different organs and disease states. We constructed a vector that drives transcription of the destabilized luciferase in eukaryotic cells under the control of the human PERIOD 2 gene promoter. This circadian reporter construct was stably transfected into human mammary epithelial cells, and circadian responsive reporter cells were selected to develop the in vitro bioluminescence assay. Here, we present a detailed protocol to establish and validate the assay. We further provide details for proof of concept experiments demonstrating the ability of our in vitro assay to recapitulate the in vivo effects of various chemicals on the cellular biological clock. The results indicate that the assay can be adapted to a variety of cell types to screen for both

Utilization of the Tango beta-arrestin recruitment technology for cell-based EDG receptor assay development and interrogation.

Science.gov (United States)

Wetter, Justin A; Revankar, Chetana; Hanson, Bonnie J

2009-10-01

Cellular assay development for the endothelial differentiation gene (EDG) family of G-protein-coupled receptors (GPCRs) and related lysophospholipid (LP) receptors is complicated by endogenous receptor expression and divergent receptor signaling. Endogenously expressed LP receptors exist in most tissue culture cell lines. These LP receptors, along with other endogenously expressed GPCRs, contribute to off-target signaling that can complicate interpretation of second-messenger-based cellular assay results. These receptors also activate a diverse and divergent set of cellular signaling pathways, necessitating the use of a variety of assay formats with mismatched procedures and functional readouts. This complicates examination and comparison of these receptors across the entire family. The Tango technology uses the conserved beta-arrestin-dependent receptor deactivation process to allow interrogation of the EDG and related receptors with a single functional assay. This method also isolates the target receptor signal, allowing the use of tissue culture cell lines regardless of their endogenous receptor expression. The authors describe the use of this technique to build cell-based receptor-specific assays for all 8 members of the EDG receptor family as well as the related LPA receptors GPR23, GPR92, and GPR87. In addition, they demonstrate the value of this technology for identification and investigation of functionally selective receptor compounds as demonstrated by the immunosuppressive compound FtY720-P and its action at the EDG(1) and EDG(3) receptors.

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...

The single-cell gel electrophoresis assay to determine apoptosis ...

African Journals Online (AJOL)

When the frequency of appearance of apoptotic cells following was observed over a period of time, there was a significant increase in appearance of apoptosis when using single cell gel electrophoresis assay. The present report demonstrates that the characteristic pattern of apoptotic comets detected by the comet assay ...

Fluorescence Adherence Inhibition Assay: A Novel Functional Assessment of Blocking Virus Attachment by Vaccine-Induced Antibodies.

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Atul Asati

Full Text Available Neutralizing antibodies induced by vaccination or natural infection play a critically important role in protection against the viral diseases. In general, neutralization of the viral infection occurs via two major pathways: pre- and post-attachment modes, the first being the most important for such infections as influenza and polio, the latter being significant for filoviruses. Neutralizing capacity of antibodies is typically evaluated by virus neutralization assays that assess reduction of viral infectivity to the target cells in the presence of functional antibodies. Plaque reduction neutralization test, microneutralization and immunofluorescent assays are often used as gold standard virus neutralization assays. However, these methods are associated with several important prerequisites such as use of live virus requiring safety precautions, tedious evaluation procedure and long assessment time. Hence, there is a need for a robust, inexpensive high throughput functional assay that can be performed rapidly using inactivated virus, without extensive safety precautions. Herein, we report a novel high throughput Fluorescence Adherence Inhibition assay (fADI using inactivated virus labeled with fluorescent secondary antibodies virus and Vero cells or erythrocytes as targets. It requires only few hours to assess pre-attachment neutralizing capacity of donor sera. fADI assay was tested successfully on donors immunized with polio, yellow fever and influenza vaccines. To further simplify and improve the throughput of the assay, we have developed a mathematical approach for calculating the 50% titers from a single sample dilution, without the need to analyze multi-point titration curves. Assessment of pre- and post-vaccination human sera from subjects immunized with IPOL®, YF-VAX® and 2013-2014 Fluzone® vaccines demonstrated high efficiency of the assay. The results correlated very well with microneutralization assay performed independently by the FDA

An indicator cell assay for blood-based diagnostics.

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Samuel A Danziger

Full Text Available We have established proof of principle for the Indicator Cell Assay Platform™ (iCAP™, a broadly applicable tool for blood-based diagnostics that uses specifically-selected, standardized cells as biosensors, relying on their innate ability to integrate and respond to diverse signals present in patients' blood. To develop an assay, indicator cells are exposed in vitro to serum from case or control subjects and their global differential response patterns are used to train reliable, disease classifiers based on a small number of features. In a feasibility study, the iCAP detected pre-symptomatic disease in a murine model of amyotrophic lateral sclerosis (ALS with 94% accuracy (p-Value = 3.81E-6 and correctly identified samples from a murine Huntington's disease model as non-carriers of ALS. Beyond the mouse model, in a preliminary human disease study, the iCAP detected early stage Alzheimer's disease with 72% cross-validated accuracy (p-Value = 3.10E-3. For both assays, iCAP features were enriched for disease-related genes, supporting the assay's relevance for disease research.

Single Cell Assay for Analyzing Single Cell Exosome and Endocrine Secretion and Cancer Markers

Science.gov (United States)

Chiu, Yu-Jui

To understand the inhomogeneity of cells in biological systems, there is a growing demand for the capability to characterize the properties of individual single cells. Since single cell studies require continuous monitoring of the cell behaviors instead of a snapshot test at a single time point, an effective single-cell assay that can support time lapsed studies in a high throughput manner is desired. Most currently available single-cell technologies cannot provide proper environments to sustain cell growth and cannot provide, for appropriate cell types, proliferation of single cells and convenient, non-invasive tests of single cell behaviors from molecular markers. In this dissertation, I present a highly versatile single-cell assay that can accommodate different cellular types, enable easy and efficient single cell loading and culturing, and be suitable for the study of effects of in-vitro environmental factors in combination with drug screening. The salient features of the assay are the non-invasive collection and surveying of single cell secretions at different time points and massively parallel translocation of single cells by user defined criteria, producing very high compatibility to the downstream process such as single cell qPCR and sequencing. Above all, the acquired information is quantitative -- for example, one of the studies is measured by the number of exosomes each single cell secretes for a given time period. Therefore, our single-cell assay provides a convenient, low-cost, and enabling tool for quantitative, time lapsed studies of single cell properties.

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

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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.

A Cancer Cell-Activatable Aptamer-Reporter System for One-Step Assay of Circulating Tumor Cells

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Zihua Zeng

2014-01-01

Full Text Available The current antibody-mediated numeration assays of circulating tumor cells (CTCs require multiple steps and are time-consuming. To overcome these technical limitations, a cancer cell-activatable aptamer-reporter was formulated by conjugating a biomarker-specific aptamer sequence with paired fluorochrome-quencher molecules. In contrast to the antibody probes, the intact aptamer-reporter was optically silent in the absence of cells of interest. However, when used in an assay, the aptamer selectively targeted cancer cells through interaction with a specific surface biomarker, which triggered internalization of the aptamer-reporter and, subsequently, into cell lysosomes. Rapid lysosomal degradation of the aptamer-reporter resulted in separation of the paired fluorochrome-quencher molecules. The released fluorochrome emitted bright fluorescent signals exclusively within the targeted cancer cells, with no background noise in the assay. Thus, the assays could be completed in a single step within minutes. By using this one-step assay, CTCs in whole blood and marrow aspirate samples of patients with lymphoma tumors were selectively highlighted and rapidly detected with no off-target signals from background blood cells. The development of the cancer cell-activatable aptamer-reporter system allows for the possibility of a simple and robust point-of-care test for CTC detection, which is currently unavailable.

Multiplex bio-assay with inductively coupled plasma mass spectrometry: Towards a massively multivariate single-cell technology

International Nuclear Information System (INIS)

Tanner, Scott D.; Ornatsky, Olga; Bandura, Dmitry R.; Baranov, Vladimir I.

2007-01-01

Recent progress in the development of massively multiplexed bioanalytical assays using element tags with inductively coupled plasma mass spectrometry detection is reviewed. Feasibility results using commercially available secondary immunolabeling reagents for leukemic cell lines are presented. Multiplex analysis of higher order is shown with first generation tag reagents based on functionalized carriers that bind lanthanide ions. DNA quantification using metallointercalation allows for cell enumeration or mitotic state differentiation. In situ hybridization permits the determination of cellular RNA. The results provide a feasibility basis for the development of a multivariate assay tool for individual cell analysis based on inductively coupled plasma mass spectrometry in a cytometer configuration

Multiplex bio-assay with inductively coupled plasma mass spectrometry: Towards a massively multivariate single-cell technology

Energy Technology Data Exchange (ETDEWEB)

Tanner, Scott D. [Institute of Biomaterials and Biomedical Engineering, University of Toronto, Room 407, 164 College Street, Toronto, Ontario, M5S 3G9 (Canada)], E-mail: sd.tanner@utoronto.ca; Ornatsky, Olga; Bandura, Dmitry R.; Baranov, Vladimir I. [Institute of Biomaterials and Biomedical Engineering, University of Toronto, Room 407, 164 College Street, Toronto, Ontario, M5S 3G9 (Canada)

2007-03-15

Recent progress in the development of massively multiplexed bioanalytical assays using element tags with inductively coupled plasma mass spectrometry detection is reviewed. Feasibility results using commercially available secondary immunolabeling reagents for leukemic cell lines are presented. Multiplex analysis of higher order is shown with first generation tag reagents based on functionalized carriers that bind lanthanide ions. DNA quantification using metallointercalation allows for cell enumeration or mitotic state differentiation. In situ hybridization permits the determination of cellular RNA. The results provide a feasibility basis for the development of a multivariate assay tool for individual cell analysis based on inductively coupled plasma mass spectrometry in a cytometer configuration.

Automated image-based assay for evaluation of HIV neutralization and cell-to-cell fusion inhibition.

Science.gov (United States)

Sheik-Khalil, Enas; Bray, Mark-Anthony; Özkaya Şahin, Gülsen; Scarlatti, Gabriella; Jansson, Marianne; Carpenter, Anne E; Fenyö, Eva Maria

2014-08-30

Standardized techniques to detect HIV-neutralizing antibody responses are of great importance in the search for an HIV vaccine. Here, we present a high-throughput, high-content automated plaque reduction (APR) assay based on automated microscopy and image analysis that allows evaluation of neutralization and inhibition of cell-cell fusion within the same assay. Neutralization of virus particles is measured as a reduction in the number of fluorescent plaques, and inhibition of cell-cell fusion as a reduction in plaque area. We found neutralization strength to be a significant factor in the ability of virus to form syncytia. Further, we introduce the inhibitory concentration of plaque area reduction (ICpar) as an additional measure of antiviral activity, i.e. fusion inhibition. We present an automated image based high-throughput, high-content HIV plaque reduction assay. This allows, for the first time, simultaneous evaluation of neutralization and inhibition of cell-cell fusion within the same assay, by quantifying the reduction in number of plaques and mean plaque area, respectively. Inhibition of cell-to-cell fusion requires higher quantities of inhibitory reagent than inhibition of virus neutralization.

A novel dendritic cell-based direct ex vivo assay for detection and enumeration of circulating antigen-specific human T cells.

Science.gov (United States)

Carrio, Roberto; Zhang, Ge; Drake, Donald R; Schanen, Brian C

2018-05-07

Although a variety of assays have been used to examine T cell responses in vitro, standardized ex vivo detection of antigen-specific CD4 + T cells from human circulatory PBMCs remains constrained by low-dimensional characterization outputs and the need for polyclonal, mitogen-induced expansion methods to generate detectable response signals. To overcome these limitations, we developed a novel methodology utilizing antigen-pulsed autologous human dendritic target cells in a rapid and sensitive assay to accurately enumerate antigen-specific CD4 + T cell precursor frequency by multiparametric flow cytometry. With this approach, we demonstrate the ability to reproducibly quantitate poly-functional T cell responses following both primary and recall antigenic stimulation. Furthermore, this approach enables more comprehensive phenotypic profiling of circulating antigen-specific CD4 + T cells, providing valuable insights into the pre-existing polarization of antigen-specific T cells in humans. Combined, this approach permits sensitive and detailed ex vivo detection of antigen-specific CD4 + T cells delivering an important tool for advancing vaccine, immune-oncology and other therapeutic studies.

An improved method for staining cell colonies in clonogenic assays

OpenAIRE

Guda, Kishore; Natale, Leanna; Markowitz, Sanford D.

2007-01-01

Clonogenic assay is a widely used experimental approach to test for the effects of drugs/genes on the growth and proliferative characteristics of cells in vitro. Accurate quantitation of treatment effects in clonogeneic assays depends on the ability to visualize and count cell colonies precisely. We report a novel method (referred as ETeB) for staining cell colonies grown on plastic and specially coated substrates like collagen. Using colon cancer cell lines grown on plastic and collagen, we ...

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)

Zhu, Xiaoming; Fu, Afu; Luo, Kathy Qian

2012-01-01

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.

Methods for transient assay of gene function in floral tissues

Directory of Open Access Journals (Sweden)

Pathirana Nilangani N

2007-01-01

Full Text Available Abstract Background There is considerable interest in rapid assays or screening systems for assigning gene function. However, analysis of gene function in the flowers of some species is restricted due to the difficulty of producing stably transformed transgenic plants. As a result, experimental approaches based on transient gene expression assays are frequently used. Biolistics has long been used for transient over-expression of genes of interest, but has not been exploited for gene silencing studies. Agrobacterium-infiltration has also been used, but the focus primarily has been on the transient transformation of leaf tissue. Results Two constructs, one expressing an inverted repeat of the Antirrhinum majus (Antirrhinum chalcone synthase gene (CHS and the other an inverted repeat of the Antirrhinum transcription factor gene Rosea1, were shown to effectively induce CHS and Rosea1 gene silencing, respectively, when introduced biolistically into petal tissue of Antirrhinum flowers developing in vitro. A high-throughput vector expressing the Antirrhinum CHS gene attached to an inverted repeat of the nos terminator was also shown to be effective. Silencing spread systemically to create large zones of petal tissue lacking pigmentation, with transmission of the silenced state spreading both laterally within the affected epidermal cell layer and into lower cell layers, including the epidermis of the other petal surface. Transient Agrobacterium-mediated transformation of petal tissue of tobacco and petunia flowers in situ or detached was also achieved, using expression of the reporter genes GUS and GFP to visualise transgene expression. Conclusion We demonstrate the feasibility of using biolistics-based transient RNAi, and transient transformation of petal tissue via Agrobacterium infiltration to study gene function in petals. We have also produced a vector for high throughput gene silencing studies, incorporating the option of using T-A cloning to

Cell-mediated cytotoxicity for melanome tumor cells: detection by a (3H)proline release assay

International Nuclear Information System (INIS)

Saal, J.G.; Rieber, E.P.; Riethmueller, G.

1976-01-01

An in vitro lymphocyte-mediated cytotoxicity assay using [ 3 H]proline-labelled target cells is described. The assay, modified from an original procedure of Bean et al., assesses the release of [ 3 H]proline by filtering the total culture fluid containing both trypsinised tumor cells and effector cells. Filtration is performed with a semiautomatic harvesting device using low suction pressure and large-diameter glass filters. Pretreatment of filters with whole serum diminishes adsorption of cell-free radioactive material considerably and thus increases the sensitivity of the assay. Nearly 100% of the radioactivity could be recovered with this harvesting device. The technique allowed the detection of cytolytic activities of lymphocytes after 6 h of incubation. Lymphocytes from patients with primary malignant melanoma showed a significantly higher cytolytic reactivity (p > 0.001) than normal donors' lymphocytes against three different melanoma cell lines. In a series of parallel experiments on 36 patients and 18 normal donors, this modification of the [ 3 ]proline test was compared with three different assays: the conventional microcytotoxicity test of Takasugi and Klein, the original [ 3 H]proline microcytotoxicity test of Bean et al., and the viability count of tumor cells. (Auth.)

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.

Limit-dilution assay and clonal expansion of all T cells capable of proliferation

International Nuclear Information System (INIS)

Chen, W.-F.; Wilson, A.; Scollay, R.; Shortman, K.

1982-01-01

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 [ 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. (Auth.)

Alkaline Comet Assay for Assessing DNA Damage in Individual Cells.

Science.gov (United States)

Pu, Xinzhu; Wang, Zemin; Klaunig, James E

2015-08-06

Single-cell gel electrophoresis, commonly called a comet assay, is a simple and sensitive method for assessing DNA damage at the single-cell level. It is an important technique in genetic toxicological studies. The comet assay performed under alkaline conditions (pH >13) is considered the optimal version for identifying agents with genotoxic activity. The alkaline comet assay is capable of detecting DNA double-strand breaks, single-strand breaks, alkali-labile sites, DNA-DNA/DNA-protein cross-linking, and incomplete excision repair sites. The inclusion of digestion of lesion-specific DNA repair enzymes in the procedure allows the detection of various DNA base alterations, such as oxidative base damage. This unit describes alkaline comet assay procedures for assessing DNA strand breaks and oxidative base alterations. These methods can be applied in a variety of cells from in vitro and in vivo experiments, as well as human studies. Copyright © 2015 John Wiley & Sons, Inc.

A novel multiparametric flow cytometry-based cytotoxicity assay simultaneously immunophenotypes effector cells: comparisons to a 4 h 51Cr-release assay.

Science.gov (United States)

Kim, G G; Donnenberg, V S; Donnenberg, A D; Gooding, W; Whiteside, T L

2007-08-31

Natural killer (NK) cell-or T cell-mediated cytotoxicity traditionally is measured in 4-16 h (51)Cr-release assays (CRA). A new four-color flow cytometry-based cytotoxicity assay (FCC) was developed to simultaneously measure NK cell cytotoxicity and NK cell phenotype (CD3(-)CD16(+)CD56(+)). Target cells, K562 or Daudi, were labeled with Cell Tracker Orange (CTO) prior to the addition of effector cells. Following co-incubation, 7 amino-actinomycin D (7-AAD) was added to measure death of target cells. The phenotype of effectors, viability of targets, the formation of tumor-effector cell conjugates and absolute numbers of all cells were measured based on light scatter (FSC/SSC), double discrimination of the fluorescence peak integral and height, and fluorescence intensity. Kinetic studies (0.5 and 1 to 4 h) at different effector to target (E:T) cell ratios (50, 25, 12, and 6) confirmed that the 3 h incubation was optimal. The FCC assay is more sensitive than the CRA, has a coefficient of variation (CV) 8-13% and reliably measures NK cell-or lymphokine-activated killer (LAK) cell-mediated killing of target cells in normal controls and subjects with cancer. The FCC assay can be used to study a range of phenotypic attributes, in addition to lytic activity of various subsets of effector cells, without radioactive tracers and thus, it is relatively inexpensive. The FCC assay has a potential for providing information about molecular interactions underlying target cell lysis and thus becoming a major tool for studies of disease pathogenesis as well as development of novel immune therapies.

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

Quantifying rates of cell migration and cell proliferation in co-culture barrier assays reveals how skin and melanoma cells interact during melanoma spreading and invasion.

Science.gov (United States)

Haridas, Parvathi; Penington, Catherine J; McGovern, Jacqui A; McElwain, D L Sean; Simpson, Matthew J

2017-06-21

Malignant spreading involves the migration of cancer cells amongst other native cell types. For example, in vivo melanoma invasion involves individual melanoma cells migrating through native skin, which is composed of several distinct subpopulations of cells. Here, we aim to quantify how interactions between melanoma and fibroblast cells affect the collective spreading of a heterogeneous population of these cells in vitro. We perform a suite of circular barrier assays that includes: (i) monoculture assays with fibroblast cells; (ii) monoculture assays with SK-MEL-28 melanoma cells; and (iii) a series of co-culture assays initiated with three different ratios of SK-MEL-28 melanoma cells and fibroblast cells. Using immunostaining, detailed cell density histograms are constructed to illustrate how the two subpopulations of cells are spatially arranged within the spreading heterogeneous population. Calibrating the solution of a continuum partial differential equation to the experimental results from the monoculture assays allows us to estimate the cell diffusivity and the cell proliferation rate for the melanoma and the fibroblast cells, separately. Using the parameter estimates from the monoculture assays, we then make a prediction of the spatial spreading in the co-culture assays. Results show that the parameter estimates obtained from the monoculture assays lead to a reasonably accurate prediction of the spatial arrangement of the two subpopulations in the co-culture assays. Overall, the spatial pattern of spreading of the melanoma cells and the fibroblast cells is very similar in monoculture and co-culture conditions. Therefore, we find no clear evidence of any interactions other than cell-to-cell contact and crowding effects. Copyright © 2017 Elsevier Ltd. All rights reserved.

Drug-permeability and transporter assays in Caco-2 and MDCK cell lines.

Science.gov (United States)

Volpe, Donna A

2011-12-01

The human colon adenocarcinoma Caco-2 and Madin-Darby canine kidney epithelial cell lines provide in vitro tools to assess a drug's permeability and transporter interactions during discovery and development. The cells, when cultured on semiporous filters, form confluent monolayers that model the intestinal epithelial barrier for permeability, transporter and drug-interaction assays. The applications of these assays in pharmaceutical research include qualitative prediction and ranking of absorption, determining mechanism(s) of permeability, formulation effects on drug permeability, and the potential for transporter-mediated drug-drug interactions. This review focuses on recent examples of Caco-2 and Madin-Darby canine kidney cells assays for drug permeability including transfected and knock-down cells, miniaturization and automation, and assay combinations to better understand and predict intestinal drug absorption.

Inter-experiment variation and dependence on culture conditions in assaying the chemosensitivity of human small cell lung cancer cell lines

DEFF Research Database (Denmark)

Roed, H; Christensen, I B; Vindeløv, L L

1987-01-01

by a logarithmic function. Even after correction for lack of proportionality the two assay systems provided significantly different dose-response curves. The stability of the chemosensitivity was tested after 25-30 weeks continuous in vitro culture or prolonged storage in liquid nitrogen. One cell line underwent...... significant changes after continuous in vitro culture whereas the cell lines tested after prolonged storage in liquid nitrogen showed only minor changes. It is concluded that instead of considering the concentration necessary to achieve a certain degree of cell kill (e.g. ID50) in one experiment on one cell...

A novel multiparametric flow cytometry-based cytotoxicity assay simultaneously immunophenotypes effector cells: Comparisons to a 4 h 51Cr-release assay

OpenAIRE

Kim, GG; Donnenberg, VS; Donnenberg, AD; Gooding, W; Whiteside, TL

2007-01-01

Natural killer (NK) cell- or T cell-mediated cytotoxicity traditionally is measured in 4-16h 51Cr-release assays (CRA). A new four-color flow cytometry-based cytotoxicity assay (FCC) was developed to simultaneously measure NK cell cytotoxicity and NK cell phenotype (CD3−CD16+CD56+). Target cells, K562 or Daudi, were labeled with Cell Tracker Orange (CTO) prior to the addition of effector cells. Following co-incubation, 7 amino-actinomycin D (7-AAD) was added to measure death of target cells. ...

Evaluation of royal jelly as an alternative to fetal bovine serum in cell culture using cell proliferation assays and live cell imaging.

Science.gov (United States)

Musa, Marahaini; Nasir, Nurul Fatihah Mohamad; Thirumulu, Kannan Ponnuraj

2014-01-01

Royal jelly is a nutritious substance produced by the young nurse bees and contains significant amounts of proteins which are important for cell growth and proliferation. The aim of this study was to evaluate the effect of royal jelly as an alternative to fetal bovine serum (FBS) in cell culture using cell proliferation assays and live cell imaging. MRC-5 cells were treated with various concentrations of royal jelly extract in MTT assay. The control groups were comprised of Alpha-Minimal Essential Medium (α-MEM) alone and α-MEM with 10% FBS. Subsequently, the cell proliferation was studied for 10 days using Alamar Blue assay and live cell imaging from 48 to 72 h. The population doubling time (PDT) was determined using trypan blue assay after live cell imaging. In MTT assay, 0.156 and 0.078 mg/ml of royal jelly produced higher cell viability compared to positive control group but were not significantly different (P > 0.05). In the Alamar Blue assay, 0.156 and 0.078 mg/ml of royal jelly produced greater percentage of reduction at day 3 even though no significant difference was found (P > 0.05). Based on live cell imaging, the PDT for positive, negative, 0.156 and 0.078 mg/ml of royal jelly groups were 29.09, 62.50, 41.67 and 41.67 h respectively. No significant difference was found in the PDT between all the groups (P > 0.05). Royal jelly does not exhibit similar ability like FBS to facilitate cell growth under the present test conditions.

Automation of cell-based drug absorption assays in 96-well format using permeable support systems.

Science.gov (United States)

Larson, Brad; Banks, Peter; Sherman, Hilary; Rothenberg, Mark

2012-06-01

Cell-based drug absorption assays, such as Caco-2 and MDCK-MDR1, are an essential component of lead compound ADME/Tox testing. The permeability and transport data they provide can determine whether a compound continues in the drug discovery process. Current methods typically incorporate 24-well microplates and are performed manually. Yet the need to generate absorption data earlier in the drug discovery process, on an increasing number of compounds, is driving the use of higher density plates. A simple, more efficient process that incorporates 96-well permeable supports and proper instrumentation in an automated process provides more reproducible data compared to manual methods. Here we demonstrate the ability to perform drug permeability and transport assays using Caco-2 or MDCKII-MDR1 cells. The assay procedure was automated in a 96-well format, including cell seeding, media and buffer exchanges, compound dispense, and sample removal using simple robotic instrumentation. Cell monolayer integrity was confirmed via transepithelial electrical resistance and Lucifer yellow measurements. Proper cell function was validated by analyzing apical-to-basolateral and basolateral-to-apical movement of rhodamine 123, a known P-glycoprotein substrate. Apparent permeability and efflux data demonstrate how the automated procedure provides a less variable method than manual processing, and delivers a more accurate assessment of a compound's absorption characteristics.

Functional diagnostics for thyrotropin hormone receptor autoantibodies: bioassays prevail over binding assays.

Science.gov (United States)

Lytton, Simon David; Schluter, Anke; Banga, Paul J

2018-06-01

Autoantibodies to the thyrotropin hormone receptor (TSH-R) are directly responsible for the hyperthyroidism in Graves' disease and mediate orbital manifestations in Graves' orbitopathy (otherwise known as thyroid eye disease). These autoantibodies are heterogeneous in their function and collectively referred to as TRAbs. Measurement of TRAbs is clinically important for diagnosis of a variety of conditions and different commercial assays with high sensitivity and specificity are available for diagnostic purposes. This review provides overwhelming evidence that the TRAbs detected in binding assays by mainly the automated electrochemical luminescence immunoassays (ECLIA) do not distinguish TRAbs that stimulate the TSH-R (called TSIs or TSAbs) and TRAbs that just inhibit the binding of TSH without stimulating the TSH-R (called TBAbs). However, TSAbs and TBAbs have divergent pathogenic roles, and depending which fraction predominates cause different clinical symptoms and engender different therapeutic regimen. Therefore, diagnostic distinction of TSAbs and TBAbs is of paramount clinical importance. To date, only bioassays such as the Mc4 TSH-R bioassay (Thyretain TM , Quidel) and the Bridge assay (Immulite 2000, Siemens) can measure TSAbs, with only the former being able to distinguish between TSAbs and TBAbs. On this note, it is strongly recommended to only use the term TSI or TSAb when reporting the results of bioassays, whereas the results of automated TRAb binding assays should be reported as TRAbs (of undetermined functional significance). This review aims to present a technical and analytical account of leading commercial diagnostic methods of anti-TSH-R antibodies, a metaanalysis of their clinical performance and a perspective for the use of cell based TSH-R bioassays in the clinical diagnostics of Graves' disease.

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.

Development and characterization of a Rift Valley fever virus cell-cell fusion assay using alphavirus replicon vectors

International Nuclear Information System (INIS)

Filone, Claire Marie; Heise, Mark; Doms, Robert W.; Bertolotti-Ciarlet, Andrea

2006-01-01

Rift Valley fever virus (RVFV), a member of the Phlebovirus genus in the Bunyaviridae family, is transmitted by mosquitoes and infects both humans and domestic animals, particularly cattle and sheep. Since primary RVFV strains must be handled in BSL-3+ or BSL-4 facilities, a RVFV cell-cell fusion assay will facilitate the investigation of RVFV glycoprotein function under BSL-2 conditions. As for other members of the Bunyaviridae family, RVFV glycoproteins are targeted to the Golgi, where the virus buds, and are not efficiently delivered to the cell surface. However, overexpression of RVFV glycoproteins using an alphavirus replicon vector resulted in the expression of the glycoproteins on the surface of multiple cell types. Brief treatment of RVFV glycoprotein expressing cells with mildly acidic media (pH 6.2 and below) resulted in rapid and efficient syncytia formation, which we quantified by β-galactosidase α-complementation. Fusion was observed with several cell types, suggesting that the receptor(s) for RVFV is widely expressed or that this acid-dependent virus does not require a specific receptor to mediate cell-cell fusion. Fusion occurred over a broad temperature range, as expected for a virus with both mosquito and mammalian hosts. In contrast to cell fusion mediated by the VSV-G glycoprotein, RVFV glycoprotein-dependent cell fusion could be prevented by treating target cells with trypsin, indicating that one or more proteins (or protein-associated carbohydrate) on the host cell surface are needed to support membrane fusion. The cell-cell fusion assay reported here will make it possible to study the membrane fusion activity of RVFV glycoproteins in a high-throughput format and to screen small molecule inhibitors for the ability to block virus-specific membrane fusion

Comparison of cell-based assays for the identification and evaluation of competitive CXCR4 inhibitors.

Directory of Open Access Journals (Sweden)

Anneleen Van Hout

Full Text Available The chemokine receptor CXCR4 is activated by its unique chemokine ligand CXCL12 and regulates many physiological and developmental processes such as hematopoietic cell trafficking. CXCR4 is also one of the main co-receptors for human immunodeficiency virus (HIV entry. Dysfunction of the CXCL12/CXCR4 axis contributes to several human pathologies, including cancer and inflammatory diseases. Consequently, inhibition of CXCR4 activation is recognized as an attractive target for therapeutic intervention. In this regard, numerous agents modifying CXCR4 activity have been evaluated in in vitro experimental studies and pre-clinical models. Here, we evaluated a CXCL12 competition binding assay for its potential as a valuable initial screen for functional and competitive CXCR4 inhibitors. In total, 11 structurally diverse compounds were included in a side-by-side comparison of in vitro CXCR4 cell-based assays, such as CXCL12 competition binding, CXCL12-induced calcium signaling, CXCR4 internalization, CXCL12-guided cell migration and CXCR4-specific HIV-1 replication experiments. Our data indicated that agents that inhibit CXCL12 binding, i.e. the anti-CXCR4 peptide analogs T22, T140 and TC14012 and the small molecule antagonists AMD3100, AMD3465, AMD11070 and IT1t showed inhibitory activity with consistent relative potencies in all further applied CXCR4-related assays. Accordingly, agents exerting no or very weak receptor binding (i.e., CTCE-9908, WZ811, Me6TREN and gambogic acid showed no or very poor anti-CXCR4 inhibitory activity. Thus, CXCL12 competition binding studies were proven to be highly valuable as an initial screening assay and indicative for the pharmacological and functional profile of competitive CXCR4 antagonists, which will help the design of new potent CXCR4 inhibitors.

In vitro Cell Viability by CellProfiler® Software as Equivalent to MTT Assay.

Science.gov (United States)

Gasparini, Luciana S; Macedo, Nayana D; Pimentel, Elisângela F; Fronza, Marcio; Junior, Valdemar L; Borges, Warley S; Cole, Eduardo R; Andrade, Tadeu U; Endringer, Denise C; Lenz, Dominik

2017-07-01

This study evaluated in vitro cell viability by the colorimetric MTT stands for 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay compared to image analysis by CellProfiler ® software. Hepatoma (Hepa-1c1c7) and fibroblast (L929) cells were exposed to isolated substances, camptothecin, lycorine, tazettine, albomaculine, 3-epimacronine, trispheridine, galanthine and Padina gymnospora , Sargassum sp. methanolic extract, and Habranthus itaobinus Ravenna ethyl acetate in different concentrations. After MTT assay, cells were stained with Panotic dye kit. Cell images were obtained with an inverted microscope equipped with a digital camera. The images were analyzed by CellProfiler ® . No cytotoxicity at the highest concentration analyzed for 3-epimacronine, albomaculine, galanthine, trispheridine, P. gymnospora extract and Sargassum sp. extract where detected. Tazettine offered cytotoxicity only against the Hepa1c1c7 cell line. Lycorine, camptothecin, and H. itaobinus extract exhibited cytotoxic effects in both cell lines. The viability methods tested were correlated demonstrated by Bland-Atman test with normal distribution with mean difference between the two methods close to zero, bias value 3.0263. The error was within the limits of the confidence intervals and these values had a narrow difference. The correlation between the two methods was demonstrated by the linear regression plotted as R 2 . CellProfiler ® image analysis presented similar results to the MTT assay in the identification of viable cells, and image analysis may assist part of biological analysis procedures. The presented methodology is inexpensive and reproducible. In vitro cell viability assessment with MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay may be replaced by image analysis by CellProfiler ® . The viability methods

Multiplex High-Throughput Targeted Proteomic Assay To Identify Induced Pluripotent Stem Cells.

Science.gov (United States)

Baud, Anna; Wessely, Frank; Mazzacuva, Francesca; McCormick, James; Camuzeaux, Stephane; Heywood, Wendy E; Little, Daniel; Vowles, Jane; Tuefferd, Marianne; Mosaku, Olukunbi; Lako, Majlinda; Armstrong, Lyle; Webber, Caleb; Cader, M Zameel; Peeters, Pieter; Gissen, Paul; Cowley, Sally A; Mills, Kevin

2017-02-21

Induced pluripotent stem cells have great potential as a human model system in regenerative medicine, disease modeling, and drug screening. However, their use in medical research is hampered by laborious reprogramming procedures that yield low numbers of induced pluripotent stem cells. For further applications in research, only the best, competent clones should be used. The standard assays for pluripotency are based on genomic approaches, which take up to 1 week to perform and incur significant cost. Therefore, there is a need for a rapid and cost-effective assay able to distinguish between pluripotent and nonpluripotent cells. Here, we describe a novel multiplexed, high-throughput, and sensitive peptide-based multiple reaction monitoring mass spectrometry assay, allowing for the identification and absolute quantitation of multiple core transcription factors and pluripotency markers. This assay provides simpler and high-throughput classification into either pluripotent or nonpluripotent cells in 7 min analysis while being more cost-effective than conventional genomic tests.

In vitro assays for predicting tumor cell response to radiation by apoptotic pathways

International Nuclear Information System (INIS)

Algan, Oe.; Hanks, G.E.; Biade, S.; Chapman, J.D.

1995-01-01

Purpose: We had previously shown that the rate of spontaneous and radiation-induced apoptosis was significantly greater in well-differentiated compared to anaplastic Dunning prostate carcinomas. The goal of this study was to define the most useful assay for quantifying radiation-induced apoptotic cell death and to determine if measured rates of radiation-induced apoptosis in tumor cell populations can predict treatment outcome. Materials and Methods: The time course and extent of radiation-induced apoptosis after single doses of Cesium-137 gamma-rays were measured by five different assays. These included gross DNA degradation, nucleosome ladder formation, labeling of 3'-OH ends in DNA with an immunofluorescence probe, immunofluorescence vital stains (LIVE/DEAD[reg] EUKOLIGHT TM ) and trypan blue. The majority of these studies were performed with DU-145 human prostate cells. Data was analyzed to determine the component of cell inactivation resulting from apoptosis with the modified linear quadratic equation, -1n (SF) = (α a + α p ) D + β p D 2 , were α a represents cell inactivation by radiation-induced apoptosis, α p and β p represent cell death by proliferative mechanisms and D represents radiation dose. Results: These studies indicated that DU-145 cell death after radiation occurs over two distinct time periods. The first phase of death begins shortly after irradiation and plateaus within 16-24 hr. This process of cell death has properties consistent with apoptosis as determined by 3'-OH DNA end-labeling and nucleosome ladder assays. The second phase of cell death (determined by viability staining) begins approximately 48 hr after irradiation and continues until the remainder of inactivated cells express their death. This longer phase of cell inactivation probably represents proliferative cell death and other non-apoptotic mechanisms. The five different assays were performed on DU-145 cells 24 hr after irradiation with 10 Gy. Significant nucleosome ladders

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.

Platelet-free shear flow assay facilitates analysis of shear-dependent functions of VWF and ADAMTS13.

Science.gov (United States)

Kraus, Emma; Kraus, Kristina; Obser, Tobias; Oyen, Florian; Klemm, Ulrike; Schneppenheim, Reinhard; Brehm, Maria A

2014-12-01

The multimeric form of von Willebrand factor (VWF), is the largest soluble protein in mammals and exhibits a multidomain structure resulting in multiple functions. Upon agonist stimulation endothelial cells secrete VWF multimers from Weibel-Palade bodies into the blood stream where VWF plays an essential role in platelet-dependent primary hemostasis. Elongation of VWF strings on the cells' surface leads to accessibility of VWF binding sites for proteins, such as platelet membrane glycoprotein Ib. The prothrombotic strings are size-regulated by the metalloprotease ADAMTS13 by shear force-activated proteolytic cleavage. VWF string formation was induced by histamine stimulation of HUVEC cells under unidirectional shear flow and VWF strings were detected employing the VWF binding peptide of platelet glycoprotein Ib coupled to latex beads. VWF strings were then used as substrate for kinetic studies of recombinant and plasma ADAMTS13. To investigate specific aspects of the shear-dependent functions of VWF and ADAMTS13, we developed a shear flow assay that allows observation of VWF string formation and their degradation by ADAMTS13 without the need for isolated platelets. Our assay specifically detects VWF strings, can be coupled with fluorescent applications and allows semi-automated, quantitative assessment of recombinant and plasma ADAMTS13 activity. Our assay may serve as a valuable research tool to investigate the biochemical characteristics of VWF and ADAMTS13 under shear flow and could complement diagnostics of von Willebrand Disease and Thrombotic Thrombocytopenic Purpura as it allows detection of shear flow-dependent dysfunction of VWD-associated VWF mutants as well as TTP-associated ADAMTS13 mutants. Copyright © 2014 Elsevier Ltd. All rights reserved.

Automatic cell cloning assay for determining the clonogenic capacity of cancer and cancer stem-like cells.

Science.gov (United States)

Fedr, Radek; Pernicová, Zuzana; Slabáková, Eva; Straková, Nicol; Bouchal, Jan; Grepl, Michal; Kozubík, Alois; Souček, Karel

2013-05-01

The clonogenic assay is a well-established in vitro method for testing the survival and proliferative capability of cells. It can be used to determine the cytotoxic effects of various treatments including chemotherapeutics and ionizing radiation. However, this approach can also characterize cells with different phenotypes and biological properties, such as stem cells or cancer stem cells. In this study, we implemented a faster and more precise method for assessing the cloning efficiency of cancer stem-like cells that were characterized and separated using a high-speed cell sorter. Cell plating onto a microplate using an automatic cell deposition unit was performed in a single-cell or dilution rank mode by the fluorescence-activated cell sorting method. We tested the new automatic cell-cloning assay (ACCA) on selected cancer cell lines and compared it with the manual approach. The obtained results were also compared with the results of the limiting dilution assay for different cell lines. We applied the ACCA to analyze the cloning capacity of different subpopulations of prostate and colon cancer cells based on the expression of the characteristic markers of stem (CD44 and CD133) and cancer stem cells (TROP-2, CD49f, and CD44). Our results revealed that the novel ACCA is a straightforward approach for determining the clonogenic capacity of cancer stem-like cells identified in both cell lines and patient samples. Copyright © 2013 International Society for Advancement of Cytometry.

Single-cell microgel electrophoresis: an in vitro assay of radiosensitivity

International Nuclear Information System (INIS)

Deeley, J.O.T.; Moore, J.L.

1993-01-01

The results obtained by a microgel electrophoresis are comparable to conventional gel electrophoresis and elution techniques (Singh et al, 1989), DNA precipitation, alkali unwinding and cell clonogenicity assays (Olive et al, 1990). Since single cells are assessed, microgel electrophoresis is particularly appropriate for end-points such as the intercell variation in response. The simplicity, low cost and rapidity of microgel electrophoresis compared with other assays makes it particularly attractive for assessing the effects on DNA of radiation and other genotoxic agents on the general population. (Author)

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.

In vivo Comet assay--statistical analysis and power calculations of mice testicular cells.

Science.gov (United States)

Hansen, Merete Kjær; Sharma, Anoop Kumar; Dybdahl, Marianne; Boberg, Julie; Kulahci, Murat

2014-11-01

The in vivo Comet assay is a sensitive method for evaluating DNA damage. A recurrent concern is how to analyze the data appropriately and efficiently. A popular approach is to summarize the raw data into a summary statistic prior to the statistical analysis. However, consensus on which summary statistic to use has yet to be reached. Another important consideration concerns the assessment of proper sample sizes in the design of Comet assay studies. This study aims to identify a statistic suitably summarizing the % tail DNA of mice testicular samples in Comet assay studies. A second aim is to provide curves for this statistic outlining the number of animals and gels to use. The current study was based on 11 compounds administered via oral gavage in three doses to male mice: CAS no. 110-26-9, CAS no. 512-56-1, CAS no. 111873-33-7, CAS no. 79-94-7, CAS no. 115-96-8, CAS no. 598-55-0, CAS no. 636-97-5, CAS no. 85-28-9, CAS no. 13674-87-8, CAS no. 43100-38-5 and CAS no. 60965-26-6. Testicular cells were examined using the alkaline version of the Comet assay and the DNA damage was quantified as % tail DNA using a fully automatic scoring system. From the raw data 23 summary statistics were examined. A linear mixed-effects model was fitted to the summarized data and the estimated variance components were used to generate power curves as a function of sample size. The statistic that most appropriately summarized the within-sample distributions was the median of the log-transformed data, as it most consistently conformed to the assumptions of the statistical model. Power curves for 1.5-, 2-, and 2.5-fold changes of the highest dose group compared to the control group when 50 and 100 cells were scored per gel are provided to aid in the design of future Comet assay studies on testicular cells. Copyright © 2014 Elsevier B.V. All rights reserved.

Assessing Clinical Outcomes in Colorectal Cancer with Assays for Invasive Circulating Tumor Cells.

Science.gov (United States)

Zhang, Yue; Zarrabi, Kevin; Hou, Wei; Madajewicz, Stefan; Choi, Minsig; Zucker, Stanley; Chen, Wen-Tien

2018-06-06

Colorectal carcinoma (CRC) is the second leading cause of cancer-related mortality. The goals of this study are to evaluate the association between levels of invasive circulating tumor cells (iCTCs) with CRC outcomes and to explore the molecular characteristics of iCTCs. Peripheral blood from 93 patients with Stage I⁻IV CRC was obtained and assessed for the detection and characterization of iCTCs using a functional collagen-based adhesion matrix (CAM) invasion assay. Patients were followed and assessed for overall survival. Tumor cells isolated by CAM were characterized using cell culture and microarray analyses. Of 93 patients, 88 (95%) had detectable iCTCs, ranging over 0⁻470 iCTCs/mL. Patients with Stage I⁻IV disease exhibited median counts of 0.0 iCTCs/mL ( n = 6), 13.0 iCTCs/mL ( n = 12), 41.0 iCTCs/mL ( n = 12), and 133.0 iCTCs/mL ( n = 58), respectively ( p < 0.001). Kaplan⁻Meier curve analysis demonstrated a significant survival benefit in patients with low iCTC counts compared with in patients with high iCTC counts (log-rank p < 0.001). Multivariable Cox model analysis revealed that iCTC count was an independent prognostic factor of overall survival ( p = 0.009). Disease stage ( p = 0.01, hazard ratio 1.66; 95% confidence interval: 1.12⁻2.47) and surgical intervention ( p = 0.03, HR 0.37; 95% CI: 0.15⁻0.92) were also independent prognostic factors. Gene expression analysis demonstrated the expression of both endothelial and tumor progenitor cell biomarkers in iCTCs. CAM-based invasion assay shows a high detection sensitivity of iCTCs that inversely correlated with overall survival in CRC patients. Functional and gene expression analyses showed the phenotypic mosaics of iCTCs, mimicking the survival capability of circulating endothelial cells in the blood stream.

Mammalian cell transformation: Mechanisms of carcinogenesis and assays for carcinogens

International Nuclear Information System (INIS)

Barrett, J.C.; Tennant, R.W.

1985-01-01

This book contains nine sections, each consisting of several papers. The section titles are: Molecular Changes in Cell Transformation; Differentiation, Growth Control, and Cell Transformation; Mutagenesis and Cell Transformation; Tumor Promotion and Cell Transformation; Mechanisms of Transformation of Human Fibroblasts; Mechanisms of Transformation of Epithelial Cells; Mechanisms of C 3 H 10T12 Cell Transformation; Mechanisms of Radiation-Induced Cell Transformation; and Use of Cell Transformation Assays for Carcinogen Testing

Human Pluripotent Stem Cell Differentiation into Functional Epicardial Progenitor Cells

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Juan Antonio Guadix

2017-12-01

Full Text Available Summary: Human pluripotent stem cells (hPSCs are widely used to study cardiovascular cell differentiation and function. Here, we induced differentiation of hPSCs (both embryonic and induced to proepicardial/epicardial progenitor cells that cover the heart during development. Addition of retinoic acid (RA and bone morphogenetic protein 4 (BMP4 promoted expression of the mesodermal marker PDGFRα, upregulated characteristic (proepicardial progenitor cell genes, and downregulated transcription of myocardial genes. We confirmed the (proepicardial-like properties of these cells using in vitro co-culture assays and in ovo grafting of hPSC-epicardial cells into chick embryos. Our data show that RA + BMP4-treated hPSCs differentiate into (proepicardial-like cells displaying functional properties (adhesion and spreading over the myocardium of their in vivo counterpart. The results extend evidence that hPSCs are an excellent model to study (proepicardial differentiation into cardiovascular cells in human development and evaluate their potential for cardiac regeneration. : The authors have shown that hPSCs can be instructed in vitro to differentiate into a specific cardiac embryonic progenitor cell population called the proepicardium. Proepicardial cells are required for normal formation of the heart during development and might contribute to the development of cell-based therapies for heart repair. Keywords: human pluripotent stem cells, proepicardium, progenitor cells, cardiovascular, differentiation

Osteocalcin protects pancreatic beta cell function and survival under high glucose conditions

Energy Technology Data Exchange (ETDEWEB)

Kover, Karen, E-mail: kkover@cmh.edu [Division of Endocrine/Diabetes, Children' s Mercy Hospital & Clinics, Kansas City, MO 64108 (United States); University of Missouri-Kansas City School of Medicine, Kansas City, MO 64108 (United States); Yan, Yun; Tong, Pei Ying; Watkins, Dara; Li, Xiaoyu [Division of Endocrine/Diabetes, Children' s Mercy Hospital & Clinics, Kansas City, MO 64108 (United States); University of Missouri-Kansas City School of Medicine, Kansas City, MO 64108 (United States); Tasch, James; Hager, Melissa [Kansas City University Medical Biosciences, Kansas City, MO (United States); Clements, Mark; Moore, Wayne V. [Division of Endocrine/Diabetes, Children' s Mercy Hospital & Clinics, Kansas City, MO 64108 (United States); University of Missouri-Kansas City School of Medicine, Kansas City, MO 64108 (United States)

2015-06-19

Diabetes is characterized by progressive beta cell dysfunction and loss due in part to oxidative stress that occurs from gluco/lipotoxicity. Treatments that directly protect beta cell function and survival in the diabetic milieu are of particular interest. A growing body of evidence suggests that osteocalcin, an abundant non-collagenous protein of bone, supports beta cell function and proliferation. Based on previous gene expression data by microarray, we hypothesized that osteocalcin protects beta cells from glucose-induced oxidative stress. To test our hypothesis we cultured isolated rat islets and INS-1E cells in the presence of normal, high, or high glucose ± osteocalcin for up to 72 h. Oxidative stress and viability/mitochondrial function were measured by H{sub 2}O{sub 2} assay and Alamar Blue assay, respectively. Caspase 3/7 activity was also measured as a marker of apoptosis. A functional test, glucose stimulated insulin release, was conducted and expression of genes/protein was measured by qRT-PCR/western blot/ELISA. Osteocalcin treatment significantly reduced high glucose-induced H{sub 2}O{sub 2} levels while maintaining viability/mitochondrial function. Osteocalcin also significantly improved glucose stimulated insulin secretion and insulin content in rat islets after 48 h of high glucose exposure compared to untreated islets. As expected sustained high glucose down-regulated gene/protein expression of INS1 and BCL2 while increasing TXNIP expression. Interestingly, osteocalcin treatment reversed the effects of high glucose on gene/protein expression. We conclude that osteocalcin can protect beta cells from the negative effects of glucose-induced oxidative stress, in part, by reducing TXNIP expression, thereby preserving beta cell function and survival. - Highlights: • Osteocalcin reduces glucose-induced oxidative stress in beta cells. • Osteocalcin preserves beta cell function and survival under stress conditions. • Osteocalcin reduces glucose

Osteocalcin protects pancreatic beta cell function and survival under high glucose conditions

International Nuclear Information System (INIS)

Kover, Karen; Yan, Yun; Tong, Pei Ying; Watkins, Dara; Li, Xiaoyu; Tasch, James; Hager, Melissa; Clements, Mark; Moore, Wayne V.

2015-01-01

Diabetes is characterized by progressive beta cell dysfunction and loss due in part to oxidative stress that occurs from gluco/lipotoxicity. Treatments that directly protect beta cell function and survival in the diabetic milieu are of particular interest. A growing body of evidence suggests that osteocalcin, an abundant non-collagenous protein of bone, supports beta cell function and proliferation. Based on previous gene expression data by microarray, we hypothesized that osteocalcin protects beta cells from glucose-induced oxidative stress. To test our hypothesis we cultured isolated rat islets and INS-1E cells in the presence of normal, high, or high glucose ± osteocalcin for up to 72 h. Oxidative stress and viability/mitochondrial function were measured by H 2 O 2 assay and Alamar Blue assay, respectively. Caspase 3/7 activity was also measured as a marker of apoptosis. A functional test, glucose stimulated insulin release, was conducted and expression of genes/protein was measured by qRT-PCR/western blot/ELISA. Osteocalcin treatment significantly reduced high glucose-induced H 2 O 2 levels while maintaining viability/mitochondrial function. Osteocalcin also significantly improved glucose stimulated insulin secretion and insulin content in rat islets after 48 h of high glucose exposure compared to untreated islets. As expected sustained high glucose down-regulated gene/protein expression of INS1 and BCL2 while increasing TXNIP expression. Interestingly, osteocalcin treatment reversed the effects of high glucose on gene/protein expression. We conclude that osteocalcin can protect beta cells from the negative effects of glucose-induced oxidative stress, in part, by reducing TXNIP expression, thereby preserving beta cell function and survival. - Highlights: • Osteocalcin reduces glucose-induced oxidative stress in beta cells. • Osteocalcin preserves beta cell function and survival under stress conditions. • Osteocalcin reduces glucose-induced TXNIP

A comparative study of Mono Mac 6 cells, isolated mononuclear cells and Limulus amoebocyte lysate assay in pyrogen testing

DEFF Research Database (Denmark)

Moesby, Lise; Jensen, S; Hansen, E W

1999-01-01

) and Staphylococcus aureus was comparable to that of MNC. Aspergillus niger and Candida albicans induced IL-6 in isolated MNC, but not in MM6. The detection limit for Salmonella typhimurium in the MM6 assay was comparable to that of the LAL assay. As expected, S. aureus and C. albicans did not show any LAL activity......Pyrogen induced secretion of interleukin 6 (IL-6) in Mono Mac 6 (MM6) cells was measured. The ability of the MM6 cell culture to detect pyrogens was compared to the Limulus amoebocyte lysate (LAL) test and isolated mononuclear cells (MNC). The detection limit of MM6 for lipopolysaccharide (LPS....... A. niger and Influenza virus showed some activity in the LAL test, but could not be detected by MM6 cells. In conclusion, the MM6 assay is a good supplement to the current pyrogen assays for detection of LPS, S. aureus and S. typhimurium, but the MM6 assay could not detect A. niger, C. albicans...

Cellular function reinstitution of offspring red blood cells cloned from the sickle cell disease patient blood post CRISPR genome editing

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Jianguo Wen

2017-06-01

Full Text Available Abstract Background Sickle cell disease (SCD is a disorder of red blood cells (RBCs expressing abnormal hemoglobin-S (HbS due to genetic inheritance of homologous HbS gene. However, people with the sickle cell trait (SCT carry a single allele of HbS and do not usually suffer from SCD symptoms, thus providing a rationale to treat SCD. Methods To validate gene therapy potential, hematopoietic stem cells were isolated from the SCD patient blood and treated with CRISPR/Cas9 approach. To precisely dissect genome-editing effects, erythroid progenitor cells were cloned from single colonies of CRISPR-treated cells and then expanded for simultaneous gene, protein, and cellular function studies. Results Genotyping and sequencing analysis revealed that the genome-edited erythroid progenitor colonies were converted to SCT genotype from SCD genotype. HPLC protein assays confirmed reinstallation of normal hemoglobin at a similar level with HbS in the cloned genome-edited erythroid progenitor cells. For cell function evaluation, in vitro RBC differentiation of the cloned erythroid progenitor cells was induced. As expected, cell sickling assays indicated function reinstitution of the genome-edited offspring SCD RBCs, which became more resistant to sickling under hypoxia condition. Conclusions This study is an exploration of genome editing of SCD HSPCs.

DNA damage evaluation of hydroxyapatite on fibroblast cell L929 using the single cell gel electrophoresis assay.

Science.gov (United States)

Rajab, N F; Yaakob, T A; Ong, B Y; Hamid, M; Ali, A M; Annuar, B O; Inayat-Hussain, S H

2004-05-01

Hydroxyapatite is the main component of the bone which is a potential biomaterial substance that can be applied in orthopaedics. In this study, the biocompatibility of this biomaterial was assessed using an in vitro technique. The cytotoxicity and genotoxicity effect of HA2 and HA3 against L929 fibroblast cell was evaluated using the MTT Assay and Alkaline Comet Assay respectively. Both HA2 and HA3 compound showed low cytotoxicity effect as determined using MTT Assay. Cells viability following 72 hours incubation at maximum concentration of both HA2 and HA3 (200 mg/ml) were 75.3 +/- 8.8% and 86.7 +/- 13.1% respectively. However, the cytotoxicity effect of ZnSO4.7H2O as a positive control showed an IC50 values of 46 mg/ml (160 microM). On the other hand, both HA2 and HA3 compound showed a slight genotoxicity effect as determined using the Alkaline Comet Assay following incubation at the concentration 200 mg/ml for 72 hours. This assay has been widely used in genetic toxicology to detect DNA strand breaks and alkali-labile site. The percentage of the cells with DNA damage for both substance was 27.7 +/- 1.3% and 15.6 +/- 1.0% for HA2 and HA3 respectively. Incubation of the cells for 24 hours with 38 microg/ml (IC25) of positive control showed an increase in percentage of cells with DNA damage (67.5 +/- 0.7%). In conclusion, our study indicated that both hydroxyapatite compounds showed a good biocompatibility in fibroblast cells.

Development And Application of Functional Assays For Freshwater Dissolved Organic Matter

Science.gov (United States)

Thacker, S.; Tipping, E.; Gondar, D.; Baker, A.

2006-12-01

Dissolved organic matter (DOM) in natural waters participates in many important ecological and geochemical reactions, including acid-base buffering, light absorption, proton binding, binding of heavy metals, organic contaminants, aluminium and radionuclides, adsorption at surfaces, aggregation and photochemical reactivity. We are studying DOM in order to understand and quantify these functional properties, so we can use the knowledge to predict the influence of DOM on the natural freshwater environment. As DOM has no readily identifiable structure, our approach is to measure what it does, rather than what it is. Thus, we have developed a series of 12 standardised, reproducible assays of physico-chemical functions of dissolved organic matter (DOM) in freshwaters. The assays provide quantitative information on light absorption, fluorescence, photochemical fading, pH buffering, copper binding, benzo(a)pyrene binding, hydrophilicity and adsorption to alumina. We have collected twenty DOM samples in total, ten samples from a eutrophic lake (Esthwaite Water) and ten samples from three stream waters. A mild isolation method was then used to concentrate the DOM samples for the assay work. When assaying the concentrates, parallel assays were also preformed with Suwannee River Fulvic Acid (SRFA), as a quality control standard. Our results showed that; (i) for eleven of the assays, the variability among the twenty DOM samples was significantly (p<0.001) greater than can be explained by analytical error, i.e. by comparison with results from the SRFA quality control; (ii) the functional properties of the DOM from Esthwaite Water are strongly influenced by the seasonally-dependent input of autochthonous DOM, derived from phytoplankton. The autochthonous DOM is less fluorescent, light absorbing, hydrophobic and has a lower acid group content and capacity to be adsorbed onto alumina than terrestrially derived allochthonous DOM; (iii) significant correlations were found between

Interleukin 21 and its receptor are involved in NK cell expansion and regulation of lymphocyte function.

Science.gov (United States)

Parrish-Novak, J; Dillon, S R; Nelson, A; Hammond, A; Sprecher, C; Gross, J A; Johnston, J; Madden, K; Xu, W; West, J; Schrader, S; Burkhead, S; Heipel, M; Brandt, C; Kuijper, J L; Kramer, J; Conklin, D; Presnell, S R; Berry, J; Shiota, F; Bort, S; Hambly, K; Mudri, S; Clegg, C; Moore, M; Grant, F J; Lofton-Day, C; Gilbert, T; Rayond, F; Ching, A; Yao, L; Smith, D; Webster, P; Whitmore, T; Maurer, M; Kaushansky, K; Holly, R D; Foster, D

2000-11-02

Cytokines are important in the regulation of haematopoiesis and immune responses, and can influence lymphocyte development. Here we have identified a class I cytokine receptor that is selectively expressed in lymphoid tissues and is capable of signal transduction. The full-length receptor was expressed in BaF3 cells, which created a functional assay for ligand detection and cloning. Conditioned media from activated human CD3+ T cells supported proliferation of the assay cell line. We constructed a complementary DNA expression library from activated human CD3+ T cells, and identified a cytokine with a four-helix-bundle structure using functional cloning. This cytokine is most closely related to IL2 and IL15, and has been designated IL21 with the receptor designated IL21 R. In vitro assays suggest that IL21 has a role in the proliferation and maturation of natural killer (NK) cell populations from bone marrow, in the proliferation of mature B-cell populations co-stimulated with anti-CD40, and in the proliferation of T cells co-stimulated with anti-CD3.

21 CFR 864.7100 - Red blood cell enzyme assay.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Red blood cell enzyme assay. 864.7100 Section 864.7100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7100 Red blood cell...

Using a medium-throughput comet assay to evaluate the global DNA methylation status of single cells

Science.gov (United States)

Lewies, Angélique; Van Dyk, Etresia; Wentzel, Johannes F.; Pretorius, Pieter J.

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 experiments described here, a medium-throughput comet assay and methylation sensitive comet assay are combined to produce a methylation sensitive medium-throughput comet assay to measure changes in the global DNA methylation pattern in individual cells under various growth conditions. PMID:25071840

Semi-automated limit-dilution assay and clonal expansion of all T-cell precursors of cytotoxic lymphocytes

Energy Technology Data Exchange (ETDEWEB)

Wilson, A.; Chen, W.F.; Scollay, R.; Shortman, K. (Walter and Eliza Hall Inst. of Medical Research, Parkville (Australia))

1982-08-13

A limit-dilution microculture system is described, where almost all precursor T cells of the cytotoxic lineage (CTL-p) develop into extended clones of cytotoxic T cells (CTL), which are then detected with a new radio-autographic /sup 111/In-release assay. The principle is to polyclonally activate all T cells with concanavalin A, to expand the resultant clones over an 8-9 day period in cultures saturated with growth factors, then to detect all clones with cytotoxic function by phytohaemagglutinin mediated lysis of P815 tumour cells. The key variables for obtaining high cloning efficiency are the use of flat-bottomed 96-well culture trays, the use of appropriately irradiated spleen filler cells, and the inclusion of a T-cell growth factor supplement. Cultures are set up at input levels of around one T cell per well. Forty percent of T cells then form CTL clones readily detected by the cytotoxic assay. The lytic activity of the average clone is equivalent to 3000 CTL, but clone size appears to be much larger. The precursor cells are predominantly if not entirely from the Lyt 2/sup +/ T-cell subclass and almost all cells of this subclass form cytolytic clones. Analysis of the frequency of positive cultures shows a good fit to the expected Poisson distribution, with no evidence of the CTL-p frequency estimates being distorted by helper or suppressor effects.

Semi-automated limit-dilution assay and clonal expansion of all T-cell precursors of cytotoxic lymphocytes

International Nuclear Information System (INIS)

Wilson, A.; Chen, W.-F.; Scollay, R.; Shortman, K.

1982-01-01

A limit-dilution microculture system is described, where almost all precursor T cells of the cytotoxic lineage (CTL-p) develop into extended clones of cytotoxic T cells (CTL), which are then detected with a new radio-autographic 111 In-release assay. The principle is to polyclonally activate all T cells with concanavalin A, to expand the resultant clones over an 8-9 day period in cultures saturated with growth factors, then to detect all clones with cytotoxic function by phytohaemagglutinin mediated lysis of P815 tumour cells. The key variables for obtaining high cloning efficiency are the use of flat-bottomed 96-well culture trays, the use of appropriately irradiated spleen filler cells, and the inclusion of a T-cell growth factor supplement. Cultures are set up at input levels of around one T cell per well. Forty percent of T cells then form CTL clones readily detected by the cytotoxic assay. The lytic activity of the average clone is equivalent to 3000 CTL, but clone size appears to be much larger. The precursor cells are predominantly if not entirely from the Lyt 2 + T-cell subclass and almost all cells of this subclass form cytolytic clones. Analysis of the frequency of positive cultures shows a good fit to the expected Poisson distribution, with no evidence of the CTL-p frequency estimates being distorted by helper or suppressor effects. (Auth.)

Cytokinesis-block micronucleus assay evolves into a 'cytome' assay of chromosomal instability, mitotic dysfunction and cell death

International Nuclear Information System (INIS)

Fenech, Michael

2006-01-01

The cytokinesis-block micronucleus (CBMN) assay was originally developed as an ideal system for measuring micronuclei (MNi) however it can also be used to measure nucleoplasmic bridges (NPBs), nuclear buds (NBUDs), cell death (necrosis or apoptosis) and nuclear division rate. Current evidence suggests that (a) NPBs originate from dicentric chromosomes in which the centromeres have been pulled to the opposite poles of the cell at anaphase and are therefore indicative of DNA mis-repair, chromosome rearrangement or telomere end-fusions, (b) NPBs may break to form MNi, (c) the nuclear budding process is the mechanism by which cells remove amplified and/or excess DNA and is therefore a marker of gene amplification and/or altered gene dosage, (d) cell cycle checkpoint defects result in micronucleus formation and (e) hypomethylation of DNA, induced nutritionally or by inhibition of DNA methyl transferase can lead to micronucleus formation either via chromosome loss or chromosome breakage. The strong correlation between micronucleus formation, nuclear budding and NPBs (r = 0.75-0.77, P < 0.001) induced by either folic acid deficiency or exposure to ionising radiation is supportive of the hypothesis that folic acid deficiency and/or ionising radiation cause genomic instability and gene amplification by the initiation of breakage-fusion-bridge cycles. In its comprehensive mode, the CBMN assay measures all cells including necrotic and apoptotic cells as well as number of nuclei per cell to provide a measure of cytotoxicity and mitotic activity. The CBMN assay has in fact evolved into a 'cytome' method for measuring comprehensively chromosomal instability phenotype and altered cellular viability caused by genetic defects and/or nutrional deficiencies and/or exogenous genotoxins thus opening up an exciting future for the use of this methodology in the emerging fields of nutrigenomics and toxicogenomics and their combinations

The glycophorin A assay for somatic cell mutations in humans

International Nuclear Information System (INIS)

Langlois, R.G.; Bigbee, W.L.; Jensen, R.H.

1989-01-01

In this report we briefly review our past experience and some new developments with the GPA assay. Particular emphasis will be placed on two areas that affect the utility of the GPA assay for human population monitoring. The first is our efforts to simplify the GPA assay to make it more generally available for large population studies. The second is to begin to understand some of the characteristics of human hemopoiesis which affect the accumulation and expression of mutant phenotype cells. 11 refs., 4 figs

Effects of selective serotonin reuptake inhibitors on three sex steroids in two versions of the aromatase enzyme inhibition assay and in the H295R cell assay

DEFF Research Database (Denmark)

Jacobsen, Naja Wessel; Hansen, Cecilie Hurup; Nellemann, Christine

2015-01-01

shown to inhibit the aromatase enzyme in both types of aromatase assays. The IC50 values ranged from 3 to 600μM. All five SSRIs, were further investigated in the H295R cell line. All compounds altered the steroid secretion from the cells, the lowest observed effect levels were 0.9μM and 3.1μ....... In this study we investigated whether the endocrine effect due to SSRI exposure could be detected in well adopted in vitro steroidogenesis assays, two versions of the aromatase enzyme inhibition assay and the H295R cell assay. The five drugs citalopram, fluoxetine, fluvoxamine, paroxetine and sertraline, were......M for sertraline and fluvoxamine, respectively. In general the H295R cell assay was more sensitive to SSRI exposure than the two aromatase assays, up to 20 times more sensitive. This indicates that the H295R cell line is a better tool for screening endocrine disrupting effects. Our findings show that the endocrine...

Use of an in vivo FTA assay to assess the magnitude, functional avidity and epitope variant cross-reactivity of T cell responses following HIV-1 recombinant poxvirus vaccination.

Directory of Open Access Journals (Sweden)

Danushka K Wijesundara

Full Text Available Qualitative characteristics of cytotoxic CD8+ T cells (CTLs are important in measuring the effectiveness of CTLs in controlling HIV-1 infections. Indeed, in recent studies patients who are naturally resistant to HIV-1 infections have been shown to possess CTLs that are of high functional avidity and have a high capacity to recognize HIV epitope variants, when compared to HIV-1 infection progressors. When developing efficacious vaccines, assays that can effectively measure CTL quality specifically in vivo are becoming increasingly important. Here we report the use of a recently developed high-throughput multi-parameter technique, known as the fluorescent target array (FTA assay, to simultaneously measure CTL killing magnitude, functional avidity and epitope variant cross-reactivity in real time in vivo. In the current study we have applied the FTA assay as a screening tool to assess a large cohort of over 20 different HIV-1 poxvirus vaccination strategies in mice. This screen revealed that heterologous poxvirus prime-boost vaccination regimes (i.e., recombinant fowlpox (FPV-HIV prime followed by a recombinant vaccinia virus (VV-HIV booster were the most effective in generating high quality CTL responses in vivo. In conclusion, we have demonstrated how the FTA assay can be utilized as a cost effective screening tool (by reducing the required number of animals by >100 fold, to evaluate a large range of HIV-1 vaccination strategies in terms of CTL avidity and variant cross-reactivity in an in vivo setting.

Use of an in vivo FTA assay to assess the magnitude, functional avidity and epitope variant cross-reactivity of T cell responses following HIV-1 recombinant poxvirus vaccination.

Science.gov (United States)

Wijesundara, Danushka K; Ranasinghe, Charani; Jackson, Ronald J; Lidbury, Brett A; Parish, Christopher R; Quah, Benjamin J C

2014-01-01

Qualitative characteristics of cytotoxic CD8+ T cells (CTLs) are important in measuring the effectiveness of CTLs in controlling HIV-1 infections. Indeed, in recent studies patients who are naturally resistant to HIV-1 infections have been shown to possess CTLs that are of high functional avidity and have a high capacity to recognize HIV epitope variants, when compared to HIV-1 infection progressors. When developing efficacious vaccines, assays that can effectively measure CTL quality specifically in vivo are becoming increasingly important. Here we report the use of a recently developed high-throughput multi-parameter technique, known as the fluorescent target array (FTA) assay, to simultaneously measure CTL killing magnitude, functional avidity and epitope variant cross-reactivity in real time in vivo. In the current study we have applied the FTA assay as a screening tool to assess a large cohort of over 20 different HIV-1 poxvirus vaccination strategies in mice. This screen revealed that heterologous poxvirus prime-boost vaccination regimes (i.e., recombinant fowlpox (FPV)-HIV prime followed by a recombinant vaccinia virus (VV)-HIV booster) were the most effective in generating high quality CTL responses in vivo. In conclusion, we have demonstrated how the FTA assay can be utilized as a cost effective screening tool (by reducing the required number of animals by >100 fold), to evaluate a large range of HIV-1 vaccination strategies in terms of CTL avidity and variant cross-reactivity in an in vivo setting.

Development of a replication-competent lentivirus assay for dendritic cell-targeting lentiviral vectors

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Daniel C Farley

Full Text Available It is a current regulatory requirement to demonstrate absence of detectable replication-competent lentivirus (RCL in lentiviral vector products prior to use in clinical trials. Immune Design previously described an HIV-1-based integration-deficient lentiviral vector for use in cancer immunotherapy (VP02. VP02 is enveloped with E1001, a modified Sindbis virus glycoprotein which targets dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN expressed on dendritic cells in vivo. Vector enveloped with E1001 does not transduce T-cell lines used in standard HIV-1-based RCL assays, making current RCL testing formats unsuitable for testing VP02. We therefore developed a novel assay to test for RCL in clinical lots of VP02. This assay, which utilizes a murine leukemia positive control virus and a 293F cell line expressing the E1001 receptor DC-SIGN, meets a series of evaluation criteria defined in collaboration with US regulatory authorities and demonstrates the ability of the assay format to amplify and detect a hypothetical RCL derived from VP02 vector components. This assay was qualified and used to test six independent GMP production lots of VP02, in which no RCL was detected. We propose that the evaluation criteria used to rationally design this novel method should be considered when developing an RCL assay for any lentiviral vector.

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

International Nuclear Information System (INIS)

Brengues, Muriel; Gu, Jian; Zenhausern, Frederic

2015-01-01

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)

Development of a partition-controlled dosing system for cell assays.

NARCIS (Netherlands)

Kramer, N.I.; Busser, F.J.M.; Oosterwijk, M.T.; Schirmer, K.; Escher, B.I.; Hermens, J.L.M.

2010-01-01

Hydrophobic and volatile chemicals have proven to be difficult to dose in cell assays. Cosolvents are often needed to dissolve these chemicals in cell culture medium. Moreover, the free concentration of these chemicals in culture medium may diminish over time due to metabolism, evaporation, and

Quantum Dot Nanotoxicity Investigations Using Human Lung Cells and TOXOR Electrochemical Enzyme Assay Methodology.

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O'Hara, Tony; Seddon, Brian; O'Connor, Andrew; McClean, Siobhán; Singh, Baljit; Iwuoha, Emmanuel; Fuku, Xolile; Dempsey, Eithne

2017-01-27

Recent studies have suggested that certain nanomaterials can interfere with optically based cytotoxicity assays resulting in underestimations of nanomaterial toxicity. As a result there has been growing interest in the use of whole cell electrochemical biosensors for nanotoxicity applications. Herein we report application of an electrochemical cytotoxicity assay developed in house (TOXOR) in the evaluation of toxic effects of mercaptosuccinic acid capped cadmium telluride quantum dots (MSA capped CdTe QDs), toward mammalian cells. MSA capped CdTe QDs were synthesized, characterized, and their cytotoxicity toward A549 human lung epithelial cells investigated. The internalization of QDs within cells was scrutinized via confocal microscopy. The cytotoxicity assay is based on the measurement of changes in cellular enzyme acid phosphatase upon 24 h exposure to QDs. Acid phosphatase catalyzes dephosphorylation of 2-naphthyl phosphate to 2-naphthol (determined by chronocoulometry) and is indicative of metabolic activity in cells. The 24 h IC50 (concentration resulting in 50% reduction in acid phosphatase activity) value for MSA capped CdTe QDs was found to be 118 ± 49 μg/mL using the TOXOR assay and was in agreement with the MTT assay (157 ± 31 μg/mL). Potential uses of this electrochemical assay include the screening of nanomaterials, environmental toxins, in addition to applications in the pharmaceutical, food, and health sectors.

Normalization methods in time series of platelet function assays

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Van Poucke, Sven; Zhang, Zhongheng; Roest, Mark; Vukicevic, Milan; Beran, Maud; Lauwereins, Bart; Zheng, Ming-Hua; Henskens, Yvonne; Lancé, Marcus; Marcus, Abraham

2016-01-01

Abstract Platelet function can be quantitatively assessed by specific assays such as light-transmission aggregometry, multiple-electrode aggregometry measuring the response to adenosine diphosphate (ADP), arachidonic acid, collagen, and thrombin-receptor activating peptide and viscoelastic tests such as rotational thromboelastometry (ROTEM). The task of extracting meaningful statistical and clinical information from high-dimensional data spaces in temporal multivariate clinical data represented in multivariate time series is complex. Building insightful visualizations for multivariate time series demands adequate usage of normalization techniques. In this article, various methods for data normalization (z-transformation, range transformation, proportion transformation, and interquartile range) are presented and visualized discussing the most suited approach for platelet function data series. Normalization was calculated per assay (test) for all time points and per time point for all tests. Interquartile range, range transformation, and z-transformation demonstrated the correlation as calculated by the Spearman correlation test, when normalized per assay (test) for all time points. When normalizing per time point for all tests, no correlation could be abstracted from the charts as was the case when using all data as 1 dataset for normalization. PMID:27428217

Genetic mouse embryo assay: improving performance and quality testing for assisted reproductive technology (ART) with a functional bioassay.

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Gilbert, Rebecca S; Nunez, Brandy; Sakurai, Kumi; Fielder, Thomas; Ni, Hsiao-Tzu

2016-03-24

Growing concerns about safety of ART on human gametes, embryos, clinical outcomes and long-term health of offspring require improved methods of risk assessment to provide functionally relevant assays for quality control testing and pre-clinical studies prior to clinical implementation. The one-cell mouse embryo assay (MEA) is the most widely used for development and quality testing of human ART products; however, concerns exist due to the insensitivity/variability of this bioassay which lacks standardization and involves subjective analysis by morphology alone rather than functional analysis of the developing embryos. We hypothesized that improvements to MEA by the use of functional molecular biomarkers could enhance sensitivity and improve detection of suboptimal materials/conditions. Fresh one-cell transgenic mouse embryos with green fluorescent protein (GFP) expression driven by Pou6f1 or Cdx2 control elements were harvested and cultured to blastocysts in varied test and control conditions to compare assessment by standard morphology alone versus the added dynamic expression of GFP for screening and selection of critical raw materials and detection of suboptimal culture conditions. Transgenic mouse embryos expressing functionally relevant biomarkers of normal early embryo development can be used to monitor the developmental impact of culture conditions. This novel approach provides a superior MEA that is more meaningful and sensitive for detection of embryotoxicity than morphological assessment alone.

Dissecting the T Cell Response: Proliferation Assays vs. Cytokine Signatures by ELISPOT

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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.

Dissecting the T Cell Response: Proliferation Assays vs. Cytokine Signatures by ELISPOT

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Anthony, Donald D.; Milkovich, Kimberly A.; Zhang, Wenji; Rodriguez, Benigno; Yonkers, Nicole L.; Tary-Lehmann, Magdalena; Lehmann, Paul V.

2012-01-01

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-γ 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. PMID:24710419

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 che...... 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...

Assessment of the predictive capacity of the optimized in vitro comet assay using HepG2 cells.

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Hong, Yoon-Hee; Jeon, Hye Lyun; Ko, Kyung Yuk; Kim, Joohwan; Yi, Jung-Sun; Ahn, Ilyoung; Kim, Tae Sung; Lee, Jong Kwon

2018-03-01

Evaluation of DNA damage is critical during the development of new drugs because it is closely associated with genotoxicity and carcinogenicity. The in vivo comet assay to assess DNA damage is globally harmonized as OECD TG 489. However, a comet test guideline that evaluates DNA damage without sacrificing animals does not yet exist. The goal of this study was to select an appropriate cell line for optimization of the in vitro comet assay to assess DNA damage. We then evaluated the predictivity of the in vitro comet assay using the selected cell line. In addition, the effect of adding S9 was evaluated using 12 test chemicals. For cell line selection, HepG2, Chinese hamster lung (CHL/IU), and TK6 cell lines were evaluated. We employed a method for the in vitro comet assay based on that for the in vivo comet assay. The most appropriate cell line was determined by% tail DNA increase after performing in vitro comet assays with 6 test chemicals. The predictivity of the in vitro comet assay using the selected cell line was measured with 10 test chemicals (8 genotoxins and 2 non-genotoxic chemicals). The HepG2 cell line was found to be the most appropriate, and in vitro comet assays using HepG2 cells exhibited a high accuracy of 90% (9/10). This study suggests that HepG2 is an optimal cell line for the in vitro comet assay to assess DNA damage. Copyright © 2018 Elsevier B.V. All rights reserved.

Stem cell-like differentiation potentials of endometrial side population cells as revealed by a newly developed in vivo endometrial stem cell assay.

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Kaoru Miyazaki

Full Text Available 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.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.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 endometrial tissue reconstitution. Using this assay, we demonstrated that ESP

Biochemical studies of immune RNA using a cell-mediated cytotoxicity assay

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Griffin, G.D.; Sellin, H.G.; Novelli, G.D.

1980-01-01

Immune RNA (iRNA), a subcellular macromolecular species usually prepared by phenol extraction of lymphoid tissue, can confer some manifestation(s) of cellular immunity on naive lymphocytes. Experiments were done to develop an assay system to detect activation of lymphocytes by iRNA to become cytotoxic toward tumor cells, and to study certain properties of iRNA using this system. Guinea pigs were immunized with human mammary carcinoma cells and the iRNA, prepared from spleens of animals shown by prior assay to have blood lymphocytes highly cytotoxic against the tumor cells, was assayed by ability of iRNA-activated lymphocytes to lyse /sup 51/Cr-labelled tumor cells. The ability of iRNA to activate lymphocytes to tumor cytotoxicity could only be differentiated from a cytotoxic activation by RNA preparations from unimmunized animals at very low doses of RNA. The most active iRNA preparations were from cytoplasmic subcellular fractions, extracted by a cold phenol procedure, while iRNA isolated by hot phenol methods was no more active than control RNA prepared by the same techniques. Attempts to demonstrate poly(A) sequences in iRNA were inconclusive.

Development of norepinephrine transporter reuptake inhibition assays using SK-N-BE(2C cells

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Ann M. Decker

2018-05-01

Full Text Available This report describes efforts to develop and validate novel norepinephrine transporter reuptake inhibition assays using human neuroblastoma SK-N-BE(2C cells in 24-well format. Before conducting the assays, the SK-N-BE(2C cells were first evaluated for their ability to uptake [3H]norepinephrine and were shown to have a saturable uptake with a KM value of 416 nM. Using this determined KM value, reuptake inhibition assays were then conducted with a variety of ligands including antidepressants, as well as piperazine and phenyltropane derivatives. The results obtained with the SK-N-BE(2C cells indicate that this model system can detect a range of ligand potencies, which compare well with other established transporter assays. Our data suggest that SK-N-BE(2C cells have potential utility to serve as another model system to detect norepinephrine reuptake inhibition activity.

Cell-patterned glass spray for direct drug assay using mass spectrometry

International Nuclear Information System (INIS)

Wu, Jing; Wang, Shiqi; Chen, Qiushui; Jiang, Hao; Liang, Shuping; Lin, Jin-Ming

2015-01-01

In this work, the establishment of a glass spray mass spectrometry (GS-MS) platform for direct cell-based drug assay was described. Cell co-culture, drug-induced cell apoptosis, proliferation analysis and intracellular drug absorption measurement were performed simultaneously on this specifically designed platform. Two groups of co-cultured cells (NIH-3T3/HepG2 and HepG2/MCF-7) were cultivated and they showed high viability within 3 days. The biocompatibility of the platform facilitated the subsequent bioassays, in which, cyclophosphamide (CPA) and genistein were used as the model drugs. The distinctions of cell apoptosis and proliferation between the mono-cultured and co-cultured cells were clearly observed and well explained by in situ GS-MS measurements. A satisfactory linearity of the calibration curve between the relative MS intensity and CPA concentrations was obtained using stable isotope labeling method (y = 0.16545 + 0.0985x, R"2 = 0.9937). The variations in the quantity of absorbed drug were detected and the results were consistent with the concentration-dependence of cell apoptosis. All the results demonstrated that direct cell-based drug assay could be performed on the stable isotope labeling assisted GS-MS platform in a facile and quantitative manner. - Highlights: • A versatile glass spray mass spectrometry (GS-MS) platform for direct cell-based drug assay was developed in this paper. • It has characteristics of the atmospheric pressure ionization method. • It is multifunctional for cell co-culture, bioassays, qualitative and quantitative intracellular drug absorption measurement. • GS-MS has the potential to increase the use of mass spectrometry in biological analysis.

EL4 cell-based colorimetric toxin neutralization activity assays for determination of neutralizing anti-ricin antibodies.

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Lindsey, Changhong Y; Brown, J Edward; Torabazar, Nahid R; Smith, Leonard A

2013-01-01

A recombinant ricin toxin A-chain 1-33/44-198 vaccine (RVEc), developed at the United States Army Medical Research Institute of Infectious Diseases as a vaccine candidate, is under investigation in a phase 1 clinical study. To effectively evaluate the immunogenicity of this ricin vaccine and to eliminate the use of radioactive material, an EL4 cell-based colorimetric toxin neutralization activity (TNA) assay using a CellTiter 96 AQueous One Solution Cell Proliferation Assay Reagent has been developed, optimized, and applied in the vaccine efficacy studies. The TNA assay measures the protective neutralizing anti-ricin antibodies in animal sera by determining the cell viability after ricin exposure in the assay system and comparing it to a purified mouse polyclonal antiricin IgG standard curve. The standard curve of the anti-ricin TNA assay closely fits a four-parameter logistic regression model. The unknown test sample concentration was expressed as microg/mL, but not the 50% effective concentration (EC50), which was determined by most TNA assays. The neutralizing endpoint titers, not the 50% effective dilution (ED50), of human specimens were measured with the TNA assay in support of the clinical study of the RVEc vaccine. The optimal amount of ricin toxin, EL4 cells, and concentration of standards used in the assay system was established to minimize false-negative and false-positive results of serum specimens from the nonclinical and clinical studies of RVEc. The testing conditions were adjusted to optimize assay performance. The colorimetric TNA assay replaced a radioactive TNA assay previously used in the ricin vaccine studies.

The toxicity study of functionalized CNT from fermented tapioca on neuroblastoma cell

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Nurulhuda, I.; Mazatulikhma, M. Z.; Alrokayan, S.; Khan, H.; Rusop, M.

2018-05-01

Carbon nanotubes known as one of the most interesting types of nanomaterials, especially use in application directly to cells. Somehow the use should take into consideration regarding the potential adverse impact on human health. Current study, the carbon nanotube was synthesized from fermented tapioca and functionalized with polyethylene glycol and directly test on the neuroblastoma cells in vitro. The toxicity effect on cells was assessed by 3(4, 5-dimethylthiazol-2-yl)-2, 5-tetrazolium bromide assays. It showed a dose-and time-dependent less toxic effect on functionalized carbon nanotube compared to non-functionalized. This leads us to the conclusion that functionalized carbon nanotube can be use for drug delivery in future.

Pertussis serology: assessment of IgG anti-PT ELISA for replacement of the CHO cell assay*

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DALBY, TINE; SØRENSEN, CHARLOTTE; PETERSEN, JESPER WESTPHAL; KROGFELT, KAREN ANGELIKI

2010-01-01

Dalby T, Sørensen C, Petersen JW, Krogfelt KA. Pertussis serology: assessment of IgG anti-PT ELISA for replacement of the CHO cell assay. APMIS 2010; 118: 968–72. Two types of serological assays are commonly used for the assessment of pertussis vaccine-induced antibodies; the Chinese hamster ovary cell (CHO cell) assay and the immunoglobulin G (IgG) anti pertussis toxin (PT) enzyme-linked immunosorbent assay (IgG anti-PT ELISA). Recently, both the techniques have been modified to improve performance with sera with interfering activity (CHO cell assay) or with heat-treated sera (IgG anti-PT ELISA). These two improved techniques were compared by the analysis of 100 individual serum samples from a previous clinical trial and 213 sera from a longitudinal serum collection from 20 Danish adults recently vaccinated with the Danish acellular pertussis vaccine. The comparison showed a significant linear correlation between the results of the two assays with a p-value of ELISA can be used as a replacement for the often troublesome and time-consuming CHO cell assay for the measurement of vaccine-induced human antibodies to PT. PMID:21091778

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.

A study on the toxicity of three radiosensitizers on retinoblastoma cells by MTT assay

International Nuclear Information System (INIS)

Yi Xianjin; Jin Yizun; Ding Li; Ni Zhou; Wang Wenji

1994-01-01

The toxicity of three radiosensitizers BSO, CM and RSU-1069 on retinoblastoma cells was determined and the efficiency of in vitro MTT assay on drug-screening for retinoblastoma was also evaluated. The results showed that the MTT assay is very useful. The toxicity of radiosensitizers on retinoblastoma cells is dependent on cell line characteristics, drug concentration and time of exposure to it

A Cell Culture Approach to Optimized Human Corneal Endothelial Cell Function

Science.gov (United States)

Bartakova, Alena; Kuzmenko, Olga; Alvarez-Delfin, Karen; Kunzevitzky, Noelia J.; Goldberg, Jeffrey L.

2018-01-01

Purpose Cell-based therapies to replace corneal endothelium depend on culture methods to optimize human corneal endothelial cell (HCEC) function and minimize endothelial-mesenchymal transition (EnMT). Here we explore contribution of low-mitogenic media on stabilization of phenotypes in vitro that mimic those of HCECs in vivo. Methods HCECs were isolated from cadaveric donor corneas and expanded in vitro, comparing continuous presence of exogenous growth factors (“proliferative media”) to media without those factors (“stabilizing media”). Identity based on canonical morphology and expression of surface marker CD56, and function based on formation of tight junction barriers measured by trans-endothelial electrical resistance assays (TEER) were assessed. Results Primary HCECs cultured in proliferative media underwent EnMT after three to four passages, becoming increasingly fibroblastic. Stabilizing the cells before each passage by switching them to a media low in mitogenic growth factors and serum preserved canonical morphology and yielded a higher number of cells. HCECs cultured in stabilizing media increased both expression of the identity marker CD56 and also tight junction monolayer integrity compared to cells cultured without stabilization. Conclusions HCECs isolated from donor corneas and expanded in vitro with a low-mitogenic media stabilizing step before each passage demonstrate more canonical structural and functional features and defer EnMT, increasing the number of passages and total canonical cell yield. This approach may facilitate development of HCEC-based cell therapies. PMID:29625488

Functional Assay of Cancer Cell Invasion Potential Based on Mechanotransduction of Focused Ultrasound

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Andrew C. Weitz

2017-08-01

Full Text Available Cancer cells undergo a number of biophysical changes as they transform from an indolent to an aggressive state. These changes, which include altered mechanical and electrical properties, can reveal important diagnostic information about disease status. Here, we introduce a high-throughput, functional technique for assessing cancer cell invasion potential, which works by probing for the mechanically excitable phenotype exhibited by invasive cancer cells. Cells are labeled with fluorescent calcium dye and imaged during stimulation with low-intensity focused ultrasound, a non-contact mechanical stimulus. We show that cells located at the focus of the stimulus exhibit calcium elevation for invasive prostate (PC-3 and DU-145 and bladder (T24/83 cancer cell lines, but not for non-invasive cell lines (BPH-1, PNT1A, and RT112/84. In invasive cells, ultrasound stimulation initiates a calcium wave that propagates from the cells at the transducer focus to other cells, over distances greater than 1 mm. We demonstrate that this wave is mediated by extracellular signaling molecules and can be abolished through inhibition of transient receptor potential channels and inositol trisphosphate receptors, implicating these proteins in the mechanotransduction process. If validated clinically, our technology could provide a means to assess tumor invasion potential in cytology specimens, which is not currently possible. It may therefore have applications in diseases such as bladder cancer, where cytologic diagnosis of tumor invasion could improve clinical decision-making.

Assaying gene function by growth competition experiment.

Science.gov (United States)

Merritt, Joshua; Edwards, Jeremy S

2004-07-01

High-throughput screening and analysis is one of the emerging paradigms in biotechnology. In particular, high-throughput methods are essential in the field of functional genomics because of the vast amount of data generated in recent and ongoing genome sequencing efforts. In this report we discuss integrated functional analysis methodologies which incorporate both a growth competition component and a highly parallel assay used to quantify results of the growth competition. Several applications of the two most widely used technologies in the field, i.e., transposon mutagenesis and deletion strain library growth competition, and individual applications of several developing or less widely reported technologies are presented.

A simple assay for the detection of antibodies to endocrine islet cell surface antigens

International Nuclear Information System (INIS)

Contreas, G.; Madsen, O.D.; Vissing, H.; Lernmark, Aa.

1986-01-01

A simple and sensitive immunoradiometric assay for the detection of islet cell surface antibodies (CIRMA) has been developed. Live, transformed islet cells derived from a liver metastasis of a transplantable islet cell tumor were grown in removable microtiter wells and incubated with antibody. Cell-bound antibodies were quantitated using 125 I-labelled second antibodies. The assay was used to detect islet cell antibodies present in sera from non-diabetic and diabetic BB rats and proved to be particularly effective for screening hybridoma supernatants in order to identify monoclonal antibodies recognizing islet cell surface antigens. (Auth.)

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.

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.

A terminal-labelling microcytotoxity assay with 125I-iododeoxyuridine as a label for target cells

International Nuclear Information System (INIS)

Stirrat, G.M.

1976-01-01

The development of a terminal-labelling microcytotoxicity assay is described in which target cells (fetal fibroblasts) were labelled with 125 I-iododeoxyuridine after effector (lymphoid) cells had been incubated with them for 24 h. The time-course for the development of cell-mediated cytotoxicity was assessed following allogeneic skin grafting. 'Non-specific' cytotoxicity detracts from the sensitivity of all microcytotoxicity assays and the terminal-labelling assay using 125 I is no exception. The non-specific effects can be reduced but not eliminated by the removal of adherent cells. The optimum target cell/effector cell ratio would seem to be between 1:100 and 1:250. Residual lymph node cells did not appear to incorporate enough label to affect the test results. In vivo correlates of in vitro findings are still not easy to determine

Comparison of multiple assays for detecting human antibodies directed against antigens on normal and malignant tissue culture cells

International Nuclear Information System (INIS)

Rosenberg, S.A.; Schwarz, S.; Anding, H.; Hyatt, C.; Williams, G.M.; Johns Hopkins Univ., Baltimore, Md.

1977-01-01

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

CYTOKINESIS-BLOCK MICRONUCLEUS ASSAY IN HUMAN GLIOMA CELLS EXPOSED TO RADIATION

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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.

A Comparison of Real-Time and Endpoint Cell Viability Assays for Improved Synthetic Lethal Drug Validation.

Science.gov (United States)

Single, Andrew; Beetham, Henry; Telford, Bryony J; Guilford, Parry; Chen, Augustine

2015-12-01

Cell viability assays fulfill a central role in drug discovery studies. It is therefore important to understand the advantages and disadvantages of the wide variety of available assay methodologies. In this study, we compared the performance of three endpoint assays (resazurin reduction, CellTiter-Glo, and nuclei enumeration) and two real-time systems (IncuCyte and xCELLigence). Of the endpoint approaches, both the resazurin reduction and CellTiter-Glo assays showed higher cell viabilities when compared directly to stained nuclei counts. The IncuCyte and xCELLigence real-time systems were comparable, and both were particularly effective at tracking the effects of drug treatment on cell proliferation at sub-confluent growth. However, the real-time systems failed to evaluate contrasting cell densities between drug-treated and control-treated cells at full growth confluency. Here, we showed that using real-time systems in combination with endpoint assays alleviates the disadvantages posed by each approach alone, providing a more effective means to evaluate drug toxicity in monolayer cell cultures. Such approaches were shown to be effective in elucidating the toxicity of synthetic lethal drugs in an isogenic pair of MCF10A breast cell lines. © 2015 Society for Laboratory Automation and Screening.

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......-polysaccharide antibodies aggregation of secreted monomeric antibody (IgG) is critical for plaque formation and increases the avidity of binding to target cells....

Structure-function Evaluation of Stem Cell Therapies for Spinal Cord Injury.

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Zhang, Fuguo

2018-02-23

Spinal cord injuries (SCI) are prevalent, devastating for quality and expectancy of life, and cause heavy economic burdens. Stem cell therapies hold promise in complete structural and functional restoration of SCI. This review focuses on the methods currently used to evaluate the stem cell therapies for SCI. Various kinds of stem cells involving embryonic stem cells (ESCs), bone marrow stromal cells (BMSCs), neural stem cells (NSCs) and induced pluripotent stem cells (iPSCs) are extensively used in regenerative research of SCI. For evaluation, the survival and integration of transplanted cells, spinal cord reconstruction and functional recovery all should be considered. Histological and histochemistrical, microscopic, and colorimetric assays, and real-time RT-PCR techniques are applied to determine the outcome. From the three main aspects-transplanted cells, spinal cord structure, and functional recovery-we summarize and discuss these methods with certain instances of applications in SCI models. Importantly, for the evaluations of function, neuronal transmitting, electrophysiological analysis and behavioral score are included. Wider conjunction of established technologies, as well as the further development of nondestructive methods might make a big difference in testing stem cell therapies. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

The loss-of-allele assay for ES cell screening and mouse genotyping.

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Frendewey, David; Chernomorsky, Rostislav; Esau, Lakeisha; Om, Jinsop; Xue, Yingzi; Murphy, Andrew J; Yancopoulos, George D; Valenzuela, David M

2010-01-01

Targeting vectors used to create directed mutations in mouse embryonic stem (ES) cells consist, in their simplest form, of a gene for drug selection flanked by mouse genomic sequences, the so-called homology arms that promote site-directed homologous recombination between the vector and the target gene. The VelociGene method for the creation of targeted mutations in ES cells employs targeting vectors, called BACVecs, that are based on bacterial artificial chromosomes. Compared with conventional short targeting vectors, BacVecs provide two major advantages: (1) their much larger homology arms promote high targeting efficiencies without the need for isogenicity or negative selection strategies; and (2) they enable deletions and insertions of up to 100kb in a single targeting event, making possible gene-ablating definitive null alleles and other large-scale genomic modifications. Because of their large arm sizes, however, BACVecs do not permit screening by conventional assays, such as long-range PCR or Southern blotting, that link the inserted targeting vector to the targeted locus. To exploit the advantages of BACVecs for gene targeting, we inverted the conventional screening logic in developing the loss-of-allele (LOA) assay, which quantifies the number of copies of the native locus to which the mutation was directed. In a correctly targeted ES cell clone, the LOA assay detects one of the two native alleles (for genes not on the X or Y chromosome), the other allele being disrupted by the targeted modification. We apply the same principle in reverse as a gain-of-allele assay to quantify the copy number of the inserted targeting vector. The LOA assay reveals a correctly targeted clone as having lost one copy of the native target gene and gained one copy of the drug resistance gene or other inserted marker. The combination of these quantitative assays makes LOA genotyping unequivocal and amenable to automated scoring. We use the quantitative polymerase chain reaction

Discriminating Different Cancer Cells Using a Zebrafish in Vivo Assay

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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

Discriminating Different Cancer Cells Using a Zebrafish in Vivo Assay

International Nuclear Information System (INIS)

Moshal, Karni S.; Ferri-Lagneau, Karine F.; Haider, Jamil; Pardhanani, Pooja; Leung, TinChung

2011-01-01

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

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

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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.

The Effect of Primary Cancer Cell Culture Models on the Results of Drug Chemosensitivity Assays: The Application of Perfusion Microbioreactor System as Cell Culture Vessel

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Chen, Yi-Dao; Huang, Shiang-Fu; Wang, Hung-Ming

2015-01-01

To precisely and faithfully perform cell-based drug chemosensitivity assays, a well-defined and biologically relevant culture condition is required. For the former, a perfusion microbioreactor system capable of providing a stable culture condition was adopted. For the latter, however, little is known about the impact of culture models on the physiology and chemosensitivity assay results of primary oral cavity cancer cells. To address the issues, experiments were performed. Results showed that minor environmental pH change could significantly affect the metabolic activity of cells, demonstrating the importance of stable culture condition for such assays. Moreover, the culture models could also significantly influence the metabolic activity and proliferation of cells. Furthermore, the choice of culture models might lead to different outcomes of chemosensitivity assays. Compared with the similar test based on tumor-level assays, the spheroid model could overestimate the drug resistance of cells to cisplatin, whereas the 2D and 3D culture models might overestimate the chemosensitivity of cells to such anticancer drug. In this study, the 3D culture models with same cell density as that in tumor samples showed comparable chemosensitivity assay results as the tumor-level assays. Overall, this study has provided some fundamental information for establishing a precise and faithful drug chemosensitivity assay. PMID:25654105

Toxcast Profiling in a Human Stem Cell Assay for Developmental Toxicity (SOT)

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We correlated the ToxCast library in a metabolic biomarker-based in vitro assay (Stemina devTOXqP) utilizing human embryonic stem (hES) cells (H9 line). This assay identifies the concentration of a chemical that disrupts cellular metabolism in a manner indicative of teratogenic...

Calcein AM release-based cytotoxic cell assay for fish leucocytes.

Science.gov (United States)

Iwanowicz, Luke R; Densmore, Christine L; Ottinger, Christopher A

2004-02-01

A non-specific cytotoxic cell assay for fish is presented that is based on the release of the activated fluorochrome calcein AM from lysed carp epithelioma papulosum cyprini (EPC) cells. To establish the suitability of treating EPC cells with calcein AM the uptake and spontaneous release of the calcein AM by the EPC cells was evaluated. Incubation of 5 microM calcein AM in culture medium with 1x10(5)EPC cells well(-1)for a minimum of 3 h provided sufficient labelling. Spontaneous release of fluorescence from the labelled EPC cells during 10 h of post labelling incubation ranged from 30 to 39% of the total observed fluorescence. Cytotoxic activity of trout leucocytes was evaluated at three leucocyte to target cell ratios (10:1, 2:1 and 1:1) following incubation (4, 6, 8, and 10 h) with calcein AM-labelled EPC cells at 15 degrees C. In some instances, the monoclonal antibody specific for the NCC surface receptor NCCRP-1 (MAb5C.6) was included in the cultures. The activity of NCC cells was significantly inhibited in the presence of 0.25 microg well(-1)of MAb5C.6 relative to no antibody (Pcell activity of approximately 18% was observed following 8 h of incubation at the 2:1 and 1:1 leucocyte to target cell ratios. Percent cytotoxic cell activity using calcein AM was similar to values reported for rainbow trout leucocytes using the 51Cr-release assay.

In vitro immunomodulation of a whole blood IFN-γ release assay enhances T cell responses in subjects with latent tuberculosis infection.

Directory of Open Access Journals (Sweden)

Rajiv L Gaur

Full Text Available Activation of innate immunity via pathogen recognition receptors (PRR modulates adaptive immune responses. PRR ligands are being exploited as vaccine adjuvants and as therapeutics, but their utility in diagnostics has not been explored. Interferon-gamma (IFN-γ release assays (IGRAs are functional T cell assays used to diagnose latent tuberculosis infection (LTBI; however, novel approaches are needed to improve their sensitivity.In vitro immunomodulation of a whole blood IGRA (QuantiFERON®-TB GOLD In-Tube with Toll-like receptor agonists poly(I:C, LPS, and imiquimod was performed on blood from subjects with LTBI and negative controls.In vitro immunomodulation significantly enhanced the response of T cells stimulated with M. tuberculosis antigens from subjects with LTBI but not from uninfected controls. Immunomodulation of IGRA revealed T cell responses in subjects with LTBI whose T cells otherwise do not respond to in vitro stimulation with antigens alone. Similar to their in vivo functions, addition of poly(I:C and LPS to whole blood induced secretion of inflammatory cytokines and IFN-α and enhanced the surface expression of antigen presenting and costimulatory molecules on antigen presenting cells.In vitro immunomodulation of whole blood IGRA may be an effective strategy for enhancing the sensitivity of T cells for diagnosis of LTBI.

Assaying Cellular Viability Using the Neutral Red Uptake Assay.

Science.gov (United States)

Ates, Gamze; Vanhaecke, Tamara; Rogiers, Vera; Rodrigues, Robim M

2017-01-01

The neutral red uptake assay is a cell viability assay that allows in vitro quantification of xenobiotic-induced cytotoxicity. The assay relies on the ability of living cells to incorporate and bind neutral red, a weak cationic dye, in lysosomes. As such, cytotoxicity is expressed as a concentration-dependent reduction of the uptake of neutral red after exposure to the xenobiotic under investigation. The neutral red uptake assay is mainly used for hazard assessment in in vitro toxicology applications. This method has also been introduced in regulatory recommendations as part of 3T3-NRU-phototoxicity-assay, which was regulatory accepted in all EU member states in 2000 and in the OECD member states in 2004 as a test guideline (TG 432). The present protocol describes the neutral red uptake assay using the human hepatoma cell line HepG2, which is often employed as an alternative in vitro model for human hepatocytes. As an example, the cytotoxicity of acetaminophen and acetyl salicylic acid is assessed.

Biomarkers of effect in endocrine disruption: how to link a functional assay to an adverse outcome pathway

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Stefano Lorenzetti

2015-06-01

Full Text Available The development of in vitro testing strategies may achieve a cost-effective generation of comprehensive datasets on a large number of chemicals, according to the requirements of the European Regulation REACH. Much emphasis is placed on in vitro methods based on subcellular mechanisms (e.g., nuclear receptor interaction, but it is necessary to define the predictive value of molecular or biochemical changes within an adverse outcome pathway (AOP. AOP pivots on the description of the flow from a molecular initiating event through a cascade of intermediate events needed to produce a specific adverse effect at organism level: downstream responses at cell level are, therefore, essential to define an AOP. Several in vitro assays are based on human cell lines representative of endocrine-targeted tissues (e.g., prostate and on functional biomarkers of clinical relevance (e.g., PSA secretion in human prostate epithelial cells. We discuss the implementation of such functional biomarkers in the AOP context.

Advances in establishment and analysis of three-dimensional tumor spheroid-based functional assays for target validation and drug evaluation

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Vinci Maria

2012-03-01

Full Text Available Abstract Background There is overwhelming evidence that in vitro three-dimensional tumor cell cultures more accurately reflect the complex in vivo microenvironment than simple two-dimensional cell monolayers, not least with respect to gene expression profiles, signaling pathway activity and drug sensitivity. However, most currently available three-dimensional techniques are time consuming and/or lack reproducibility; thus standardized and rapid protocols are urgently needed. Results To address this requirement, we have developed a versatile toolkit of reproducible three-dimensional tumor spheroid models for dynamic, automated, quantitative imaging and analysis that are compatible with routine high-throughput preclinical studies. Not only do these microplate methods measure three-dimensional tumor growth, but they have also been significantly enhanced to facilitate a range of functional assays exemplifying additional key hallmarks of cancer, namely cell motility and matrix invasion. Moreover, mutual tissue invasion and angiogenesis is accommodated by coculturing tumor spheroids with murine embryoid bodies within which angiogenic differentiation occurs. Highly malignant human tumor cells were selected to exemplify therapeutic effects of three specific molecularly-targeted agents: PI-103 (phosphatidylinositol-3-kinase (PI3K-mammalian target of rapamycin (mTOR inhibitor, 17-N-allylamino-17-demethoxygeldanamycin (17-AAG (heat shock protein 90 (HSP90 inhibitor and CCT130234 (in-house phospholipase C (PLCγ inhibitor. Fully automated analysis using a Celigo cytometer was validated for tumor spheroid growth and invasion against standard image analysis techniques, with excellent reproducibility and significantly increased throughput. In addition, we discovered key differential sensitivities to targeted agents between two-dimensional and three-dimensional cultures, and also demonstrated enhanced potency of some agents against cell migration

Technical Advance: New in vitro method for assaying the migration of primary B cells using an endothelial monolayer as substrate.

Science.gov (United States)

Stewart-Hutchinson, Phillip J; Szasz, Taylor P; Jaeger, Emily R; Onken, Michael D; Cooper, John A; Morley, Sharon Celeste

2017-09-01

Migration of B cells supports their development and recruitment into functional niches. Therefore, defining factors that control B cell migration will lead to a better understanding of adaptive immunity. In vitro cell migration assays with B cells have been limited by poor adhesion of cells to glass coated with adhesion molecules. We have developed a technique using monolayers of endothelial cells as the substrate for B cell migration and used this technique to establish a robust in vitro assay for B cell migration. We use TNF-α to up-regulate surface expression of the adhesion molecule VCAM-1 on endothelial cells. The ligand VLA-4 is expressed on B cells, allowing them to interact with the endothelial monolayer and migrate on its surface. We tested our new method by examining the role of L-plastin (LPL), an F-actin-bundling protein, in B cell migration. LPL-deficient (LPL -/- ) B cells displayed decreased speed and increased arrest coefficient compared with wild-type (WT) B cells, following chemokine stimulation. However, the confinement ratios for WT and LPL -/- B cells were similar. Thus, we demonstrate how the use of endothelial monolayers as a substrate will support future interrogation of molecular pathways essential to B cell migration. © Society for Leukocyte Biology.

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

Science.gov (United States)

Ayehunie, Seyoum; Snell, Maureen; Child, Matthew; Klausner, Mitchell

2009-01-01

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 2-5 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. PMID:19665512

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

International Nuclear Information System (INIS)

Ayehunie, Seyoum; Snell, Maureen; Child, Matthew; Klausner, Mitchell

2009-01-01

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.

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. Copyright © 2014 Elsevier B.V. All rights reserved.

Cell viability and PSA secretion assays in LNCaP cells: a tiered in vitro approach to screen chemicals with a prostate-mediated effect on male reproduction within the ReProTect project.

Science.gov (United States)

Lorenzetti, Stefano; Marcoccia, Daniele; Narciso, Laura; Mantovani, Alberto

2010-08-01

Prostate function is critical for male fertility; nevertheless, prostate was so far overlooked in reproductive toxicity assays. Within the EU project ReProTect, the human prostate cell line LNCaP was utilized to identify molecules targeting prostate function by the integrated assessment of cell viability (MTS assay) and prostate-specific antigen (PSA) secretion as specific marker; a training set - five (anti)androgenic chemicals - and a ReProTect feasibility set - ten chemicals - were used. Several compounds reduced PSA only at cytotoxic concentrations. Androgens (DHT, MT) markedly increased PSA as did the herbicide glufosinate ammonium, not known as androgen agonist. Anti-androgens (2OH-flutamide, linuron, vinclozolin, di-n-butyl phthalate) also increased PSA, but the effect of magnitude was much lower than for androgens. The ER-binder bisphenol A reduced PSA, while increasing cell viability. At this stage, the approach can identify chemicals able to interfere with prostate function: further refinements may allow to include prostate effects in reproductive toxicity in vitro testing. Copyright 2010 Elsevier Inc. All rights reserved.

CellAnimation: an open source MATLAB framework for microscopy assays.

Science.gov (United States)

Georgescu, Walter; Wikswo, John P; Quaranta, Vito

2012-01-01

Advances in microscopy technology have led to the creation of high-throughput microscopes that are capable of generating several hundred gigabytes of images in a few days. Analyzing such wealth of data manually is nearly impossible and requires an automated approach. There are at present a number of open-source and commercial software packages that allow the user to apply algorithms of different degrees of sophistication to the images and extract desired metrics. However, the types of metrics that can be extracted are severely limited by the specific image processing algorithms that the application implements, and by the expertise of the user. In most commercial software, code unavailability prevents implementation by the end user of newly developed algorithms better suited for a particular type of imaging assay. While it is possible to implement new algorithms in open-source software, rewiring an image processing application requires a high degree of expertise. To obviate these limitations, we have developed an open-source high-throughput application that allows implementation of different biological assays such as cell tracking or ancestry recording, through the use of small, relatively simple image processing modules connected into sophisticated imaging pipelines. By connecting modules, non-expert users can apply the particular combination of well-established and novel algorithms developed by us and others that are best suited for each individual assay type. In addition, our data exploration and visualization modules make it easy to discover or select specific cell phenotypes from a heterogeneous population. CellAnimation is distributed under the Creative Commons Attribution-NonCommercial 3.0 Unported license (http://creativecommons.org/licenses/by-nc/3.0/). CellAnimationsource code and documentation may be downloaded from www.vanderbilt.edu/viibre/software/documents/CellAnimation.zip. Sample data are available at www

A Functional High-Throughput Assay of Myelination in Vitro

Science.gov (United States)

2014-07-01

Human induced pluripotent stem cells, hydrogels, 3D culture, electrophysiology, high-throughput assay 16. SECURITY CLASSIFICATION OF: 17...image the 3D rat dorsal root ganglion ( DRG ) cultures with sufficiently low background as to detect electrically-evoked depolarization events, as...of voltage-sensitive dyes. 8    We have made substantial progress in Task 4.1. We have fabricated neural fiber tracts from DRG explants and

A High-Content Live-Cell Viability Assay and Its Validation on a Diverse 12K Compound Screen.

Science.gov (United States)

Chiaravalli, Jeanne; Glickman, J Fraser

2017-08-01

We have developed a new high-content cytotoxicity assay using live cells, called "ImageTOX." We used a high-throughput fluorescence microscope system, image segmentation software, and the combination of Hoechst 33342 and SYTO 17 to simultaneously score the relative size and the intensity of the nuclei, the nuclear membrane permeability, and the cell number in a 384-well microplate format. We then performed a screen of 12,668 diverse compounds and compared the results to a standard cytotoxicity assay. The ImageTOX assay identified similar sets of compounds to the standard cytotoxicity assay, while identifying more compounds having adverse effects on cell structure, earlier in treatment time. The ImageTOX assay uses inexpensive commercially available reagents and facilitates the use of live cells in toxicity screens. Furthermore, we show that we can measure the kinetic profile of compound toxicity in a high-content, high-throughput format, following the same set of cells over an extended period of time.

An improved method for staining cell colonies in clonogenic assays.

Science.gov (United States)

Guda, Kishore; Natale, Leanna; Markowitz, Sanford D

2007-06-01

Clonogenic assay is a widely used experimental approach to test for the effects of drugs/genes on the growth and proliferative characteristics of cells in vitro. Accurate quantitation of treatment effects in clonogeneic assays depends on the ability to visualize and count cell colonies precisely. We report a novel method (referred as ETeB) for staining cell colonies grown on plastic and specially coated substrates like collagen. Using colon cancer cell lines grown on plastic and collagen, we compared the colony staining efficiencies of the widely used methylene blue, and Ethidium bromide (ETeB) stains. Results show that the ETeB protocol works well on plastic and is extremely effective for staining colonies on collagen when compared to methylene blue. The key features and advantages of ETeB technique are; (a) reduction in background for colonies grown on collagen and possibly other substrates, (b) the whole procedure takes less than a minute, (c) no post-stain washing step is required which eliminates colony losses for cell lines that are loosely adherent, (d) colony visualization and counting can be done immediately following the staining procedure using a standard UV illuminator and software, and (e) the method works across a wide variety of cell lines. The simplicity and robustness of this procedure should warrant its usage in both small and large-scale clonogenic experiments.

Comparison of cell-based and non-cell-based assay platforms for the detection of clinically relevant anti-drug neutralizing antibodies for immunogenicity assessment of therapeutic proteins.

Science.gov (United States)

Hu, Jenny; Wala, Iwona; Han, Hong; Nagatani, Janice; Barger, Troy; Civoli, Francesca; Kaliyaperumal, Arunan; Zhuang, Yao; Gupta, Shalini

2015-04-01

Anti-drug neutralizing antibodies (NAbs) formed due to unwanted immunogenicity of a therapeutic protein point towards a mature immune response. NAb detection is important in interpreting the therapeutic's efficacy and safety in vivo. In vitro cell-based NAb assays provide a physiological system for NAb detection, however are complex assays. Non-cell-based competitive ligand binding (CLB) approaches are also employed for NAb detection. Instead of cells, CLB assays use soluble receptor and conjugated reagents and are easier to perform, however have reduced physiological relevance. The aim of this study was to compare the performance of CLB assays to established cell-based assays to determine the former's ability to detect clinically relevant NAbs towards therapeutics that (i) acted as an agonist or (ii) acted as antagonists by binding to a target receptor. We performed a head-to-head comparison of the performance of cell-based and CLB NAb assays for erythropoietin (EPO) and two anti-receptor monoclonal antibodies (AMG-X and AMG 317). Clinically relevant NAb-positive samples identified previously by a cell-based assay were assessed in the corresponding CLB format(s). A panel of 12 engineered fully human anti-EPO monoclonal antibodies (MAbs) was tested in both EPO NAb assay formats. Our results showed that the CLB format was (i) capable of detecting human anti-EPO MAbs of differing neutralizing capabilities and affinities and (ii) provided similar results as the cell-based assay for detecting NAbs in patient samples. The cell-based and CLB assays also behaved comparably in detecting NAbs in clinical samples for AMG-X. In the case of anti-AMG 317 NAbs, the CLB format failed to detect NAbs in more than 50% of the tested samples. We conclude that assay sensitivity, drug tolerance and the selected assay matrix played an important role in the inability of AMG 317 CLB assays to detect clinically relevant NAbs. Copyright © 2015 Elsevier B.V. All rights reserved.

Radiopeptide internalisation and externalisation assays: Cell viability and radioligand integrity

International Nuclear Information System (INIS)

Raza Naqvi, Syed Ali; Sosabowski, Jane K.; Ahamad Nagra, Saeed; Ishfaq, Malik M.; Mather, Stephen J.; Matzow, Torkjel

2011-01-01

Various aspects of radiopeptide receptor-mediated cell internalisation and externalisation assays were assessed, including the integrity of externalised peptides and the effect of varying the pH and incubation time of the acid wash step (to remove surface receptor-bound ligand) on efficacy and cell viability. The observed intact proportion of externalised peptide was 5-10%, and acid wash buffers with pH 2.8 or below were found to be detrimental to cell viability and integrity, particularly following prolonged incubation times.

In vitro cell-mediated immunity assay using 125I-iododeoxyuridine

International Nuclear Information System (INIS)

Morris, J.E.; Graham, T.M.

1979-01-01

We investigated an in vitro cell-mediated immunity assay using incorporation of 125 I-iododeoxyuridine as an indicator of lymphocyte responsiveness to mitogen stimulation. The system permits the use of whole-blood cultures in rats and dogs

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

A functional assay-based strategy for nanomaterial risk forecasting

International Nuclear Information System (INIS)

Hendren, Christine Ogilvie; Lowry, Gregory V.; Unrine, Jason M.; Wiesner, Mark R.

2015-01-01

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

Fundamental and clinical evaluation of ''SCC RIABEAD'' kit for immunoradiometric assay of squamous cell carcinoma related antigen

International Nuclear Information System (INIS)

Koizumi, Mitsuru; Endo, Keigo; Nakajima, Kotoko

1987-01-01

A commercial ''SCC RIABEAD'' kit for immunoradiometric assay of squamous cell carcinoma related antigen (SCC antigen) was fundamentally and clinically evaluated. Laboratory performance was satisfactory for intra-assay and inter-assay reproducibility, recovery, and dilution, with rapid and simple measurement techniques. Seropositivity for SCC antigen was significantly higher for squamous cell carcinoma of the liver and uterine cervix than the other histology types. In the case of cervical squamous cell carcinoma, it increased with progressing disease. Post-treatment serum levels of SCC antigen returned to negative. SCC antigen is considered to be a useful tumor marker for these diseases. There was a good correlation between the measurement values obtained from the present and conventional (SCC RIAKIT) assays. The present assay remarkably decreased false-positive cases of pulmonary benign diseases. The results showed a ''SCC RIABEAD'' to be a favorable kit for immunoradiometric assay of SSC antigen, as compared with conventional assay kit. (Namekawa, K.)

The physical and functional interaction of NDRG2 with MSP58 in cells

International Nuclear Information System (INIS)

Zhang Jing; Liu Junye; Li Xia; Li Fuyang; Wang Lifeng; Zhang Jian; Liu Xinping; Shen Lan; Liu Na; Deng Yanchun; Yang Angang; Han Hua; Zhao Mujun; Yao Libo

2007-01-01

NDRG2, a member of N-Myc downstream regulated gene family, exerts the important functions in cell differentiation and tumor suppression. Although the ectopic expressed Ndrg2 inhibits the proliferation of tumor cells, its intracellular signal transduction pathway is hardly known. Here, we identified MSP58, a 58-kDa microspherule protein, as an interacting partner of human Ndrg2 by using yeast two-hybrid screening. The interaction was confirmed by glutathione S-transferase pull-down assay in vitro and by co-immune-precipitation assay in vivo. The forkhead associated domain of MSP58 is essential for its interaction with Ndrg2. Ndrg2 could co-localize with MSP58 in nuclear of HeLa cell during cell stress. Furthermore, the modulation of Ndrg2 level influences the cell cycle process together with MSP58. In conclusion, the findings offered a novel insight into the physiological roles of Ndrg2

Marrow stem cell release in the autorepopulation assay

Energy Technology Data Exchange (ETDEWEB)

Maloney, M A; Patt, H M [California Univ., San Francisco (USA). Lab. of Radiobiology

1978-01-01

The early migration of stem cells from shielded marrow to an irradiated spleen has been re-evaluated, and the findings have been compared with the results of earlier studies. The composite data reveal a constant rate during the first 24 h after irradiation, with a slope of 1.6 cells per h and an intercept of 2.4. The positive intercept is interpreted to signify an immediate brief perturbation of CFU/sub s/ release. The low concentration of CFU/sub s/ in the bloodstream, despite their continuous migration from the shielded marrow, is indicative of a rapid, and probably greatly increased, blood turnover. Despite the constancy of stem cell seeding, it is not yet possible to determine whether the rate of stem cell release is different in shielded marrow than in normal marrow. The resolution of this question requires more precise information about spleen seeding efficiency in the autorepopulation assay and about the normal turnover rate of stem cells in the bloodstream.

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

Directory of Open Access Journals (Sweden)

Yi Huan

2013-04-01

Full Text Available 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-ylamino]2-deoxyglucose (2-NBDG as a glucose analog, we have developed a nonradioactive, cell-based assay for the discovery and characterization of SGLT2 inhibitors.

Using a split luciferase assay (SLA) to measure the kinetics of cell-cell fusion mediated by herpes simplex virus glycoproteins.

Science.gov (United States)

Saw, Wan Ting; Matsuda, Zene; Eisenberg, Roselyn J; Cohen, Gary H; Atanasiu, Doina

2015-11-15

Herpes simplex virus (HSV) entry and cell-cell fusion require the envelope proteins gD, gH/gL and gB. We propose that receptor-activated conformational changes to gD activate gH/gL, which then triggers gB (the fusogen) into an active form. To study this dynamic process, we have adapted a dual split protein assay originally developed to study the kinetics of human immunodeficiency virus (HIV) mediated fusion. This assay uses a chimera of split forms of renilla luciferase (RL) and green fluorescent protein (GFP). Effector cells are co-transfected with the glycoproteins and one of the split reporters. Receptor-bearing target cells are transfected with the second reporter. Co-culture results in fusion and restoration of RL, which can convert a membrane permeable substrate into a luminescent product, thereby enabling one to monitor initiation and extent of fusion in live cells in real time. Restoration of GFP can also be studied by fluorescence microscopy. Two sets of split reporters have been developed: the original one allows one to measure fusion kinetics over hours whereas the more recent version was designed to enhance the sensitivity of RL activity allowing one to monitor both initiation and rates of fusion in minutes. Here, we provide a detailed, step-by-step protocol for the optimization of the assay (which we call the SLA for split luciferase assay) using the HSV system. We also show several examples of the power of this assay to examine both the initiation and kinetics of cell-cell fusion by wild type forms of gD, gB, gH/gL of both serotypes of HSV as well as the effect of mutations and antibodies that alter the kinetics of fusion. The SLA can be applied to other viral systems that carry out membrane fusion. Copyright © 2015 Elsevier Inc. All rights reserved.

Development of a surrogate angiogenic potency assay for clinical-grade stem cell production.

Science.gov (United States)

Lehman, Nicholas; Cutrone, Rochelle; Raber, Amy; Perry, Robert; Van't Hof, Wouter; Deans, Robert; Ting, Anthony E; Woda, Juliana

2012-09-01

Clinical results from acute myocardial infarction (AMI) patients treated with MultiStem®, a large-scale expanded adherent multipotent progenitor cell population (MAPC), have demonstrated a strong safety and benefit profile for these cells. The mechanism of benefit with MAPC treatment is a result, in part, of its ability to induce neovascularization through trophic support. Production of clinical-grade stem cell products requires the development of lot-release criteria based on potency assays that directly reflect the fundamental mechanistic pathway underlying the therapeutic response to verify manufacturing process consistency and product potency. Using an in vitro endothelial tube formation assay, a potency assay has been developed that reflects MAPC pro-angiogenic activity. Serum-free conditioned media collected from MAPC culture induced endothelial tube formation. A proteomic survey of angiogenic factors produced by the cells in vitro revealed candidate factors linked to angiogenic potency. Three cytokines, chemokine (C-X-C motif) ligand 5 (CXCL5), interleukin 8 (IL-8) and vascular endothelial growth factor (VEGF), were required for this angiogenic activity. Depletion of any of these factors from the media prevented tube formation, while adding back increasing amounts of these cytokines into the depleted serum-free conditioned media established the lower limits of each of the cytokines required to induce angiogenesis. A necessary threshold of angiogenic factor expression was established using an in vitro angiogenesis assay. By correlating the levels of the cytokines required to induce tube formation in vitro with levels of the factors found in the spent media from manufacturing production runs, detection of these factors was identified as a surrogate potency assay with defined pass/fail criteria.

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

Cell based assays for anti-Plasmodium activity evaluation.

Science.gov (United States)

Mokgethi-Morule, Thabang; N'Da, David D

2016-03-10

Malaria remains one of the most common and deadly infectious diseases worldwide. The severity of this global public health challenge is reflected by the approximately 198 million people, who were reportedly infected in 2013 and by the more than 584,000 related deaths in that same year. The rising emergence of drug resistance towards the once effective artemisinin combination therapies (ACTs) has become a serious concern and warrants more robust drug development strategies, with the objective of eradicating malaria infections. The intricate biology and life cycle of Plasmodium parasites complicate the understanding of the disease in such a way that would enhance the development of more effective chemotherapies that would achieve radical clinical cure and that would prevent disease relapse. Phenotypic cell based assays have for long been a valuable approach and involve the screening and analysis of diverse compounds with regards to their activities towards whole Plasmodium parasites in vitro. To achieve the Millennium Development Goal (MDG) of malaria eradication by 2020, new generation drugs that are active against all parasite stages (erythrocytic (blood), exo-erythrocytic (liver stages and gametocytes)) are needed. Significant advances are being made in assay development to overcome some of the practical challenges of assessing drug efficacy, particularly in the liver and transmission stage Plasmodium models. This review discusses primary screening models and the fundamental progress being made in whole cell based efficacy screens of anti-malarial activity. Ongoing challenges and some opportunities for improvements in assay development that would assist in the discovery of effective, safe and affordable drugs for malaria treatments are also discussed. Copyright © 2016 Elsevier B.V. All rights reserved.

An In Vitro Potency Assay for Monitoring the Immunomodulatory Potential of Stromal Cell-Derived Extracellular Vesicles

Directory of Open Access Journals (Sweden)

Karin Pachler

2017-07-01

Full Text Available The regenerative and immunomodulatory activity of mesenchymal stromal cells (MSCs is partially mediated by secreted vesicular factors. Extracellular vesicles (EVs exocytosed by MSCs are gaining increased attention as prospective non-cellular therapeutics for a variety of diseases. However, the lack of suitable in vitro assays to monitor the therapeutic potential of EVs currently restricts their application in clinical studies. We have evaluated a dual in vitro immunomodulation potency assay that reproducibly reports the inhibitory effect of MSCs on induced T-cell proliferation and the alloantigen-driven mixed leukocyte reaction of pooled peripheral blood mononuclear cells in a dose-dependent manner. Phytohemagglutinin-stimulated T-cell proliferation was inhibited by MSC-derived EVs in a dose-dependent manner comparable to MSCs. In contrast, inhibition of alloantigen-driven mixed leukocyte reaction was only observed for MSCs, but not for EVs. Our results support the application of a cell-based in vitro potency assay for reproducibly determining the immunomodulatory potential of EVs. Validation of this assay can help establish reliable release criteria for EVs for future clinical studies.

Development of assay platforms for in vitro screening of Treg modulating potential of pharmacological compounds

DEFF Research Database (Denmark)

Pedersen, Anders Elm; Holmstrøm, Kim; Jørgensen, Flemming

2015-01-01

that investigates Treg modulation by current drugs. For such research as well as for novel cell based therapies based on Treg infusions, rapid in vitro assays as well as functional assays based on inhibitory capacity of Tregs are required. Here, we report on such assays using highly pure fluorescence-activated cell...... and TNF-α. In conclusion, these assays have the potential for use in pharmacological screening and discovery in relation to drug development in immunology....

Radiopeptide internalisation and externalization assays: cell viability and radioligand integrity.

Science.gov (United States)

Naqvi, Syed Ali Raza; Sosabowski, Jane K; Nagra, Saeed Ahamad; Ishfaq, Malik M; Mather, Stephen J; Matzow, Torkjel

2011-01-01

Various aspects of radiopeptide receptor-mediated cell internalisation and externalization assays were assessed, including the integrity of externalized peptides and the effect of varying the pH and incubation time of the acid wash step (to remove surface receptor-bound ligand) on efficacy and cell viability. The observed intact proportion of externalized peptide was 5-10%, and acid wash buffers with pH 2.8 or below were found to be detrimental to cell viability and integrity, particularly following prolonged incubation times. Copyright © 2010 Elsevier Ltd. All rights reserved.

Dendritic cell migration assay: a potential prediction model for identification of contact allergens.

Science.gov (United States)

Gibbs, Susan; Spiekstra, Sander; Corsini, Emanuela; McLeod, Julie; Reinders, Judith

2013-04-01

This manuscript describes methodology and a prediction model for the MUTZ-LC migration assay. The assay represents the physiological change in Langerhans cell (LC) behavior after exposure to a sensitizing chemical, resulting in LC migration from the epidermis to the dermis. MUTZ-LC are derived from the commercially available MUTZ-3 cell line. Upon exposure to a sensitizer MUTZ-LC migrate preferentially towards CXCL12 whereas upon exposure to a non-sensitizer MUTZ-LC migrate towards CCL5. A CXCL12/CCL5 ratio >1.10 in 2/3 independent experiments is indicative of a sensitizer, whereas a CXCL12/CCL5 ratio ≤1.10 is indicative of a non-sensitizer. At non cytotoxic chemical concentrations 9 sensitizers (2,4-dinitrochlorobenzene, paraphenylendiamine, cinnamaldehyde, isoeugenol, nickel-sulfate, tetramethylthiuram disulfide, eugenol, cinnamic-alcohol, ammonium-hexachloroplatinate) were distinguished from 4 non sensitizers (sodium lauryl sulfate, salicylic acid, phenol, octanoic acid). Critical points in assay performance are (i) MUTZ-3 passage number after thawing (p6-p40); (ii) cell viability (>80%); (iii) standard curve to optimize correlation of fluorescence with cell number; and (iv) optimization of the concentration of rhCXCL12 and rhCCL5 in transwell. The protocol has been tested in three European laboratories and results suggest that it may provide working conditions for performing the DC migration assay which is aimed at distinguishing sensitizers from non sensitizers. Copyright © 2012 Elsevier Ltd. All rights reserved.

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

International Nuclear Information System (INIS)

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

1988-01-01

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

Cell Painting, a high-content image-based assay for morphological profiling using multiplexed fluorescent dyes

Science.gov (United States)

Bray, Mark-Anthony; Singh, Shantanu; Han, Han; Davis, Chadwick T.; Borgeson, Blake; Hartland, Cathy; Kost-Alimova, Maria; Gustafsdottir, Sigrun M.; Gibson, Christopher C.; Carpenter, Anne E.

2016-01-01

In morphological profiling, quantitative data are extracted from microscopy images of cells to identify biologically relevant similarities and differences among samples based on these profiles. This protocol describes the design and execution of experiments using Cell Painting, a morphological profiling assay multiplexing six fluorescent dyes imaged in five channels, to reveal eight broadly relevant cellular components or organelles. Cells are plated in multi-well plates, perturbed with the treatments to be tested, stained, fixed, and imaged on a high-throughput microscope. Then, automated image analysis software identifies individual cells and measures ~1,500 morphological features (various measures of size, shape, texture, intensity, etc.) to produce a rich profile suitable for detecting subtle phenotypes. Profiles of cell populations treated with different experimental perturbations can be compared to suit many goals, such as identifying the phenotypic impact of chemical or genetic perturbations, grouping compounds and/or genes into functional pathways, and identifying signatures of disease. Cell culture and image acquisition takes two weeks; feature extraction and data analysis take an additional 1-2 weeks. PMID:27560178

Expression of assayable residual stem cell damage in erythroid differentiation

International Nuclear Information System (INIS)

Huebner, G.E.; Miller, M.E.; Cronkite, E.P.

1985-01-01

In rodents, residual damage is inducible in hematopoietic stem cells by exposure to ionizing radiation or alkylating agents. This damage can b e assayed in mice by transferring bone marrow into lethally irradiated syngeneic recipients and subsequently measuring the incremental increase of-( 125 I)iodo-2'-deoxyuridine incorporation in spleens. In this study, bone marrow from mice treated 3 weeks previously with Methylnitrosourea (50 mg/kg) or 450 rad was injected into recipients in order to determine possible residual effects of treatment of erythroid cell differentiation following stem cell seeding. Such effects were detected by a reduced amount of 59 Fe incorporation into spleens, thus indicatin g transfer of residual stem cell damage to differentiating cells. (orig.)

Small Molecule Agonists of Cell Adhesion Molecule L1 Mimic L1 Functions In Vivo.

Science.gov (United States)

Kataria, Hardeep; Lutz, David; Chaudhary, Harshita; Schachner, Melitta; Loers, Gabriele

2016-09-01

Lack of permissive mechanisms and abundance of inhibitory molecules in the lesioned central nervous system of adult mammals contribute to the failure of functional recovery after injury, leading to severe disabilities in motor functions and pain. Peripheral nerve injury impairs motor, sensory, and autonomic functions, particularly in cases where nerve gaps are large and chronic nerve injury ensues. Previous studies have indicated that the neural cell adhesion molecule L1 constitutes a viable target to promote regeneration after acute injury. We screened libraries of known drugs for small molecule agonists of L1 and evaluated the effect of hit compounds in cell-based assays in vitro and in mice after femoral nerve and spinal cord injuries in vivo. We identified eight small molecule L1 agonists and showed in cell-based assays that they stimulate neuronal survival, neuronal migration, and neurite outgrowth and enhance Schwann cell proliferation and migration and myelination of neurons in an L1-dependent manner. In a femoral nerve injury mouse model, enhanced functional regeneration and remyelination after application of the L1 agonists were observed. In a spinal cord injury mouse model, L1 agonists improved recovery of motor functions, being paralleled by enhanced remyelination, neuronal survival, and monoaminergic innervation, reduced astrogliosis, and activation of microglia. Together, these findings suggest that application of small organic compounds that bind to L1 and stimulate the beneficial homophilic L1 functions may prove to be a valuable addition to treatments of nervous system injuries.

Diesel-Enriched Particulate Matter Functionally Activates Human Dendritic Cells

Science.gov (United States)

Porter, Michael; Karp, Matthew; Killedar, Smruti; Bauer, Stephen M.; Guo, Jia; Williams, D'Ann; Breysse, Patrick; Georas, Steve N.; Williams, Marc A.

2007-01-01

Epidemiologic studies have associated exposure to airborne particulate matter (PM) with exacerbations of asthma. It is unknown how different sources of PM affect innate immunity. We sought to determine how car- and diesel exhaust–derived PM affects dendritic cell (DC) activation. DC development was modeled using CD34+ hematopoietic progenitors. Airborne PM was collected from exhaust plenums of Fort McHenry Tunnel providing car-enriched particles (CEP) and diesel-enriched particles (DEP). DC were stimulated for 48 hours with CEP, DEP, CD40-ligand, or lipopolysaccharide. DC activation was assessed by flow cytometry, enzyme-linked immunosorbent assay, and standard culture techniques. DEP increased uptake of fluorescein isothiocyanate–dextran (a model antigen) by DC. Diesel particles enhanced cell-surface expression of co-stimulatory molecules (e.g., CD40 [P < 0.01] and MHC class II [P < 0.01]). By contrast, CEP poorly affected antigen uptake and expression of cell surface molecules, and did not greatly affect cytokine secretion by DC. However, DEP increased production of TNF, IL-6, and IFN-γ (P < 0.01), IL-12 (P < 0.05), and vascular endothelial growth factor (P < 0.001). In co-stimulation assays of PM-exposed DC and alloreactive CD4+ T cells, both CEP and DEP directed a Th2-like pattern of cytokine production (e.g., enhanced IL-13 and IL-18 and suppressed IFN-γ production). CD4+ T cells were not functionally activated on exposure to either DEP or CEP. Car- and diesel-enriched particles exert a differential effect on DC activation. Our data support the hypothesis that DEP (and to a lesser extent CEP) regulate important functional aspects of human DC, supporting an adjuvant role for this material. PMID:17630318

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 .

Automation of the ELISpot assay for high-throughput detection of antigen-specific T-cell responses.

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Almeida, Coral-Ann M; Roberts, Steven G; Laird, Rebecca; McKinnon, Elizabeth; Ahmed, Imran; Pfafferott, Katja; Turley, Joanne; Keane, Niamh M; Lucas, Andrew; Rushton, Ben; Chopra, Abha; Mallal, Simon; John, Mina

2009-05-15

The enzyme linked immunospot (ELISpot) assay is a fundamental tool in cellular immunology, providing both quantitative and qualitative information on cellular cytokine responses to defined antigens. It enables the comprehensive screening of patient derived peripheral blood mononuclear cells to reveal the antigenic restriction of T-cell responses and is an emerging technique in clinical laboratory investigation of certain infectious diseases. As with all cellular-based assays, the final results of the assay are dependent on a number of technical variables that may impact precision if not highly standardised between operators. When studies that are large scale or using multiple antigens are set up manually, these assays may be labour intensive, have many manual handling steps, are subject to data and sample integrity failure and may show large inter-operator variability. Here we describe the successful automated performance of the interferon (IFN)-gamma ELISpot assay from cell counting through to electronic capture of cytokine quantitation and present the results of a comparison between automated and manual performance of the ELISpot assay. The mean number of spot forming units enumerated by both methods for limiting dilutions of CMV, EBV and influenza (CEF)-derived peptides in six healthy individuals were highly correlated (r>0.83, pautomated system compared favourably with the manual ELISpot and further ensured electronic tracking, increased through-put and reduced turnaround time.

Studies on the utility and mechanism of the V-79 cell metabolic cooperation assay for tumor promoters

International Nuclear Information System (INIS)

Hartman, T.G.

1985-01-01

Cigarette smoke condensate and its fractions were tested for activity in the V-79 Metabolic Cooperation Assay to determine the usefulness of the assay for analysis of a complex mixture and to compare the results obtained with previously conducted in vivo promoter assays. The whole condensate and several of its fractions were positive in the assay. In general, the Metabolic Cooperation Assay results were comparable to previously published results obtained on mouse skin. The effect of cell density, phorbol 12-myrystate-13-acetate (PMA) exposure time, concentration, pre-exposure and binding activity on the recovery of mutant V-79 Chinese hamster lung fibroblasts in the Metabolic Cooperation Assay was determined. A PMA exposure interval of only 1 minute resulted in maximum recovery of mutant cells. PMA began to inhibit metabolic cooperation at an exposure concentration of 0.1 ng/ml. Pre-exposure of cells to PMA increased the recovery of both post-PMA-treated and non-treated mutant cells in a dose-dependent manner. 3 H-PMA was rapidly bound to or taken up by the V-79 cells under assay conditions. The effect of calcium antagonists and representative compounds from several classes of anti-promoters including anti-inflammatory sterols, protease inhibitors, retinoids and cyclic nucleotides on metabolic determined. Each compound was tested for its effect on metabolic cooperation and also for its ability to reverse or modify the inhibitory properties of PMA on inter-cellular communication. Of all the compounds tested only cyclic adenosine monophosphate (cAMP) was able to antagonize the inhibitory effect of PMA

Peptide array-based interaction assay of solid-bound peptides and anchorage-dependant cells and its effectiveness in cell-adhesive peptide design.

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Kato, Ryuji; Kaga, Chiaki; Kunimatsu, Mitoshi; Kobayashi, Takeshi; Honda, Hiroyuki

2006-06-01

Peptide array, the designable peptide library covalently synthesized on cellulose support, was applied to assay peptide-cell interaction, between solid-bound peptides and anchorage-dependant cells, to study objective peptide design. As a model case, cell-adhesive peptides that could enhance cell growth as tissue engineering scaffold material, was studied. On the peptide array, the relative cell-adhesion ratio of NIH/3T3 cells was 2.5-fold higher on the RGDS (Arg-Gly-Asp-Ser) peptide spot as compared to the spot with no peptide, thus indicating integrin-mediated peptide-cell interaction. Such strong cell adhesion mediated by the RGDS peptide was easily disrupted by single residue substitution on the peptide array, thus indicating that the sequence recognition accuracy of cells was strictly conserved in our optimized scheme. The observed cellular morphological extension with active actin stress-fiber on the RGD motif-containing peptide supported our strategy that peptide array-based interaction assay of solid-bound peptide and anchorage-dependant cells (PIASPAC) could provide quantitative data on biological peptide-cell interaction. The analysis of 180 peptides obtained from fibronectin type III domain (no. 1447-1629) yielded 18 novel cell-adhesive peptides without the RGD motif. Taken together with the novel candidates, representative rules of ineffective amino acid usage were obtained from non-effective candidate sequences for the effective designing of cell-adhesive peptides. On comparing the amino acid usage of the top 20 and last 20 peptides from the 180 peptides, the following four brief design rules were indicated: (i) Arg or Lys of positively charged amino acids (except His) could enhance cell adhesion, (ii) small hydrophilic amino acids are favored in cell-adhesion peptides, (iii) negatively charged amino acids and small amino acids (except Gly) could reduce cell adhesion, and (iv) Cys and Met could be excluded from the sequence combination since they have

Hydrocortisone Increases the Vinblastine-Induced Chromosomal Damages in L929 Cells Investigated by the Micronucleus Assay on Cytokinesis-Blocked Binucleated Cells

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Tahere Ebrahimipour

2017-03-01

Full Text Available Background: Stress may cause damages to DNA or/and change the ability of the cells to overcome these damages. It may also cause irregularities in the cell cycle and induce abnormal cell divisions through glucocorticoid-dependent functions. The abnormal cell divisions, in turn, lead to chromosomal mal-segregation and aneuploidy. In this study, the effects of the stress hormone, hydrocortisone (HYD, were investigated on the induced chromosomal abnormalities by vinblastine (VIN during cell cycle in L929 cells. Methods: This work was performed in winter 2013 at Department of Biology, University of Ferdowsi, Mashhad, Iran. Cultured cells were divided into different groups including control, VIN-treated, HYD treated and VIN+HYD co-treated cells. The induced chromosomal damages were investigated by micronucleus assay in cytokinesis-blocked binucleated cells. Results: Although HYD by itself did not increase the micronuclei (Mn frequency, co-treatment of cells with VIN and HYD led to significant increase (P<0.05 in the frequency of Mn in comparison to control and VIN treated groups. Conclusion: Cells treated with stress hormone are more sensitive to damages induced by VIN. Therefore, stress may not directly result in genetic instability, it can increase the harmful effects associated with other genotoxic agents.

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

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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.

Investigating evolutionary conservation of dendritic cell subset identity and functions

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Thien-Phong eVu Manh

2015-06-01

Full Text Available Dendritic cells (DC were initially defined as mononuclear phagocytes with a dendritic morphology and an exquisite efficiency for naïve T cell activation. DC encompass several subsets initially identified by their expression of specific cell surface molecules and later shown to excel in distinct functions and to develop under the instruction of different transcription factors or cytokines. Very few cell surface molecules are expressed in a specific manner on any immune cell type. Hence, to identify cell types, the sole use of a small number of cell surface markers in classical flow cytometry can be deceiving. Moreover, the markers currently used to define mononuclear phagocyte subsets vary depending on the tissue and animal species studied and even between laboratories. This has led to confusion in the definition of DC subset identity and in their attribution of specific functions. There is a strong need to identify a rigorous and consensus way to define mononuclear phagocyte subsets, with precise guidelines potentially applicable throughout tissues and species. We will discuss the advantages, drawbacks and complementarities of different methodologies: cell surface phenotyping, ontogeny, functional characterization and molecular profiling. We will advocate that gene expression profiling is a very rigorous, largely unbiased and accessible method to define the identity of mononuclear phagocyte subsets, which strengthens and refines surface phenotyping. It is uniquely powerful to yield new, experimentally testable, hypotheses on the ontogeny or functions of mononuclear phagocyte subsets, their molecular regulation and their evolutionary conservation. We propose defining cell populations based on a combination of cell surface phenotyping, expression analysis of hallmark genes and robust functional assays, in order to reach a consensus and integrate faster the huge but scattered knowledge accumulated by different laboratories on different cell types

Autoradiographic assay of mutants resistant to diphtheria toxin in mammalian cells in vitro

International Nuclear Information System (INIS)

Ronen, A.; Gingerich, J.D.; Duncan, A.M.V.; Heddle, J.A.

1984-01-01

Diptheria toxin kills mammalian cells by ribosylating elongation factor 2, a protein factor necessary for protein synthesis. The frequency of cells able to form colonies in the presence of the toxin can be used as an assay for mutation to diphtheria toxin resistance. Resistance to diphtheria toxin can also be detected autoradiographically in cells exposed to [ 3 H]leucine after treatment with the toxin. In cultures of Chinese hamster ovary cells, the frequency of such resistant cells is increased by exposure of the cells to γ-rays, ultraviolet light, ethylnitrosourea, mitomycin c, ethidium bromide, and 5-bromo-2'-deoxyuridine in a dose- and time-dependent manner. The resistant cells form discrete microcolonies if they are allowed to divide several times before intoxication which indicates that they are genuine mutants. The assay is potentially adaptable to any cell population that can be intoxicated with diphtheria toxin and labeled with [ 3 H]leucine, whether or not the cells can form colonies. It may be useful, therefore, for measuring mutation rates in slowly growing or nondividing cell populations such as breast, brain, and liver, as well as in cells that do divide but cannot be readily cloned, such as the colonic epithelium. 23 references, 6 figures

An HPLC-UV method for the measurement of permeability of marker drugs in the Caco-2 cell assay

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J.M. Kratz

2011-06-01

Full Text Available The Caco-2 cell line has been used as a model to predict the in vitro permeability of the human intestinal barrier. The predictive potential of the assay relies on an appropriate in-house validation of the method. The objective of the present study was to develop a single HPLC-UV method for the identification and quantitation of marker drugs and to determine the suitability of the Caco-2 cell permeability assay. A simple chromatographic method was developed for the simultaneous determination of both passively (propranolol, carbamazepine, acyclovir, and hydrochlorothiazide and actively transported drugs (vinblastine and verapamil. Separation was achieved on a C18 column with step-gradient elution (acetonitrile and aqueous solution of ammonium acetate, pH 3.0 at a flow rate of 1.0 mL/min and UV detection at 275 nm during the total run time of 35 min. The method was validated and found to be specific, linear, precise, and accurate. This chromatographic system can be readily used on a routine basis and its utilization can be extended to other permeability models. The results obtained in the Caco-2 bi-directional transport experiments confirmed the validity of the assay, given that high and low permeability profiles were identified, and P-glycoprotein functionality was established.

Significance of the proportion of binucleate cells in the micronucleus assay

International Nuclear Information System (INIS)

Imamura, Masahiro; Edgren, M.R.

1994-01-01

Using treatment with cytochalasin-B (Cyt-B) for the induction of a cytokinetic block, the significance of the proportion of binucleate cells (BNC) in the micronucleus (MN) assay was investigated in a methodological study. A Chinese hamster cell line V79 was used in which MN were induced by radiation. In complementary tests the radiation effect in inducing MN was enhanced by depletion of the cellular glutathione content with buthionine sulfoximine (BSO). The data indicated that the concentration of Cyt-B is the major factor which determines the proportion of BNC. This proportion was shown to be independent of radiation dose and of BSO. Furthermore, the MN frequency was not related to the percentage of BNC. Therefore, a high proportion of BNC may be practical for the MN assay, but may not make the technique more accurate. (author)

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

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Maria Charzyńska

2014-01-01

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.

Direct microculture enzyme-linked immunosorbent assay for studying neural cells: oligodendrocytes.

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Gard, A L; Warrington, A E; Pfeiffer, S E

1988-05-01

Oligodendrocyte development has been studied in a standardized primary microculture system initiated from day 20-21 fetal rat brain using a solid-phase enzyme-linked immunosorbent assay (ELISA) carried out directly on fixed cells (direct microculture ELISA). A highly reproducible dissociation procedure is described that allows careful control of the number of cells seeded per culture. At a seeding density of 1 x 10(5) cells/culture, up to 250 oligodendrocyte-generating microcultures consisting of 10-12% oligodendrocytes can be prepared from a single fetal rat brain, thereby permitting the simultaneous assay of multiple developmental parameters in sibling cultures. The validity of this method for quantifying myelinogenesis was established by comparing the results obtained by direct microculture ELISA with immunocytochemical counting of cells in parallel cultures. As few as 200 oligodendrocytes could be detected using a biotinylated anti-Ig and an avidin-urease conjugate detection system; CNP immunoreactivity measured by ELISA was linearly proportional to the number of immunolabeled cells between 6 and 34 days in culture; the developmental time courses of 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) and myelin basic protein (MBP) expression determined by the two methods were very similar. Finally, cell suspensions were seeded at increasing dilution to determine the number of cells required to generate cultures that tested positive for oligodendrocytes by ELISA. As few as 9,000 cells were sufficient, predicting a minimum of 8,000 oligoprogenitors per 20-21 day fetal rat brain. The application of direct microculture ELISA for studying oligodendrocyte population size and myelinogenesis is discussed.

Effects of mesenchymal stem cells from human induced pluripotent stem cells on differentiation, maturation, and function of dendritic cells.

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Gao, Wen-Xiang; Sun, Yue-Qi; Shi, Jianbo; Li, Cheng-Lin; Fang, Shu-Bin; Wang, Dan; Deng, Xue-Quan; Wen, Weiping; Fu, Qing-Ling

2017-03-02

Mesenchymal stem cells (MSCs) have potent immunomodulatory effects on multiple immune cells and have great potential in treating immune disorders. Induced pluripotent stem cells (iPSCs) serve as an unlimited and noninvasive source of MSCs, and iPSC-MSCs have been reported to have more advantages and exhibit immunomodulation on T lymphocytes and natural killer cells. However, the effects of iPSC-MSCs on dendritic cells (DCs) are unclear. The aim of this study is to investigate the effects of iPSC-MSCs on the differentiation, maturation, and function of DCs. Human monocyte-derived DCs were induced and cultured in the presence or absence of iPSC-MSCs. Flow cytometry was used to analyze the phenotype and functions of DCs, and enzyme-linked immunosorbent assay (ELISA) was used to study cytokine production. In this study, we successfully induced MSCs from different clones of human iPSCs. iPSC-MSCs exhibited a higher proliferation rate with less cell senescence than BM-MSCs. iPSC-MSCs inhibited the differentiation of human monocyte-derived DCs by both producing interleukin (IL)-10 and direct cell contact. Furthermore, iPSC-MSCs did not affect immature DCs to become mature DCs, but modulated their functional properties by increasing their phagocytic ability and inhibiting their ability to stimulate proliferation of lymphocytes. More importantly, iPSC-MSCs induced the generation of IL-10-producing regulatory DCs in the process of maturation, which was mostly mediated by a cell-cell contact mechanism. Our results indicate an important role for iPSC-MSCs in the modulation of DC differentiation and function, supporting the clinical application of iPSC-MSCs in DC-mediated immune diseases.

A Sensitive Chemotaxis Assay Using a Novel Microfluidic Device

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Chen Zhang

2013-01-01

Full Text Available Existing chemotaxis assays do not generate stable chemotactic gradients and thus—over time—functionally measure only nonspecific random motion (chemokinesis. In comparison, microfluidic technology has the capacity to generate a tightly controlled microenvironment that can be stably maintained for extended periods of time and is, therefore, amenable to adaptation for assaying chemotaxis. We describe here a novel microfluidic device for sensitive assay of cellular migration and show its application for evaluating the chemotaxis of smooth muscle cells in a chemokine gradient.

Optimization and validation of a neutralizing antibody assay for HIV-1 in A3R5 cells.

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Sarzotti-Kelsoe, Marcella; Daniell, Xiaoju; Todd, Christopher A; Bilska, Miroslawa; Martelli, Amanda; LaBranche, Celia; Perez, Lautaro G; Ochsenbauer, Christina; Kappes, John C; Rountree, Wes; Denny, Thomas N; Montefiori, David C

2014-07-01

A3R5 is a human CD4(+) lymphoblastoid cell line that was engineered to express CCR5 and is useful for the detection of weak neutralizing antibody responses against tier 2 strains of HIV-1. Here we describe the optimization and validation of the HIV-1 neutralizing antibody assay that utilizes A3R5 cells, performed in compliance with Good Clinical Laboratory Practice (GCLP) guidelines. The assay utilizes Renilla luciferase-expressing replication competent infectious molecular clones (IMC) encoding heterologous env genes from different HIV-1 clades. Key assay validation parameters tested included specificity, accuracy, precision, limit of detection and quantitation, specificity, linearity and range, and robustness. Plasma samples demonstrated higher non-specific activity than serum samples in the A3R5 assay. This assay can tolerate a wide range of virus input but is more sensitive to cell concentration. The higher sensitivity of the A3R5 assay in neutralization responses to tier 2 strains of HIV-1 makes it complementary to, but not a substitute for the TZM-bl assay. The validated A3R5 assay is employed as an endpoint immunogenicity test for vaccine-elicited neutralizing antibodies against tier 2 strains of HIV-1, and to identify correlates of protection in HIV-1 vaccine trials conducted globally. Copyright © 2014 Elsevier B.V. All rights reserved.

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

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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.

Effects of Multivitamins and Known Teratogens on Chick Cardiomyocytes Micromass Culture Assay

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Samreen Memon

2013-09-01

Full Text Available   Objective(s: This study aimed to find out whether the chick cardiomyocyte micromass (MM system could be employed to predict the teratogenecity of common environmental factors. Different multivitamins and over the counter drugs were used in this study.   Materials and Methods: White Leghorn 5-day-old embryo hearts were dissected and trypsinized to produce a cardiomyocyte cell suspension in Dulbecco's Modified Eagle's Medium. The cultures were incubated at 370C in 5% CO2 in air, and observations were made at 24, 48 and 144 hr, for the detection of cell beating. Cellular viability was assessed using the resazurin assay and cell protein content was assessed by the kenacid blue assay. It was observed that while not affecting total cell number folic acid, vitamin C, sodium fluoride and ginseng did not significantly reduced cell activity and beating. However cadmium chloride significantly reduced the beating, cell viability and cell protein content in micromass cultures. Results: The results demonstrate the potential of the chick cardiomyocyte MM culture assay to identify teratogens/embryotoxins that alter morphology and function, which may result in either teratogenic outcome or cytotoxicity. Conclusion: This could form part of a screen for developmental toxicity related to cardiac function

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

International Nuclear Information System (INIS)

Heo, D.S.; Park, J.G.; Hata, K.; Day, R.; Herberman, R.B.; Whiteside, T.L.

1990-01-01

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

In vitro effects of piracetam on the radiosensitivity of hypoxic cells (adaptation of MTT assay to hypoxic conditions)

International Nuclear Information System (INIS)

Gheuens, E.E.O.; Bruijn, E.A. de; Van der Heyden, S.; Van Oosterom, A.T.; Lagarde, P.; Pooter, C.M.J. de; Chomy, F.

1995-01-01

This paper describes the adaptation of the MTT assay to hypoxic conditions in order to test the in vitro effect of piracetam on hypoxic cells and particularly on the radiosensitivity of hypoxic cells since this drug has shown clinical effect on acute and chronic hypoxia. The V79 cell line was selected by reference to preliminary hypoxic experiments using clonogenic assay and euoxic experiments using clonogenic and MTT assays. Cell growth and survival in our hypoxic conditions were assessed using MTT assay with an enclosure and special 48-well plates both made of glass. Growth curves on glass plates after 1-hour exposure to nitrogen versus air were comparable, so there is no bias effect due to gas composition. Survival curves using MTT versus reference clonogenic assay were comparable after radiation exposure in eu- and hypoxic conditions, and confirm the validity of our original technique for creating hypoxia. The Oxygen Enhancement Ratio was of about 3 for 1-hour hypoxic exposure. Piracetam gave no cytotoxic effect up to 10 mM of piracetam. Growth curves after continuous drug exposure and 1-hour euoxic versus hypoxic exposure gave no cytotoxic effect up to 10 mM of piracetam. Survival curves after continuous drug exposure to 10 mM of piracetam gave no significant effect on the radiosensitivity of hypoxic V79 cells using MTT or clonogenic assay. (author). 32 refs., 6 figs

Comparative study on collagen-binding enzyme-linked immunosorbent assay and ristocetin cofactor activity assays for detection of functional activity of von Willebrand factor.

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Turecek, Peter L; Siekmann, Jürgen; Schwarz, Hans Peter

2002-04-01

For more than two decades, the ristocetin cofactor (RCo) assay, which measures the von Willebrand factor (vWF)-mediated agglutination of platelets in the presence of the antibiotic ristocetin, has been the most common method for measuring the functional activity of vWF. There is, however, general agreement among clinical analysts that this method has major practical disadvantages in performance and reproducibility. Today, collagen-binding assays (CBA) based on the enzyme-linked immunosorbent assay (ELISA) technique that measure the interaction of vWF and collagen are an alternative analytic procedure based on a more physiological function than that of the RCo procedure. We used both assay systems in a comparative study to assess the functional activity of vWF in plasma as well as in therapeutic preparations. We measured RCo activities of plasma from healthy donors and patients with different types of von Willebrand disease (vWD) and of vWF as a drug substance in factor (F) VIII/vWF concentrates using both the aggregometric and the macroscopic methods. In addition, we measured collagen-binding activity (vWF:CB) using a recently developed commercially available CBA system. To investigate the relation between the structure and the functional activity of vWF, we isolated vWF species with different numbers of multimers from FVIII/vWF concentrates by affinity chromatography on immobilized heparin. The vWF:RCo and vWF:CB of the different fractions were measured, and the multimeric structure of vWF was analyzed by sodium dodecyl sulfate (SDS) agarose gel electrophoresis. (vWF:CB and vWF:RCo are part of the nomenclature proposed by the International Society on Thrombosis and Hemostasis Scientific and Standardization Committee [ISTH SSC] subcommittee on von Willebrand factor, in Maastricht, Germany, June 16, 2000.) Measurement of functional vWF activity by CBA can be carried out with substantially higher interassay reproducibility than can measurement of RCo. Both assay

Protein subcellular localization assays using split fluorescent proteins

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Waldo, Geoffrey S [Santa Fe, NM; Cabantous, Stephanie [Los Alamos, NM

2009-09-08

The invention provides protein subcellular localization assays using split fluorescent protein systems. The assays are conducted in living cells, do not require fixation and washing steps inherent in existing immunostaining and related techniques, and permit rapid, non-invasive, direct visualization of protein localization in living cells. The split fluorescent protein systems used in the practice of the invention generally comprise two or more self-complementing fragments of a fluorescent protein, such as GFP, wherein one or more of the fragments correspond to one or more beta-strand microdomains and are used to "tag" proteins of interest, and a complementary "assay" fragment of the fluorescent protein. Either or both of the fragments may be functionalized with a subcellular targeting sequence enabling it to be expressed in or directed to a particular subcellular compartment (i.e., the nucleus).

Radiosensitivity evaluation of human tumor cell lines by detecting 4977 bp deletion in mitochondrial DNA and comet assay

International Nuclear Information System (INIS)

Chu Liping; Liu Qiang; Wang Qin; Li Jin; Yue Jingyin; Mu Chuanjie; Fan Feiyue

2008-01-01

Objective: To explore the feasibility of determining radiosensitivity of human tumor cell lines in vitro using the assay of mtDNA 4977 bp deletion and comet assay. Methods: Three human tumor cell lines were selected in this study, HepG 2 , EC-9706 and MCF-7. The surviving fraction(SF), the ratio of mtDNA 4977 bp deletion and DNA damage were detected by MTY assay, nested PCR technique and comet assay, respectively. Results: The results of MTT assay showed that the radiosensitivity of HepG 2 and EC-9706 was higher than that of MCF-7. The ratio of mtDNA 4977 bp deletion of HepG 2 and EC-9706 was higher significantly than that of MCF-7 (P 2 and EC-9706 was higher than that of MCF-7. The difference of radiosensitivity among these three tumor cell lines was significant after 8 Gy γ-ray irradiation. Conclusions: Combination of many biological parameter is helpful to evaluate the radiosensitivity of tumor cells more accurately. (authors)

Functional neuromuscular junctions formed by embryonic stem cell-derived motor neurons.

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Joy A Umbach

Full Text Available A key objective of stem cell biology is to create physiologically relevant cells suitable for modeling disease pathologies in vitro. Much progress towards this goal has been made in the area of motor neuron (MN disease through the development of methods to direct spinal MN formation from both embryonic and induced pluripotent stem cells. Previous studies have characterized these neurons with respect to their molecular and intrinsic functional properties. However, the synaptic activity of stem cell-derived MNs remains less well defined. In this study, we report the development of low-density co-culture conditions that encourage the formation of active neuromuscular synapses between stem cell-derived MNs and muscle cells in vitro. Fluorescence microscopy reveals the expression of numerous synaptic proteins at these contacts, while dual patch clamp recording detects both spontaneous and multi-quantal evoked synaptic responses similar to those observed in vivo. Together, these findings demonstrate that stem cell-derived MNs innervate muscle cells in a functionally relevant manner. This dual recording approach further offers a sensitive and quantitative assay platform to probe disorders of synaptic dysfunction associated with MN disease.

Adhesion kinetics of human primary monocytes, dendritic cells, and macrophages: Dynamic cell adhesion measurements with a label-free optical biosensor and their comparison with end-point assays.

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Orgovan, Norbert; Ungai-Salánki, Rita; Lukácsi, Szilvia; Sándor, Noémi; Bajtay, Zsuzsa; Erdei, Anna; Szabó, Bálint; Horvath, Robert

2016-09-01

Monocytes, dendritic cells (DCs), and macrophages (MFs) are closely related immune cells that differ in their main functions. These specific functions are, to a considerable degree, determined by the differences in the adhesion behavior of the cells. To study the inherently and essentially dynamic aspects of the adhesion of monocytes, DCs, and MFs, dynamic cell adhesion assays were performed with a high-throughput label-free optical biosensor [Epic BenchTop (BT)] on surfaces coated with either fibrinogen (Fgn) or the biomimetic copolymer PLL-g-PEG-RGD. Cell adhesion profiles typically reached their maximum at ∼60 min after cell seeding, which was followed by a monotonic signal decrease, indicating gradually weakening cell adhesion. According to the biosensor response, cell types could be ordered by increasing adherence as monocytes, MFs, and DCs. Notably, all three cell types induced a larger biosensor signal on Fgn than on PLL-g-PEG-RGD. To interpret this result, the molecular layers were characterized by further exploiting the potentials of the biosensor: by measuring the adsorption signal induced during the surface coating procedure, the authors could estimate the surface density of adsorbed molecules and, thus, the number of binding sites potentially presented for the adhesion receptors. Surfaces coated with PLL-g-PEG-RGD presented less RGD sites, but was less efficient in promoting cell spreading than those coated with Fgn; hence, other binding sites in Fgn played a more decisive role in determining cell adherence. To support the cell adhesion data obtained with the biosensor, cell adherence on Fgn-coated surfaces 30-60 min after cell seeding was measured with three complementary techniques, i.e., with (1) a fluorescence-based classical adherence assay, (2) a shear flow chamber applying hydrodynamic shear stress to wash cells away, and (3) an automated micropipette using vacuum-generated fluid flow to lift cells up. These techniques confirmed the results

Cytotoxicity Test Based on Human Cells Labeled with Fluorescent Proteins: Fluorimetry, Photography, and Scanning for High-Throughput Assay.

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Kalinina, Marina A; Skvortsov, Dmitry A; Rubtsova, Maria P; Komarova, Ekaterina S; Dontsova, Olga A

2018-06-01

High- and medium-throughput assays are now routine methods for drug screening and toxicology investigations on mammalian cells. However, a simple and cost-effective analysis of cytotoxicity that can be carried out with commonly used laboratory equipment is still required. The developed cytotoxicity assays are based on human cell lines stably expressing eGFP, tdTomato, mCherry, or Katushka2S fluorescent proteins. Red fluorescent proteins exhibit a higher signal-to-noise ratio, due to less interference by medium autofluorescence, in comparison to green fluorescent protein. Measurements have been performed on a fluorescence scanner, a plate fluorimeter, and a camera photodocumentation system. For a 96-well plate assay, the sensitivity per well and the measurement duration were 250 cells and 15 min for the scanner, 500 cells and 2 min for the plate fluorimeter, and 1000 cells and less than 1 min for the camera detection. These sensitivities are similar to commonly used MTT (tetrazolium dye) assays. The used scanner and the camera had not been previously applied for cytotoxicity evaluation. An image processing scheme for the high-resolution scanner is proposed that significantly diminishes the number of control wells, even for a library containing fluorescent substances. The suggested cytotoxicity assay has been verified by measurements of the cytotoxicity of several well-known cytotoxic drugs and further applied to test a set of novel bacteriotoxic compounds in a medium-throughput format. The fluorescent signal of living cells is detected without disturbing them and adding any reagents, thus allowing to investigate time-dependent cytotoxicity effects on the same sample of cells. A fast, simple and cost-effective assay is suggested for cytotoxicity evaluation based on mammalian cells expressing fluorescent proteins and commonly used laboratory equipment.

Cell treatment and surface functionalization using a miniature atmospheric pressure glow discharge plasma torch

International Nuclear Information System (INIS)

Yonson, S; Coulombe, S; Leveille, V; Leask, R L

2006-01-01

A miniature atmospheric pressure glow discharge plasma torch was used to detach cells from a polystyrene Petri dish. The detached cells were successfully transplanted to a second dish and a proliferation assay showed the transplanted cells continued to grow. Propidium iodide diffused into the cells, suggesting that the cell membrane had been permeabilized, yet the cells remained viable 24 h after treatment. In separate experiments, hydrophobic, bacteriological grade polystyrene Petri dishes were functionalized. The plasma treatment reduced the contact angle from 93 0 to 35 0 , and promoted cell adhesion. Two different torch nozzles, 500 μm and 150 μm in internal diameter, were used in the surface functionalization experiments. The width of the tracks functionalized by the torch, as visualized by cell adhesion, was approximately twice the inside diameter of the nozzle. These results indicate that the miniature plasma torch could be used in biological micropatterning, as it does not use chemicals like the present photolithographic techniques. Due to its small size and manouvrability, the torch also has the ability to pattern complex 3D surfaces

Determination of Interference During In Vitro Pyrogen Detection: Development and Characterization of a Cell-Based Assay.

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Palma, Linda; Rossetti, Francesca; Dominici, Sabrina; Buondelmonte, Costantina; Rocchi, Marco B L; Rizzardi, Gian P; Vallanti, Giuliana; Magnani, Mauro

Contamination of pharmaceutical products and medical devices with pyrogens such as endotoxins is the most common cause of systemic inflammation and, in worst cases, of septic shock. Thus, quantification of pyrogens is crucial. The limulus amebocyte lysate (LAL)-based assays are the reference tests for in vitro endotoxin detection, in association with the in vivo rabbit pyrogen test (RPT), according to European Pharmacopoeia (EP 2.6.14), and U.S. Pharmacopoeia (USP ). However, several substances interfere with LAL assay, while RPT is not accurate, not quantitative, and raises ethical limits. Biological assays, as monocyte activation tests, have been developed and included in European Pharmacopoeia (EP 7.0; 04/2010:20630) guidelines as an alternative to RPT and proved relevant to the febrile reaction in vivo. Because this reaction is carried out by endogenous mediators under the transcriptional control of nuclear factor-kappaB (NF-kappaB), we sought to determine whether a NF-kappaB reporter-gene assay, based on MonoMac-6 (MM6) cells, could reconcile the basic mechanism of innate immune response with the relevance of monocytoid cell lines to the organism reaction to endotoxins. This article describes both optimization and characterization of the reporter cells-based assay, which overall proved the linearity, accuracy, and precision of the test, and demonstrated the sensitivity of the assay to 0.24 EU/mL endotoxin, close to the pyrogenic threshold in humans. Moreover, the assay was experimentally compared to the LAL test in the evaluation of selected interfering samples. The good performance of the MM6 reporter test demonstrates the suitability of this assay to evaluate interfering or false-positive samples.

Hemocytes of zebra mussels (Dreissena polymorpha are relevant cells for the monitoring of environmental genotoxicity by the comet assay.

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Marc Bonnard

2015-06-01

Full Text Available The measure of DNA integrity by the single cell gel electrophoresis (SCGE or comet assay is especially recommended for its sensitivity and its capacity for detecting different types of damages. Therefore, it has been applied in environmental genotoxicity in a variety of organisms. It appears today necessary to define both reference and threshold levels of DNA damage, for their application in in situ biomonitoring. However, little is known about the influence of both biological (sex, reproduction status or external (temperature… confounding factors on the measure of DNA damage by the comet assay. These variables need to be taken into account if the robustness of the assay is to be established (Jha, 2008. In the zebra mussel Dreissena polymorpha (recommended as a sentinel species in the evaluation of freshwater quality the measure of DNA damage by the comet assay is mainly performed on hemocytes, which are circulating cells involved in key physiological functions such as immunity, homeostasis, detoxication…. This communication will present and discuss results from an innovative study about the variability of the baseline level of DNA damage in hemocytes of mussels encaged for one year in the canal de l’Aisne à la Marne (Reims, according to their sex and their reproductive status. The sensitivity and the suitability of hemocytes in the evaluation of environmental genotoxicity will also be discussed, referring to observations during a 6 month-exposure of mussels in mesocosms to environmentally realistic concentrations of carbamazepine.

The fluorometric microculture cytotoxicity assay.

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Lindhagen, Elin; Nygren, Peter; Larsson, Rolf

2008-01-01

The fluorometric microculture cytotoxicity assay (FMCA) is a nonclonogenic microplate-based cell viability assay used for measurement of the cytotoxic and/or cytostatic effect of different compounds in vitro. The assay is based on hydrolysis of the probe, fluorescein diacetate (FDA) by esterases in cells with intact plasma membranes. The assay is available as both a semiautomated 96-well plate setup and a 384-well plate version fully adaptable to robotics. Experimental plates are prepared with a small amount of drug solution and can be stored frozen. Cells are seeded on the plates and cell viability is evaluated after 72 h. The protocol described here is applicable both for cell lines and freshly prepared tumor cells from patients and is suitable both for screening in drug development and as a basis for a predictive test for individualization of anticancer drug therapy.

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

Milk contains many different proteins of which the larger constituents like the caseins and major whey constituents are well characterized. We have for some time been studying the structure and function of proteins associated with the milk fat globule membrane like lactadherin, MUC1/15, xanthine...... 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...

Curcumin Modulates Pancreatic Adenocarcinoma Cell-Derived Exosomal Function

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Osterman, Carlos J. Diaz; Lynch, James C.; Leaf, Patrick; Gonda, Amber; Ferguson Bennit, Heather R.; Griffiths, Duncan; Wall, Nathan R.

2015-01-01

Pancreatic cancer has the highest mortality rates of all cancer types. One potential explanation for the aggressiveness of this disease is that cancer cells have been found to communicate with one another using membrane-bound vesicles known as exosomes. These exosomes carry pro-survival molecules and increase the proliferation, survival, and metastatic potential of recipient cells, suggesting that tumor-derived exosomes are powerful drivers of tumor progression. Thus, to successfully address and eradicate pancreatic cancer, it is imperative to develop therapeutic strategies that neutralize cancer cells and exosomes simultaneously. Curcumin, a turmeric root derivative, has been shown to have potent anti-cancer and anti-inflammatory effects in vitro and in vivo. Recent studies have suggested that exosomal curcumin exerts anti-inflammatory properties on recipient cells. However, curcumin’s effects on exosomal pro-tumor function have yet to be determined. We hypothesize that curcumin will alter the pro-survival role of exosomes from pancreatic cancer cells toward a pro-death role, resulting in reduced cell viability of recipient pancreatic cancer cells. The main objective of this study was to determine the functional alterations of exosomes released by pancreatic cancer cells exposed to curcumin compared to exosomes from untreated pancreatic cancer cells. We demonstrate, using an in vitro cell culture model involving pancreatic adenocarcinoma cell lines PANC-1 and MIA PaCa-2, that curcumin is incorporated into exosomes isolated from curcumin-treated pancreatic cancer cells as observed by spectral studies and fluorescence microscopy. Furthermore, curcumin is delivered to recipient pancreatic cancer cells via exosomes, promoting cytotoxicity as demonstrated by Hoffman modulation contrast microscopy as well as AlamarBlue and Trypan blue exclusion assays. Collectively, these data suggest that the efficacy of curcumin may be enhanced in pancreatic cancer cells through

Menadione-mediated WST1 reduction assay for the determination of metabolic activity of cultured neural cells.

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Stapelfeldt, Karsten; Ehrke, Eric; Steinmeier, Johann; Rastedt, Wiebke; Dringen, Ralf

2017-12-01

Cellular reduction of tetrazolium salts to their respective formazans is frequently used to determine the metabolic activity of cultured cells as an indicator of cell viability. For membrane-impermeable tetrazolium salts such as WST1 the application of a membrane-permeable electron cycler is usually required to mediate the transfer of intracellular electrons for extracellular WST1 reduction. Here we demonstrate that in addition to the commonly used electron cycler M-PMS, menadione can also serve as an efficient electron cycler for extracellular WST1 reduction in cultured neural cells. The increase in formazan absorbance in glial cell cultures for the WST1 reduction by menadione involves enzymatic menadione reduction and was twice that recorded for the cytosolic enzyme-independent WST1 reduction in the presence of M-PMS. The optimized WST1 reduction assay allowed within 30 min of incubation a highly reliable detection of compromised cell metabolism caused by 3-bromopyruvate and impaired membrane integrity caused by Triton X-100, with a sensitivity as good as that of spectrophotometric assays which determine cellular MTT reduction or lactate dehydrogenase release. The short incubation period of 30 min and the observed good sensitivity make this optimized menadione-mediated WST1 reduction assay a quick and reliable alternative to other viability and toxicity assays. Copyright © 2017 Elsevier Inc. All rights reserved.

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

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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.

Biomonitoring of genotoxic risk in radar facility workers: comparison of the comet assay with micronucleus assay and chromatid breakage assay

International Nuclear Information System (INIS)

Garaj-Vrhovac, V.; Kopjar, N.

2003-01-01

Genotoxic risks of occupational exposure in a radar facility were evaluated by using alkaline comet assay, micronucleus assay and chromatid breakage assay on peripheral blood leukocytes in exposed subjects and corresponding controls. Results show that occupational exposure to microwave radiation correlates with an increase of genome damage in somatic cells. The levels of DNA damage in exposed subjects determined by using alkaline comet assay were increased compared to control and showed interindividual variations. Incidence of micronuclei was also significantly increased compared to baseline control values. After short exposure of cultured lymphocytes to bleomycin, cells of occupationally exposed subjects responded with high numbers of chromatid breaks. Although the level of chromosome damage generated by bleomycin varied greatly between individuals, in exposed subjects a significantly elevated number of chromatid breaks was observed. Our results support data reported in literature indicating that microwave radiation represents a potential DNA-damaging hazard. Alkaline comet assay is confirmed as a sensitive and highly reproducible technique for detection of primary DNA damage inflicted in somatic cells. Micronucleus assay was confirmed as reliable bio-markers of effect and chromatid breakage assay as sensitive bio-marker of individual cancer susceptibility. The results obtained also confirm the necessity to improve measures and to perform accurate health surveillance of individuals occupationally exposed to microwave radiation

Heterogeneity of functional properties of Clone 66 murine breast cancer cells expressing various stem cell phenotypes.

Science.gov (United States)

Mukhopadhyay, Partha; Farrell, Tracy; Sharma, Gayatri; McGuire, Timothy R; O'Kane, Barbara; Sharp, J Graham

2013-01-01

Breast cancer grows, metastasizes and relapses from rare, therapy resistant cells with a stem cell phenotype (cancer stem cells/CSCs). However, there is a lack of studies comparing the functions of CSCs isolated using different phenotypes in order to determine if CSCs are homogeneous or heterogeneous. Cells with various stem cell phenotypes were isolated by sorting from Clone 66 murine breast cancer cells that grow orthotopically in immune intact syngeneic mice. These populations were compared by in vitro functional assays for proliferation, growth, sphere and colony formation; and in vivo limiting dilution analysis of tumorigenesis. The proportion of cells expressing CD44(high)CD24(low/neg), side population (SP) cells, ALDH1(+), CD49f(high), CD133(high), and CD34(high) differed, suggesting heterogeneity. Differences in frequency and size of tumor spheres from these populations were observed. Higher rates of proliferation of non-SP, ALDH1(+), CD34(low), and CD49f(high) suggested properties of transit amplifying cells. Colony formation was higher from ALDH1(-) and non-SP cells than ALDH1(+) and SP cells suggesting a progenitor phenotype. The frequency of clonal colonies that grew in agar varied and was differentially altered by the presence of Matrigel™. In vivo, fewer cells with a stem cell phenotype were needed for tumor formation than "non-stem" cells. Fewer SP cells were needed to form tumors than ALDH1(+) cells suggesting further heterogeneities of cells with stem phenotypes. Different levels of cytokines/chemokines were produced by Clone 66 with RANTES being the highest. Whether the heterogeneity reflects soluble factor production remains to be determined. These data demonstrate that Clone 66 murine breast cancer cells that express stem cell phenotypes are heterogeneous and exhibit different functional properties, and this may also be the case for human breast cancer stem cells.

Biomarkers in chronic fatigue syndrome: evaluation of natural killer cell function and dipeptidyl peptidase IV/CD26.

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Mary A Fletcher

2010-05-01

Full Text Available Chronic Fatigue Syndrome (CFS studies from our laboratory and others described decreased natural killer cell cytotoxicity (NKCC and elevated proportion of lymphocytes expressing the activation marker, dipeptidyl peptidase IV (DPPIV also known as CD26. However, neither these assays nor other laboratory tests are widely accepted for the diagnosis or prognosis of CFS. This study sought to determine if NKCC or DPPIV/CD26 have diagnostic accuracy for CFS.Subjects included female and male CFS cases and healthy controls. NK cell function was measured with a bioassay, using K562 cells and (51Cr release. Lymphocyte associated DPPIV/CD26 was assayed by qualitative and quantitative flow cytometry. Serum DPPIV/CD26 was measured by ELISA. Analysis by receiver operating characteristic (ROC curve assessed biomarker potential. Cytotoxic function of NK cells for 176 CFS subjects was significantly lower than in the 230 controls. According to ROC analysis, NKCC was a good predictor of CFS status. There was no significant difference in NK cell counts between cases and controls. Percent CD2+ lymphocytes (T cells and NK cells positive for DPPIV/C26 was elevated in CFS cases, but there was a decrease in the number of molecules (rMol of DPPIV/C26 expressed on T cells and NK cells and a decrease in the soluble form of the enzyme in serum. Analyses by ROC curves indicated that all three measurements of DPPIV/CD26 demonstrated potential as biomarkers for CFS. None of the DPPIV/C26 assays were significantly correlated with NKCC.By ROC analysis, NKCC and three methods of measuring DPPIV/C26 examined in this study had potential as biomarkers for CFS. Of these, NKCC, %CD2+CD26+ lymphocytes and rMol CD26/CD2+ lymphocyte, required flow cytometry, fresh blood and access to a high complexity laboratory. Soluble DPPIV/C26 in serum is done with a standard ELISA assay, or with other soluble factors in a multiplex type of ELISA. Dipeptidyl peptidase IV on lymphocytes or in serum was

ToxCast Profiling in a Human Stem Cell Assay for ...

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Standard practice for assessing disruptions in embryogenesis involves testing pregnant animals of two species, typically rats and rabbits, exposed during major organogenesis and evaluated just prior to term. Under this design the major manifestations of developmental toxicity are observed as one or more apical endpoints including intrauterine death, fetal growth retardation, structural malformations and variations. Alternative approaches to traditional developmental toxicity testing have been proposed in the form of in vitro data (e.g., embryonic stem cells, zebrafish embryos, HTS assays) and in silico models (e.g., computational toxicology). To increase the diversity of assays used to assess developmental toxicity in EPA’s ToxCast program, we tested the chemicals in Stemina’s metabolomics-based platform that utilizes the commecrially available H9 human embryonic stem cell line. The devTOXqP dataset for ToxCast of high-quality based on replicate samples and model performance (82% balanced accuracy, 0.71 sensitivity and 1.00 specificity). To date, 136 ToxCast chemicals (12.8% of 1065 tested) were positive in this platform; 48 triggered the biomarker signal without any change in hESC viability and 88 triggered activity concurrent with effects on cell viability. Work is in progress to complete the STM dataset entry into the TCPL, compare data with results from zFish and mESC platforms, profile bioactivity (ToxCastDB), endpoints (ToxRefDB), chemotypes (DSSTox)

Sensitivity of neuroprogenitor cells to chemical-induced apoptosis using a multiplexed assay suitable for high-throughput screening

International Nuclear Information System (INIS)

Druwe, Ingrid; Freudenrich, Theresa M.; Wallace, Kathleen; Shafer, Timothy J.; Mundy, William R.

2015-01-01

High-throughput methods are useful for rapidly screening large numbers of chemicals for biological activity, including the perturbation of pathways that may lead to adverse cellular effects. In vitro assays for the key events of neurodevelopment, including apoptosis, may be used in a battery of tests for detecting chemicals that could result in developmental neurotoxicity. Apoptosis contributes to nervous system development by regulating the size of the neuroprogenitor cell pool, and the balance between cellular proliferation and apoptosis during neuroprogenitor cell proliferation helps to determine the size and shape of the nervous system. Therefore, chemicals that affect apoptosis during neuronal development can have deleterious effects on the developing brain. The present study examined the utility of a high-throughput assay to detect chemical-induced apoptosis in mouse or human neuroprogenitor cells, as well as differentiated human neurons derived from induced pluripotent stem cells. Apoptosis was assessed using an assay that measures enzymatic activity of caspase-3/7 in a rapid and cost efficient manner. The results show that all three commercially available models generated a robust source of proliferating neuroprogenitor cells, and that the assay was sensitive and reproducible when used in a multi-well plate format. There were differences in the response of rodent and human neuroprogenitor cells to a set of chemicals previously shown to induce apoptosis in vitro. Neuroprogenitor cells were more sensitive to chemical-induced apoptosis than differentiated neurons, suggesting that neuroprogenitor cells are one of the cell models that should be considered for use in a developmental neurotoxicity screening battery

Peptide deformylase as an antibacterial drug target: assays for detection of its inhibition in Escherichia coli cell homogenates and intact cells.

Science.gov (United States)

Apfel, C M; Evers, S; Hubschwerlen, C; Pirson, W; Page, M G; Keck, W

2001-04-01

An assay was developed to determine the activity of peptide deformylase (PDF) inhibitors under conditions as close as possible to the physiological situation. The assay principle is the detection of N-terminal [35S]methionine labeling of a protein that contains no internal methionine. If PDF is active, the deformylation of the methionine renders the peptide a substrate for methionine aminopeptidase, resulting in the removal of the N-terminal methionine label. In the presence of a PDF inhibitor, the deformylation is blocked so that the N-formylated peptide is not processed and the label is detected. Using this assay, it is possible to determine the PDF activity under near-physiological conditions in a cell-free transcription-translation system as well as in intact bacterial cells.

The cell-based L-glutathione protection assays to study endocytosis and recycling of plasma membrane proteins.

Science.gov (United States)

Cihil, Kristine M; Swiatecka-Urban, Agnieszka

2013-12-13

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 ºC - a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 ºC. Next, the temperature is decreased again to 4 ºC to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 °C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 ºC as in the endocytic assay. Next, cells are incubated again at 37 °C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 ºC, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.

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......-quality microtiter plates and the addition of dilute hydrochloric acid to the samples. In an experiment on different digester samples, the test demonstrated a unique pattern of different methanogenic strains present in each sample. The limited preparatory work required for the assay and the simple assay design make...... in samples from anaerobic digesters in a reproducible manner. Polyclonal antisera against eight strains of methanogens were employed in the test, The specificities of the antisera were increased by adsorption with cross-reacting cells. The reproducibility of the assay depended on the use of high...

Comparison of Assays for Sensitive and Reproducible Detection of Cell Culture-Infectious Cryptosporidium parvum and Cryptosporidium hominis in Drinking Water

Science.gov (United States)

Di Giovanni, George D.; Rochelle, Paul A.

2012-01-01

This study compared the three most commonly used assays for detecting Cryptosporidium sp. infections in cell culture: immunofluorescent antibody and microscopy assay (IFA), PCR targeting Cryptosporidium sp.-specific DNA, and reverse transcriptase PCR (RT-PCR) targeting Cryptosporidium sp.-specific mRNA. Monolayers of HCT-8 cells, grown in 8-well chamber slides or 96-well plates, were inoculated with a variety of viable and inactivated oocysts to assess assay performance. All assays detected infection with low doses of flow cytometry-enumerated Cryptosporidium parvum oocysts, including infection with one oocyst and three oocysts. All methods also detected infection with Cryptosporidium hominis. The RT-PCR assay, IFA, and PCR assay detected infection in 23%, 25%, and 51% of monolayers inoculated with three C. parvum oocysts and 10%, 9%, and 16% of monolayers inoculated with one oocyst, respectively. The PCR assay was the most sensitive, but it had the highest frequency of false positives with mock-infected cells and inactivated oocysts. IFA was the only infection detection assay that did not produce false positives with mock-infected monolayers. IFA was also the only assay that detected infections in all experiments with spiked oocysts recovered from Envirochek capsules following filtration of 1,000 liters of treated water. Consequently, cell culture with IFA detection is the most appropriate method for routine and sensitive detection of infectious Cryptosporidium parvum and Cryptosporidium hominis in drinking water. PMID:22038611

Interdependence of initial cell density, drug concentration and exposure time revealed by real-time impedance spectroscopic cytotoxicity assay

DEFF Research Database (Denmark)

Caviglia, Claudia; Zor, Kinga; Canepa, Silvia

2015-01-01

We investigated the combined effect of the initial cell density (12 500, 35 000, 75 000, and 100 000 cells cm−2) and concentration of the anti-cancer drug doxorubicin on HeLa cells by performing timedependent cytotoxicity assays using real-time electrochemical impedance spectroscopy. A correlation...... between the rate of cell death and the initial cell seeding density was found at 2.5 μM doxorubicin concentration, whereas this was not observed at 5 or 100 μM. By sensing the changes in the cell–substrate interaction using impedance spectroscopy under static conditions, the onset of cytotoxicity...... was observed 5 h earlier than when using a standard colorimetric end-point assay (MTS) which measures changes in the mitochondrial metabolism. Furthermore, with the MTS assay no cytotoxicity was observed after 15 h of incubation with 2.5 μM doxorubicin, whereas the impedance showed at this time point cell...

Development and optimization of a cell-based assay for the selection of synthetic compounds that potentiate bone morphogenetic protein-2 activity.

Science.gov (United States)

Okada, Motohiro; Sangadala, Sreedhara; Liu, Yunshan; Yoshida, Munehito; Reddy, Boojala Vijay B; Titus, Louisa; Boden, Scott D

2009-12-01

reliability of our cell-based assay. Direct delivery of synthesized protein can be limited by high cost, instability or inadequate post-translational modifications. Thus, there would be a clear benefit for a low cost, cell penetrable chemical compound. We successfully used our gene expression-based assay to choose an active compound from a select group of compounds that were identified by computational screenings as the most likely candidates for mimicking the function of LMP-1. Among them, we selected SVAK-3, a compound that showed a dose-dependent potentiation of BMP-2 activity in inducing osteoblastic differentiation of C2C12 cells. We show that either the full length LMP-1 protein or its potential mimetic compound consistently exhibit similar potentiation of BMP-2 activity even when multiple markers of the osteoblastic phenotype were parallely monitored.

A Simple and Sensitive High-Content Assay for the Characterization of Antiproliferative Therapeutic Antibodies.

Science.gov (United States)

Stengl, Andreas; Hörl, David; Leonhardt, Heinrich; Helma, Jonas

2017-03-01

Monoclonal antibodies (mAbs) have become a central class of therapeutic agents in particular as antiproliferative compounds. Their often complex modes of action require sensitive assays during early, functional characterization. Current cell-based proliferation assays often detect metabolites that are indicative of metabolic activity but do not directly account for cell proliferation. Measuring DNA replication by incorporation of base analogues such as 5-bromo-2'-deoxyuridine (BrdU) fills this analytical gap but was previously restricted to bulk effect characterization in enzyme-linked immunosorbent assay formats. Here, we describe a cell-based assay format for the characterization of antiproliferative mAbs regarding potency and mode of action in a single experiment. The assay makes use of single cell-based high-content-analysis (HCA) for the reliable quantification of replicating cells and DNA content via 5-ethynyl-2'-deoxyuridine (EdU) and 4',6-diamidino-2-phenylindole (DAPI), respectively, as sensitive measures of antiproliferative mAb activity. We used trastuzumab, an antiproliferative therapeutic antibody interfering with HER2 cell surface receptor-mediated growth signal transduction, and HER2-overexpressing cell lines BT474 and SKBR3 to demonstrate up to 10-fold signal-to-background (S/B) ratios for treated versus untreated cells and a shift in cell cycle profiles indicating antibody-induced cell cycle arrest. The assay is simple, cost-effective, and sensitive, providing a cell-based format for preclinical characterization of therapeutic mAbs.

STANDARDIZATION OF A FLUORESCENT-BASED QUANTITATIVE ADHESION ASSAY TO STUDY ATTACHMENT OF Taenia solium ONCOSPHERE TO EPITHELIAL CELLS In Vitro

Science.gov (United States)

Chile, Nancy; Evangelista, Julio; Gilman, Robert H.; Arana, Yanina; Palma, Sandra; Sterling, Charles R; Garcia, Hector H.; Gonzalez, Armando; Verastegui, Manuela

2012-01-01

To fully understand the preliminary stages of Taenia solium oncosphere attachment in the gut, adequate tools and assays are necessary to observe and quantify this event that leads to infection. A fluorescent-based quantitative adhesion assay, using biotinylated activated-oncospheres and monolayers of Chinese hamster ovary cells (CHO-K1) or human intestinal monolayer cells (INT-407, HCT-8 or HT-29), was developed to study initial events during the infection of target cells and to rapidly quantify the in vitro adhesion of T. solium oncospheres. Fluorescein streptavidin was used to identify biotinylated activated-oncospheres adhered to cells. This adherence was quantified using an automated fluorescence plate reader, and the results were expressed as fluorescence intensity values. A series of three assays were performed. The first was to identify the optimum number of biotinylated activated-oncospheres to be used in the adhesion assay. The goal of the second assay was to validate this novel method with the established oncosphere-binding system using the immunofluorescent-antibody assay (IFA) method to quantify oncosphere adhesion. A total of 10,000 biotinylated activated-oncospheres were utilized to assess the role of sera and laminin (LM) in oncosphere adherence to a CHO-K1 cell monolayer. The findings that sera and LM increase the adhesion of oncospheres to monolayer cells were similar to results that were previously obtained using the IFA method. The third assay compared the adherence of biotinylated activated-oncospheres to different types of human intestinal monolayer cells. In this case, the fluorescence intensity was greatest when using the INT-407 cell monolayer. We believe this new method of quantification offers the potential for rapid, large-scale screening to study and elucidate specific molecules and mechanisms involved in oncosphere-host cell attachment. PMID:22178422

Blimp-1 impairs T cell function via upregulation of TIGIT and PD-1 in patients with acute myeloid leukemia.

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Zhu, Liuluan; Kong, Yaxian; Zhang, Jianhong; Claxton, David F; Ehmann, W Christopher; Rybka, Witold B; Palmisiano, Neil D; Wang, Ming; Jia, Bei; Bayerl, Michael; Schell, Todd D; Hohl, Raymond J; Zeng, Hui; Zheng, Hong

2017-06-19

T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif (ITIM) domain (TIGIT) and programmed cell death protein 1 (PD-1) are important inhibitory receptors that associate with T cell exhaustion in acute myeloid leukemia (AML). In this study, we aimed to determine the underlying transcriptional mechanisms regulating these inhibitory pathways. Specifically, we investigated the role of transcription factor B lymphocyte-induced maturation protein 1 (Blimp-1) in T cell response and transcriptional regulation of TIGIT and PD-1 in AML. Peripheral blood samples collected from patients with AML were used in this study. Blimp-1 expression was examined by flow cytometry. The correlation of Blimp-1 expression to clinical characteristics of AML patients was analyzed. Phenotypic and functional studies of Blimp-1-expressing T cells were performed using flow cytometry-based assays. Luciferase reporter assays and ChIP assays were applied to assess direct binding and transcription activity of Blimp-1. Using siRNA to silence Blimp-1, we further elucidated the regulatory role of Blimp-1 in the TIGIT and PD-1 expression and T cell immune response. Blimp-1 expression is elevated in T cells from AML patients. Consistent with exhaustion, Blimp-1 + T cells upregulate multiple inhibitory receptors including PD-1 and TIGIT. In addition, they are functionally impaired manifested by low cytokine production and decreased cytotoxicity capacity. Importantly, the functional defect is reversed by inhibition of Blimp-1 via siRNA knockdown. Furthermore, Blimp-1 binds to the promoters of PD-1 and TIGIT and positively regulates their expression. Our study demonstrates an important inhibitory effect of Blimp-1 on T cell response in AML; thus, targeting Blimp-1 and its regulated molecules to improve the immune response may provide effective leukemia therapeutics.

Blimp-1 impairs T cell function via upregulation of TIGIT and PD-1 in patients with acute myeloid leukemia

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Liuluan Zhu

2017-06-01

Full Text Available Abstract Background T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif (ITIM domain (TIGIT and programmed cell death protein 1 (PD-1 are important inhibitory receptors that associate with T cell exhaustion in acute myeloid leukemia (AML. In this study, we aimed to determine the underlying transcriptional mechanisms regulating these inhibitory pathways. Specifically, we investigated the role of transcription factor B lymphocyte-induced maturation protein 1 (Blimp-1 in T cell response and transcriptional regulation of TIGIT and PD-1 in AML. Methods Peripheral blood samples collected from patients with AML were used in this study. Blimp-1 expression was examined by flow cytometry. The correlation of Blimp-1 expression to clinical characteristics of AML patients was analyzed. Phenotypic and functional studies of Blimp-1-expressing T cells were performed using flow cytometry-based assays. Luciferase reporter assays and ChIP assays were applied to assess direct binding and transcription activity of Blimp-1. Using siRNA to silence Blimp-1, we further elucidated the regulatory role of Blimp-1 in the TIGIT and PD-1 expression and T cell immune response. Results Blimp-1 expression is elevated in T cells from AML patients. Consistent with exhaustion, Blimp-1+ T cells upregulate multiple inhibitory receptors including PD-1 and TIGIT. In addition, they are functionally impaired manifested by low cytokine production and decreased cytotoxicity capacity. Importantly, the functional defect is reversed by inhibition of Blimp-1 via siRNA knockdown. Furthermore, Blimp-1 binds to the promoters of PD-1 and TIGIT and positively regulates their expression. Conclusions Our study demonstrates an important inhibitory effect of Blimp-1 on T cell response in AML; thus, targeting Blimp-1 and its regulated molecules to improve the immune response may provide effective leukemia therapeutics.

Cell-free assay measuring repair DNA synthesis in human fibroblasts

International Nuclear Information System (INIS)

Ciarrocchi, G.; Linn, S.

1978-01-01

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

Analysis of Chemopredictive Assay for Targeting Cancer Stem Cells in Glioblastoma Patients

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Candace M. Howard

2017-04-01

Full Text Available Introduction: The prognosis of glioblastoma (GBM treated with standard-of-care maximal surgical resection and concurrent adjuvant temozolomide (TMZ/radiotherapy remains very poor (less than 15 months. GBMs have been found to contain a small population of cancer stem cells (CSCs that contribute to tumor propagation, maintenance, and treatment resistance. The highly invasive nature of high-grade gliomas and their inherent resistance to therapy lead to very high rates of recurrence. For these reasons, not all patients with similar diagnoses respond to the same chemotherapy, schedule, or dose. Administration of ineffective anticancer therapy is not only costly but more importantly burdens the patient with unnecessary toxicity and selects for the development of resistant cancer cell clones. We have developed a drug response assay (ChemoID that identifies the most effective chemotherapy against CSCs and bulk of tumor cells from of a panel of potential treatments, offering great promise for individualized cancer management. Providing the treating physician with drug response information on a panel of approved drugs will aid in personalized therapy selections of the most effective chemotherapy for individual patients, thereby improving outcomes. A prospective study was conducted evaluating the use of the ChemoID drug response assay in GBM patients treated with standard of care. Methods: Forty-one GBM patients (mean age 54 years, 59% male, all eligible for a surgical biopsy, were enrolled in an Institutional Review Board–approved protocol, and fresh tissue samples were collected for drug sensitivity testing. Patients were all treated with standard-of-care TMZ plus radiation with or without maximal surgery, depending on the status of the disease. Patients were prospectively monitored for tumor response, time to recurrence, progression-free survival (PFS, and overall survival (OS. Odds ratio (OR associations of 12-month recurrence, PFS, and OS outcomes

A dual reporter cell assay for identifying serotype and drug susceptibility of herpes simplex virus.

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Lu, Wen-Wen; Sun, Jun-Ren; Wu, Szu-Sian; Lin, Wan-Hsuan; Kung, Szu-Hao

2011-08-15

A dual reporter cell assay (DRCA) that allows real-time detection of herpes simplex virus (HSV) infection was developed. This was achieved by stable transfection of cells with an expression cassette that contains the dual reporter genes, secreted alkaline phosphatase (SEAP) and enhanced green fluorescent protein (EGFP), under the control of an HSV early gene promoter. Baby hamster kidney (BHK) and Chinese hamster ovary (CHO) cell lines were used as parental cell lines because the former is permissive for both HSV serotypes, HSV-1 and HSV-2, whereas the latter is susceptible to infection only by HSV-2. The DRCA permitted differential detection of HSV-1 and HSV-2 by observation of EGFP-positive cells, as substantiated by screening a total of 35 samples. The BHK-based cell line is sensitive to a viral titer as low as a single plaque-forming unit with a robust assay window as measured by a chemiluminescent assay. Evaluations of the DRCA with representative acyclovir-sensitive and acyclovir-resistant HSV strains demonstrated that their drug susceptibilities were accurately determined by a 48-h format. In summary, this novel DRCA is a useful means for serotyping of HSV in real time as well as a rapid screening method for determining anti-HSV susceptibilities. Copyright © 2011 Elsevier Inc. All rights reserved.

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

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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.

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

International Nuclear Information System (INIS)

Kodaka, Manami; Yang, Zeyu; Nakagawa, Kentaro; Maruyama, Junichi; Xu, Xiaoyin; Sarkar, Aradhan; Ichimura, Ayana; Nasu, Yusuke; Ozawa, Takeaki; Iwasa, Hiroaki; Ishigami-Yuasa, Mari; Ito, Shigeru; Kagechika, Hiroyuki

2015-01-01

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

Pharmacology of functional endogenous IP prostanoid receptors in NCB-20 cells: comparison with binding data from human platelets.

Science.gov (United States)

Crider, J Y; Xu, S X; Sharif, N A

2001-01-01

The objective of these studies was to characterize the effects of a broad range of prostanoid agonists upon the stimulation of cAMP production in National Cancer Bank (NCB-20; mouse neuroblastoma/hamster brain hybridoma) cells. The pharmacology of these functional responses in NCB-20 cells was compared with that of the classic endogenous IP receptor present on human platelets using [3H]-iloprost binding techniques. In both assay systems, agonists from the IP prostanoid class exhibited the highest affinities and functional potencies. Specific prostanoids exhibited the following rank order of potency (EC50 +/- SEM) in stimulating cAMP production in the NCB-20 cells: carbaprostacyclin (4.3 +/- 0.9 nM) = PGI2 (6.6 +/-1.5 nM) > iloprost (75+/-13 nM) > 11-deoxy PGE, (378+/-138 nM) > misoprostol (1,243+/-48) > PGE2 (3020+/-700 nM) > ZK-118182 (7265+/-455 nM). Iloprost wasthe most potent compound in the human platelet binding assay while prostanoidsfromthe DPand EP receptor classes showed modest affinity. These studies provide functional and binding information for a broad range of both natural and synthetic prostanoid receptor ligands at the endogenous IP receptor in two different cell types.

A cell impedance measurement device for the cytotoxicity assay dependent on the velocity of supplied toxic fluid

Science.gov (United States)

Kang, Yoon-Tae; Kim, Min-Ji; Cho, Young-Ho

2018-04-01

We present a cell impedance measurement chip capable of characterizing the toxic response of cells depending on the velocity of the supplied toxic fluid. Previous impedance-based devices using a single open-top chamber have been limited to maintaining a constant supply velocity, and devices with a single closed-top chamber present difficulties in simultaneous cytotoxicity assay for varying levels of supply velocities. The present device, capable of generating constant and multiple levels of toxic fluid velocity simultaneously within a single stepwise microchannel, performs a cytotoxicity assay dependent on toxic fluid velocity, in order to find the effective velocity of toxic fluid to cells for maximizing the cytotoxic effect. We analyze the cellular toxic response of 5% ethanol media supplied to cancer cells within a toxic fluid velocity range of 0-8.3 mm s-1. We observe the velocity-dependent cell detachment rate, impedance, and death rate. We find that the cell detachment rate decreased suddenly to 2.4% at a velocity of 4.4 mm s-1, and that the change rates of cell resistance and cell capacitance showed steep decreases to 8% and 41%, respectively, at a velocity of 5.7 mm s-1. The cell death rate and impedance fell steeply to 32% at a velocity of 5.7 mm s-1. We conclude that: (1) the present device is useful in deciding on the toxic fluid velocity effective to cytotoxicity assay, since the cellular toxic response is dependent on the velocity of toxic fluid, and; (2) the cell impedance analysis facilitates a finer cellular response analysis, showing better correlation with the cell death rate, compared to conventional visual observation. The present device, capable of performing the combinational analysis of toxic fluid velocity and cell impedance, has potential for application to the fine cellular toxicity assay of drugs with proper toxic fluid velocity.

Transformation assay in Bhas 42 cells: a model using initiated cells to study mechanisms of carcinogenesis and predict carcinogenic potential of chemicals.

Science.gov (United States)

Sasaki, Kiyoshi; Umeda, Makoto; Sakai, Ayako; Yamazaki, Shojiro; Tanaka, Noriho

2015-01-01

Transformation assays using cultured cells have been applied to the study of carcinogenesis. Although various cell systems exist, few cell types such as BALB/c 3T3 subclones and Syrian hamster embryo cells have been used to study chemically induced two-stage carcinogenesis. Bhas 42 cells were established as a clone by the transfection with the v-Ha-ras gene into mouse BALB/c 3T3 A31-1-1 cells and their subsequent selection based on their sensitivity to 12-O-tetradecanoylphorbol-13-acetate. Using Bhas 42 cells, transformed foci were induced by the treatment with nongenotoxic carcinogens, most of which act as tumor promoters. Therefore, Bhas 42 cells were considered to be a model of initiated cells. Subsequently, not only nongenotoxic carcinogens but also genotoxic carcinogens, most of which act as tumor initiators, were found to induce transformed foci by the modification of the protocol. Furthermore, transformation of Bhas 42 cells was induced by the transfection with genes of oncogenic potential. We interpret this high sensitivity of Bhas 42 cells to various types of carcinogenic stimuli to be related to the multistage model of carcinogenesis, as the transfection of v-Ha-ras gene further advances the parental BALB/c 3T3 A31-1-1 cells toward higher transforming potential. Thus, we propose that Bhas 42 cells are a novel and sensitive cell line for the analysis of carcinogenesis and can be used for the detection of not only carcinogenic substances but also gene alterations related to oncogenesis. This review will address characteristics of Bhas 42 cells, the transformation assay protocol, validation studies, and the various chemicals tested in this assay.

Gain-of-function assays in the axolotl (Ambystoma mexicanum) to identify signaling pathways that induce and regulate limb regeneration.

Science.gov (United States)

Lee, Jangwoo; Aguilar, Cristian; Gardiner, David

2013-01-01

The adult salamander has been studied as a model for regeneration of complex tissues for many decades. Only recently with the development of gain-of-function assays for regeneration, has it been possible to screen for and assay the function of the multitude of signaling factors that have been identified in studies of embryonic development and tumorigenesis. Given the conservation of function of these regulatory pathways controlling growth and pattern formation, it is now possible to use the functional assays in the salamander to test the ability of endogenous as well as small-molecule signaling factors to induce a regenerative response.

ELISPOT Assay for Monitoring Cytotoxic T Lymphocytes (CTL Activity in Cancer Vaccine Clinical Trials

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Thomas J. Sayers

2012-05-01

Full Text Available The profiling and monitoring of immune responses are key elements in the evaluation of the efficacy and development of new biotherapies, and a number of assays have been introduced for analyzing various immune parameters before, during, and after immunotherapy. The choice of immune assays for a given clinical trial depends on the known or suggested immunomodulating mechanisms associated with the tested therapeutic modality. Cell-mediated cytotoxicity represents a key mechanism in the immune response to various pathogens and tumors. Therefore, the selection of monitoring methods for the appropriate assessment of cell-mediated cytotoxicity is thought to be crucial. Assays that can detect both cytotoxic T lymphocytes (CTL frequency and function, such as the IFN-γ enzyme-linked immunospot assay (ELISPOT have gained increasing popularity for monitoring clinical trials and in basic research. Results from various clinical trials, including peptide and whole tumor cell vaccination and cytokine treatment, have shown the suitability of the IFN-γ ELISPOT assay for monitoring T cell responses. However, the Granzyme B ELISPOT assay and Perforin ELISPOT assay may represent a more direct analysis of cell-mediated cytotoxicity as compared to the IFN-γ ELISPOT, since Granzyme B and perforin are the key mediators of target cell death via the granule-mediated pathway. In this review we analyze our own data and the data reported by others with regard to the application of various modifications of ELISPOT assays for monitoring CTL activity in clinical vaccine trials.

High-Throughput Flow Cytometric Method for the Simultaneous Measurement of CAR-T Cell Characterization and Cytotoxicity against Solid Tumor Cell Lines.

Science.gov (United States)

Martinez, Emily M; Klebanoff, Samuel D; Secrest, Stephanie; Romain, Gabrielle; Haile, Samuel T; Emtage, Peter C R; Gilbert, Amy E

2018-04-01

High-throughput flow cytometry is an attractive platform for the analysis of adoptive cellular therapies such as chimeric antigen receptor T cell therapy (CAR-T) because it allows for the concurrent measurement of T cell-dependent cellular cytotoxicity (TDCC) and the functional characterization of engineered T cells with respect to percentage of CAR transduction, T cell phenotype, and measurement of T cell function such as activation in a single assay. The use of adherent tumor cell lines can be challenging in these flow-based assays. Here, we present the development of a high-throughput flow-based assay to measure TDCC for a CAR-T construct co-cultured with multiple adherent tumor cell lines. We describe optimal assay conditions (such as adherent cell dissociation techniques to minimize impact on cell viability) that result in robust cytotoxicity assays. In addition, we report on the concurrent use of T cell transduction and activation antibody panels (CD25) that provide further dissection of engineered T cell function. In conclusion, we present the development of a high-throughput flow cytometry method allowing for in vitro interrogation of solid tumor, targeting CAR-T cell-mediated cytotoxicity, CAR transduction, and engineered T cell characterization in a single assay.

A novel cell-based assay to measure activity of Venezuelan equine encephalitis virus nsP2 protease

International Nuclear Information System (INIS)

Campos-Gomez, Javier; Ahmad, Fahim; Rodriguez, Efrain; Saeed, Mohammad F.

2016-01-01

The encephalitic alphaviruses encode nsP2 protease (nsP2pro), which because of its vital role in virus replication, represents an attractive target for therapeutic intervention. To facilitate the discovery of nsP2 inhibitors we have developed a novel assay for quantitative measurement of nsP2pro activity in a cell-based format. The assay is based on a substrate fusion protein consisting of eGFP and Gaussia luciferase (Gluc) linked together by a small peptide containing a VEEV nsp2pro cleavage sequence. The expression of the substrate protein in cells along with recombinant nsP2pro results in cleavage of the substrate protein resulting in extracellular release of free Gluc. The Gluc activity in supernatants corresponds to intracellular nsP2pro-mediated substrate cleavage; thus, providing a simple and convenient way to quantify nsP2pro activity. Here, we demonstrate potential utility of the assay in identification of nsP2pro inhibitors, as well as in investigations related to molecular characterization of nsP2pro. - Highlights: • A novel cell-based assay to measure VEEV nsP2 protease activity was developed. • Assay utility was demonstrated for antiviral screening. • .The assay also proved to be useful in basic mechanistic studies of nsP2 protease.

A novel cell-based assay to measure activity of Venezuelan equine encephalitis virus nsP2 protease

Energy Technology Data Exchange (ETDEWEB)

Campos-Gomez, Javier; Ahmad, Fahim; Rodriguez, Efrain; Saeed, Mohammad F., E-mail: saeed@southernresearch.org

2016-09-15

The encephalitic alphaviruses encode nsP2 protease (nsP2pro), which because of its vital role in virus replication, represents an attractive target for therapeutic intervention. To facilitate the discovery of nsP2 inhibitors we have developed a novel assay for quantitative measurement of nsP2pro activity in a cell-based format. The assay is based on a substrate fusion protein consisting of eGFP and Gaussia luciferase (Gluc) linked together by a small peptide containing a VEEV nsp2pro cleavage sequence. The expression of the substrate protein in cells along with recombinant nsP2pro results in cleavage of the substrate protein resulting in extracellular release of free Gluc. The Gluc activity in supernatants corresponds to intracellular nsP2pro-mediated substrate cleavage; thus, providing a simple and convenient way to quantify nsP2pro activity. Here, we demonstrate potential utility of the assay in identification of nsP2pro inhibitors, as well as in investigations related to molecular characterization of nsP2pro. - Highlights: • A novel cell-based assay to measure VEEV nsP2 protease activity was developed. • Assay utility was demonstrated for antiviral screening. • .The assay also proved to be useful in basic mechanistic studies of nsP2 protease.

Prospects for cellular mutational assays in human populations

International Nuclear Information System (INIS)

Mendelsohn, M.L.

1984-01-01

Practical, sensitive, and effective human cellular assays for detecting somatic and germinal mutations would have great value in environmental mutagenesis and carcinogenesis studies. Such assays would fill the void between human mutagenicity and the data that exist from short-term tests and from mutagenicity in other species. This paper discusses the following possible human cellular assays: (1) HPRT (hypoxanthine phosphoribosyltransferase) somatic cell mutation based on 6-thioguanine resistance; (2) hemoglobin somatic cell mutation assay; (3) glycophorin somatic cell mutation assay; and (4) LDH-X sperm cell mutation assay. 18 references

Prospects for cellular mutational assays in human populations

Energy Technology Data Exchange (ETDEWEB)

Mendelsohn, M.L.

1984-06-29

Practical, sensitive, and effective human cellular assays for detecting somatic and germinal mutations would have great value in environmental mutagenesis and carcinogenesis studies. Such assays would fill the void between human mutagenicity and the data that exist from short-term tests and from mutagenicity in other species. This paper discusses the following possible human cellular assays: (1) HPRT (hypoxanthine phosphoribosyltransferase) somatic cell mutation based on 6-thioguanine resistance; (2) hemoglobin somatic cell mutation assay; (3) glycophorin somatic cell mutation assay; and (4) LDH-X sperm cell mutation assay. 18 references.

Application of the Single Cell Gel Electrophoresis (SCGE) Assay to Genotoxicity Evaluation in Plants and Animals

International Nuclear Information System (INIS)

Kim, Jin Kyu

2007-10-01

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

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.

[3H]uridine uptake by target monolayers as a terminal label in an in vitro cell-mediated cytotoxicity assay

International Nuclear Information System (INIS)

Smith, G.; Nicklin, S.

1979-01-01

A terminal labelling method is described for measuring cell-mediated cytotoxicity based on the ability of surviving target cells to incorporate [ 3 H]uridine into their RNA precursor pools. Parameters of the system were examined using whole and damaged embryonic mouse fibroblast monolayers. This assay is less laborious than direct cell counting and gives increased sensitivity at low target to effector cell ratios. The labelling time is short and, unlike similar techniques, it allows target cell monolayers to remain intact after completion of the radioassay and available for histological examination. This is important where heterogeneous target populations are employed since it allows assessment of differential cell killing and eliminates the need for duplicate cultures. The assay was used in conjunction with a well defined mouse popliteal lymph node assay to investigate the appearance of cytotoxic cells during a localised graft versus host response. Results showed a direct correlation between proliferative index and the development of highly specific cell-mediated cytotoxicity. (Auth.)

High-throughput screening of cellulase F mutants from multiplexed plasmid sets using an automated plate assay on a functional proteomic robotic workcell

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Qureshi Nasib

2006-05-01

F mutants and corresponding optimized clones in expression-ready plasmids. Conclusion The multiplex method using an integrated automated platform for high-throughput screening in a functional proteomic assay allows rapid identification of plasmids containing optimized clones ready for use in subsequent applications including transformations to produce improved strains or cell lines.

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.

The functions of the variable lipoprotein family of Mycoplasma hyorhinis in adherence to host cells.

Science.gov (United States)

Xiong, Qiyan; Wang, Jia; Ji, Yan; Ni, Bo; Zhang, Bixiong; Ma, Qinghong; Wei, Yanna; Xiao, Shaobo; Feng, Zhixin; Liu, Maojun; Shao, Guoqing

2016-04-15

Mycoplasma hyorhinis (M. hyorhinis) is a swine pathogen that is associated with various human cancers and contamination in cell cultures. However, no studies on the adhesion molecules of this pathogen have yet been reported. The variable lipoprotein (Vlp) family is an important surface component of M. hyorhinis. Herein, we performed several experiments to identify the function of the Vlp family in adherence to host cells. Seven recombinant Vlp (rVlp) proteins were expressed in Escherichia coli and purified by affinity chromatography. The potential role of rVlp adherence to pig kidney (PK-15) and swine tracheal epithelial (STEC) cells was then studied by indirect immunofluorescence assay and microtiter plate adherence assay. Adhesion of M. hyorhinis to PK-15 and STEC cells was specifically inhibited by the addition of a cocktail of rVlp proteins. The rVlp protein mixture was shown to bind to both PK-15 and STEC cells. The binding increased in a dose-dependent manner and could be blocked by antisera against the rVlp proteins. Most of the rVlp proteins could bind individually to both PK-15 and STEC cells except for rVlpD and rVlpF, which bound only to STEC cells. Because Vlp members vary in size among different strains and generations, they may vary in their cytoadhesion capabilities in various strains. In summary, the present results indicate that the Vlp family functions as adhesins of M. hyorhinis. Copyright © 2016. Published by Elsevier B.V.

The potential value of the neutral comet assay and the expression of genes associated with DNA damage in assessing the radiosensitivity of tumor cells.

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Jayakumar, Sundarraj; Bhilwade, Hari N; Pandey, Badri N; Sandur, Santosh K; Chaubey, Ramesh C

2012-10-09

The assessment of tumor radiosensitivity would be particularly useful in optimizing the radiation dose during radiotherapy. Therefore, the degree of correlation between radiation-induced DNA damage, as measured by the alkaline and the neutral comet assays, and the clonogenic survival of different human tumor cells was studied. Further, tumor radiosensitivity was compared with the expression of genes associated with the cellular response to radiation damage. Five different human tumor cell lines were chosen and the radiosensitivity of these cells was established by clonogenic assay. Alkaline and neutral comet assays were performed in γ-irradiated cells (2-8Gy; either acute or fractionated). Quantitative PCR was performed to evaluate the expression of DNA damage response genes in control and irradiated cells. The relative radiosensitivity of the cell lines assessed by the extent of DNA damage (neutral comet assay) immediately after irradiation (4Gy or 6Gy) was in agreement with radiosensitivity pattern obtained by the clonogenic assay. The survival fraction of irradiated cells showed a better correlation with the magnitude of DNA damage measured by the neutral comet assay (r=-0.9; Pcomet assay (r=-0.73; Pcomet assay was better than alkaline comet assay for assessment of radiosensitivities of tumor cells after acute or fractionated doses of irradiation. © 2012 Elsevier B.V. All rights reserved.

Standardization of the CFU-GM assay: Advantages of plating a fixed number of CD34+ cells in collagen gels.

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Dobo, Irène; Pineau, Danielle; Robillard, Nelly; Geneviève, Frank; Piard, Nicole; Zandecki, Marc; Hermouet, Sylvie

2003-10-01

We investigated whether plating a stable amount of CD34(+) cells improves the CFU-GM assay. Data of CFU-GM assays performed with leukaphereses products in two transplant centers using a commercial collagen-based medium and unified CFU-GM scoring criteria were pooled and analyzed according to the numbers of CD34(+) cells plated. A first series of 113 CFU-GM assays was performed with a fixed number of mononuclear cells (i.e., a variable number of CD34(+) cells). In these cultures the CFU-GM/CD34 ratio varied according to the number of CD34(+) cells plated: median CFUGM/CD34 ratios were 1/6.2 to 1/6.6 for grafts containing or =2% CD34(+) cells. The median CFU-GM/CD34 ratio also varied depending on pathology: 1/9.3 for multiple myeloma (MM), 1/6.8 for Hodgkin's disease (HD), 1/6.5 for non-Hodgkin lymphoma (NHL), and 1/4.5 for solid tumors (ST). A second series of 95 CFU-GM assays was performed with a fixed number of CD34(+) cells (220/ml). The range of median CFU-GM/CD34 ratios was narrowed to 1/7.0 to 1/5.2, and coefficients of variation for CFU-GM counts decreased by half to 38.1% (NHL), 36.1% (MM), 49.9% (HD), and 22.4% (ST). In addition, CFU-GM scoring was facilitated as the percentages of cultures with >50 CFU/GM/ml decreased from 6.7% to 43.8% when a variable number of CD34(+) cells was plated, to 4.5% to 16.7% when 220 CD34(+) cells/ml were plated. Hence, plating a fixed number of CD34(+) cells in collagen gels improves the CFU-GM assay by eliminating cell number-related variability and reducing pathology-related variability in colony growth.

Use of the microculture kinetic assay of apoptosis to determine chemosensitivities of leukemias.

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Kravtsov, V D; Greer, J P; Whitlock, J A; Koury, M J

1998-08-01

Chemotherapeutic agents exert their antitumor effects by inducing apoptosis. The microculture kinetic (MiCK) assay provides an automated, continuous means of monitoring apoptosis in a cell population. We used the MiCK assay to determine the chemosensitivities of the human promyelocytic HL-60 and lymphoblastic CEM cell lines and leukemia cells freshly isolated from patients with acute nonlymphocytic (ANLL) or acute lymphocytic (ALL) leukemias. Continuous monitoring of apoptosis in the MiCK assay permits determination of the time to the maximum apoptosis (Tm) and its two components which are initiation time (Ti) and development time (Td). Duration of the three timing components of apoptosis varies from hours to days depending on the drug, drug concentration, and type of target cells. In the MiCK assay, the extent of apoptosis is reported in kinetic units of apoptosis. Kinetic units are determined by the slope of the curve created when optical density caused by cell blebbing is plotted as a function of time. Using the leukemia cell lines, we define the relationship between kinetic units determined by the MiCK assay and the percentage of morphologically apoptotic cells in the culture. Flow cytometry analysis of apoptosis in Annexin-V-fluorescein isothiocyanate-labeled preparations of HL-60 and CEM cells was also used to compare with data obtained by the MiCK assay. The feasibility of the MiCK assay of apoptosis as a chemosensitivity test was confirmed by its comparison with a 3H-thymidine incorporation assay. We show that samples from 10 ANLL and ALL patients patients tested for sensitivity to various doses of idarubicin (IDR), daunorubicin (DNR), or mitoxantrone (MTA) gave the same percentages of apoptotic cells when calculated by the MiCK assay as when determined by morphological analysis. The MiCK assay was used for dose-response analyses of the sensitivities to IDR, DNR, and MTA of leukemia cells from 4 other patients (2 ANLL and 2 ALL). The results from both cell

Maturation and upregulation of functions of murine dendritic cells (DCs) under the influence of purified aromatic-turmerone (AR).

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Yonggang, Tan; Yiming, Meng; Heying, Zhang; Cheng, Sun; Qiushi, Wang; Xianghong, Yang; Wei, Zheng; Huawei, Zhou; Shan, Fengping

2012-10-01

The aim of this work is to evaluate the effects of purified aromatic-turmerone (ar-turmerione, AR) on murine dendritic cells (DCs). These impacts of AR on DCs from bone marrow derived DCs(BMDCs) were assessed with use of conventional scanning electron microscopy (SEM), fluorescence activated cell sorting (FACS), transmission electron microscopy (TEM), cytochemistry assay, FITC-dextran, bio-assay and enzyme linked immunosorbent assay (ELISA). We found that AR induced phenotypic maturation as evidenced by increased expression of CD86, CD40, CD83, CD80 and major histocompatibility complex II (MHC II). The functional tests showed the activity of acidic phosphatase (ACP) inside the DCs were downregulated after treatment with AR (which occurs when phagocytosis of DCs were decreased). Finally, we proved that AR increased the production of IL-12 and tumor necrosis factor α (TNF-α). These data suggested that AR could promote phenotypic and functional maturation of DCs and this adjuvant-like activity may have potential therapeutic value. It is therefore concluded that AR could exert positive modulation on murine DCs.

Functionality of Dengue Virus Specific Memory T Cell Responses in Individuals Who Were Hospitalized or Who Had Mild or Subclinical Dengue Infection

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Jeewandara, Chandima; Adikari, Thiruni N.; Gomes, Laksiri; Fernando, Samitha; Fernando, R. H.; Perera, M. K. T.; Ariyaratne, Dinuka; Kamaladasa, Achala; Salimi, Maryam; Prathapan, Shamini

2015-01-01

Background Although antibody responses to dengue virus (DENV) in naturally infected individuals have been extensively studied, the functionality of DENV specific memory T cell responses in relation to clinical disease severity is incompletely understood. Methodology/Principal findings Using ex vivo IFNγ ELISpot assays, and by determining cytokines produced in ELISpot supernatants, we investigated the functionality of DENV-specific memory T cell responses in a large cohort of individuals from Sri Lanka (n=338), who were naturally infected and were either hospitalized due to dengue or had mild or sub clinical dengue infection. We found that T cells of individuals with both past mild or sub clinical dengue infection and who were hospitalized produced multiple cytokines when stimulated with DENV-NS3 peptides. However, while DENV-NS3 specific T cells of those with mild/sub clinical dengue infection were more likely to produce only granzyme B (p=0.02), those who were hospitalized were more likely to produce both TNFα and IFNγ (p=0.03) or TNFα alone. We have also investigated the usefulness of a novel T cell based assay, which can be used to determine the past infecting DENV serotype. 92.4% of DENV seropositive individuals responded to at least one DENV serotype of this assay and none of the seronegatives responded. Individuals who were seronegative, but had received the Japanese encephalitis vaccine too made no responses, suggesting that the peptides used in this assay did not cross react with the Japanese encephalitis virus. Conclusions/significance The types of cytokines produced by DENV-specific memory T cells appear to influence the outcome of clinical disease severity. The novel T cell based assay, is likely to be useful in determining the past infecting DENV serotype in immune-epidemiological studies and also in dengue vaccine trials. PMID:25875020

Cell transformation assays for prediction of carcinogenic potential: state of the science and future research needs

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Creton, Stuart; Aardema, Marilyn J.; Carmichael, Paul L.; Harvey, James S.; Martin, Francis L.; Newbold, Robert F.; O’Donovan, Michael R.; Pant, Kamala; Poth, Albrecht; Sakai, Ayako; Sasaki, Kiyoshi; Scott, Andrew D.; Schechtman, Leonard M.; Shen, Rhine R.; Tanaka, Noriho; Yasaei, Hemad

2012-01-01

Cell transformation assays (CTAs) have long been proposed as in vitro methods for the identification of potential chemical carcinogens. Despite showing good correlation with rodent bioassay data, concerns over the subjective nature of using morphological criteria for identifying transformed cells and a lack of understanding of the mechanistic basis of the assays has limited their acceptance for regulatory purposes. However, recent drivers to find alternative carcinogenicity assessment methodologies, such as the Seventh Amendment to the EU Cosmetics Directive, have fuelled renewed interest in CTAs. Research is currently ongoing to improve the objectivity of the assays, reveal the underlying molecular changes leading to transformation and explore the use of novel cell types. The UK NC3Rs held an international workshop in November 2010 to review the current state of the art in this field and provide directions for future research. This paper outlines the key points highlighted at this meeting. PMID:21852270

Bioluminescence-based cytotoxicity assay for simultaneous evaluation of cell viability and membrane damage in human hepatoma HepG2 cells.

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Uno, Katsuhiro; Murotomi, Kazutoshi; Kazuki, Yasuhiro; Oshimura, Mitsuo; Nakajima, Yoshihiro

2018-05-01

We have developed a bioluminescence-based non-destructive cytotoxicity assay in which cell viability and membrane damage are simultaneously evaluated using Emerald luciferase (ELuc) and endoplasmic reticulum (ER)-targeted copepod luciferase (GLuc-KDEL), respectively, by using multi-integrase mouse artificial chromosome (MI-MAC) vector. We have demonstrated that the time-dependent concentration response curves of ELuc luminescence intensity and WST-1 assay, and GLuc-KDEL luminescence intensity and lactate dehydrogenase (LDH) activity in the culture medium accompanied by cytotoxicity show good agreement in toxicant-treated ELuc- and GLuc-KDEL-expressing HepG2 stable cell lines. We have clarified that the increase of GLuc-KDEL luminescence intensity in the culture medium reflects the type of cell death, including necrosis and late apoptosis, but not early apoptosis. We have also uncovered a strong correlation between GLuc-KDEL luminescence intensity in the culture medium and the extracellular release of high mobility group box 1 (HMGB1), a representative damage-associated molecular pattern (DAMP) molecule. The bioluminescence measurement assay using ELuc and GLuc-KDEL developed in this study can simultaneously monitor cell viability and membrane damage, respectively, and the increase of GLuc-KDEL luminescence intensity in the culture medium accompanied by the increase of cytotoxicity is an index of necrosis and late apoptosis associated with the extracellular release of DAMP molecules. Copyright © 2018 John Wiley & Sons, Ltd.

Controlled cell morphology and liver-specific function of engineered primary hepatocytes by fibroblast layer cell densities.

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Sakai, Yusuke; Koike, Makiko; Kawahara, Daisuke; Hasegawa, Hideko; Murai, Tomomi; Yamanouchi, Kosho; Soyama, Akihiko; Hidaka, Masaaki; Takatsuki, Mitsuhisa; Fujita, Fumihiko; Kuroki, Tamotsu; Eguchi, Susumu

2018-03-05

Engineered primary hepatocytes, including co-cultured hepatocyte sheets, are an attractive to basic scientific and clinical researchers because they maintain liver-specific functions, have reconstructed cell polarity, and have high transplantation efficiency. However, co-culture conditions regarding engineered primary hepatocytes were suboptimal in promoting these advantages. Here we report that the hepatocyte morphology and liver-specific function levels are controlled by the normal human diploid fibroblast (TIG-118 cell) layer cell density. Primary rat hepatocytes were plated onto TIG-118 cells, previously plated 3 days before at 1.04, 5.21, and 26.1×10 3  cells/cm 2 . Hepatocytes plated onto lower TIG-118 cell densities expanded better during the early culture period. The hepatocytes gathered as colonies and only exhibited small adhesion areas because of the pushing force from proliferating TIG-118 cells. The smaller areas of each hepatocyte result in the development of bile canaliculi. The highest density of TIG-118 cells downregulated albumin synthesis activity of hepatocytes. The hepatocytes may have undergone apoptosis associated with high TGF-β1 concentration and necrosis due to a lack of oxygen. These occurrences were supported by apoptotic chromatin condensation and high expression of both proteins HIF-1a and HIF-1b. Three types of engineered hepatocyte/fibroblast sheets comprising different TIG-118 cell densities were harvested after 4 days of hepatocyte culture and showed a complete cell sheet format without any holes. Hepatocyte morphology and liver-specific function levels are controlled by TIG-118 cell density, which helps to design better engineered hepatocytes for future applications such as in vitro cell-based assays and transplantable hepatocyte tissues. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Development and Fit-for-Purpose Validation of a Soluble Human Programmed Death-1 Protein Assay.

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Ni, Yan G; Yuan, Xiling; Newitt, John A; Peterson, Jon E; Gleason, Carol R; Haulenbeek, Jonathan; Santockyte, Rasa; Lafont, Virginie; Marsilio, Frank; Neely, Robert J; DeSilva, Binodh; Piccoli, Steven P

2015-07-01

Programmed death-1 (PD-1) protein is a co-inhibitory receptor which negatively regulates immune cell activation and permits tumors to evade normal immune defense. Anti-PD-1 antibodies have been shown to restore immune cell activation and effector function-an exciting breakthrough in cancer immunotherapy. Recent reports have documented a soluble form of PD-1 (sPD-1) in the circulation of normal and disease state individuals. A clinical assay to quantify sPD-1 would contribute to the understanding of sPD-1-function and facilitate the development of anti-PD-1 drugs. Here, we report the development and validation of a sPD-1 protein assay. The assay validation followed the framework for full validation of a biotherapeutic pharmacokinetic assay. A purified recombinant human PD-1 protein was characterized extensively and was identified as the assay reference material which mimics the endogenous analyte in structure and function. The lower limit of quantitation (LLOQ) was determined to be 100 pg/mL, with a dynamic range spanning three logs to 10,000 pg/mL. The intra- and inter-assay imprecision were ≤15%, and the assay bias (percent deviation) was ≤10%. Potential matrix effects were investigated in sera from both normal healthy volunteers and selected cancer patients. Bulk-prepared frozen standards and pre-coated Streptavidin plates were used in the assay to ensure consistency in assay performance over time. This assay appears to specifically measure total sPD-1 protein since the human anti-PD-1 antibody, nivolumab, and the endogenous ligands of PD-1 protein, PDL-1 and PDL-2, do not interfere with the assay.

Transferability study of CHO cell clustering assays for monitoring of pertussis toxin activity in acellular pertussis vaccines.

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Isbrucker, R; Daas, A; Wagner, L; Costanzo, A

2016-01-01

Current regulations for acellular pertussis (aP) vaccines require that they are tested for the presence of residual or reversion-derived pertussis toxin (PTx) activity using the mouse histamine sensitisation test (HIST). Although a CHO cell clustering assay can be used by manufacturers to verify if sufficient inactivation of the substance has occurred in-process, this assay cannot be used at present for the final product due to the presence of aluminium adjuvants which interfere with mammalian cell cultures. Recently, 2 modified CHO cell clustering assays which accommodate for the adjuvant effects have been proposed as alternatives to the HIST. These modified assays eliminate the adjuvant-induced cytotoxicity either through dilution of the vaccine (called the Direct Method) or by introducing a porous barrier between the adjuvant and the cells (the Indirect Method). Transferability and suitability of these methods for testing of products present on the European market were investigated during a collaborative study organised by the European Directorate for the Quality of Medicines & HealthCare (EDQM). Thirteen laboratories participated in this study which included 4 aP-containing vaccines spiked by addition of PTx. This study also assessed the transferability of a standardised CHO cell clustering assay protocol for use with non-adjuvanted PTx preparations. Results showed that the majority of laboratories were able to detect the PTx spike in all 4 vaccines at concentrations of 4 IU/mL or lower using the Indirect Method. This sensitivity is in the range of the theoretical sensitivity of the HIST. The Direct Method however did not show the expected results and would need additional development work.

Antibacterial agent triclosan suppresses RBL-2H3 mast cell function

International Nuclear Information System (INIS)

Palmer, Rachel K.; Hutchinson, Lee M.; Burpee, Benjamin T.; Tupper, Emily J.; Pelletier, Jonathan H.; Kormendy, Zsolt; Hopke, Alex R.; Malay, Ethan T.; Evans, Brieana L.; Velez, Alejandro; Gosse, Julie A.

2012-01-01

Triclosan is a broad-spectrum antibacterial agent, which has been shown previously to alleviate human allergic skin disease. The purpose of this study was to investigate the hypothesis that the mechanism of this action of triclosan is, in part, due to effects on mast cell function. Mast cells play important roles in allergy, asthma, parasite defense, and carcinogenesis. In response to various stimuli, mast cells degranulate, releasing allergic mediators such as histamine. In order to investigate the potential anti-inflammatory effect of triclosan on mast cells, we monitored the level of degranulation in a mast cell model, rat basophilic leukemia cells, clone 2H3. Having functional homology to human mast cells, as well as a very well defined signaling pathway leading to degranulation, this cell line has been widely used to gain insight into mast-cell driven allergic disorders in humans. Using a fluorescent microplate assay, we determined that triclosan strongly dampened the release of granules from activated rat mast cells starting at 2 μM treatment, with dose-responsive suppression through 30 μM. These concentrations were found to be non-cytotoxic. The inhibition was found to persist when early signaling events (such as IgE receptor aggregation and tyrosine phosphorylation) were bypassed by using calcium ionophore stimulation, indicating that the target for triclosan in this pathway is likely downstream of the calcium signaling event. Triclosan also strongly suppressed F-actin remodeling and cell membrane ruffling, a physiological process that accompanies degranulation. Our finding that triclosan inhibits mast cell function may explain the clinical data mentioned above and supports the use of triclosan or a mechanistically similar compound as a topical treatment for allergic skin disease, such as eczema. -- Highlights: ►The effects of triclosan on mast cell function using a murine mast cell model. ►Triclosan strongly inhibits degranulation of mast cells. �

The use of real-time cell analyzer technology in drug discovery: defining optimal cell culture conditions and assay reproducibility with different adherent cellular models.

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Atienzar, Franck A; Tilmant, Karen; Gerets, Helga H; Toussaint, Gaelle; Speeckaert, Sebastien; Hanon, Etienne; Depelchin, Olympe; Dhalluin, Stephane

2011-07-01

The use of impedance-based label-free technology applied to drug discovery is nowadays receiving more and more attention. Indeed, such a simple and noninvasive assay that interferes minimally with cell morphology and function allows one to perform kinetic measurements and to obtain information on proliferation, migration, cytotoxicity, and receptor-mediated signaling. The objective of the study was to further assess the usefulness of a real-time cell analyzer (RTCA) platform based on impedance in the context of quality control and data reproducibility. The data indicate that this technology is useful to determine the best coating and cellular density conditions for different adherent cellular models including hepatocytes, cardiomyocytes, fibroblasts, and hybrid neuroblastoma/neuronal cells. Based on 31 independent experiments, the reproducibility of cell index data generated from HepG2 cells exposed to DMSO and to Triton X-100 was satisfactory, with a coefficient of variation close to 10%. Cell index data were also well reproduced when cardiomyocytes and fibroblasts were exposed to 21 compounds three times (correlation >0.91, p technology appears to be a powerful and reliable tool in drug discovery because of the reasonable throughput, rapid and efficient performance, technical optimization, and cell quality control.

Phenotypic and functional profiling of CD4 T cell compartment in distinct populations of healthy adults with different antigenic exposure.

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Sophie Roetynck

Full Text Available Multiparameter flow cytometry has revealed extensive phenotypic and functional heterogeneity of CD4 T cell responses in mice and humans, emphasizing the importance of assessing multiple aspects of the immune response in correlation with infection or vaccination outcome. The aim of this study was to establish and validate reliable and feasible flow cytometry assays, which will allow us to characterize CD4 T cell population in humans in field studies more fully.We developed polychromatic flow cytometry antibody panels for immunophenotyping the major CD4 T cell subsets as well as broadly characterizing the functional profiles of the CD4 T cells in peripheral blood. We then validated these assays by conducting a pilot study comparing CD4 T cell responses in distinct populations of healthy adults living in either rural or urban Kenya. This study revealed that the expression profile of CD4 T cell activation and memory markers differed significantly between African and European donors but was similar amongst African individuals from either rural or urban areas. Adults from rural Kenya had, however, higher frequencies and greater polyfunctionality among cytokine producing CD4 T cells compared to both urban populations, particularly for "Th1" type of response. Finally, endemic exposure to malaria in rural Kenya may have influenced the expansion of few discrete CD4 T cell populations with specific functional signatures.These findings suggest that environmentally driven T cell activation does not drive the dysfunction of CD4 T cells but is rather associated with greater magnitude and quality of CD4 T cell response, indicating that the level or type of microbial exposure and antigenic experience may influence and shape the functionality of CD4 T cell compartment. Our data confirm that it is possible and mandatory to assess multiple functional attributes of CD4 T cell response in the context of infection.

Proliferation assay of mouse embryonic stem (ES) cells exposed to atmospheric-pressure plasmas at room temperature

International Nuclear Information System (INIS)

Miura, Taichi; Hirano, Kazumi; Ogura, Chika; Ikeguchi, Masamichi; Seki, Atsushi; Nishihara, Shoko; Ando, Ayumi; Kanazawa, Tatsuya; Hamaguchi, Satoshi

2014-01-01

Proliferation assays of mouse embryonic stem (ES) cells have been performed with cell culture media exposed to atmospheric-pressure plasmas (APPs), which generate reactive species in the media at room temperature. It is found that serum in cell culture media functions as a scavenger of highly reactive species and tends to protect cells in the media against cellular damage. On the other hand, if serum is not present in a cell culture medium when it is exposed to APP, the medium becomes cytotoxic and cannot be detoxified by serum added afterwards. Plasma-induced cytotoxic media hinder proliferation of mouse ES cells and may even cause cell death. It is also shown by nuclear magnetic resonance spectroscopy that organic compounds in cell culture media are in general not significantly modified by plasma exposure. These results indicate that if there is no serum in media when they are exposed to APPs, highly reactive species (such as OH radicals) generated in the media by the APP exposure are immediately converted to less reactive species (such as H 2 O 2 ), which can no longer readily react with serum that is added to the medium after plasma exposure. This study has clearly shown that it is these less reactive species, rather than highly reactive species, that make the medium cytotoxic to mouse ES cells. (paper)

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.

Surface functionalization of a polymeric lipid bilayer for coupling a model biological membrane with molecules, cells, and microstructures.

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Morigaki, Kenichi; Mizutani, Kazuyuki; Saito, Makoto; Okazaki, Takashi; Nakajima, Yoshihiro; Tatsu, Yoshiro; Imaishi, Hiromasa

2013-02-26

We describe a stable and functional model biological membrane based on a polymerized lipid bilayer with a chemically modified surface. A polymerized lipid bilayer was formed from a mixture of two diacetylene-containing phospholipids, 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DiynePC) and 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphoethanolamine (DiynePE). DiynePC formed a stable bilayer structure, whereas the ethanolamine headgroup of DiynePE enabled functional molecules to be grafted onto the membrane surface. Copolymerization of DiynePC and DiynePE resulted in a robust bilayer. Functionalization of the polymeric bilayer provided a route to a robust and biomimetic surface that can be linked with biomolecules, cells, and three-dimensional (3D) microstructures. Biotin and peptides were grafted onto the polymeric bilayer for attaching streptavidin and cultured mammalian cells by molecular recognition, respectively. Nonspecific adsorption of proteins and cells on polymeric bilayers was minimum. DiynePE was also used to attach a microstructure made of an elastomer (polydimethylsiloxan: PDMS) onto the membrane, forming a confined aqueous solution between the two surfaces. The microcompartment enabled us to assay the activity of a membrane-bound enzyme (cyochrome P450). Natural (fluid) lipid bilayers were incorporated together with membrane-bound proteins by lithographically polymerizing DiynePC/DiynePE bilayers. The hybrid membrane of functionalized polymeric bilayers and fluid bilayers offers a novel platform for a wide range of biomedical applications including biosensor, bioassay, cell culture, and cell-based assay.

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

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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.

A simple, specific high-throughput enzyme-linked immunosorbent assay (ELISA) for quantitative determination of melatonin in cell culture medium.

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Li, Ye; Cassone, Vincent M

2015-09-01

A simple, specific, high-throughput enzyme-linked immunosorbent assay (ELISA) for quantitative determination of melatonin was developed for directly measuring melatonin in cell culture medium with 10% FBS. This assay adopts a commercial monoclonal melatonin antibody and melatonin-HRP conjugate, so it can be applied in multiple labs rapidly with low cost compared with commercial RIA and ELISA kits. In addition, the procedure is much simpler with only four steps: 1) sample/conjugate incubation, 2) plate washing, 3) TMB color reaction and 4) reading of results. The standards of the assay cover a wide working range from 100 pg/mL to 10 ng/mL. The sensitivity was 68 pg/mL in cell culture medium with 10% FBS and 26 pg/mL in PBS with as little as 25 μL sample volume. The recovery of melatonin from cell culture medium was 101.0%. The principal cross-reacting compound was 5-methoxytryptophol (0.1%). The variation coefficients of the assay, within and between runs, ranged between 6.68% and 15.76% in cell culture medium. The mean linearity of a series diluted cell culture medium sample was 105% (CV=5%), ranging between 98% and 111%, y=5.5263x+0.0646, R(2)=0.99. The assay enables small research and teaching labs to reliably measure this important neurohormone. Copyright © 2015 Elsevier B.V. All rights reserved.

Discovery and validation of the tumor-suppressive function of long noncoding RNA PANDA in human diffuse large B-cell lymphoma through the inactivation of MAPK/ERK signaling pathway.

Science.gov (United States)

Wang, Yingjun; Zhang, Mingzhi; Xu, Huanan; Wang, Yifei; Li, Zhaoming; Chang, Yu; Wang, Xinhuan; Fu, Xiaorui; Zhou, Zhiyuan; Yang, Siyuan; Wang, Bei; Shang, Yufeng

2017-09-22

Diffuse large B-cell lymphoma (DLBCL) is one of the leading causes of cancer-related mortality, and responds badly to existing treatment. Thus, it is of urgent need to identify novel prognostic markers and therapeutic targets of DLBCL. Recent studies have shown that long non-coding RNAs (lncRNAs) play an important role in the development of cancer. By using the next generation HiSeq sequencing assay, we determined lncRNAs exhibiting differential expression between DLBCL patients and healthy controls. Then, RT-qPCR was performed for identification in clinical samples and cell materials, and lncRNA PANDA was verified to be down-regulated in DLBCL patients and have considerable diagnostic potential. In addition, decreased serum PANDA level was correlated to poorer clinical outcome and lower overall survival in DLBCL patients. Subsequently, we determined the experimental role of lncRNA PANDA in DLBCL progression. Luciferase reporter assay and chromatin immunoprecipitation assay suggested that lncRNA PANDA was induced by p53 and p53 interacts with the promoter region of PANDA. Cell functional assay further indicated that PANDA functioned as a tumor suppressor gene through the suppression of cell growth by a G0/G1 cell cycle arrest in DLBCL. More importantly, Cignal Signal Transduction Reporter Array and western blot assay showed that lncRNA PANDA inactivated the MAPK/ERK signaling pathway. In conclusion, our integrated approach demonstrates that PANDA in DLBCL confers a tumor suppressive function through inhibiting cell proliferation and silencing MAPK/ERK signaling pathway. Thus, PANDA may be a promising therapeutic target for patients with DLBCL.

Quantum dot-based molecular imaging of cancer cell growth using a clone formation assay.

Science.gov (United States)

Geng, Xia-Fei; Fang, Min; Liu, Shao-Ping; Li, Yan

2016-10-01

This aim of the present study was to investigate clonal growth behavior and analyze the proliferation characteristics of cancer cells. The MCF‑7 human breast cancer cell line, SW480 human colon cancer cell line and SGC7901 human gastric cancer cell line were selected to investigate the morphology of cell clones. Quantum dot‑based molecular targeted imaging techniques (which stained pan‑cytokeratin in the cytoplasm green and Ki67 in the cell nucleus yellow or red) were used to investigate the clone formation rate, cell morphology, discrete tendency, and Ki67 expression and distribution in clones. From the cell clone formation assay, the MCF‑7, SW480 and SGC7901 cells were observed to form clones on days 6, 8 and 12 of cell culture, respectively. These three types of cells had heterogeneous morphology, large nuclear:cytoplasmic ratios, and conspicuous pathological mitotic features. The cells at the clone periphery formed multiple pseudopodium. In certain clones, cancer cells at the borderline were separated from the central cell clusters or presented a discrete tendency. With quantum dot‑based molecular targeted imaging techniques, cells with strong Ki67 expression were predominantly shown to be distributed at the clone periphery, or concentrated on one side of the clones. In conclusion, cancer cell clones showed asymmetric growth behavior, and Ki67 was widely expressed in clones of these three cell lines, with strong expression around the clones, or aggregated at one side. Cell clone formation assay based on quantum dots molecular imaging offered a novel method to study the proliferative features of cancer cells, thus providing a further insight into tumor biology.

An automated high throughput screening-compatible assay to identify regulators of stem cell neural differentiation.

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Casalino, Laura; Magnani, Dario; De Falco, Sandro; Filosa, Stefania; Minchiotti, Gabriella; Patriarca, Eduardo J; De Cesare, Dario

2012-03-01

The use of Embryonic Stem Cells (ESCs) holds considerable promise both for drug discovery programs and the treatment of degenerative disorders in regenerative medicine approaches. Nevertheless, the successful use of ESCs is still limited by the lack of efficient control of ESC self-renewal and differentiation capabilities. In this context, the possibility to modulate ESC biological properties and to obtain homogenous populations of correctly specified cells will help developing physiologically relevant screens, designed for the identification of stem cell modulators. Here, we developed a high throughput screening-suitable ESC neural differentiation assay by exploiting the Cell(maker) robotic platform and demonstrated that neural progenies can be generated from ESCs in complete automation, with high standards of accuracy and reliability. Moreover, we performed a pilot screening providing proof of concept that this assay allows the identification of regulators of ESC neural differentiation in full automation.

Limitations of a hemolytic plaque assay for IgG-anti-IgG rheumatoid factor-producing cells.

Science.gov (United States)

Venn, A J; Dresser, D W

1987-09-24

An attempt has been made to develop a hemolytic plaque assay capable of detecting homophile IgG rheumatoid factor (RF)-producing cells. Anti-immunoglobulin allotype-developing reagents were used to distinguish between target and effector IgG. The hemolytic assay has been used to demonstrate an apparently high level of homophile IgM and IgG RF-producing cells in the spleens and lymph nodes of mice stimulated by LPS. However, it appears that a large proportion of the plaques obtained in these assays are due to an artefact resulting from cross-linking of target and effector molecules by the developing reagents. In the case of IgM RF the artefact depends on the presence of a small contamination of the target IgG by IgM, allowing cross-linking of target and effector IgM by the anti-mu-specific developing reagent. With the IgG RF, cross-reactivity of the rabbit anti-Ighb allotype-developing serum for the 'wrong' (Igha) allotype, normally undetectable, becomes sufficient to be biologically relevant when the developing antibody is complexed by being bound to its target (Ighb) allotype. Nevertheless anti-allotype reagents may afford an accurate means of detecting homophile IgG RF producing cells using other assay systems.

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)

Galvao dos Santos, G.; Reinders, J.; Ouwehand, K.; Rustemeyer, T.; Scheper, R.J.; Gibbs, S.

2009-01-01

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.

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells.

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Casanova, Alain; Low, Shyan H; Emmenlauer, Mario; Conde-Alvarez, Raquel; Salcedo, Suzana P; Gorvel, Jean-Pierre; Dehio, Christoph

2016-08-05

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by direct contact with infected animals or by ingestion of contaminated food and can lead to severe chronic infections. Brucella can invade professional and non-professional phagocytic cells and replicates within endoplasmic reticulum (ER)-derived vacuoles. The host factors required for Brucella entry into host cells, avoidance of lysosomal degradation, and replication in the ER-like compartment remain largely unknown. Here we describe two assays to identify host factors involved in Brucella entry and replication in HeLa cells. The protocols describe the use of RNA interference, while alternative screening methods could be applied. The assays are based on the detection of fluorescently labeled bacteria in fluorescently labeled host cells using automated wide-field microscopy. The fluorescent images are analyzed using a standardized image analysis pipeline in CellProfiler which allows single cell-based infection scoring. In the endpoint assay, intracellular replication is measured two days after infection. This allows bacteria to traffic to their replicative niche where proliferation is initiated around 12 hr after bacterial entry. Brucella which have successfully established an intracellular niche will thus have strongly proliferated inside host cells. Since intracellular bacteria will greatly outnumber individual extracellular or intracellular non-replicative bacteria, a strain constitutively expressing GFP can be used. The strong GFP signal is then used to identify infected cells. In contrast, for the entry assay it is essential to differentiate between intracellular and extracellular bacteria. Here, a strain encoding for a tetracycline-inducible GFP is used. Induction of GFP with simultaneous inactivation of extracellular bacteria by gentamicin enables the differentiation between intracellular and extracellular

Radioreceptor assays: plasma membrane receptors and assays for polypeptide and glycoprotein hormones

International Nuclear Information System (INIS)

Schulster, D.

1977-01-01

Receptors for peptide, protein and glycoprotein hormones, and the catecholamines are located on the plasma membranes of their target cells. Preparations of the receptors may be used as specific, high-affinity binding agents for these hormones in assay methodology akin to that for radioimmunoassay. A particular advantage of the radioreceptor assay is that it has a specificity directed towards the biologically active region of the hormone, rather than to some immunologically active region that may have little (or no) involvement in the expression of hormonal activity. Methods for hormone receptor preparation vary greatly, and range from the use of intact cells (as the source of hormone receptor) to the use of purified or solubilized membrane receptors. Receptors isolated from plasma membranes have proved to be of variable stability, and may be damaged during preparation and/or storage. Moreover, since they are present in relatively low concentration in the cell, their preparation in sufficient quantity for use in a radioreceptor assay may present technical problems. In general, there is good correlation between radioreceptor assays and in-vitro bioassays; differences between results from radioreceptor assays and radioimmunoassays are similar to those noted between in-vitro bioassays and radioimmunoassays. The sensitivity of the method is such that normal plasma concentrations of various hormones have been assayed by this technique. (author)

Relationship between the radioisotopic footpad assay and other immunological assays in tumor bearing rats

International Nuclear Information System (INIS)

Mizushima, Yutaka; Takeichi, Noritoshi; Minami, Akio; Kasai, Masaharu; Itaya, Toshiyuki

1981-01-01

KMT-17, a fibrosarcoma induced by 3-methylcholanthrene in a WKA rat, is a sensitive tumor to various kinds of immunological assays and is a suitable model tumor for the study of the immune status in tumor bearing hosts. The antitumor immune response of KMT-17 bearing rats was studied by a radioisotopic footpad assay (FPA) in comparison with other in vivo and in vitro assays. Delayed hypersensitivity to tumor antigens measured by the FPA was observed from the 8th day after transplantation of KMT-17 cells, reached a peak on the 12 - 15th day, and then declined in the late stage on the 17th day. The kinetics of the FPA correlated well with those of an in vivo Winn assay and of an in vitro lymphocyte cytotoxicity assay ( 51 Cr-release assay). The appearance of an antitumor antibody detected by a complement dependent cytotoxicity test also correlated well with the kinetics of the FPA. A growth inhibition assay (GIA) for non-specific cell-mediated immunity also showed similar kinetics to that of the FPA. The delayed hypersensitivity footpad reaction to tumor cell extracts measured by this FPA was tumor-specific. These results suggest that the FPA is a simple and reliable in vivo assay for evaluating antitumor immunity in tumor bearing hosts. (author)

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.

Improvement of Intestinal Immune Cell Function by Lactic Acid Bacteria for Dairy Products

Directory of Open Access Journals (Sweden)

Tomonori Kamiya

2016-12-01

Full Text Available Lactic acid bacteria (LAB form a major component of gut microbiota and are often used as probiotics for fermented foods, such as yoghurt. In this study, we aimed to evaluate immunomodulatory activity of LAB, especially that of Lactobacillus bulgaricus ME-552 (ME552 and Streptococcus thermophilus ME-553 (ME553. In vivo/in vitro assay was performed in order to investigate their effects on T cell function. After oral administration of ME553 to C57BL/6 mice, the amount of both interferon γ (IFN-γ and interleukin 17 (IL-17 produced by cluster of differentiation (CD 4+ T cells from Peyer’s patches (PPs were significantly enhanced. On the other hand, ME552 only up-regulated the production of IL-17 from PP cells. The extent of induction for IFN-γ production differed between ME552 and ME553. These results suggest that LAB modulate T cell effector functions and mucosal immunity.

Improvement of Intestinal Immune Cell Function by Lactic Acid Bacteria for Dairy Products.

Science.gov (United States)

Kamiya, Tomonori; Watanabe, Yohei; Makino, Seiya; Kano, Hiroshi; Tsuji, Noriko M

2016-12-23

Lactic acid bacteria (LAB) form a major component of gut microbiota and are often used as probiotics for fermented foods, such as yoghurt. In this study, we aimed to evaluate immunomodulatory activity of LAB, especially that of Lactobacillus bulgaricus ME-552 (ME552) and Streptococcus thermophilus ME-553 (ME553). In vivo/in vitro assay was performed in order to investigate their effects on T cell function. After oral administration of ME553 to C57BL/6 mice, the amount of both interferon γ (IFN-γ) and interleukin 17 (IL-17) produced by cluster of differentiation (CD) 4⁺ T cells from Peyer's patches (PPs) were significantly enhanced. On the other hand, ME552 only up-regulated the production of IL-17 from PP cells. The extent of induction for IFN-γ production differed between ME552 and ME553. These results suggest that LAB modulate T cell effector functions and mucosal immunity.

A quantitative comet infection assay for influenza virus

Science.gov (United States)

Lindsay, Stephen M.; Timm, Andrea; Yin, John

2011-01-01

Summary The virus comet assay is a cell-based virulence assay used to evaluate an antiviral drug or antibody against a target virus. The comet assay differs from the plaque assay in allowing spontaneous flows in 6-well plates to spread virus. When implemented quantitatively the comet assay has been shown to have an order-of-magnitude greater sensitivity to antivirals than the plaque assay. In this study, a quantitative comet assay for influenza virus is demonstrated, and is shown to have a 13-fold increase in sensitivity to ribavirin. AX4 cells (MDCK cells with increased surface concentration of α2–6 sialic acid, the influenza virus receptor) have reduced the comet size variability relative to MDCK cells, making them a better host cell for use in this assay. Because of enhanced antiviral sensitivity in flow-based assays, less drug is required, which could lead to lower reagent costs, reduced cytotoxicity, and fewer false-negative drug screen results. The comet assay also serves as a readout of flow conditions in the well. Observations from comets formed at varying humidity levels indicate a role for evaporation in the mechanism of spontaneous fluid flow in wells. PMID:22155578

Functional activity of human sodium/iodide symporter in tumor cell lines

International Nuclear Information System (INIS)

Petrich, T.; Knapp, W.H.; Poetter, E.

2003-01-01

Aim: The sodium/iodide symporter (NIS) actively transports iodide into thyrocytes. Thus, NIS represents a key protein for diagnosis and radioiodine therapy of differentiated thyroid cancer. Additionally, in the future the NIS gene may be used for cancer gene therapy of non-thyroid-derived malignancies. In this study we evaluated the functionality of NIS with respect to iodide uptake in a panel of tumor cell lines and compared this to gene transfer efficiency. Methods: A human NIS-containing expression vector and reporter-gene vectors encoding and beta;-Galactosidase- or EGFP were used for transient transfection of 13 tumor cell lines. Following transfection measurements of NIS-mediated radioiodide uptake using Na 125 I and of transfection efficiency were performed. The latter included β;-Galactosidase activity measurements using a commercial kit and observation by fluorescence microscopy for EGFP expression. Results: In contrast to respective parental cells, most NIS-transfected cell lines displayed high, perchlorate-sensitive radioiodide uptake. Differences in radioiodide uptake between cell lines apparently corresponded to transfection efficiencies, as judged from reporter-gene assays. Conclusion: With respect to iodide uptake we provide evidence that NIS is functional in different cellular context. As iodide uptake capacity appears to be well correlated to gene transfer efficiency, cell type-specific actions on NIS (e. g. post-translational modification such as glycosylation) are not inhibitory to NIS function. Our data support the promising role of NIS in cancer gene therapy strategies. (orig.)

Immunobiology of T cell responses to Mls-locus-disparate stimulator cells. III. Helper and cytolytic functions of cloned, Mls-reactive T cell lines

International Nuclear Information System (INIS)

Katz, M.E.; Tite, J.P.; Janeway, C.A. Jr.

1986-01-01

Mls-specific T cell clones derived by limiting dilution were tested for cytotoxic activity in a lectin-dependent 51 Cr-release assay. All the T cell clones tested were cytotoxic in such an assay in apparent contrast to previous reports (1, 2). However, only those target cells sensitive to cytolysis by other L3T4a + cytolytic T cells (3) were killed by Mls-specific T cell clones in short term 51 Cr-release assays, possibly explaining this discrepancy. All the T cell clones tested were L3T4a + ,Lyt-2 - and stimulated B cells from Mls strains of mice to proliferate and secrete immunoglobulin. Furthermore, lysis of innocent bystander targets was observed when the T cells were stimulated with Mls-disparate stimulator cells. These results are consistent with those obtained with L3T4a - T cells specific for protein antigen:self Ia and that express cytotoxic potential (3)

In vivo Comet assay – statistical analysis and power calculations of mice testicular cells

DEFF Research Database (Denmark)

Hansen, Merete Kjær; Sharma, Anoop Kumar; Dybdahl, Marianne

2014-01-01

is to provide curves for this statistic outlining the number of animals and gels to use. The current study was based on 11 compounds administered via oral gavage in three doses to male mice: CAS no. 110-26-9, CAS no. 512-56-1, CAS no. 111873-33-7, CAS no. 79-94-7, CAS no. 115-96-8, CAS no. 598-55-0, CAS no. 636....... A linear mixed-effects model was fitted to the summarized data and the estimated variance components were used to generate power curves as a function of sample size. The statistic that most appropriately summarized the within-sample distributions was the median of the log-transformed data, as it most...... consistently conformed to the assumptions of the statistical model. Power curves for 1.5-, 2-, and 2.5-fold changes of the highest dose group compared to the control group when 50 and 100 cells were scored per gel are provided to aid in the design of future Comet assay studies on testicular cells....

Evaluation of radio-induced DNA damage and their repair in human lymphocytes by comet assay or single cell gel electrophoresis

International Nuclear Information System (INIS)

Nascimento, Patricia A. do; Suzuki, Miriam F.; Okazaki, Kayo

1997-01-01

The comet assay, also called single cell gel electrophoresis technique, permits to evaluate quantitatively DNA breakage induced by chemical and physical agents at the level of the single cell. The present paper refers to the construction of dose-response curves to DNA damage and repair studies in human peripheral lymphocytes, utilizing the comet assay for the radiosensitivity analysis. So, the blood samples were obtained from healthy donors (40-50 year old), irradiated in a 60 Co source (GAMMACEL 220) with doses of 0.17, 0.25, 0.57, 1.10, 2.12 and 4.22 Gy (0.59 Gy/min.) and processed 1 and 24 hours after the exposition. Results obtained showed a increase in the total lenght of comet (DNA migration) as a function of radiation dose in samples processed 1 and 24 hours after the treatment. The DNA lesion in irradiated lymphocytes with 4.22 Gy (means value of 101.4 μm) were 3.4 times higher than in the untreated lymphocytes (mean value of 30 μm) instead of 24 hours after the irradiation were 1.5 times higher (mean value of 46.3 μm). This reduction on DNA repair occurred in these cells. It was also possible visualized the presence of subpopulations of the cells with different sensitivity and repair capacity to ionizing radiation in these donors. (author). 8 refs., 3 figs

Polymerase chain reaction assay for the detection of Bacillus cereus group cells

DEFF Research Database (Denmark)

Hansen, Bjarne Munk; Leser, Thomas D.; Hendriksen, Niels Bohse

2001-01-01

of the B. cereus group in food and in the environment. Using 16S rDNA as target, a PCR assay for the detection of B. cereus group cells has been developed. Primers specific for the 16S rDNA of the B. cereus group bacteria were selected and used in combination with consensus primers for 165 rDNA as internal...... PCR procedure control. The PCR procedure was optimized with respect to annealing temperature. When DNA from the B. cereus group bacteria was present, the PCR assay yielded a B. cereus specific fragment, while when non-B. cereus prokaryotic DNA was present, the consensus 165 rDNA primers directed...

An Evolutionary-Conserved Function of Mammalian Notch Family Members as Cell Adhesion Molecules

Science.gov (United States)

Murata, Akihiko; Yoshino, Miya; Hikosaka, Mari; Okuyama, Kazuki; Zhou, Lan; Sakano, Seiji; Yagita, Hideo; Hayashi, Shin-Ichi

2014-01-01

Notch family members were first identified as cell adhesion molecules by cell aggregation assays in Drosophila studies. However, they are generally recognized as signaling molecules, and it was unclear if their adhesion function was restricted to Drosophila. We previously demonstrated that a mouse Notch ligand, Delta-like 1 (Dll1) functioned as a cell adhesion molecule. We here investigated whether this adhesion function was conserved in the diversified mammalian Notch ligands consisted of two families, Delta-like (Dll1, Dll3 and Dll4) and Jagged (Jag1 and Jag2). The forced expression of mouse Dll1, Dll4, Jag1, and Jag2, but not Dll3, on stromal cells induced the rapid and enhanced adhesion of cultured mast cells (MCs). This was attributed to the binding of Notch1 and Notch2 on MCs to each Notch ligand on the stromal cells themselves, and not the activation of Notch signaling. Notch receptor-ligand binding strongly supported the tethering of MCs to stromal cells, the first step of cell adhesion. However, the Jag2-mediated adhesion of MCs was weaker and unlike other ligands appeared to require additional factor(s) in addition to the receptor-ligand binding. Taken together, these results demonstrated that the function of cell adhesion was conserved in mammalian as well as Drosophila Notch family members. Since Notch receptor-ligand interaction plays important roles in a broad spectrum of biological processes ranging from embryogenesis to disorders, our finding will provide a new perspective on these issues from the aspect of cell adhesion. PMID:25255288

Nitric oxide and TGF-β1 inhibit HNF-4α function in HEPG2 cells

International Nuclear Information System (INIS)

Lucas, Susana de; Lopez-Alcorocho, Juan Manuel; Bartolome, Javier; Carreno, Vicente

2004-01-01

This study analyzes if the profibrogenic factors nitric oxide and transforming growth factor-β1 (TGF-β1) affect hepatocyte nuclear factor-4α (HNF-4α) function. For this purpose, HepG2 cells were treated with TGF-β1 or with a nitric oxide donor to determine mRNA levels of coagulation factor VII and HNF-4α. Treatment effect on factor VII gene promoter was assessed by chloramphenicol acetyl-transferase assays in cells transfected with the pFVII-CAT plasmid. HNF-4α binding and protein levels were determined by gel shift assays and Western blot. TGF-β1 and nitric oxide downregulated factor VII mRNA levels by inhibiting its gene promoter activity. This inhibition is caused by a decrease in the DNA binding of HNF-4α. TGF-β1 induces degradation of HNF-4α in the proteasome while nitric oxide provokes nitrosylation of cysteine residues in this factor. TGF-β1 and nitric oxide inhibit HNF-4α activity. These findings may explain the loss of liver functions that occurs during fibrosis progression

Lack of inhibitory effects of the anti-fibrotic drug imatinib on endothelial cell functions in vitro and in vivo.

Science.gov (United States)

Venalis, Paulius; Maurer, Britta; Akhmetshina, Alfiya; Busch, Nicole; Dees, Clara; Stürzl, Michael; Zwerina, Jochen; Jüngel, Astrid; Gay, Steffen; Schett, Georg; Distler, Oliver; Distler, Jörg H W

2009-10-01

Systemic sclerosis (SSc) is a systemic autoimmune disease that is characterized by microangiopathy with progressive loss of capillaries and tissue fibrosis. Imatinib exerts potent anti-fibrotic effects and is currently evaluated in clinical trials. The aim of the present study was to exclude that the anti-fibrotic effects of imatinib are complicated by inhibitory effects on endothelial cell functions, which might augment vascular disease in SSc. Endothelial cells and mice were treated with pharmacologically relevant concentrations of imatinib. The expression of markers of vascular activation was assessed with real-time PCR. Proliferation was analysed with the cell counting experiments and the MTT assay. Apoptosis was quantified with caspase 3 assays, annexin V in vitro and with TUNEL staining in vivo. Migration was studied with scratch and transwell assays. Tube forming was investigated with the matrigel assay. Imatinib did not alter the expression of markers of vascular activation. Imatinib did not increase the percentage of annexin V positive cells or the activity of caspase 3. No reduction in proliferation or metabolic activity of endothelial cells was observed. Imatinib did not affect migration of endothelial cells and did not reduce the formation of capillary tubes. Consistent with the in vitro data, no difference in the number of apoptotic endothelial cells was observed in vivo in mice treated with imatinib. Imatinib does not inhibit activation, viability, proliferation, migration or tube forming of endothelial cells in vitro and in vivo. Thus, treatment with imatinib might not augment further endothelial cell damage in SSc.

Single-Cell Based Quantitative Assay of Chromosome Transmission Fidelity.

Science.gov (United States)

Zhu, Jin; Heinecke, Dominic; Mulla, Wahid A; Bradford, William D; Rubinstein, Boris; Box, Andrew; Haug, Jeffrey S; Li, Rong

2015-03-30

Errors in mitosis are a primary cause of chromosome instability (CIN), generating aneuploid progeny cells. Whereas a variety of factors can influence CIN, under most conditions mitotic errors are rare events that have been difficult to measure accurately. Here we report a green fluorescent protein-based quantitative chromosome transmission fidelity (qCTF) assay in budding yeast that allows sensitive and quantitative detection of CIN and can be easily adapted to high-throughput analysis. Using the qCTF assay, we performed genome-wide quantitative profiling of genes that affect CIN in a dosage-dependent manner and identified genes that elevate CIN when either increased (icCIN) or decreased in copy number (dcCIN). Unexpectedly, qCTF screening also revealed genes whose change in copy number quantitatively suppress CIN, suggesting that the basal error rate of the wild-type genome is not minimized, but rather, may have evolved toward an optimal level that balances both stability and low-level karyotype variation for evolutionary adaptation. Copyright © 2015 Zhu et al.

Ligand-receptor assay for evaluation of functional activity of human recombinant VEGF and VEGFR-1 extracellular fragment.

Science.gov (United States)

Leopol'd, A V; Baklaushev, V P; Korchagina, A A; Shein, S A; Grinenko, N F; Pavlov, K A; Ryabukhin, I A; Chekhonin, V P

2012-04-01

cDNA encoding VEGF and Ig-like extracellular domains 2-4 of VEGFR-1 (sFlt-1(2-4)) were cloned into prokaryotic expression vectors pET32a and pQE60. Recombinant proteins were purified (metal affinity chromatography) and renatured. Chemiluminescent study for the interaction of recombinant VEGF and sFlt-1(2-4) showed that biotinylated VEGF specifically binds to the polystyrene-immobilized receptor extracellular fragment. Biotinylated recombinant sFlt-1 interacts with immobilized VEGF. Analysis of the interaction of immobilized recombinant VEGFR-1 and VEGF with C6 glioma cells labeled with CFDA-SE (vital fluorescent dye) showed that recombinant VEGFR-1 also binds to native membrane-associated VEGF. Recombinant VEGF was shown to bind to specific receptors expressed on the surface of C6 glioma cells. Functional activity of these proteins was confirmed by ligand-receptor assay for VEGF and VEGFR-1 (sFlt-1) and quantitative chemiluminescent detection.

Inhibition of neuronal cell–cell adhesion measured by the microscopic aggregation assay and impedance sensing

NARCIS (Netherlands)

Wiertz, Remy; Marani, Enrico; Rutten, Wim

2010-01-01

Microscopic aggregation assay and impedance sensing (IS) were used to monitor a change in in vitro neuron–neuron adhesion in response to blocking of cell adhesion molecules. By blocking neuron–neuron adhesion, migration and aggregation of neuronal cells can be inhibited. This leads to better control

Schwann Cell Glycogen Selectively Supports Myelinated Axon Function

Science.gov (United States)

Brown, Angus M; Evans, Richard D; Black, Joel; Ransom, Bruce R

2012-01-01

Objectives Interruption of energy supply to peripheral axons is a cause of axon loss. We determined if glycogen was present in mammalian peripheral nerve, and if it supported axon conduction during aglycemia. Methods We used biochemical assay and electron microscopy to determine the presence of glycogen, and electrophysiology to monitor axon function. Results Glycogen was present in sciatic nerve, its concentration varying directly with ambient [glucose]. Electron microscopy detected glycogen granules primarily in myelinating Schwann cell cytoplasm and these diminished after exposure to aglycemia. During aglycemia, conduction failure in large myelinated axons (A fibers) mirrored the time-course of glycogen loss. Latency to CAP failure was directly related to nerve glycogen content at aglycemia onset. Glycogen did not benefit the function of slow-conducting, small diameter unmyelinated axons (C fibers) during aglycemia. Blocking glycogen breakdown pharmacologically accelerated CAP failure during aglycemia in A fibers, but not in C fibers. Lactate was as effective as glucose in supporting sciatic nerve function, and was continuously released into the extracellular space in the presence of glucose and fell rapidly during aglycemia. Interpretation Our findings indicated that glycogen is present in peripheral nerve, primarily in myelinating Schwann cells, and exclusively supports large diameter, myelinated axon conduction during aglycemia. Available evidence suggests that peripheral nerve glycogen breaks down during aglycemia and is passed, probably as lactate, to myelinated axons to support function. Unmyelinated axons are not protected by glycogen and are more vulnerable to dysfunction during periods of hypoglycemia. PMID:23034913

Quantification of circulating mature endothelial cells using a whole blood four-color flow cytometric assay.

Science.gov (United States)

Jacques, Nathalie; Vimond, Nadege; Conforti, Rosa; Griscelli, Franck; Lecluse, Yann; Laplanche, Agnes; Malka, David; Vielh, Philippe; Farace, Françoise

2008-09-15

Circulating endothelial cells (CEC) are currently proposed as a potential biomarker for measuring the impact of anti-angiogenic treatments in cancer. However, the lack of consensus on the appropriate method of CEC measurement has led to conflicting data in cancer patients. A validated assay adapted for evaluating the clinical utility of CEC in large cohorts of patients undergoing anti-angiogenic treatments is needed. We developed a four-color flow cytometric assay to measure CEC as CD31(+), CD146(+), CD45(-), 7-amino-actinomycin-D (7AAD)(-) events in whole blood. The distinctive features of the assay are: (1) staining of 1 ml whole blood, (2) use of a whole blood IgPE control to measure accurately background noise, (3) accumulation of a large number of events (almost 5 10(6)) to ensure statistical analysis, and (4) use of 10 microm fluorescent microbeads to evaluate the event size. Assay reproducibility was determined in duplicate aliquots of samples drawn from 20 metastatic cancer patients. Assay linearity was tested by spiking whole blood with low numbers of HUVEC. Five-color flow cytometric experiments with CD144 were performed to confirm the endothelial origin of the cells. CEC were measured in 20 healthy individuals and 125 patients with metastatic cancer. Reproducibility was good between duplicate aliquots (r(2)=0.948, mean difference between duplicates of 0.86 CEC/ml). Detected HUVEC correlated with spiked HUVEC (r(2)=0.916, mean recovery of 100.3%). Co-staining of CD31, CD146 and CD144 confirmed the endothelial nature of cells identified as CEC. Median CEC levels were 6.5/ml (range, 0-15) in healthy individuals and 15.0/ml (range, 0-179) in patients with metastatic carcinoma (p<0.001). The assay proposed here allows reproducible and sensitive measurement of CEC by flow cytometry and could help evaluate CEC as biomarkers of anti-angiogenic therapies in large cohorts of patients.

Human Cell Assays for Synthesis-Dependent Strand Annealing and Crossing over During Double-Strand Break Repair.

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Zapotoczny, Grzegorz; Sekelsky, Jeff

2017-04-03

DNA double-strand breaks (DSBs) are one of the most deleterious types of lesions to the genome. Synthesis-dependent strand annealing (SDSA) is thought to be a major pathway of DSB repair, but direct tests of this model have only been conducted in budding yeast and Drosophila To better understand this pathway, we developed an SDSA assay for use in human cells. Our results support the hypothesis that SDSA is an important DSB repair mechanism in human cells. We used siRNA knockdown to assess the roles of a number of helicases suggested to promote SDSA. None of the helicase knockdowns reduced SDSA, but knocking down BLM or RTEL1 increased SDSA. Molecular analysis of repair products suggests that these helicases may prevent long-tract repair synthesis. Since the major alternative to SDSA (repair involving a double-Holliday junction intermediate) can lead to crossovers, we also developed a fluorescent assay that detects crossovers generated during DSB repair. Together, these assays will be useful in investigating features and mechanisms of SDSA and crossover pathways in human cells. Copyright © 2017 Zapotoczny and Sekelsky.

Human Cell Assays for Synthesis-Dependent Strand Annealing and Crossing over During Double-Strand Break Repair

Directory of Open Access Journals (Sweden)

Grzegorz Zapotoczny

2017-04-01

Full Text Available DNA double-strand breaks (DSBs are one of the most deleterious types of lesions to the genome. Synthesis-dependent strand annealing (SDSA is thought to be a major pathway of DSB repair, but direct tests of this model have only been conducted in budding yeast and Drosophila. To better understand this pathway, we developed an SDSA assay for use in human cells. Our results support the hypothesis that SDSA is an important DSB repair mechanism in human cells. We used siRNA knockdown to assess the roles of a number of helicases suggested to promote SDSA. None of the helicase knockdowns reduced SDSA, but knocking down BLM or RTEL1 increased SDSA. Molecular analysis of repair products suggests that these helicases may prevent long-tract repair synthesis. Since the major alternative to SDSA (repair involving a double-Holliday junction intermediate can lead to crossovers, we also developed a fluorescent assay that detects crossovers generated during DSB repair. Together, these assays will be useful in investigating features and mechanisms of SDSA and crossover pathways in human cells.

In vitro generation of functional insulin-producing cells from lipoaspirated human adipose tissue-derived stem cells.

Science.gov (United States)

Mohamad Buang, Mohamad Lizan; Seng, Heng Kien; Chung, Lee Han; Saim, Aminuddin Bin; Idrus, Ruszymah Bt Hj

2012-01-01

Tissue engineering strategy has been considered as an alternative treatment for diabetes mellitus due to lack of permanent pharmaceutical treatment and islet donors for transplantation. Various cell lines have been used to generate functional insulin-producing cells (IPCs) including progenitor pancreatic cell lines, embryonic stem cells (ESCs), umbilical cord blood stem cells (UCB-SCs), adult bone marrow stem cells (BMSCs), and adipose tissue-derived stem cells (ADSCs). Human ADSCs from lipoaspirated abdominal fat tissue was differentiated into IPCs following a two-step induction protocol based on a combination of alternating high and low glucose, nicotinamide, activin A and glucagon-like peptide 1 (GLP-1) for a duration of 3 weeks. During differentiation, histomorphological changes of the stem cells towards pancreatic β-islet characteristics were observed via light microscope and transmission electron microscope (TEM). Dithizone (DTZ) staining, which is selective towards IPCs, was used to stain the new islet-like cells. Production of insulin hormone by the cells was analyzed via enzyme-linked immunosorbent assay (ELISA), whereas its hormonal regulation was tested via a glucose challenge test. Histomorphological changes of the differentiated cells were noted to resemble pancreatic β-cells, whereas DTZ staining positively stained the cells. The differentiated cells significantly produced human insulin as compared to the undifferentiated ADSCs, and its production was increased with an increase of glucose concentration in the culture medium. These initial data indicate that human lipoaspirated ADSCs have the potential to differentiate into functional IPCs, and could be used as a therapy to treat diabetes mellitus in the future. Copyright © 2012 IMSS. Published by Elsevier Inc. All rights reserved.

A buccal cell model comet assay: Development and evaluation for human biomonitoring and nutritional studies

International Nuclear Information System (INIS)

Szeto, Y.T.; Benzie, I.F.F.; Collins, A.R.; Choi, S.W.; Cheng, C.Y.; Yow, C.M.N.; Tse, M.M.Y.

2005-01-01

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 H 2 O 2 ) increased DNA strand breaks in a dose related manner, and incubation of cells in Trolox

A buccal cell model comet assay: Development and evaluation for human biomonitoring and nutritional studies

Energy Technology Data Exchange (ETDEWEB)

Szeto, Y.T. [Department of Health Technology and Informatics, Hong Kong Polytechnic University, Kowloon, Hong Kong (China); School of Health Sciences, Macao Polytechnic Institute, Macao (China); Benzie, I.F.F. [Department of Health Technology and Informatics, Hong Kong Polytechnic University, Kowloon, Hong Kong (China)]. E-mail: iris.benzie@inet.polyu.edu.hk; Collins, A.R. [Department of Nutrition, University of Oslo, Oslo (Norway); Choi, S.W. [Department of Health Technology and Informatics, Hong Kong Polytechnic University, Kowloon, Hong Kong (China); Cheng, C.Y. [Department of Health Technology and Informatics, Hong Kong Polytechnic University, Kowloon, Hong Kong (China); Yow, C.M.N. [Department of Health Technology and Informatics, Hong Kong Polytechnic University, Kowloon, Hong Kong (China); Tse, M.M.Y. [School of Nursing, Hong Kong Polytechnic University, Kowloon, Hong Kong (China)

2005-10-15

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 H{sub 2}O{sub 2}) increased DNA strand breaks in a dose related manner, and incubation of cells in

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

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 mimic sensitization mechanisms in vivo. These assays should be an alternative approach to the LLNA. The NCTC...... 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...

Development of a functional cell-based assay that probes the specific interaction between influenza A virus NP and its packaging signal sequence RNA.

Science.gov (United States)

Woo, Jiwon; Yu, Kyung Lee; Lee, Sun Hee; You, Ji Chang

2015-02-06

Although cis-acting packaging signal RNA sequences for the influenza virus NP encoding vRNA have been identified recently though genetic studies, little is known about the interaction between NP and the vRNA packaging signals either in vivo or in vitro. Here, we provide evidence that NP is able to interact specifically with the vRNA packaging sequence RNA within living cells and that the specific RNA binding activity of NP in vivo requires both the N-terminal and central region of the protein. This assay established would be a valuable tool for further detailed studies of the NP-packaging signal RNA interaction in living cells. Copyright © 2014 Elsevier Inc. All rights reserved.

Fluorescence-based high-throughput functional profiling of ligand-gated ion channels at the level of single cells.

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Sahil Talwar

Full Text Available Ion channels are involved in many physiological processes and are attractive targets for therapeutic intervention. Their functional properties vary according to their subunit composition, which in turn varies in a developmental and tissue-specific manner and as a consequence of pathophysiological events. Understanding this diversity requires functional analysis of ion channel properties in large numbers of individual cells. Functional characterisation of ligand-gated channels involves quantitating agonist and drug dose-response relationships using electrophysiological or fluorescence-based techniques. Electrophysiology is limited by low throughput and high-throughput fluorescence-based functional evaluation generally does not enable the characterization of the functional properties of each individual cell. Here we describe a fluorescence-based assay that characterizes functional channel properties at single cell resolution in high throughput mode. It is based on progressive receptor activation and iterative fluorescence imaging and delivers >100 dose-responses in a single well of a 384-well plate, using α1-3 homomeric and αβ heteromeric glycine receptor (GlyR chloride channels as a model system. We applied this assay with transiently transfected HEK293 cells co-expressing halide-sensitive yellow fluorescent protein and different GlyR subunit combinations. Glycine EC50 values of different GlyR isoforms were highly correlated with published electrophysiological data and confirm previously reported pharmacological profiles for the GlyR inhibitors, picrotoxin, strychnine and lindane. We show that inter and intra well variability is low and that clustering of functional phenotypes permits identification of drugs with subunit-specific pharmacological profiles. As this method dramatically improves the efficiency with which ion channel populations can be characterized in the context of cellular heterogeneity, it should facilitate systems

Metal-Carbon Interactions on Reduced Graphene Oxide under Facile Thermal Treatment: Microbiological and Cell Assay

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N. L. V. Carreño

2017-01-01

Full Text Available Silver-functionalized reduced graphene oxide (Ag-rGO nanosheets were prepared by single chemical and thermal processes, with very low concentration of silver. The resulting carbon framework consists of reduced graphene oxide (rGO sheets or 3D networks, decorated with anchored silver nanoparticles. The Ag-rGO nanosheets were dispersed into a polymer matrix and the composites evaluated for use as biological scaffolds. The rGO material in poly(dimethylsiloxane (PDMS has been tested for antimicrobial activity against Gram-positive Staphylococcus aureus (S. Aureus bacteria, after exposure times of 24 and 120 hours, as well as in the determination of cell viability on cultures of fibroblast cells (NIH/3T3. Using 1 mL of Ag-rGO in PDMS the antibacterial effectiveness against Staphylococcus aureus was limited, showing an increased amount of Colony Forming Units (CFU, after 24 hours of contact. In the cell viability assay, after 48 hours of contact, the group of 1 mL of Ag-rGO with PDMS was the only group that increased cell viability when compared to the control group. In this context, it is believed these behaviors are due to the increase in cell adhesion capacity promoted by the rGO. Thus, the Ag-rGO/PDMS hybrid nanocomposite films can be used as scaffolds for tissue engineering, as they limit antimicrobial activity.

Cyquant cell proliferation assay as a fluorescence-based method for in vitro screening of antimalarial activity.

Science.gov (United States)

Sriwilaijaroen, Nongluk; Kelly, Jane Xu; Riscoe, Michael; Wilairat, Prapon

2004-12-01

The appearance of drug resistant parasites and the absence of an effective vaccine have resulted in the need for new effective antimalarial drugs. Consequently, a convenient method for in vitro screening of large numbers of antimalarial drug candidates has become apparent. The CyQUANT cell proliferation assay is a highly sensitive fluorescence-based method for quantitation of cell number by measuring the strong fluorescence produced when green GR dye binds to nucleic acids. We have applied the CyQUANT assay method to evaluate the growth of Plasmodium falciparum D6 strain in culture. The GR-nucleic acid fluorescence linearly correlated with percent parasitemia at both 0.75 or 1 percent hematocrit with the same correlation coefficient of r2 = 0.99. The sensitivity of P. falciparum D6 strain to chloroquine and to 3,6-bis-omega-diethylaminoamyloxyxanthone, a novel antimalarial, determined by the CyQUANT assay were comparable to those obtained by the traditional [3H]-ethanolamine assay: IC50 value of chloroquine was 54 nM and 51 nM by the CyQUANT and [3H]-ethanolamine assay, respectively; IC50 value for 3,6-bis-omega-diethylaminoamyloxyxanthone was 254 nM and 223 nM by the CyQUANT and [3H]-ethanolamine assay, respectively. This procedure requires no radioisotope, uses simple equipment, and is an easy and convenient procedure, with no washing and harvesting steps. Moreover, all procedures can be set up continuously and thus, the CyQUANT assay is suitable in automatic high through-put drug screening of antimalarial drugs.

Monitoring T-Cell Responses in Translational Studies: Optimization of Dye-Based Proliferation Assay for Evaluation of Antigen-Specific Responses

Directory of Open Access Journals (Sweden)

Anja Ten Brinke

2017-12-01

Full Text Available Adoptive therapy with regulatory T cells or tolerance-inducing antigen (Ag-presenting cells is innovative and promising therapeutic approach to control undesired and harmful activation of the immune system, as observed in autoimmune diseases, solid organ and bone marrow transplantation. One of the critical issues to elucidate the mechanisms responsible for success or failure of these therapies and define the specificity of the therapy is the evaluation of the Ag-specific T-cell responses. Several efforts have been made to develop suitable and reproducible assays. Here, we focus on dye-based proliferation assays. We highlight with practical examples the fundamental issues to take into consideration for implementation of an effective and sensitive dye-based proliferation assay to monitor Ag-specific responses in patients. The most critical points were used to design a road map to set up and analyze the optimal assay to assess Ag-specific T-cell responses in patients undergoing different treatments. This is the first step to optimize monitoring of tolerance induction, allowing comparison of outcomes of different clinical studies. The road map can also be applied to other therapeutic interventions, not limited to tolerance induction therapies, in which Ag-specific T-cell responses are relevant such as vaccination approaches and cancer immunotherapy.

Genotoxicity of waterpipe smoke in buccal cells and peripheral blood leukocytes as determined by comet assay.

Science.gov (United States)

Al-Amrah, Hadba Jar-Allah; Aboznada, Osama Abdullah; Alam, Mohammad Zubair; ElAssouli, M-Zaki Mustafa; Mujallid, Mohammad Ibrahim; ElAssouli, Sufian Mohamad

2014-12-01

Waterpipe smoke causes DNA damage in peripheral blood leukocytes and in buccal cells of smokers. To determine the exposure effect of waterpipe smoke on buccal cells and peripheral blood leukocytes in regard to DNA damage using comet assay. The waterpipe smoke condensates were analyzed by gas chromatography-mass spectrometry (GC-MS). The study was performed on 20 waterpipe smokers. To perform comet assay on bucaal cells of smokers, 10 µl of cell suspension was mixed with 85 µl of pre-warmed 1% low melting agarose, applied to comet slide and electrophoresed. To analyze the effect of smoke condensate in vitro, 1 ml of peripheral blood was mixed with 10 µl of smoke condensate and subjected for comet assay. The GC-MS analysis revealed the presence of 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4on, nicotine, hydroxymethyl furancarboxaldehyde and 3-ethoxy-4-hydroxybenzaldehyde in the smoke condensates. Waterpipe smoking caused DNA damage in vivo in buccal cells of smokers. The tail moment and tail length in buccal cells of smokers were 186 ± 26 and 456 ± 71, respectively, which are higher than control. The jurak and moassel smoke condensates were found to cause DNA damage in peripheral blood leukocytes. The moassel smoke condensate was more damaging. There is wide misconception that waterpipe smoking is not as harmful as cigarette smoking. This study demonstrated that waterpipe smoke induced DNA damage in exposed cells. Waterpipe smokes cause DNA damage in buccal cells. The smoke condensate of both jurak and moassel caused comet formation suggesting DNA damage in peripheral blood leukocytes.

Neutral Red versus MTT assay of cell viability in the presence of copper compounds.

Science.gov (United States)

Gomez Perez, Mariela; Fourcade, Lyvia; Mateescu, Mircea Alexandru; Paquin, Joanne

2017-10-15

Copper is essential for numerous physiological functions, and copper compounds may display therapeutic as well as cytotoxic effects. The MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay is a standard test largely used in cytotoxicity studies. This report shows that low micromolar levels of copper compounds such as Cu(II)Urea 2 , Cu(II)Ser 2 and CuCl 2 can interfere with the MTT assay making improper the detection of formazan product of MTT reduction. Comparatively, the Neutral Red assay appears to be sensitive and showing no interference with these compounds. The lactate dehydrogenase alternative assay cannot be used because of inhibitory effect of these copper compounds on the enzyme activity. Copyright © 2017 Elsevier Inc. All rights reserved.

Advanced glycation endproducts alter functions and promote apoptosis in endothelial progenitor cells through receptor for advanced glycation endproducts mediate overpression of cell oxidant stress.

Science.gov (United States)

Chen, Jianfei; Song, Minbao; Yu, Shiyong; Gao, Pan; Yu, Yang; Wang, Hong; Huang, Lan

2010-02-01

Endothelial progenitor cells (EPCs) play an important role in preventing atherosclerosis. The factors that regulate the function of EPCs are not completely clear. Increased formation of advanced glycation endproducts (AGEs) is generally regarded as one of the main mechanisms responsible for vascular damage in patients with diabetes and atherosclerosis. AGEs lead to the generation of reactive oxygen species (ROS) and part of the regenerative capacity of EPCs seems to be due to their low baseline ROS levels and reduced sensitivity to ROS-induced cell apoptosis. Therefore, we tested the hypothesis that AGEs can alter functions and promote apoptosis in EPCs through overpress cell oxidant stress. EPCs, isolated from bone marrow, were cultured in the absence or presence of AGEs (50, 100, and 200 microg/ml). A modified Boyden's chamber was used to assess the migration of EPCs and the number of recultured EPCs was counted to measure the adhesiveness function. MTT assay was used to determine the proliferation function. ROS were analyzed using the ROS assay kit. A spectrophotometer was used to assess superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) activity, and PCR was used to test mRNA expression of SOD and GSH-PX. SiRNA was used to block receptor for advanced glycation endproducts (RAGEs) expression. Apoptosis was evaluated by Annexin V immunostaining and TUNEL staining. Co-culturing with AGEs increases ROS production, decreases anti-oxidant defenses, overpresses oxidant stress, inhibits the proliferation, migration, and adhesion of EPCs, and induces EPCs apoptosis. In addition, these effects were attenuated during block RAGE protein expression by siRNA. AGEs may serve to impair EPCs functions through RAGE-mediate oxidant stress, and promote EPCs sensitivity toward oxidative-stress-mediated apoptosis, which indicates a new pathophysiological mechanism of disturbed vascular adaptation in atherosclerosis and suggests that lower levels of AGEs might improve the

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. Copyright © 2013 Elsevier Ltd. All rights reserved.

An improved microculture-hemolytic spot assay for the study of carrier-specific antibody responses.

Science.gov (United States)

Kotkes, P; Weisman, Z; Mozes, E; Bentwich, Z

1984-11-30

A microculture system based on limiting dilution and a hemolytic spot assay was adapted for study of the carrier-specific anti-hapten response in vitro. Spleen or lymph node cells from normal mice or mice immunized with NIP-ovalbumin (NIP-OVA) or NIP-human thyroglobulin (NIP-Tg) were cultured for 5 days by the microculture technique. The anti-hapten (anti-NIP) response was measured by assaying the supernatants of the microcultures in a hemolytic spot test with NIP coupled to sheep red blood cells. A micro-ELISA reader was adapted to read the degree of lysis in the spot assay which gives an objective quantitation of the degree of lysis and thus reduces the number of culture replicates. In vivo induced specific helper cells in mice immunized with the carrier protein, human thyroglobulin, as well as carrier-specific T cell factors, gave rise to carrier-specific anti-NIP responses. The microculture system may enhance the expression of T-cell helper function when suppressor cells or their precursors are present in the initial cell preparation.

Herpes simplex virus produces larger plaques when assayed on ultraviolet irradiated CV1 cells

International Nuclear Information System (INIS)

Coohill, T.P.; Babich, M.A.; Taylor, W.D.; Snipes, W.

1980-01-01

Plaque development for either untreated or UV treated irradiated Herpes simplex virus Type 1 was faster when assayed on UV irradiated CV1 cells. This Large Plaque Effect only occurred if a minimum delay of 12h between cell irradiation and viral inoculation was allowed. Shorter delays gave plaques that were smaller than controls (unirradiated virus-unirradiated cells). The effect was maximal for a 48-h delay and remained unchanged for delays as long as 84h. The effect was greatest for cell exposures of 10Jm -2 . (author)

Development of a Rapid Insulin Assay by Homogenous Time-Resolved Fluorescence.

Directory of Open Access Journals (Sweden)

Zachary J Farino

Full Text Available Direct measurement of insulin is critical for basic and clinical studies of insulin secretion. However, current methods are expensive and time-consuming. We developed an insulin assay based on homogenous time-resolved fluorescence that is significantly more rapid and cost-effective than current commonly used approaches. This assay was applied effectively to an insulin secreting cell line, INS-1E cells, as well as pancreatic islets, allowing us to validate the assay by elucidating mechanisms by which dopamine regulates insulin release. We found that dopamine functioned as a significant negative modulator of glucose-stimulated insulin secretion. Further, we showed that bromocriptine, a known dopamine D2/D3 receptor agonist and newly approved drug used for treatment of type II diabetes mellitus, also decreased glucose-stimulated insulin secretion in islets to levels comparable to those caused by dopamine treatment.

A micromethod for the assay of cellular secretory physiology: Application to rabbit parietal cells

International Nuclear Information System (INIS)

Adrian, T.E.; Goldenring, J.R.; Oddsdottir, M.; Zdon, M.J.; Zucker, K.A.; Lewis, J.J.; Modlin, I.M.

1989-01-01

A micromethod for investigating secretory physiology in isolated cells was evaluated. The method utilized a specially designed polycarbonate incubation chamber to provide constant oxygenation to cells incubating in a 96-well microtiter plate. Cells were rapidly separated from media by vacuum filtration. Isolated parietal cells were utilized to demonstrate the versatility of the method for assay of intracellular accumulation of [ 14 C]-aminopyrine, secretion of intrinsic factor into the medium, and assay of intracellular cAMP. Histamine stimulated the uptake of [ 14 C]aminopyrine and intrinsic factor secretion in a sustained and linear fashion. At the end of the 2-h period uptake of aminopyrine and secretion of intrinsic factor were increased 17- and 5-fold, respectively. This response to histamine was accompanied by a rapid and sustained 3-fold rise in intracellular cyclic AMP. In contrast, carbamylcholine caused a transient increase in [ 14 C]aminopyrine accumulation and intrinsic factor secretion which was most pronounced during the first 10 min and had almost ceased by 30 min. Carbamylcholine had no effect on intracellular cAMP levels. This new method, which can handle 400 replicates using parietal cells from the fundic mucosa of a single rabbit, is suitable for studying the time course of intracellular events which accompany general secretory processes

Regulation of dendritic cell function by insulin/IGF-1/PI3K/Akt signaling through klotho expression.

Science.gov (United States)

Xuan, Nguyen Thi; Hoang, Nguyen Huy; Nhung, Vu Phuong; Duong, Nguyen Thuy; Ha, Nguyen Hai; Hai, Nong Van

2017-06-01

Insulin or insulin-like growth factor 1 (IGF-1) promotes the activation of phosphoinositide 3 kinase (PI3K)/Akt signaling in immune cells including dendritic cells (DCs), the most potent professional antigen-presenting cells for naive T cells. Klotho, an anti-aging protein, participates in the regulation of the PI3K/Akt signaling, thus the Ca 2+ -dependent migration is reduced in klotho-deficient DCs. The present study explored the effects of insulin/IGF-1 on DC function through klotho expression. To this end, the mouse bone marrow cells were isolated and cultured with GM-CSF to attain bone marrow-derived DCs (BMDCs). Cells were treated with insulin or IGF-1 and followed by stimulating with lipopolysaccharides (LPS). Tumor necrosis factor (TNF)-α formation was examined by enzyme-linked immunosorbent assay (ELISA). Phagocytosis was analyzed by FITC-dextran uptake assay. The expression of klotho was determined by quantitative PCR, immunoprecipitation and western blotting. As a result, treatment of the cells with insulin/IGF-1 resulted in reducing the klotho expression as well as LPS-stimulated TNF-α release and increasing the FITC-dextran uptake but unaltering reactive oxygen species (ROS) production in BMDCs. The effects were abolished by using pharmacological inhibition of PI3K/Akt with LY294002 and paralleled by transfecting DCs with klotho siRNA. In conclusion, the regulation of klotho sensitive DC function by IGF-1 or insulin is mediated through PI3K/Akt signaling pathway in BMDCs.

Antibacterial agent triclosan suppresses RBL-2H3 mast cell function

Energy Technology Data Exchange (ETDEWEB)

Palmer, Rachel K., E-mail: rachel.palmer@maine.edu [Graduate School of Biomedical Sciences, University of Maine, Orono, ME 04469 (United States); Department of Molecular and Biomedical Sciences, University of Maine, Orono, ME 04469 (United States); Hutchinson, Lee M.; Burpee, Benjamin T.; Tupper, Emily J.; Pelletier, Jonathan H.; Kormendy, Zsolt; Hopke, Alex R.; Malay, Ethan T.; Evans, Brieana L.; Velez, Alejandro [Department of Molecular and Biomedical Sciences, University of Maine, Orono, ME 04469 (United States); Gosse, Julie A., E-mail: julie.gosse@umit.maine.edu [Graduate School of Biomedical Sciences, University of Maine, Orono, ME 04469 (United States); Department of Molecular and Biomedical Sciences, University of Maine, Orono, ME 04469 (United States)

2012-01-01

Triclosan is a broad-spectrum antibacterial agent, which has been shown previously to alleviate human allergic skin disease. The purpose of this study was to investigate the hypothesis that the mechanism of this action of triclosan is, in part, due to effects on mast cell function. Mast cells play important roles in allergy, asthma, parasite defense, and carcinogenesis. In response to various stimuli, mast cells degranulate, releasing allergic mediators such as histamine. In order to investigate the potential anti-inflammatory effect of triclosan on mast cells, we monitored the level of degranulation in a mast cell model, rat basophilic leukemia cells, clone 2H3. Having functional homology to human mast cells, as well as a very well defined signaling pathway leading to degranulation, this cell line has been widely used to gain insight into mast-cell driven allergic disorders in humans. Using a fluorescent microplate assay, we determined that triclosan strongly dampened the release of granules from activated rat mast cells starting at 2 μM treatment, with dose-responsive suppression through 30 μM. These concentrations were found to be non-cytotoxic. The inhibition was found to persist when early signaling events (such as IgE receptor aggregation and tyrosine phosphorylation) were bypassed by using calcium ionophore stimulation, indicating that the target for triclosan in this pathway is likely downstream of the calcium signaling event. Triclosan also strongly suppressed F-actin remodeling and cell membrane ruffling, a physiological process that accompanies degranulation. Our finding that triclosan inhibits mast cell function may explain the clinical data mentioned above and supports the use of triclosan or a mechanistically similar compound as a topical treatment for allergic skin disease, such as eczema. -- Highlights: ►The effects of triclosan on mast cell function using a murine mast cell model. ►Triclosan strongly inhibits degranulation of mast cells. �

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.

A functional assay for detection of the mitoxantrone resistance protein, MXR (ABCG2)

DEFF Research Database (Denmark)

Robey, R W; Honjo, Y; van de Laar, A

2001-01-01

The fluorescent compounds rhodamine 123, LysoTracker Green DMD-26, mitoxantrone, and BODIPY-prazosin were used with the antagonist fumitremorgin C (FTC) in order to develop functional assays for the half-transporter, MXR/BCRP/ABCP1. A measure of FTC-inhibitable efflux was generated for each compo...

Sensitive cell-based assay for determination of human immunodeficiency virus type 1 coreceptor tropism.

Science.gov (United States)

Weber, Jan; Vazquez, Ana C; Winner, Dane; Gibson, Richard M; Rhea, Ariel M; Rose, Justine D; Wylie, Doug; Henry, Kenneth; Wright, Alison; King, Kevin; Archer, John; Poveda, Eva; Soriano, Vicente; Robertson, David L; Olivo, Paul D; Arts, Eric J; Quiñones-Mateu, Miguel E

2013-05-01

CCR5 antagonists are a powerful new class of antiretroviral drugs that require a companion assay to evaluate the presence of CXCR4-tropic (non-R5) viruses prior to use in human immunodeficiency virus (HIV)-infected individuals. In this study, we have developed, characterized, verified, and prevalidated a novel phenotypic test to determine HIV-1 coreceptor tropism (VERITROP) based on a sensitive cell-to-cell fusion assay. A proprietary vector was constructed containing a near-full-length HIV-1 genome with the yeast uracil biosynthesis (URA3) gene replacing the HIV-1 env coding sequence. Patient-derived HIV-1 PCR products were introduced by homologous recombination using an innovative yeast-based cloning strategy. The env-expressing vectors were then used in a cell-to-cell fusion assay to determine the presence of R5 and/or non-R5 HIV-1 variants within the viral population. Results were compared with (i) the original version of Trofile (Monogram Biosciences, San Francisco, CA), (ii) population sequencing, and (iii) 454 pyrosequencing, with the genotypic data analyzed using several bioinformatics tools, i.e., the 11/24/25 rule, Geno2Pheno (2% to 5.75%, 3.5%, or 10% false-positive rate [FPR]), and webPSSM. VERITROP consistently detected minority non-R5 variants from clinical specimens, with an analytical sensitivity of 0.3%, with viral loads of ≥1,000 copies/ml, and from B and non-B subtypes. In a pilot study, a 73.7% (56/76) concordance was observed with the original Trofile assay, with 19 of the 20 discordant results corresponding to non-R5 variants detected using VERITROP and not by the original Trofile assay. The degree of concordance of VERITROP and Trofile with population and deep sequencing results depended on the algorithm used to determine HIV-1 coreceptor tropism. Overall, VERITROP showed better concordance with deep sequencing/Geno2Pheno at a 0.3% detection threshold (67%), whereas Trofile matched better with population sequencing (79%). However, 454

Fluorescence Polarization Screening Assays for Small Molecule Allosteric Modulators of ABL Kinase Function.

Science.gov (United States)

Grover, Prerna; Shi, Haibin; Baumgartner, Matthew; Camacho, Carlos J; Smithgall, Thomas E

2015-01-01

The ABL protein-tyrosine kinase regulates intracellular signaling pathways controlling diverse cellular processes and contributes to several forms of cancer. The kinase activity of ABL is repressed by intramolecular interactions involving its regulatory Ncap, SH3 and SH2 domains. Small molecules that allosterically regulate ABL kinase activity through its non-catalytic domains may represent selective probes of ABL function. Here we report a screening assay for chemical modulators of ABL kinase activity that target the regulatory interaction of the SH3 domain with the SH2-kinase linker. This fluorescence polarization (FP) assay is based on a purified recombinant ABL protein consisting of the N-cap, SH3 and SH2 domains plus the SH2-kinase linker (N32L protein) and a short fluorescein-labeled probe peptide that binds to the SH3 domain. In assay development experiments, we found that the probe peptide binds to the recombinant ABL N32L protein in vitro, producing a robust FP signal that can be competed with an excess of unlabeled peptide. The FP signal is not observed with control N32L proteins bearing either an inactivating mutation in the SH3 domain or enhanced SH3:linker interaction. A pilot screen of 1200 FDA-approved drugs identified four compounds that specifically reduced the FP signal by at least three standard deviations from the untreated controls. Secondary assays showed that one of these hit compounds, the antithrombotic drug dipyridamole, enhances ABL kinase activity in vitro to a greater extent than the previously described ABL agonist, DPH. Docking studies predicted that this compound binds to a pocket formed at the interface of the SH3 domain and the linker, suggesting that it activates ABL by disrupting this regulatory interaction. These results show that screening assays based on the non-catalytic domains of ABL can identify allosteric small molecule regulators of kinase function, providing a new approach to selective drug discovery for this important

Fluorescence Polarization Screening Assays for Small Molecule Allosteric Modulators of ABL Kinase Function.

Directory of Open Access Journals (Sweden)

Prerna Grover

Full Text Available The ABL protein-tyrosine kinase regulates intracellular signaling pathways controlling diverse cellular processes and contributes to several forms of cancer. The kinase activity of ABL is repressed by intramolecular interactions involving its regulatory Ncap, SH3 and SH2 domains. Small molecules that allosterically regulate ABL kinase activity through its non-catalytic domains may represent selective probes of ABL function. Here we report a screening assay for chemical modulators of ABL kinase activity that target the regulatory interaction of the SH3 domain with the SH2-kinase linker. This fluorescence polarization (FP assay is based on a purified recombinant ABL protein consisting of the N-cap, SH3 and SH2 domains plus the SH2-kinase linker (N32L protein and a short fluorescein-labeled probe peptide that binds to the SH3 domain. In assay development experiments, we found that the probe peptide binds to the recombinant ABL N32L protein in vitro, producing a robust FP signal that can be competed with an excess of unlabeled peptide. The FP signal is not observed with control N32L proteins bearing either an inactivating mutation in the SH3 domain or enhanced SH3:linker interaction. A pilot screen of 1200 FDA-approved drugs identified four compounds that specifically reduced the FP signal by at least three standard deviations from the untreated controls. Secondary assays showed that one of these hit compounds, the antithrombotic drug dipyridamole, enhances ABL kinase activity in vitro to a greater extent than the previously described ABL agonist, DPH. Docking studies predicted that this compound binds to a pocket formed at the interface of the SH3 domain and the linker, suggesting that it activates ABL by disrupting this regulatory interaction. These results show that screening assays based on the non-catalytic domains of ABL can identify allosteric small molecule regulators of kinase function, providing a new approach to selective drug discovery

Filtration assay for quantitation of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) specific binding to whole cells in culture

International Nuclear Information System (INIS)

Dold, K.M.; Greenlee, W.F.

1990-01-01

A rapid and sensitive filtration assay for quantitating the specific binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to whole cells in culture is described. Cell monolayers are incubated with [3H]TCDD in the presence or absence of excess unlabeled ligand, detached from the culture dish with trypsin, filtered, and washed with cold (-78 degrees C) acetone to separate free and nonspecifically bound TCDD from specifically bound TCDD. TCDD receptor binding parameters were characterized in the murine hepatoma cell line Hepa1c1c7. The lower limit of detection of TCDD specific binding was in a sample equivalent to 10 micrograms of total cell protein. The equilibrium dissociation constant and stereospecificity for binding to the TCDD receptor were the same as those previously reported with other TCDD receptor assays on broken cell preparations. Analysis of binding in the murine hepatoma TCDD receptor variants TAO-c1BPrc1 and BPrc1 indicated that this assay will detect receptor number or affinity variants, but will not detect nuclear transfer deficient variants. The major advantage of the whole cell binding assay is that it provides the means to rapidly and reproducibly quantitate TCDD specific binding in small samples of whole cells in culture. In addition, this method eliminates loss or degradation of the receptor protein during the fractionation of cells required in previously reported methods. This method should prove useful in screening clonal cell populations for TCDD receptor number and affinity variants, and in screening for TCDD receptor binding activity in complementation studies of receptor deficient cells

Variation in assessment of oxidatively damaged DNA in mononuclear blood cells by the comet assay with visual scoring

DEFF Research Database (Denmark)

Forchhammer, Lykke; Bräuner, Elvira Vaclavik; Folkmann, Janne Kjaersgaard

2008-01-01

The comet assay is popular for assessments of genotoxicity, but the comparison of results between studies is challenging because of differences in experimental procedures and reports of DNA damage in different units. We investigated the variation of DNA damage in mononuclear blood cells (MNBCs......) measured by the comet assay with focus on the variation related to alkaline unwinding and electrophoresis time, number of cells scored, as well as the putative benefits of transforming the primary end points to common units by the use of reference standards and calibration curves. Eight experienced......, our results indicate that inter-investigator difference in scoring is a strong determinant of DNA damage levels measured by the comet assay....

Aminopropyltriethoxysilane-mediated surface functionalization of hydroxyapatite nanoparticles: synthesis, characterization, and in vitro toxicity assay

Directory of Open Access Journals (Sweden)

Wang S

2011-12-01

Full Text Available Shige Wang1, Shihui Wen2, Mingwu Shen2, Rui Guo2, Xueyan Cao2, Jianhua Wang3, Xiangyang Shi1,2,41State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, 2College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, 3Department of Biochemistry and Molecular Cell Biology, School of Medicine, Shanghai Jiao Tong University, Shanghai, People's Republic of China; 4Centro de Química da Madeira, Universidade da Madeira, Campus da Penteada, Funchal, PortugalBackground: We report on aminopropyltriethoxysilane (APTS-mediated surface modification of nanohydroxyapatite with different surface functional groups for potential biomedical applications. In this study, nanohydroxyapatite covalently linked with APTS (n-HA-APTS was reacted with acetic anhydride or succinic anhydride to produce neutralized (n-HA-APTS.Ac or negatively charged (n-HA-APTS.SAH nanohydroxyapatite, respectively. Nanohydroxyapatite formed with amine, acetyl, and carboxyl groups was extensively characterized using Fourier transform infrared spectroscopy, transmission electron microscopy, 1H nuclear magnetic resonance spectroscopy, X-ray diffraction, inductively coupled plasma-atomic emission spectroscopy, and zeta potential measurements.Results: In vitro 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide colorimetric assay revealed that the slight toxicity of the amine-functionalized n-HA-APTS could be eliminated by post-functionalization of APTS amines to form acetyl and carboxyl groups. Blood compatibility assessment demonstrated that the negligible hemolytic activity of the pristine nanohydroxyapatite particles did not appreciably change after APTS-mediated surface functionalization.Conclusion: APTS-mediated functionalization of nanohydroxyapatite with different surface groups may be useful for further functionalization of nanohydroxyapatite with biologically active materials, thereby providing possibilities for a broad range of

Tandem assays of protein and glucose with functionalized core/shell particles based on magnetic separation and surface-enhanced Raman scattering.

Science.gov (United States)

Kong, Xianming; Yu, Qian; Lv, Zhongpeng; Du, Xuezhong

2013-10-11

Tandem assays of protein and glucose in combination with mannose-functionalized Fe3 O4 @SiO2 and Ag@SiO2 tag particles have promising potential in effective magnetic separation and highly sensitive and selective SERS assays of biomaterials. It is for the first time that tandem assay of glucose is developed using SERS based on the Con A-sandwiched microstructures between the functionalized magnetic and tag particles. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay.

Science.gov (United States)

Cui, Heying; Loftus, Kyle M; Noell, Crystal R; Solmaz, Sozanne R

2018-05-03

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; however, the number of identified targets for Cdk1, particularly in human cells is still low. The identification of Cdk1-specific phosphorylation sites is important, as they provide mechanistic insights into how Cdk1 controls the cell cycle. Cell cycle regulation is critical for faithful chromosome segregation, and defects in this complicated process lead to chromosomal aberrations and cancer. Here, we describe an in vitro kinase assay that is used to identify Cdk1-specific phosphorylation sites. In this assay, a purified protein is phosphorylated in vitro by commercially available human Cdk1/cyclin B. Successful phosphorylation is confirmed by SDS-PAGE, and phosphorylation sites are subsequently identified by mass spectrometry. We also describe purification protocols that yield highly pure and homogeneous protein preparations suitable for the kinase assay, and a binding assay for the functional verification of the identified phosphorylation sites, which probes the interaction between a classical nuclear localization signal (cNLS) and its nuclear transport receptor karyopherin α. To aid with experimental design, we review approaches for the prediction of Cdk1-specific phosphorylation sites from protein sequences. Together these protocols present a very powerful approach that yields Cdk1-specific phosphorylation sites and enables mechanistic studies into how Cdk1 controls the cell cycle. Since this method relies on purified proteins, it can be applied to any model organism and yields reliable results, especially when combined with cell functional studies.

A simple clot based assay for detection of procoagulant cell-derived microparticles.

Science.gov (United States)

Patil, Rucha; Ghosh, Kanjaksha; Shetty, Shrimati

2016-05-01

Cell-derived microparticles (MPs) are important biomarkers in many facets of medicine. However, the MP detection methods used till date are costly and time consuming. The main aim of this study was to standardize an in-house clot based screening method for MP detection which would not only be specific and sensitive, but also inexpensive. Four different methods of MP assessment were performed and the results correlated. Using the flow cytometry technique as the gold standard, 25 samples with normal phosphatidylserine (PS) expressing MP levels and 25 samples with elevated levels were selected, which was cross checked by the commercial STA Procoag PPL clotting time (CT) assay. A simple recalcification time and an in-house clot assay were the remaining two tests. The in-house test measures the CT after the addition of calcium chloride to MP rich plasma, following incubation with Russell viper venom and phospholipid free plasma. The CT obtained by the in-house assay significantly correlated with the results obtained by flow cytometry (R2=0.87, p<0.01). Though preliminary, the in-house assay seems to be efficient, inexpensive and promising. It could definitely be utilized routinely for procoagulant MP assessment in various clinical settings.

Chemosensitivity and radiosensitivity of small cell lung cancer cell lines studied by a newly developed 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) hybrid assay

International Nuclear Information System (INIS)

Hida, T.; Ueda, R.; Takahashi, T.; Watanabe, H.; Kato, T.; Suyama, M.; Sugiura, T.; Ariyoshi, Y.

1989-01-01

The 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) hybrid assay was developed by technically combining the human tumor clonogenic assay and the MTT assay to make the most of both assays. This assay was able to estimate the in vitro growth of cultured cell lines and of tumor cells in pleural effusion, suggesting the possibility of its use for assessment of chemosensitivity and radiosensitivity of fresh tumor samples. Multiple cell lines [including morphological and/or phenotypical in vitro converters and cisplatin (CDDP)-resistant lines] were established from three patients with small cell lung cancer at different stages of the disease. Chemosensitivity of these cell lines to four commonly used chemotherapeutic drugs was tested by the MTT hybrid assay. SK1 and SK3 lines were established from Patient S. K. before and after chemotherapy and radiotherapy, respectively. SK3/CDDP, a CDDP-resistant line derived from the SK3 line, was 30-fold more resistant to CDDP [50% inhibiting dose (IC50), 21.5 micrograms/ml] than the SK1 line. In Patient M. O., MOA2/CDDP, a CDDP-resistant line derived from MOA2 (an in vitro converter from the MO line), was 41-fold more resistant to CDDP (IC50, 37 micrograms/ml) than the parent MO line. From Patient T. M., TM1 and TM2 lines were established before and after chemotherapy, respectively. The latter showed 6-fold more resistance to CDDP than the former. Chemosensitivity of these lines to three other drugs, 4-hydroperoxycyclophosphamide, Adriamycin, and etoposide, suggested cross-resistance between CDDP and 4-hydroperoxycyclophosphamide. Radiosensitivity study was also carried out with the MTT hybrid assay. The MOA2 line was more resistant [Do, 3.0 Gy; extrapolation number (n), 4.0] than the parental MO line (Do, 1.6 Gy; n, 2.1). There was no clear difference in radiosensitivity between the cell lines established before and after radiation therapy in Patient S. K

Inhibition of clone formation as an assay for T cell-mediated cytotoxicity: short-term kinetics and comparison with 51Cr release

International Nuclear Information System (INIS)

Lees, R.K.; MacDonald, H.R.; Sinclair, N.R.; University of Western Ontario London

1977-01-01

The short-term kinetics of T cell-mediated cytotoxicity was investigated using a cloning inhibition assay. Murine cytotoxic thymus-derived lymphocytes generated in vitro in mixed leukocyte cultures were incubated for various periods of time at 37degC with allogeneic mastocytoma target cells. The mixtures were then plated in soft agar, and mastocytoma clone formation was assessed after 5-7 days incubation. Using this technique, it was demonstrated that events leading to the loss of cloning ability could be detected after 1-3 min incubation at 37degC, and after 20-30 min, 95% of the clone forming cells had been inactivated. When these results were compared directly with those obtained using the conventional 51 Cr-release assay, it was found that the events leading to loss of cloning ability occurred more rapidly than indicated by the isotope assay. However, a modification of the 51 Cr-release assay involving EDTA addition gave comparable result to the cloning inhibition assay. These results raise the possibility that the events leading to 51 Cr-release of tumor target cells may be related in time to those leading to the loss of cloning ability

Altered effector function of peripheral cytotoxic cells in COPD

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Corne Jonathan M

2009-06-01

Full Text Available Abstract Background There is mounting evidence that perforin and granzymes are important mediators in the lung destruction seen in COPD. We investigated the characteristics of the three main perforin and granzyme containing peripheral cells, namely CD8+ T lymphocytes, natural killer (NK; CD56+CD3- cells and NKT-like (CD56+CD3+ cells. Methods Peripheral blood mononuclear cells (PBMCs were isolated and cell numbers and intracellular granzyme B and perforin were analysed by flow cytometry. Immunomagnetically selected CD8+ T lymphocytes, NK (CD56+CD3- and NKT-like (CD56+CD3+ cells were used in an LDH release assay to determine cytotoxicity and cytotoxic mechanisms were investigated by blocking perforin and granzyme B with relevant antibodies. Results The proportion of peripheral blood NKT-like (CD56+CD3+ cells in smokers with COPD (COPD subjects was significantly lower (0.6% than in healthy smokers (smokers (2.8%, p +CD3- cells from COPD subjects were significantly less cytotoxic than in smokers (16.8% vs 51.9% specific lysis, p +CD3+ cells (16.7% vs 52.4% specific lysis, p +CD3- and NKT-like (CD56+CD3+ cells from smokers and HNS. Conclusion In this study, we show that the relative numbers of peripheral blood NK (CD56+CD3- and NKT-like (CD56+CD3+ cells in COPD subjects are reduced and that their cytotoxic effector function is defective.

Gamma-ray imaging and holdup assays of 235-F PuFF cells 1 & 2

Energy Technology Data Exchange (ETDEWEB)

Aucott, T. [Savannah River Site (SRS), Aiken, SC (United States). Savannah River National Lab. (SRNL)

2017-12-20

Savannah River National Laboratory (SRNL) Nuclear Measurements (L4120) was tasked with performing enhanced characterization of the holdup in the PuFF shielded cells. Assays were performed in accordance with L16.1-ADS-2460 using two high-resolution gamma-ray detectors. The first detector, an In Situ Object Counting System (ISOCS)-characterized detector, was used in conjunction with the ISOCS Geometry Composer software to quantify grams of holdup. The second detector, a Germanium Gamma-ray Imager (GeGI), was used to visualize the location and relative intensity of the holdup in the cells. Carts and collimators were specially designed to perform optimum assays of the cells. Thick, pencil-beam tungsten collimators were fabricated to allow for extremely precise targeting of items of interest inside the cells. Carts were designed with a wide range of motion to position and align the detectors. A total of 24 measurements were made, each typically 24 hours or longer to provide sufficient statistical precision. This report presents the results of the enhanced characterization for cells 1 and 2. The measured gram values agree very well with results from the 2014 study. In addition, images were created using both the 2014 data and the new GeGI data. The GeGI images of the cells walls reveal significant Pu-238 holdup on the surface of the walls in cells 1 and 2. Additionally, holdup is visible in the two pass-throughs from cell 1 to the wing cabinets. This report documents the final element (exterior measurements coupled with gamma-ray imaging and modeling) of the enhanced characterization of cells 1-5 (East Cell Line).

Significance of the proportion of binucleate cells in the micronucleus assay; A methodological study

Energy Technology Data Exchange (ETDEWEB)

Imamura, Masahiro; Edgren, M.R. (Karolinska Inst., Stockholm (Sweden). Dept. of Radiation Physics)

1994-03-01

Using treatment with cytochalasin-B (Cyt-B) for the induction of a cytokinetic block, the significance of the proportion of binucleate cells (BNC) in the micronucleus (MN) assay was investigated in a methodological study. A Chinese hamster cell line V79 was used in which MN were induced by radiation. In complementary tests the radiation effect in inducing MN was enhanced by depletion of the cellular glutathione content with buthionine sulfoximine (BSO). The data indicated that the concentration of Cyt-B is the major factor which determines the proportion of BNC. This proportion was shown to be independent of radiation dose and of BSO. Furthermore, the MN frequency was not related to the percentage of BNC. Therefore, a high proportion of BNC may be practical for the MN assay, but may not make the technique more accurate. (author).

Assays of D-Amino Acid Oxidase Activity

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Elena Rosini

2018-01-01

Full Text Available D-amino acid oxidase (DAAO is a well-known flavoenzyme that catalyzes the oxidative FAD-dependent deamination of D-amino acids. As a result of the absolute stereoselectivity and broad substrate specificity, microbial DAAOs have been employed as industrial biocatalysts in the production of semi-synthetic cephalosporins and enantiomerically pure amino acids. Moreover, in mammals, DAAO is present in specific brain areas and degrades D-serine, an endogenous coagonist of the N-methyl-D-aspartate receptors (NMDARs. Dysregulation of D-serine metabolism due to an altered DAAO functionality is related to pathological NMDARs dysfunctions such as in amyotrophic lateral sclerosis and schizophrenia. In this protocol paper, we describe a variety of direct assays based on the determination of molecular oxygen consumption, reduction of alternative electron acceptors, or α-keto acid production, of coupled assays to detect the hydrogen peroxide or the ammonium production, and an indirect assay of the α-keto acid production based on a chemical derivatization. These analytical assays allow the determination of DAAO activity both on recombinant enzyme preparations, in cells, and in tissue samples.

MicroRNA-144 inhibits hepatocellular carcinoma cell proliferation

Indian Academy of Sciences (India)

MiR-144 was shown to besignificantly down-regulated in HCC tissues and cell lines. Subsequently, overexpression of miR-144 was transfectedinto HCC cell lines so as to investigate its biological function, including MTT, colony formation, and transwell assays.Gain of function assay revealed miR-144 remarkably inhibited ...

A Functional Assay for GPR55: Envision Protocol.

Science.gov (United States)

Anavi-Goffer, Sharon; Ross, Ruth A

2016-01-01

AlphaScreen(®) SureFire(®) assay is a novel technology that combines luminescent oxygen channeling technology, nano-beads, and monocloncal antibodies to detect the level of a selected protein in a volume lower than 5 μl. This method is more sensitive compared with the traditional enzyme-linked immunosorbent assays (ELISA), and can detect an increasing number of new targets. Here, we described a method for AlphaScreen(®) SureFire(®) assay that targets ERK1/2 phosphorylation, a primary downstream signaling pathway that conveys activation of GPR55 by L-α-lysophosphatidylinositol (LPI) and certain cannabinoids.