WorldWideScience

Sample records for cell culture assay

  1. 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

  2. In Vitro Cell Culture Infectivity Assay for Human Noroviruses

    Energy Technology Data Exchange (ETDEWEB)

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

    2007-01-30

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

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

    Science.gov (United States)

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

    2015-02-17

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

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

    Science.gov (United States)

    Thiel, William H; Giangrande, Paloma H

    2016-07-01

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

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

    Science.gov (United States)

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

    2010-01-01

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

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

    Science.gov (United States)

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

    2010-01-01

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

  7. Development of a quantal assay in primary shrimp cell culture for yellow head baculovirus (YBV) of penaeid shrimp.

    Science.gov (United States)

    Lu, Y; Tapay, L M; Loh, P C; Brock, J A; Gose, R

    1995-03-01

    A 50% tissue culture infectious dose assay (TCID50) using primary culture of shrimp lymphoid organ (Oka) cells was developed for the quantitative titration of yellow-head baculovirus (YBV), a newly isolated virus of penaeid shrimp. The assay protocol includes the use of Primaria-grade 96-well tissue culture plates to grow the primary lymphoid organ cells of penaeid shrimp. A 15% gill suspension from YBV-infected shrimp was determined to have an infectious virus titer of 5 x 10(5.75) TCID50/ml. This report represents the first convenient assay protocol using cell culture derived from penaeid shrimp to titer a shrimp virus.

  8. Cell culture-Taqman PCR assay for evaluation of Cryptosporidium parvum disinfection.

    Science.gov (United States)

    Keegan, Alexandra R; Fanok, Stella; Monis, Paul T; Saint, Christopher P

    2003-05-01

    Cryptosporidium parvum represents a challenge to the water industry and a threat to public health. In this study, we developed a cell culture-quantitative PCR assay to evaluate the inactivation of C. parvum with disinfectants. The assay was validated by using a range of disinfectants in common use in the water industry, including low-pressure UV light (LP-UV), ozone, mixed oxidants (MIOX), and chlorine. The assay was demonstrated to be reliable and sensitive, with a lower detection limit of a single infectious oocyst. Effective oocyst inactivation was achieved (>2 log(10) units) with LP-UV (20 mJ/cm(2)) or 2 mg of ozone/liter (for 10 min). MIOX and chlorine treatments of oocysts resulted in minimal effective disinfection, with disinfection systems for drinking water and recycled water.

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

    Science.gov (United States)

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

    2002-03-01

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

  10. Comparison of in vitro cell culture and a mouse assay for measuring infectivity of Cryptosporidium parvum.

    Science.gov (United States)

    Rochelle, Paul A; Marshall, Marilyn M; Mead, Jan R; Johnson, Anne M; Korich, Dick G; Rosen, Jeffrey S; De Leon, Ricardo

    2002-08-01

    In vitro cell cultures were compared to neonatal mice for measuring the infectivity of five genotype 2 isolates of Cryptosporidium parvum. Oocyst doses were enumerated by flow cytometry and delivered to animals and cell monolayers by using standardized procedures. Each dose of oocysts was inoculated into up to nine replicates of 9 to 12 mice or 6 to 10 cell culture wells. Infections were detected by hematoxylin and eosin staining in CD-1 mice, by reverse transcriptase PCR in HCT-8 and Caco-2 cells, and by immunofluorescence microscopy in Madin-Darby canine kidney (MDCK) cells. Infectivity was expressed as a logistic transformation of the proportion of animals or cell culture wells that developed infection at each dose. In most instances, the slopes of the dose-response curves were not significantly different when we compared the infectivity models for each isolate. The 50% infective doses for the different isolates varied depending on the method of calculation but were in the range from 16 to 347 oocysts for CD-1 mice and in the ranges from 27 to 106, 31 to 629, and 13 to 18 oocysts for HCT-8, Caco-2, and MDCK cells, respectively. The average standard deviations for the percentages of infectivity for all replicates of all isolates were 13.9, 11.5, 13.2, and 10.7% for CD-1 mice, HCT-8 cells, Caco-2 cells, and MDCK cells, respectively, demonstrating that the levels of variability were similar in all assays. There was a good correlation between the average infectivity for HCT-8 cells and the results for CD-1 mice across all isolates for untreated oocysts (r = 0.85, n = 25) and for oocysts exposed to ozone and UV light (r = 0.89, n = 29). This study demonstrated that in vitro cell culture was equivalent to the "gold standard," mouse infectivity, for measuring the infectivity of C. parvum and should therefore be considered a practical and accurate alternative for assessing oocyst infectivity and inactivation. However, the high levels of variability displayed by all

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

    Institute of Scientific and Technical Information of China (English)

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

    2008-01-01

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

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

    International Nuclear Information System (INIS)

    Four separate assays of human antibody reactivity to four separate normal and malignant human tissue culture cells lines from two patients have been evaluated using a single highly-reactive allogeneic serum. The visual end-point cytolysis assay and the chromium-51 release assay were equally sensitive in measuring complement mediated antibody cytotoxicity and both were far more sensitive than a trypan blue dye exclusion assay. The assay of antibody reactivity by hemadsorption technique was about 10 times more sensitive than any of the cytotoxicity assays. This latter assay measures only IgG antibody however. These assays showed that cell lines from different patients may differ greatly in 'reactivity' to an allogeneic serum and emphasized the importance of utilizing tumor and normal cells from the same patient when using tissue culture cells to search for tumor specific reactivity. These observations emphasize the importance of utilizing multiple assays against paired normal and malignant cells from the same patient to be certain of the specificity and magnitude of the measured antibody

  13. Radiomodifying effect of resveratrol in human rhabdomyosarcoma (RD) cell culture applying the comet assay

    Energy Technology Data Exchange (ETDEWEB)

    Magalhaes, Vanessa D.; Rogero, Sizue O.; Vieira, Daniel P.; Okazaki, Kayo; Rogero, Jose R., E-mail: van.biologa@gmail.com [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil); Cruz, Aurea S., E-mail: aurcruz@ial.sp.gov.br [Instituto Adolfo Lutz (IAL-SP), Sao Paulo, SP (Brazil)

    2013-07-01

    Cancer is considered a worldwide public health problem. Resveratrol is a defense polyphenol, synthesized naturally by a wide variety of plants according to response of ultraviolet radiation (UV) exposition or according to mechanical stress resulting of pathogens or chemical and physical agents. In vines this substance is found in elevated concentration. Thus, resveratrol is present in grape juice and wines, especially red wine. Red wines are the best dietary source of resveratrol.The protective effects performed by resveratrol during the process of cell damage, produced by oxidative effects of free radicals, are anti-inflammatory, anti-platelet and anti-carcinogenic activity, prevent or inhibit degenerative diseases, decrease incidence of cardiovascular diseases. Moreover, resveratrol is considered as a cell radioprotector. On the other hand, in some elevated concentrations resveratrol is considered as a radiosensitizing compound. The aim of this work was study in vitro the radiomodifying effect of resveratrol in human rhabdomyosarcoma (RD) cells applying the comet assay to evaluate the cellular damage and its repair capacity. In this study RD cells culture was irradiated by gamma radiation at 50 Gy and 100 Gy doses and the used resveratrol concentrations was from 15 μM to 60 μM. The protective and radioprotective effects were observed at 15 μM and 30 μM resveratrol concentrations. The resveratrol concentration of 60 μM showed cytotoxic effect to RD tumor cells and with gamma radiation presence this concentration showed no statistically significant radiosensitizing effects. (author)

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

    DEFF Research Database (Denmark)

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

    2013-01-01

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

  15. Susceptibility of adherent versus suspension target cells derived from adherent tissue culture lines to cell-mediated cytotoxicity in rapid 51Cr-release assays

    International Nuclear Information System (INIS)

    Preparation of target cells from tissue culture lines which grow adherent to tissue culture vessels is often desirable for tests of cell-mediated cytotoxicity (CMC). In the present study the authors used cells derived from adherent tissue culture lines to compare the merits of suspension vs. adherent target cells in short-term 51Cr-release assays. Cytotoxic activity of murine spleen cells sensitized in vitro against allogeneic spleen cells or syngeneic sarcoma cells was tested with fibroblast or sarcoma target cells. In parallel tests, aliquots of tissue culture lines were detached and used as either suspension or adherent target cells in CMC assays, matching the concentrations of suspension and adherent target cells. In both allogeneic and syngeneic combinations adherent target cells released less 51Cr spontaneously and were more susceptible to CMC than their suspension counterparts. (Auth.)

  16. Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation.

    Science.gov (United States)

    Côté, Marie-France; Turcotte, Audrey; Doillon, Charles; Gobeil, Stephane

    2016-01-01

    Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells often generates unwanted artifacts. Therefore, cell culture in a three-dimensional (3D) environment, often using extracellular matrix components, emerged as an interesting alternative due to its close similarity to the native in vivo tissue or organ. We developed a 3D cell culture system using two compartments, namely (i) a central compartment containing cancer cells embedded in a collagen gel acting as a pseudo-primary macrospherical tumor and (ii) a peripheral cell-free compartment made of a fibrin gel, i.e. an extracellular matrix component different from that used in the center, in which cancer cells can migrate (invasion front) and/or form microspherical tumors representing secondary or satellite tumors. The formation of satellite tumors in the peripheral compartment is remarkably correlated to the known aggressiveness or metastatic origin of the native tumor cells, which makes this 3D culture system unique. This cell culture approach might be considered to assess cancer cell invasiveness and motility, cell-extracellular matrix interactions and as a method to evaluate anti-cancer drug properties. PMID:27585303

  17. Development of an in vitro quantal assay in primary cell cultures for a non-occluded baculo-like virus of penaeid shrimp.

    Science.gov (United States)

    Tapay, L M; Lu, Y; Gose, R B; Nadala, E C; Brock, J A; Loh, P C

    1997-02-01

    An in vitro quantal assay (TCID50) for a non-occluded baculo-like virus isolate from naturally infected Penaeus japonicus obtained from China and experimentally infected P. stylirostris was developed using primary shrimp lymphoid cell cultures in Primaria 24-well tissue culture plates. The virus caused cytopathogenic effect (CPE) in the cell cultures as early as 2 day post-infection (p.i.). Initially, the cells rounded up and finally detached from the culture vessel as the infection progressed. At the present time, there is no established quantitative in vitro cell culture protocol for the assay of this baculo-like virus which has been reported by our laboratory to be highly pathogenic for P. stylirostris and P. vannamei, the two species of penaeid shrimp commercially cultured in Hawaii and the Western hemisphere. This quantal assay thus provides a simple and convenient method for the detection and assay of infectious virus in cultured penaeid shrimp.

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

    Directory of Open Access Journals (Sweden)

    Beatriz A Santillan

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

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

    Directory of Open Access Journals (Sweden)

    Mahajan Supriya D.

    2003-01-01

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

  20. IDENTIFICATION OF GLUCOSE TRANSPORTER-1 AND ITS FUNCTIONAL ASSAY IN MOUSE GLOMERULAR MESANGIAL CELLS CULTURED IN VITRO

    Institute of Scientific and Technical Information of China (English)

    章精; 刘志红; 刘栋; 黎磊石

    2001-01-01

    Objective. To evaluate the role of glucose transporter-l (GLUT1) in the glucose uptake of glomerular mesangial cells. Methods. Cultured C57/SJL mouse mesangial cells were used in the study. The expression of GLUT1 mRNA was detected by RT-PCR. The expression of GLUT1 protein was detected by immunofluorescence and flow cytometry. The uptake of glucose and its kinetics were determined by 2-deoxy-[3H] -D-glucose uptake. Results. Both GLUT1 mRNA and protein were found in mouse glomerular mesangial cells. 2-deoxy-D-glucose uptake and kinetics assay showed that this glucose transporter had high affinity for glucose and the glucose uptake specificity was further confirmed by phloretin. Conclusion. Functional GLUT1 did present in mouse mesangial cells cultured in vitro and it might be the predominant transporter mediated the uptake of glucose into mesangial cells.

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

    OpenAIRE

    Mahajan Supriya D.; Schwartz Stanley A; Nair Madhavan P.N.

    2003-01-01

    Herein we review the various methods currently in use for determining the expression of chemokines by CNS cells in vitro. Chemokine detection assays are used in conjuction with one another to provide a comprehensive, biologically relevant assessment of the chemokines which is necessary for correct data interpretation of a specific observed biological effect. The methods described include bioassays for soluble chemokine receptors, RNA extraction, RT-PCR, Real - time quantitative PCR, gene arra...

  2. Metabolic response of environmentally isolated microorganisms to industrial effluents: Use of a newly described cell culture assay

    Science.gov (United States)

    Ferebee, Robert N.

    1992-01-01

    An environmental application using a microtiter culture assay to measure the metabolic sensitivity of microorganisms to petrochemical effluents will be tested. The Biomedical Operations and Research Branch at NASA JSC has recently developed a rapid and nondestructive method to measure cell growth and metabolism. Using a colorimetric procedure the uniquely modified assay allows the metabolic kinetics of prokaryotic and eukaryotic cells to be measured. Use of such an assay if adapted for the routine monitoring of waste products, process effluents, and environmentally hazardous substances may prove to be invaluable to the industrial community. The microtiter method as described will be tested using microorganisms isolated from the Galveston Bay aquatic habitat. The microbial isolates will be identified prior to testing using the automated systems available at JSC. Sodium dodecyl sulfate (SDS), cadmium, and lead will provide control toxic chemicals. The toxicity of industrial effluent from two industrial sites will be tested. An effort will be made to test the efficacy of this assay for measuring toxicity in a mixed culture community.

  3. Evaluation of an in vitro cell culture assay for the potency assessment of recombinant human erythropoietin.

    Science.gov (United States)

    Machado, Francine T; Maldaner, Fernanda P S; Perobelli, Rafaela F; Xavier, Bruna; da Silva, Francielle S; de Freitas, Guilherme W; Bartolini, Paolo; Ribela, M Tereza C P; Dalmora, Sérgio L

    2016-05-01

    Recombinant human erythropoietin is a sialoglycoprotein that stimulates erythropoiesis. To assess potency of human erythropoietin produced by recombinant technology, we investigated an in vitro TF-1 cell proliferation assay, which was applied in conjunction with a reversed-phase liquid chromatography method for the determination of the content of sialic acids. The results obtained, which were higher than 126.8ng/μg, were compared with those obtained with the in vivo normocythaemic mouse bioassay. The in vitro assay resulted in a non-significant lower mean difference of the estimated potencies (0.61% ± 0.026, p > 0.05). The use of this combination of methods represents an advance toward the establishment of alternative in vitro approaches, in the context of the Three Rs, for the potency assessment of biotechnology-derived medicines. PMID:27256453

  4. Quantitation of viable Coxiella burnetii in milk using an integrated cell culture-polymerase chain reaction (ICC-PCR) assay.

    Science.gov (United States)

    Stewart, Diana; Shieh, Y-Carol; Tortorello, Mary; Kukreja, Ankush; Shazer, Arlette; Schlesser, Joseph

    2015-11-01

    The obligate intracellular pathogen Coxiella burnetii has long been considered the most heat resistant pathogen in raw milk, making it the reference pathogen for determining pasteurisation conditions for milk products. New milk formulations and novel non-thermal processes require validation of effectiveness which requires a more practical method for analysis than using the currently used animal model for assessing Coxiella survival. Also, there is an interest in better characterising thermal inactivation of Coxiella in various milk formulations. To avoid the use of the guinea pig model for evaluating Coxiella survival, an Integrated Cell Culture-PCR (ICC-PCR) method was developed for determining Coxiella viability in milk. Vero cell cultures were directly infected from Coxiella-contaminated milk in duplicate 24-well plates. Viability of the Coxiella in milk was shown by a ≥ 0.5 log genome equivalent (ge)/ml increase in the quantity of IS111a gene from the baseline post-infection (day 0) level after 9-11 d propagation. Coxiella in skim, 2%, and whole milk, and half and half successfully infected Vero cells and increased in number by at least 2 logs using a 48-h infection period followed by 9-d propagation time. As few as 125 Coxiella ge/ml in whole milk was shown to infect and propagate at least 2 logs in the optimised ICC-PCR assay, though variable confirmation of propagation was shown for as low as 25 Coxiella ge/ml. Applicability of the ICC-PCR method was further proven in an MPN format to quantitate the number of viable Coxiella remaining in whole milk after 60 °C thermal treatment at 0, 20, 40, 60 and 90 min.

  5. Comparison of Rapid Centrifugation Assay with Conventional Tissue Culture Method for Isolation of Dengue 2 Virus in C6/36-HT Cells

    OpenAIRE

    Roche, Rosmari Rodríguez; Alvarez, Mayling; María G. Guzmán; Morier, Luis; Kourí, Gustavo

    2000-01-01

    A rapid centrifugation assay was compared with conventional tube cell culture for dengue virus isolation in both sera and autopsy samples from dengue and dengue hemorrhagic fever/dengue shock syndrome fatal cases. The rapid centrifugation assay allowed isolation of virus from 16.6% more samples than the conventional method, and it shortened the time for dengue virus detection. Finally, it allowed the isolation of dengue 2 virus in 42.8% of tissue samples from five fatal cases. Our results sug...

  6. An indirect immunofluorescence assay using a cell culture-derived antigen for detection of antibodies to the agent of human granulocytic ehrlichiosis.

    OpenAIRE

    Nicholson, W L; Comer, J A; Sumner, J W; Gingrich-Baker, C; Coughlin, R T; Magnarelli, L A; Olson, J G; Childs, J. E.

    1997-01-01

    An indirect immunofluorescence assay for the detection of human antibodies to the agent of human granulocytic ehrlichiosis (HGE) was developed and standardized. Antigen was prepared from a human promyelocytic leukemia cell line (HL-60) infected with a tick-derived isolate of the HGE agent (USG3). Suitable antigen presentation and preservation of cellular morphology were obtained when infected cells were applied and cultured on the slide, excess medium was removed, and cells were fixed with ac...

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

    Science.gov (United States)

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

    2016-01-01

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

  8. Evaluation of a multiple-cycle, recombinant virus, growth competition assay that uses flow cytometry to measure replication efficiency of human immunodeficiency virus type 1 in cell culture.

    Science.gov (United States)

    Dykes, Carrie; Wang, Jiong; Jin, Xia; Planelles, Vicente; An, Dong Sung; Tallo, Amanda; Huang, Yangxin; Wu, Hulin; Demeter, Lisa M

    2006-06-01

    Human immunodeficiency virus type 1 (HIV-1) replication efficiency or fitness, as measured in cell culture, has been postulated to correlate with clinical outcome of HIV infection, although this is still controversial. One limitation is the lack of high-throughput assays that can measure replication efficiency over multiple rounds of replication. We have developed a multiple-cycle growth competition assay to measure HIV-1 replication efficiency that uses flow cytometry to determine the relative proportions of test and reference viruses, each of which expresses a different reporter gene in place of nef. The reporter genes are expressed on the surface of infected cells and are detected by commercially available fluorescence-labeled antibodies. This method is less labor-intensive than those that require isolation and amplification of nucleic acids. The two reporter gene products are detected with similar specificity and sensitivity, and the proportion of infected cells in culture correlates with the amount of viral p24 antigen produced in the culture supernatant. HIV replication efficiencies of six different drug-resistant site-directed mutants were reproducibly quantified and were similar to those obtained with a growth competition assay in which the relative proportion of each variant was measured by sequence analysis, indicating that recombination between the pol and reporter genes was negligible. This assay also reproducibly quantified the relative fitness conferred by protease and reverse transcriptase sequences containing multiple drug resistance mutations, amplified from patient plasma. This flow cytometry-based growth competition assay offers advantages over current assays for HIV replication efficiency and should prove useful for the evaluation of patient samples in clinical trials.

  9. Advantages of a single-cycle production assay to study cell culture-adaptive mutations of hepatitis C virus

    DEFF Research Database (Denmark)

    Russell, Rodney S; Meunier, Jean-Christophe; Takikawa, Shingo;

    2008-01-01

    mutations that were selected during serial passage in Huh-7.5 cells were studied. Recombinant genomes containing all five mutations produced 3-4 logs more infectious virions than did wild type. Neither a coding mutation in NS5A nor a silent mutation in E2 was adaptive, whereas coding mutations in E2, p7......, and NS2 all increased virus production. A single-cycle replication assay in CD81-deficient cells was developed to study more precisely the effect of the adaptive mutations. The E2 mutation had minimal effect on the amount of infectious virus released but probably enhanced entry into cells. In contrast...

  10. The Culture Repopulation Ability (CRA) Assay and Incubation in Low Oxygen to Test Antileukemic Drugs on Imatinib-Resistant CML Stem-Like Cells.

    Science.gov (United States)

    Cheloni, Giulia; Tanturli, Michele

    2016-01-01

    Chronic myeloid leukemia (CML) is a stem cell-driven disorder caused by the BCR/Abl oncoprotein, a constitutively active tyrosine kinase (TK). Chronic-phase CML patients are treated with impressive efficacy with TK inhibitors (TKi) such as imatinib mesylate (IM). However, rather than definitively curing CML, TKi induces a state of minimal residual disease, due to the persistence of leukemia stem cells (LSC) which are insensitive to this class of drugs. LSC persistence may be due to different reasons, including the suppression of BCR/Abl oncoprotein. It has been shown that this suppression follows incubation in low oxygen under appropriate culture conditions and incubation times.Here we describe the culture repopulation ability (CRA) assay, a non-clonogenic assay capable - together with incubation in low oxygen - to reveal in vitro stem cells endowed with marrow repopulation ability (MRA) in vivo. The CRA assay can be used, before moving to animal tests, as a simple and reliable method for the prescreening of drugs potentially active on CML and other leukemias with respect to their activity on the more immature leukemia cell subsets. PMID:27581140

  11. [Cell cultures].

    Science.gov (United States)

    Cipro, Simon; Groh, Tomáš

    2014-01-01

    Cell or tissue cultures (both terms are interchangeable) represent a complex process by which eukaryotic cells are maintained in vitro outside their natural environment. They have a broad usage covering not only scientific field but also diagnostic one since they represent the most important way of monoclonal antibodies production which are used for both diagnostic and therapeutic purposes. Cell cultures are also used as a "cultivation medium" in virology and for establishing proliferating cells in cytodiagnostics. They are well-established and easy-to-handle models in the area of research, e.g. as a precious source of nucleic acids or proteins. This paper briefly summarizes their importance and methods as well as the pitfalls of the cultivation and new trends in this field. PMID:24624984

  12. Cytotoxicity test of 40, 50 and 60% citric acid as dentin conditioner by using MTT assay on culture cell line

    Directory of Open Access Journals (Sweden)

    Christian Khoswanto

    2008-09-01

    Full Text Available Background: Open dentin is always covered by smear layer, therefore before restoration is performed, cavity or tooth which has been prepared should be clean from dirt. The researchers suggested that clean dentin surface would reach effective adhesion between resin and tooth structure, therefore dentin conditioner like citric acid was used to reach the condition. Even though citric acid is not strong acid but it can be very erosive to oral mucous. Several requirements should be fulfilled for dental product such as non toxic, non irritant, biocompatible and should not have negative effect against local, systemic or biological environment. Cytotoxicity test was apart of biomaterial evaluation and needed for standard screening. Purpose: This study was to know the cytotoxicity of 40, 50, 60% citric acid as dentin conditioner using MTT assay. Method: This study is an experimental research using the Post-Test Only Control Group Design. Six samples of each 40, 50 and 60% citric acid for citotoxicity test using MTT assay. The density of optic formazan indicated the number of living cells. All data were statistically analyzed by one way ANOVA. Result: The percentage of living cells in 40, 50 and 60% citric acid were 95.14%, 93.42% and 93.14%. Conclusion: Citric acid is non toxic and safe to be used as dentine conditioner.

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

    International Nuclear Information System (INIS)

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

  14. Comparison of a frozen human foreskin fibroblast cell assay to an enzyme immunoassay and toxigenic culture for the detection of toxigenic Clostridium difficile.

    Science.gov (United States)

    Strachan, Alastair J; Evans, Natalie E; Williams, O Martin; Spencer, Robert C; Greenwood, Rosemary; Probert, Chris J

    2013-01-01

    This study set out to validate the Hs27 ReadyCell assay (RCCNA) as an alternative CCNA method compared against a commonly used commercial enzyme immunoassay (EIA) method and toxigenic culture (TC) reference standard. A total of 860 samples were identified from those submitted to the Health Protection Agency microbiology laboratories over a 30-week period. RCCNA performed much better than EIA when using TC as a gold standard, with sensitivities of 90.8% versus 78.6% and positive predictive value of 87.3% to 81.9%, respectively. The Hs27 Human Foreskin Fibroblast ReadyCells are an easy-to-use and a sensitive CCNA method for the detection of toxigenic Clostridium difficile directly from stool. A turnaround time of up to 48 h for a negative result and possible need for repeat testing make it an unsuitable method to be used in most clinical laboratory setting. PMID:23107315

  15. Comparison of a frozen human foreskin fibroblast cell assay to an enzyme immunoassay and toxigenic culture for the detection of toxigenic Clostridium difficile.

    Science.gov (United States)

    Strachan, Alastair J; Evans, Natalie E; Williams, O Martin; Spencer, Robert C; Greenwood, Rosemary; Probert, Chris J

    2013-01-01

    This study set out to validate the Hs27 ReadyCell assay (RCCNA) as an alternative CCNA method compared against a commonly used commercial enzyme immunoassay (EIA) method and toxigenic culture (TC) reference standard. A total of 860 samples were identified from those submitted to the Health Protection Agency microbiology laboratories over a 30-week period. RCCNA performed much better than EIA when using TC as a gold standard, with sensitivities of 90.8% versus 78.6% and positive predictive value of 87.3% to 81.9%, respectively. The Hs27 Human Foreskin Fibroblast ReadyCells are an easy-to-use and a sensitive CCNA method for the detection of toxigenic Clostridium difficile directly from stool. A turnaround time of up to 48 h for a negative result and possible need for repeat testing make it an unsuitable method to be used in most clinical laboratory setting.

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

    Directory of Open Access Journals (Sweden)

    Sebastian Gisder

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

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

    Science.gov (United States)

    Gisder, Sebastian; Genersch, Elke

    2015-01-01

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

  18. 21 CFR 866.2350 - Microbiological assay culture medium.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Microbiological assay culture medium. 866.2350... Microbiological assay culture medium. (a) Identification. A microbiological assay culture medium is a device that... (general controls). The device is exempt from the premarket notification procedures in subpart E of...

  19. Detection of toxigenic Clostridium difficile: comparison of the cell culture neutralization, Xpert C. difficile, Xpert C. difficile/Epi, and Illumigene C. difficile assays.

    Science.gov (United States)

    Pancholi, P; Kelly, C; Raczkowski, M; Balada-Llasat, J M

    2012-04-01

    Clostridium difficile is the most important cause of nosocomial diarrhea. Several laboratory techniques are available to detect C. difficile toxins or the genes that encode them in fecal samples. We evaluated the Xpert C. difficile and Xpert C. difficile/Epi (Cepheid, CA) that detect the toxin B gene (tcdB) and tcdB, cdt, and a deletion in tcdC associated with the 027/NAP1/BI strain, respectively, by real-time PCR, and the Illumigene C. difficile (Meridian Bioscience, Inc.) that detects the toxin A gene (tcdA) by loop-mediated isothermal amplification in stool specimens. Toxigenic culture was used as the reference method for discrepant stool specimens. Two hundred prospective and fifty retrospective diarrheal stool specimens were tested simultaneously by the cell cytotoxin neutralization assay (CCNA) and the Xpert C. difficile, Xpert C. difficile/Epi, and Illumigene C. difficile assays. Of the 200 prospective stools tested, 10.5% (n = 23) were determined to be positive by CCNA, 17.5% (n = 35) were determined to be positive by Illumigene C. difficile, and 21.5% (n = 43) were determined to be positive by Xpert C. difficile and Xpert C. difficile/Epi. Of the 50 retrospective stools, previously determined to be positive by CCNA, 94% (n = 47) were determined to be positive by Illumigene C. difficile and 100% (n = 50) were determined to be positive by Xpert C. difficile and Xpert C. difficile/Epi. Of the 11 discrepant results (i.e., negative by Illumigene C. difficile but positive by Xpert C. difficile and Xpert C. difficile/Epi), all were determined to be positive by the toxigenic culture. A total of 21% of the isolates were presumptively identified by the Xpert C. difficile/Epi as the 027/NAP1/BI strain. The Xpert C. difficile and Xpert C. difficile/Epi assays were the most sensitive, rapid, and easy-to use assays for the detection of toxigenic C. difficile in stool specimens. PMID:22278839

  20. Detection of toxigenic Clostridium difficile: comparison of the cell culture neutralization, Xpert C. difficile, Xpert C. difficile/Epi, and Illumigene C. difficile assays.

    Science.gov (United States)

    Pancholi, P; Kelly, C; Raczkowski, M; Balada-Llasat, J M

    2012-04-01

    Clostridium difficile is the most important cause of nosocomial diarrhea. Several laboratory techniques are available to detect C. difficile toxins or the genes that encode them in fecal samples. We evaluated the Xpert C. difficile and Xpert C. difficile/Epi (Cepheid, CA) that detect the toxin B gene (tcdB) and tcdB, cdt, and a deletion in tcdC associated with the 027/NAP1/BI strain, respectively, by real-time PCR, and the Illumigene C. difficile (Meridian Bioscience, Inc.) that detects the toxin A gene (tcdA) by loop-mediated isothermal amplification in stool specimens. Toxigenic culture was used as the reference method for discrepant stool specimens. Two hundred prospective and fifty retrospective diarrheal stool specimens were tested simultaneously by the cell cytotoxin neutralization assay (CCNA) and the Xpert C. difficile, Xpert C. difficile/Epi, and Illumigene C. difficile assays. Of the 200 prospective stools tested, 10.5% (n = 23) were determined to be positive by CCNA, 17.5% (n = 35) were determined to be positive by Illumigene C. difficile, and 21.5% (n = 43) were determined to be positive by Xpert C. difficile and Xpert C. difficile/Epi. Of the 50 retrospective stools, previously determined to be positive by CCNA, 94% (n = 47) were determined to be positive by Illumigene C. difficile and 100% (n = 50) were determined to be positive by Xpert C. difficile and Xpert C. difficile/Epi. Of the 11 discrepant results (i.e., negative by Illumigene C. difficile but positive by Xpert C. difficile and Xpert C. difficile/Epi), all were determined to be positive by the toxigenic culture. A total of 21% of the isolates were presumptively identified by the Xpert C. difficile/Epi as the 027/NAP1/BI strain. The Xpert C. difficile and Xpert C. difficile/Epi assays were the most sensitive, rapid, and easy-to use assays for the detection of toxigenic C. difficile in stool specimens.

  1. Characterization of Listeria monocytogenes isolates of food and human origins from Brazil using molecular typing procedures and in vitro cell culture assays.

    Science.gov (United States)

    Bueno, Valter F; Banerjee, Pratik; Banada, Padmapriya P; José de Mesquita, Albenones; Lemes-Marques, Eneida G; Bhunia, Arun K

    2010-02-01

    The spreading of diseases through foods is a worldwide concern. Here, molecular and in vitro cell-culture assays were employed to characterize 63 Brazilian Listeria monocytogenes isolates (food, 47; clinical, 16). Serotype 4b was the most predominant (49%) followed by (1/2)b (30%), (1/2)a (10%), (1/2)c (6%), 3c (3%) and 3b (2%). Ribotyping yielded 17 ribopatterns, which were grouped into four phylogenetic clusters. Cluster A comprised of 39/63 isolates primarily of food origin, and clusters B, C and D contained both food and clinical isolates. Isolates were positive for virulence determinants prfA, hlyA and inlA: clinical isolates were more invasive to Caco-2 cells and expressed high levels of inlA transcripts than the food isolates. Highly invasive isolates also provoked more Ped-2E9 cells to die by apoptosis than the weakly-invasive strains. These data demonstrate a strong genetic relatedness among clinical and food isolates and suggest transmission of a subset of L. monocytogenes strains from food to humans.

  2. Live-cell luciferase assay of drug resistant cells

    OpenAIRE

    sprotocols

    2015-01-01

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

  3. In Situ Proximity Ligation Assay (PLA) Analysis of Protein Complexes Formed Between Golgi-Resident, Glycosylation-Related Transporters and Transferases in Adherent Mammalian Cell Cultures.

    Science.gov (United States)

    Maszczak-Seneczko, Dorota; Sosicka, Paulina; Olczak, Teresa; Olczak, Mariusz

    2016-01-01

    In situ proximity ligation assay (PLA) is a novel, revolutionary technique that can be employed to visualize protein complexes in fixed cells and tissues. This approach enables demonstration of close (i.e., up to 40 nm) proximity between any two proteins of interest that can be detected using a pair of specific antibodies that have been raised in distinct species. Primary antibodies bound to the target proteins are subsequently recognized by two PLA probes, i.e., secondary antibodies conjugated with oligonucleotides that anneal when brought into close proximity in the presence of two connector oligonucleotides and a DNA ligase forming a circular DNA molecule. In the next step, the resulting circular DNA is amplified by a rolling circle polymerase. Finally, fluorescent oligonucleotide probes hybridize to complementary fragments of the amplified DNA molecule, forming a typical, spot-like pattern of PLA signal that reflects subcellular localization of protein complexes. Here we describe the use of in situ PLA in adherent cultures of mammalian cells in order to visualize interactions between Golgi-resident, functionally related glycosyltransferases and nucleotide sugar transporters relevant to N-glycan biosynthesis. PMID:27632007

  4. Optimization of cell motility evaluation in scratch assay

    Directory of Open Access Journals (Sweden)

    Gotsulyak N. Ya.

    2014-05-01

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

  5. Proteasome Assay in Cell Lysates

    Science.gov (United States)

    Maher, Pamela

    2016-01-01

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

  6. Fish Stem Cell Cultures

    OpenAIRE

    Ni Hong, Zhendong Li, Yunhan Hong

    2011-01-01

    Stem cells have the potential for self-renewal and differentiation. First stem cell cultures were derived 30 years ago from early developing mouse embryos. These are pluripotent embryonic stem (ES) cells. Efforts towards ES cell derivation have been attempted in other mammalian and non-mammalian species. Work with stem cell culture in fish started 20 years ago. Laboratory fish species, in particular zebrafish and medaka, have been the focus of research towards stem cell cultures. Medaka is th...

  7. An Experiment on Standardized Cell Culture Assay in Assessing the Activities of Composite Artemisia Capillaris Tablets against Hepatitis B Virus Replication in vitro

    Institute of Scientific and Technical Information of China (English)

    HAN Jin; ZHAO Yan-ling; SHAN Li-mei; HUANG Feng-jiao; XIAO Xiao-he

    2005-01-01

    Objective:To explore the activities of Composite Artemisia Capillaris Tablet (复方茵陈片,CACT) against hepatitis B virus replication in vitro. Methods: By means of radioimmunoassay (RIA), Dot blot and Southern blot, the surface and e antigen production of 2.2.15 cells, HBV DNA in 2.2.15 cell culture medium and that in 2.2.15 cells were examined respectively. Results: HBsAg, HBeAg values of 2.2.15 cells treated by CACT were lower than those of the control, the HBV DNA quantities in culture medium and in 2.2.15 cells decreased as compared with those cells with no treatment by CACT given to them. Conclusion:CACT could inhibit HBV DNA replication, showing its potential antiviral activity in hepatitis B treatment.

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

    Science.gov (United States)

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

    2013-04-01

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

  9. Optimizing stem cell culture.

    Science.gov (United States)

    van der Sanden, Boudewijn; Dhobb, Mehdi; Berger, François; Wion, Didier

    2010-11-01

    Stem cells always balance between self-renewal and differentiation. Hence, stem cell culture parameters are critical and need to be continuously refined according to progress in our stem cell biology understanding and the latest technological developments. In the past few years, major efforts have been made to define more precisely the medium composition in which stem cells grow or differentiate. This led to the progressive replacement of ill-defined additives such as serum or feeder cell layers by recombinant cytokines or growth factors. Another example is the control of the oxygen pressure. For many years cell cultures have been done under atmospheric oxygen pressure which is much higher than the one experienced by stem cells in vivo. A consequence of cell metabolism is that cell culture conditions are constantly changing. Therefore, the development of high sensitive monitoring processes and control algorithms is required for ensuring cell culture medium homeostasis. Stem cells also sense the physical constraints of their microenvironment. Rigidity, stiffness, and geometry of the culture substrate influence stem cell fate. Hence, nanotopography is probably as important as medium formulation in the optimization of stem cell culture conditions. Recent advances include the development of synthetic bioinformative substrates designed at the micro- and nanoscale level. On going research in many different fields including stem cell biology, nanotechnology, and bioengineering suggest that our current way to culture cells in Petri dish or flasks will soon be outdated as flying across the Atlantic Ocean in the Lindbergh's plane. PMID:20803548

  10. Direct Detection and Identification of Enteroviruses from Faeces of Healthy Nigerian Children Using a Cell-Culture Independent RT-Seminested PCR Assay

    Science.gov (United States)

    Adewumi, Moses Olubusuyi; Coker, Bamidele Atinuke; Nudamajo, Felix Yasha; Adeniji, Johnson Adekunle

    2016-01-01

    Recently, a cell-culture independent protocol for detection of enteroviruses from clinical specimen was recommended by the WHO for surveillance alongside the previously established protocols. Here, we investigated whether this new protocol will show the same enterovirus diversity landscape as the established cell-culture dependent protocols. Faecal samples were collected from sixty apparently healthy children in Ibadan, Nigeria. Samples were resuspended in phosphate buffered saline, RNA was extracted, and the VP1 gene was amplified using WHO recommended RT-snPCR protocol. Amplicons were sequenced and sequences subjected to phylogenetic analysis. Fifteen (25%) of the 60 samples yielded the expected band size. Of the 15 amplicons sequenced, 12 were exploitable. The remaining 3 had electropherograms with multiple peaks and were unexploitable. Eleven of the 12 exploitable sequences were identified as Coxsackievirus A1 (CVA1), CVA3, CVA4, CVA8, CVA20, echovirus 32 (E32), enterovirus 71 (EV71), EVB80, and EVC99. Subsequently, the last exploitable sequence was identified as enterobacteriophage baseplate gene by nucleotide BLAST. The results of this study document the first description of molecular sequence data on CVA1, CVA8, and E32 strains present in Nigeria. The result further showed that species A enteroviruses were more commonly detected in the region when cell-culture bias is bypassed. PMID:27087810

  11. Direct Detection and Identification of Enteroviruses from Faeces of Healthy Nigerian Children Using a Cell-Culture Independent RT-Seminested PCR Assay.

    Science.gov (United States)

    Faleye, Temitope Oluwasegun Cephas; Adewumi, Moses Olubusuyi; Coker, Bamidele Atinuke; Nudamajo, Felix Yasha; Adeniji, Johnson Adekunle

    2016-01-01

    Recently, a cell-culture independent protocol for detection of enteroviruses from clinical specimen was recommended by the WHO for surveillance alongside the previously established protocols. Here, we investigated whether this new protocol will show the same enterovirus diversity landscape as the established cell-culture dependent protocols. Faecal samples were collected from sixty apparently healthy children in Ibadan, Nigeria. Samples were resuspended in phosphate buffered saline, RNA was extracted, and the VP1 gene was amplified using WHO recommended RT-snPCR protocol. Amplicons were sequenced and sequences subjected to phylogenetic analysis. Fifteen (25%) of the 60 samples yielded the expected band size. Of the 15 amplicons sequenced, 12 were exploitable. The remaining 3 had electropherograms with multiple peaks and were unexploitable. Eleven of the 12 exploitable sequences were identified as Coxsackievirus A1 (CVA1), CVA3, CVA4, CVA8, CVA20, echovirus 32 (E32), enterovirus 71 (EV71), EVB80, and EVC99. Subsequently, the last exploitable sequence was identified as enterobacteriophage baseplate gene by nucleotide BLAST. The results of this study document the first description of molecular sequence data on CVA1, CVA8, and E32 strains present in Nigeria. The result further showed that species A enteroviruses were more commonly detected in the region when cell-culture bias is bypassed. PMID:27087810

  12. Direct Detection and Identification of Enteroviruses from Faeces of Healthy Nigerian Children Using a Cell-Culture Independent RT-Seminested PCR Assay

    Directory of Open Access Journals (Sweden)

    Temitope Oluwasegun Cephas Faleye

    2016-01-01

    Full Text Available Recently, a cell-culture independent protocol for detection of enteroviruses from clinical specimen was recommended by the WHO for surveillance alongside the previously established protocols. Here, we investigated whether this new protocol will show the same enterovirus diversity landscape as the established cell-culture dependent protocols. Faecal samples were collected from sixty apparently healthy children in Ibadan, Nigeria. Samples were resuspended in phosphate buffered saline, RNA was extracted, and the VP1 gene was amplified using WHO recommended RT-snPCR protocol. Amplicons were sequenced and sequences subjected to phylogenetic analysis. Fifteen (25% of the 60 samples yielded the expected band size. Of the 15 amplicons sequenced, 12 were exploitable. The remaining 3 had electropherograms with multiple peaks and were unexploitable. Eleven of the 12 exploitable sequences were identified as Coxsackievirus A1 (CVA1, CVA3, CVA4, CVA8, CVA20, echovirus 32 (E32, enterovirus 71 (EV71, EVB80, and EVC99. Subsequently, the last exploitable sequence was identified as enterobacteriophage baseplate gene by nucleotide BLAST. The results of this study document the first description of molecular sequence data on CVA1, CVA8, and E32 strains present in Nigeria. The result further showed that species A enteroviruses were more commonly detected in the region when cell-culture bias is bypassed.

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

    OpenAIRE

    sprotocols

    2015-01-01

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

  14. HTS compatible assay for antioxidative agents using primary cultured hepatocytes.

    Science.gov (United States)

    Gaunitz, Frank; Heise, Kerstin

    2003-06-01

    We have used primary cultured rat hepatocytes to establish a system that is compatible with HTS for screening substance libraries for biologically active compounds. The hepatocytes were treated with t-BHP to induce oxidative stress, leading to the formation ROS. The involvement of ROS in oxidative stress and pathological alterations has been of major interest in recent years, and there is great demand to identify new compounds with antioxidant potential. In most HTS programs each compound is tested in duplicate, and may only be tested once. Because of this it is important to develop assays that can identify candidate compounds accurately and with high confidence. Using newly available cell-based assay systems, we have developed a system that can detect active compounds (hits) with a high degree of confidence. As an example of an agent that can be detected from a substance library, we analyzed the effect of fisetin as an antioxidative compound using this system. All measurements were performed using the newly developed and highly versatile Multilabel-Reader Mithras LB 940 (Berthold Technologies, Bad Wildbad, Germany). The data presented show that all Z' factors determined were highly reliable. Although the protocol is primarily designed to screen for substances with antioxidative potential, it can easily be adapted to screen for other biologically active substances.

  15. Comet assay on tetraploid yeast cells

    DEFF Research Database (Denmark)

    Rank, Jette; Syberg, Kristian; Jensen, Klara

    2009-01-01

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

  16. Assay of mast cell mediators

    DEFF Research Database (Denmark)

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

    2015-01-01

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

  17. Bacterial cell culture

    OpenAIRE

    sprotocols

    2014-01-01

    ### Materials 1. Glass culture tubes with metal caps and labels - Growth medium, from media room or customized - Glass pipette tubes - Parafilm ### Equipment 1. Vortexer - Fireboy or Bunsen burner - Motorized pipette - Micropipettes and sterile tips ### Procedure For a typical liquid culture, use 5 ml of appropriate medium. The amount in each tube does not have to be exact if you are just trying to culture cells for their precious DNA. 1. Streak an a...

  18. Fish Stem Cell Cultures

    Directory of Open Access Journals (Sweden)

    Ni Hong, Zhendong Li, Yunhan Hong

    2011-01-01

    Full Text Available Stem cells have the potential for self-renewal and differentiation. First stem cell cultures were derived 30 years ago from early developing mouse embryos. These are pluripotent embryonic stem (ES cells. Efforts towards ES cell derivation have been attempted in other mammalian and non-mammalian species. Work with stem cell culture in fish started 20 years ago. Laboratory fish species, in particular zebrafish and medaka, have been the focus of research towards stem cell cultures. Medaka is the second organism that generated ES cells and the first that gave rise to a spermatogonial stem cell line capable of test-tube sperm production. Most recently, the first haploid stem cells capable of producing whole animals have also been generated from medaka. ES-like cells have been reported also in zebrafish and several marine species. Attempts for germline transmission of ES cell cultures and gene targeting have been reported in zebrafish. Recent years have witnessed the progress in markers and procedures for ES cell characterization. These include the identification of fish homologs/paralogs of mammalian pluripotency genes and parameters for optimal chimera formation. In addition, fish germ cell cultures and transplantation have attracted considerable interest for germline transmission and surrogate production. Haploid ES cell nuclear transfer has proven in medaka the feasibility of semi-cloning as a novel assisted reproductive technology. In this special issue on “Fish Stem Cells and Nuclear Transfer”, we will focus our review on medaka to illustrate the current status and perspective of fish stem cells in research and application. We will also mention semi-cloning as a new development to conventional nuclear transfer.

  19. Fish stem cell cultures.

    Science.gov (United States)

    Hong, Ni; Li, Zhendong; Hong, Yunhan

    2011-04-13

    Stem cells have the potential for self-renewal and differentiation. First stem cell cultures were derived 30 years ago from early developing mouse embryos. These are pluripotent embryonic stem (ES) cells. Efforts towards ES cell derivation have been attempted in other mammalian and non-mammalian species. Work with stem cell culture in fish started 20 years ago. Laboratory fish species, in particular zebrafish and medaka, have been the focus of research towards stem cell cultures. Medaka is the second organism that generated ES cells and the first that gave rise to a spermatogonial stem cell line capable of test-tube sperm production. Most recently, the first haploid stem cells capable of producing whole animals have also been generated from medaka. ES-like cells have been reported also in zebrafish and several marine species. Attempts for germline transmission of ES cell cultures and gene targeting have been reported in zebrafish. Recent years have witnessed the progress in markers and procedures for ES cell characterization. These include the identification of fish homologs/paralogs of mammalian pluripotency genes and parameters for optimal chimera formation. In addition, fish germ cell cultures and transplantation have attracted considerable interest for germline transmission and surrogate production. Haploid ES cell nuclear transfer has proven in medaka the feasibility of semi-cloning as a novel assisted reproductive technology. In this special issue on "Fish Stem Cells and Nuclear Transfer", we will focus our review on medaka to illustrate the current status and perspective of fish stem cells in research and application. We will also mention semi-cloning as a new development to conventional nuclear transfer.

  20. Comet assay on mice testicular cells

    Directory of Open Access Journals (Sweden)

    Anoop Kumar Sharma

    2015-05-01

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

  1. Optimizing stem cell culture.

    OpenAIRE

    van der Sanden, Boudewijn; Dhobb, Mehdi; Berger, François; Wion, Didier

    2010-01-01

    International audience Stem cells always balance between self-renewal and differentiation. Hence, stem cell culture parameters are critical and need to be continuously refined according to progress in our stem cell biology understanding and the latest technological developments. In the past few years, major efforts have been made to define more precisely the medium composition in which stem cells grow or differentiate. This led to the progressive replacement of ill-defined additives such a...

  2. Digital Microfluidic Cell Culture.

    Science.gov (United States)

    Ng, Alphonsus H C; Li, Bingyu Betty; Chamberlain, M Dean; Wheeler, Aaron R

    2015-01-01

    Digital microfluidics (DMF) is a droplet-based liquid-handling technology that has recently become popular for cell culture and analysis. In DMF, picoliter- to microliter-sized droplets are manipulated on a planar surface using electric fields, thus enabling software-reconfigurable operations on individual droplets, such as move, merge, split, and dispense from reservoirs. Using this technique, multistep cell-based processes can be carried out using simple and compact instrumentation, making DMF an attractive platform for eventual integration into routine biology workflows. In this review, we summarize the state-of-the-art in DMF cell culture, and describe design considerations, types of DMF cell culture, and cell-based applications of DMF. PMID:26643019

  3. Evaluation of the suitability of six host genes as internal control in real-time RT-PCR assays in chicken embryo cell cultures infected with infectious bursal disease virus

    DEFF Research Database (Denmark)

    Li, Yiping; Bang, Dang Duong; Handberg, Kurt;

    2005-01-01

    and GAPDH had a lower expression level in CE cell cultures. Also, beta-actin showed no significant variation in both normalized and non-normalized assays and virus dose-independent of inoculation, while other genes did. beta-Actin was further successfully used as an internal control to quantitate Bursine-2......Infectious bursal disease virus (IBDV) can cause disease in chickens characterized by immunosuppression and high mortality. Currently, real-time RT-PCR has been used to quantitate virus-specific RNA and to better understand host response to infection. However, normalization of quantitative real...... following a 7-day IBDV infection. The CE cells were inoculated with various multiplicity of infection (MOI) of IBDV vaccine strain Bursine-2, the expression of genes was measured by quantitative real-time PCR-based on cDNA synthesized from either normalized (100 ng) or non-normalized (10 mu l) total RNA...

  4. Comet assay on mice testicular cells

    OpenAIRE

    Anoop Kumar Sharma

    2015-01-01

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

  5. Assay of neutralizing antibody against variola virus by the degree of focus reduction on HeLa cell cultures and its application to revaccination with smallpox vaccines of various potencies.

    Science.gov (United States)

    Kitamura, T; Shinjo, N

    1972-01-01

    A method for assaying neutralizing antibody against variola virus was established by focus counting on HeLa cell cultures. The ND(50) titre, i.e., the serum dilution endpoint to give a 50% reduction in the number of foci, was determined with excellent reproducibility.Groups of students 19-20 years of age were revaccinated by the multiple pressure method with serial 10-fold dilutions of a smallpox vaccine and their neutralizing antibody response was assayed by the focus counting assay system and was related to the local skin reactions on the seventh day after inoculation and to the potency of the vaccine administered. There was a significant rise in the antibody level even after inoculation with a vaccine whose potency was as low as 1.3 x 10(5) pock-forming units/ml. In general, the rise in the log antibody level was proportional to the diameter of the reddening, but a significant rise was found among individuals who had no detectable skin reaction. The skin reaction was greater among individuals with a lower initial antibody level when the vaccine administered had a potency lower than 1.3 x 10(6) pock-forming units/ml.

  6. High-Throughput Cell Toxicity Assays.

    Science.gov (United States)

    Murray, David; McWilliams, Lisa; Wigglesworth, Mark

    2016-01-01

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

  7. Human hematopoietic cell culture, transduction, and analyses

    DEFF Research Database (Denmark)

    Bonde, Jesper; Wirthlin, Louisa; Kohn, Donald B;

    2008-01-01

    This unit provides methods for introducing genes into human hematopoietic progenitor cells. The Basic Protocol describes isolation of CD34(+) cells, transduction of these cells with a retroviral vector on fibronectin-coated plates, assaying the efficiency of transduction, and establishing long......-term cultures. Support protocols describe methods for maintenance of vector-producing fibroblasts (VPF) and supernatant collection from these cells, screening medium components for the ability to support hematopoietic cell growth, and establishing colonies from long-term cultures. Other protocols provide PCR...

  8. Cell Culturing of Cytoskeleton

    Science.gov (United States)

    2004-01-01

    Biomedical research offers hope for a variety of medical problems, from diabetes to the replacement of damaged bone and tissues. Bioreactors, which are used to grow cells and tissue cultures, play a major role in such research and production efforts. Cell culturing, such as this bone cell culture, is an important part of biomedical research. The BioDyn payload includes a tissue engineering investigation. The commercial affiliate, Millenium Biologix, Inc. has been conducting bone implant experiments to better understand how synthetic bone can be used to treat bone-related illnesses and bone damaged in accidents. On STS-95, the BioDyn payload will include a bone cell culture aimed to help develop this commercial synthetic bone product. Millenium Biologix, Inc. is exploring the potential for making human bone implantable materials by seeding its proprietary artificial scaffold material with human bone cells. The product of this tissue engineering experiment using the Bioprocessing Modules (BPMs) on STS-95 is space-grown bone implants, which could have potential for dental implants, long bone grafts, and coating for orthopedic implants such as hip replacements.

  9. Oscillating Cell Culture Bioreactor

    Science.gov (United States)

    Freed, Lisa E.; Cheng, Mingyu; Moretti, Matteo G.

    2010-01-01

    To better exploit the principles of gas transport and mass transport during the processes of cell seeding of 3D scaffolds and in vitro culture of 3D tissue engineered constructs, the oscillatory cell culture bioreactor provides a flow of cell suspensions and culture media directly through a porous 3D scaffold (during cell seeding) and a 3D construct (during subsequent cultivation) within a highly gas-permeable closed-loop tube. This design is simple, modular, and flexible, and its component parts are easy to assemble and operate, and are inexpensive. Chamber volume can be very low, but can be easily scaled up. This innovation is well suited to work with different biological specimens, particularly with cells having high oxygen requirements and/or shear sensitivity, and different scaffold structures and dimensions. The closed-loop changer is highly gas permeable to allow efficient gas exchange during the cell seeding/culturing process. A porous scaffold, which may be seeded with cells, is fixed by means of a scaffold holder to the chamber wall with scaffold/construct orientation with respect to the chamber determined by the geometry of the scaffold holder. A fluid, with/without biological specimens, is added to the chamber such that all, or most, of the air is displaced (i.e., with or without an enclosed air bubble). Motion is applied to the chamber within a controlled environment (e.g., oscillatory motion within a humidified 37 C incubator). Movement of the chamber induces relative motion of the scaffold/construct with respect to the fluid. In case the fluid is a cell suspension, cells will come into contact with the scaffold and eventually adhere to it. Alternatively, cells can be seeded on scaffolds by gel entrapment prior to bioreactor cultivation. Subsequently, the oscillatory cell culture bioreactor will provide efficient gas exchange (i.e., of oxygen and carbon dioxide, as required for viability of metabolically active cells) and controlled levels of fluid

  10. Enzyme-linked immunosorbent assay detection of trichothecenes produced by the Bioherbicide Myrothecium verrucaria in cell cultures, extracts, and plant tissues

    Science.gov (United States)

    Commercially available enzyme linked immunosorbent assay (ELISA) plates for trichothecene detection, possessing cross-reactivity with several trichothecene mycotoxins (e.g., verrucarin A, and J, roridin A, L-2, E, and H), were tested for their ability to detect trichothecenes produced by a strain of...

  11. Application of long-term cultured interferon-gamma enzyme-linked immunospot assay for assessing effector and memory T cell responses in cattle

    Science.gov (United States)

    Effector and memory T cells are generated through developmental programing of naïve cells following antigen recognition. If the infection is controlled, up to 95% of the T cells generated during the expansion phase are eliminated (i.e., contraction phase) and memory T cells remain, sometimes for a l...

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

    Science.gov (United States)

    Shabrish, Snehal; Gupta, Maya; Madkaikar, Manisha

    2016-01-01

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

  13. 21 CFR 864.7100 - Red blood cell enzyme assay.

    Science.gov (United States)

    2010-04-01

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

  14. Perfusion based cell culture chips

    DEFF Research Database (Denmark)

    Heiskanen, Arto; Emnéus, Jenny; Dufva, Martin

    2010-01-01

    Performing cell culture in miniaturized perfusion chambers gives possibilities to experiment with cells under near in vivo like conditions. In contrast to traditional batch cultures, miniaturized perfusion systems provide precise control of medium composition, long term unattended cultures...... and tissue like structuring of the cultures. However, as this chapter illustrates, many issues remain to be identified regarding perfusion cell culture such as design, material choice and how to use these systems before they will be widespread amongst biomedical researchers....

  15. Evaluation of the radioprotective effect of Carissa carandas Linn. fruit extract in cultured human peripheral blood lymphocytes exposed to electron beam radiation by Single Cell Gel Electrophoresis (Comet Assay)

    International Nuclear Information System (INIS)

    Radiation is a well-known inducer of free radicals and compounds that can scavenge free radicals may reduce radiation-induced DNA damage. Carissa carandas commonly known as Karanda belongs to family Apocynaceae. Traditionally, whole plant and its parts were used in the treatment of various ailments. The aim of the present study was to assess the radioprotective effect of ethanolic extract of Carissa carandas fruit (ECF) in cultured human peripheral blood lymphocytes (HPBLs) by comet assay. The optimum protective dose of the extract was selected by treating HPBLs with 50 and 100 μg/ml ECF after exposure to 2 Gy electron beam radiation and then evaluating the frequency of DNA damage in HPBLs using Single cell gel electrophoresis (Comet Assay). To understand the mechanism of action of ECF separate experiments were conducted to evaluate the free radical scavenging of DPPH, and Fe3+ in vitro. ECF was found to inhibit free radicals in a dose dependent manner up to a dose of 1000 μg/ml for the majority of radicals as observed by the in vitro free radical scavenging assays. The irradiation of HPBLs with 2 Gy dose of electron beam radiation caused an increase in the frequency of DNA damage while treatment of HPBLs with different concentrations of ECF reduced the frequency of DNA damage significantly with the greatest reduction being observed for 100 μg/ml when compared with the irradiated control. Our study demonstrates the potential of ECF as an effective agent against radiation induced DNA damage. (author)

  16. Microfluidic Cell Culture Device

    Science.gov (United States)

    Takayama, Shuichi (Inventor); Cabrera, Lourdes Marcella (Inventor); Heo, Yun Seok (Inventor); Smith, Gary Daniel (Inventor)

    2014-01-01

    Microfluidic devices for cell culturing and methods for using the same are disclosed. One device includes a substrate and membrane. The substrate includes a reservoir in fluid communication with a passage. A bio-compatible fluid may be added to the reservoir and passage. The reservoir is configured to receive and retain at least a portion of a cell mass. The membrane acts as a barrier to evaporation of the bio-compatible fluid from the passage. A cover fluid may be added to cover the bio-compatible fluid to prevent evaporation of the bio-compatible fluid.

  17. Survival response of RIF tumor cells to heat-x-radiation combinations: Parallel measurements in culture and by the excision assay

    International Nuclear Information System (INIS)

    The cytotoxicity of heat-radiation (hX) combinations in vivo may differ from that measured in vitro. The authors have used the RIF tumor, grown in mouse feet, to compare the survival response after in situ hX-treatments with identical hX in vitro. The radiation survival curve, determined by the excision assay showed a slightly larger D/sub o/ than that measured in vitro (250, 200 rad, respective) and survival measurements appeared independent of excision time after irradiation. The 450-heat survival curve was similar in both assays, but only when the excision followed immediately after h. A 24-hr delayed excision removed the shoulder and lowered survival 30-fold after either 20 or 30 min, 450. Similar survival values were measured after 10 min, 450+X (hX) in vitro and with immediate excision, although the excision survival curve had no shoulder and a D/sub o/ of 180 rad vs. 120 rad in vitro. The survival curve with delayed excision (24 hr) also appeared as a simple exponential curve with an apparent D/sub o/ of 310 rad (n=0.02). Two fractions of combined hX, separated by 24 hr (hx+24+hX), yielded D/sub o/=90 rad, D/sub q/=230 rad in vitro but 370 and 400 rad, respectively, when measured by delayed excision. The apparent radioresistance in vivo is consistent with data by Song of increased hypoxic fractions after heating in vivo and argues against combining hx in every fraction for optimal tumor control

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

    International Nuclear Information System (INIS)

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

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

    OpenAIRE

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

    2012-01-01

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

  20. The Extended Cell Panel Assay Characterizes the Relationship of Prion Strains RML, 79A, and 139A and Reveals Conversion of 139A to 79A-Like Prions in Cell Culture

    OpenAIRE

    Oelschlegel, Anja M.; Fallahi, Mohammad; Ortiz-Umpierre, Shannon; Weissmann, Charles

    2012-01-01

    Three commonly used isolates of murine prions, 79A, 139A, and RML, were derived from the so-called Chandler isolate, which was obtained by propagating prions from scrapie-infected goat brain in mice. RML is widely believed to be identical with 139A; however, using the extended cell panel assay (ECPA), we here show that 139A and RML isolates are distinct, while 79A and RML could not be distinguished. We undertook to clone 79A and 139A prions by endpoint dilution in murine neuroblastoma-derived...

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

    Science.gov (United States)

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

    2008-01-01

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

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

    NARCIS (Netherlands)

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

    1994-01-01

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

  3. Cell culture's spider silk road.

    Science.gov (United States)

    Perkel, Jeffrey

    2014-06-01

    A number of synthetic and natural materials have been tried in cell culture and tissue engineering applications in recent years. Now Jeffrey Perkel takes a look at one new culture component that might surprise you-spider silk.

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

    International Nuclear Information System (INIS)

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

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

    Science.gov (United States)

    Hematti, Peiman

    2016-04-01

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

  6. Wnt-Dependent Control of Cell Polarity in Cultured Cells.

    Science.gov (United States)

    Runkle, Kristin B; Witze, Eric S

    2016-01-01

    The secreted ligand Wnt5a regulates cell polarity and polarized cell movement during development by signaling through the poorly defined noncanonical Wnt pathway. Cell polarity regulates most aspects of cell behavior including the organization of apical/basolateral membrane domains of epithelial cells, polarized cell divisions along a directional plane, and front rear polarity during cell migration. These characteristics of cell polarity allow coordinated cell movements required for tissue formation and organogenesis during embryonic development. Genetic model organisms have been used to identify multiple signaling pathways including Wnt5a that are required to establish cell polarity and regulate polarized cell behavior. However, the downstream signaling events that regulate these complex cellular processes are still poorly understood. The methods below describe assays to study Wnt5a-induced cell polarity in cultured cells, which may facilitate our understanding of these complex signaling pathways. PMID:27590152

  7. Epithelial cells as alternative human biomatrices for comet assay

    OpenAIRE

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

    2014-01-01

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

  8. Revisiting the IFN-γ release assay: Whole blood or PBMC cultures? - And other factors of influence.

    Science.gov (United States)

    Hartmann, Sofie Bruun; Emnéus, Jenny; Wolff, Anders; Jungersen, Gregers

    2016-07-01

    The interferon-γ release assay (IGRA) is a widely used test for the presence of a cell-mediated immune (CMI) response in vitro. This measure is used to test for infection with intracellular pathogens or for validating vaccine efficacy, and it is a widely used test for both human as well as cattle. However, there is no consensus whether to use whole blood cultures or purified PBMCs for the assay, and both cell populations are being used and results compared. Therefore the aim of this study was to compare different culture settings using immune cells from previously vaccinated calves, and to shed light on external factors that could influence the read out in terms of IFN-γ levels. It was found that optimal culture conditions varied between individual animals; when polyclonal activated, cells from whole blood cultures were most responsive, but when activated specifically, the optimal cell concentration/population varied with whole blood, 10×10(6)cells/ml PBMC and 5×10(6)cells/ml PBMC being the highest performing conditions. A further investigation of the distribution of cell populations in PBMCs compared to whole blood was conducted, and a significant (pcultures from five calves. Six plates (a-f) were tested and no significant difference in absolute levels of IFN-γ was detected in the six plates when cells were polyclonal and specifically activated. However, we observed a significant (pculture population, the concentration of cells being cultured, and the plastic ware used for the in vitro culture. These findings stress the importance of documenting the precise assay conditions when publishing results of in vitro IFN-γ release assays.

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

    Directory of Open Access Journals (Sweden)

    Stefan Weinandy

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

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

    Directory of Open Access Journals (Sweden)

    Traver Hart

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

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

    OpenAIRE

    sprotocols

    2015-01-01

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

  12. 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.

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

    Science.gov (United States)

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

    2014-07-01

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

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

    Directory of Open Access Journals (Sweden)

    Keren I. Hulkower

    2011-03-01

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

  15. Three-dimensional cell culturing by magnetic levitation.

    Science.gov (United States)

    Haisler, William L; Timm, David M; Gage, Jacob A; Tseng, Hubert; Killian, T C; Souza, Glauco R

    2013-10-01

    Recently, biomedical research has moved toward cell culture in three dimensions to better recapitulate native cellular environments. This protocol describes one method for 3D culture, the magnetic levitation method (MLM), in which cells bind with a magnetic nanoparticle assembly overnight to render them magnetic. When resuspended in medium, an external magnetic field levitates and concentrates cells at the air-liquid interface, where they aggregate to form larger 3D cultures. The resulting cultures are dense, can synthesize extracellular matrix (ECM) and can be analyzed similarly to the other culture systems using techniques such as immunohistochemical analysis (IHC), western blotting and other biochemical assays. This protocol details the MLM and other associated techniques (cell culture, imaging and IHC) adapted for the MLM. The MLM requires 45 min of working time over 2 d to create 3D cultures that can be cultured in the long term (>7 d). PMID:24030442

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

    OpenAIRE

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

    2014-01-01

    The comet assay is a simple and cost effective technique, commonly used to analyze and quantify DNA damage in individual cells. The versatility of the comet assay allows introduction of various modifications to the basic technique. The difference in the methylation sensitivity of the isoschizomeric restriction enzymes HpaII and MspI are used to demonstrate the ability of the comet assay to measure the global DNA methylation level of individual cells when using cell cultures. In the experiment...

  17. Revisiting the IFN-γ release assay: Whole blood or PBMC cultures? - And other factors of influence.

    Science.gov (United States)

    Hartmann, Sofie Bruun; Emnéus, Jenny; Wolff, Anders; Jungersen, Gregers

    2016-07-01

    The interferon-γ release assay (IGRA) is a widely used test for the presence of a cell-mediated immune (CMI) response in vitro. This measure is used to test for infection with intracellular pathogens or for validating vaccine efficacy, and it is a widely used test for both human as well as cattle. However, there is no consensus whether to use whole blood cultures or purified PBMCs for the assay, and both cell populations are being used and results compared. Therefore the aim of this study was to compare different culture settings using immune cells from previously vaccinated calves, and to shed light on external factors that could influence the read out in terms of IFN-γ levels. It was found that optimal culture conditions varied between individual animals; when polyclonal activated, cells from whole blood cultures were most responsive, but when activated specifically, the optimal cell concentration/population varied with whole blood, 10×10(6)cells/ml PBMC and 5×10(6)cells/ml PBMC being the highest performing conditions. A further investigation of the distribution of cell populations in PBMCs compared to whole blood was conducted, and a significant (psecreted IFN-γ in whole blood cultures from five calves. Six plates (a-f) were tested and no significant difference in absolute levels of IFN-γ was detected in the six plates when cells were polyclonal and specifically activated. However, we observed a significant (pexpression on plate b, and the relative-to-maximum level on this plate was significant (p<0.05) compared to plate a. Altogether these findings highlight the potential weaknesses of the IFN-γ release assay in terms of the many variables that can influence the results, including the cell culture population, the concentration of cells being cultured, and the plastic ware used for the in vitro culture. These findings stress the importance of documenting the precise assay conditions when publishing results of in vitro IFN-γ release assays. PMID:27073172

  18. Isolation, Culture, and Maintenance of Mouse Intestinal Stem Cells

    Science.gov (United States)

    O’Rourke, Kevin P.; Ackerman, Sarah; Dow, Lukas E; Lowe, Scott W

    2016-01-01

    In this protocol we describe our modifications to a method to isolate, culture and maintain mouse intestinal stem cells as crypt-villus forming organoids. These cells, isolated either from the small or large intestine, maintain self-renewal and multilineage differentiation potential over time. This provides investigators a tool to culture wild type or transformed intestinal epithelium, and a robust assay for stem cell tissue homeostasis in vitro.

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

    Directory of Open Access Journals (Sweden)

    Sandra A. Calarota

    2013-01-01

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

  20. Reference cells and ploidy in the comet assay

    OpenAIRE

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

    2015-01-01

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

  1. Evaluation on Sensitivity of the Human Sperm Motility Assay for Detecting Endotoxin in Culture Medium

    Institute of Scientific and Technical Information of China (English)

    Wei-jie ZHU; Jing LI; Wen-hong ZHANG; Kang-shou YAO

    2003-01-01

    Objective To investigate the sensitivity of the human sperm motility assay for detecting endotoxin in culture medium Materials & Methods Motile sperm were separated and exposed to different concentrations of endotoxin (0.5 ng/mL, 1 ng/mL, 10 ng/mL, 1 000 ng/mL, 10 000 ng/mL, and 50 000 ng/mL), and sperm motility was determined after incubation. Effects of endotoxin on sperm motility in media without albumin were also examined. In addition, at the same concentrations of endotoxin (0.5 ng/mL, 1 ng/mL, and 10 ng/mL), the sensitivity of the human sperm motility assay was compared to those of 1-cell and 2-cell mouse embryo bioassays.Results At levels of 0.5 ng/mL~1 000 ng/mL endotoxin in media with 2 mg/mL albumin, sperm did not show significant change in motility during 24 h of incubation when compared with the control (P>0.05). However, the sperm motility was significantly inhibited at endotoxin dosages of 10 000 and 50 000 ng/mL. In the absence of albumin supplementation, at endotoxin levels of 50 000 ng/mL, and 1 000 ng/mL, there was a marked decrease in sperm motility compared with the control after 2 h or 8 h of incubation, respectively (P<0.01). In media containing 0.5 ng/mL and 1 ng/mL endotoxin, 1-cell and 2-cell mouse embryos had significantly reduced developmental rates in all developmental stages, and at the level of 10 ng/mL, the development of the embryos was arrested.Conclusion The human sperm motility assay could detect high levels of endotoxin in culture medium but its sensitivity to endotoxin would be inferior to that of the 1-cell or 2-cell mouse embryo bioassay. In the absence of albumin supplementation, the sensitivity of the sperm motility assay could be improved.

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

    Science.gov (United States)

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

    2009-01-01

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

  3. Early detection of influenza virus by using a fluorometric assay of infected tissue culture.

    OpenAIRE

    Pachucki, C T; Creticos, C

    1988-01-01

    A fluorometric substrate, 4-methylumbelliferyl-alpha-ketoside of N-acetylneuramide, was used directly on clinical specimens and infected tissue culture 24 h after inoculation for the detection of influenza viral neuraminidase. Viral neuraminidase was detected in infected tissue culture but not in clinical specimens. The sensitivity of the assay on tissue culture was 92%, and the specificity was 96%.

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

    OpenAIRE

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

    1984-01-01

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

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

    Science.gov (United States)

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

    1993-05-01

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

  6. Revisiting the IFN-γ release assay: Whole blood or PBMC cultures? - And other factors of influence

    DEFF Research Database (Denmark)

    Hartmann, Sofie Bruun; Emnéus, Jenny; Wolff, Anders;

    2016-01-01

    The interferon-γ release assay (IGRA) is a widely used test for the presence of a cell-mediated immune (CMI) response in vitro. This measure is used to test for infection with intracellular pathogens or for validating vaccine efficacy, and it is a widely used test for both human as well as cattle....... However, there is no consensus whether to use whole blood cultures or purified PBMCs for the assay, and both cell populations are being used and results compared. Therefore the aim of this study was to compare different culture settings using immune cells from previously vaccinated calves, and to shed...... observed a significant (p cat# 3595) (plate d) compared to two different flat-bottom plates from Corning® (cat# 3596) (plate b) and Nunc™ (cat# 167008) (plate a). Furthermore 4 out of 5 calves had maximum specific IFN-γ expression...

  7. Aseptic technique for cell culture.

    Science.gov (United States)

    Coté, R J

    2001-05-01

    This unit describes some of the ways that a laboratory can deal with the constant threat of microbial contamination in cell cultures. A protocol on aseptic technique is described first. This catch-all term universally appears in any set of instructions pertaining to procedures in which noncontaminating conditions must be maintained. In reality, aseptic technique encompasses all aspects of environmental control, personal hygiene, equipment and media sterilization, and associated quality control procedures needed to ensure that a procedure is, indeed, performed with aseptic, noncontaminating technique. Although cell culture can theoretically be carried out on an open bench in a low-traffic area, most cell culture work is carried out using a horizontal laminar-flow clean bench or a vertical laminar-flow biosafety cabinet. Both are described here. PMID:18228291

  8. Reference cells and ploidy in the comet assay

    Directory of Open Access Journals (Sweden)

    Gunnar eBrunborg

    2015-02-01

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

  9. Cell culture compositions

    Science.gov (United States)

    Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yiao, Jian

    2014-03-18

    The present invention provides a novel endoglucanase nucleic acid sequence, designated egl6 (SEQ ID NO:1 encodes the full length endoglucanase; SEQ ID NO:4 encodes the mature form), and the corresponding endoglucanase VI amino acid sequence ("EGVI"; SEQ ID NO:3 is the signal sequence; SEQ ID NO:2 is the mature sequence). The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVI, recombinant EGVI proteins and methods for producing the same.

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

    Directory of Open Access Journals (Sweden)

    Zaira E Arellano-Anaya

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

  11. Epithelial cells as alternative human biomatrices for comet assay

    Directory of Open Access Journals (Sweden)

    Emilio eRojas

    2014-11-01

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

  12. Mesenchymal Progenitor Cells: Tissue Origin, Isolation and Culture.

    Science.gov (United States)

    Bourin, Philippe; Gadelorge, Mélanie; Peyrafitte, Julie-Anne; Fleury-Cappellesso, Sandrine; Gomez, Marilyn; Rage, Christine; Sensebé, Luc

    2008-01-01

    SUMMARY: Since the pioneering work of Alexander Friedenstein on multipotent mesenchymal stromal cells (MSCs), a tremendous amount of work has been done to isolate, characterize and culture such cells. Assay of colony forming unit-fibroblasts (CFU-Fs), the hallmark of MSCs, is used to estimate their frequency in tissue. MSCs are adherent cells, so they are easy to isolate, and they show contact inhibition. Thus, several parameters must be taken into account for culture: cell density, number of passages, culture medium, and growth factors used. The purity of the initial material is not a limiting parameter. Similar but not identical cell populations are found in almost all mammal or human tissues. MSCs seem to be very abundant in adipose tissue but at low frequency in blood from umbilical cord or in adult tissue. The culture conditions are very similar, whatever the source of cells. Because of their favorable properties, MSCs are very promising tools for regenerative medicine.

  13. Insect Cell Culture and Biotechnology

    Institute of Scientific and Technical Information of China (English)

    Robert R.Granados; Guoxun Li; G.W.Blissard

    2007-01-01

    The continued development of new cell culture technology is essential for the future growth and application of insect cell and baculovirus biotechnology. The use of cell lines for academic research and for commercial applications is currently dominated by two cell lines; the Spodoptera frugiperda line, SF21 (and its clonal isolate, SF9), and the Trichoplusia ni line, BTI 5B1-4, commercially known as High Five cells. The long perceived prediction that the immense potential application of the baculovirus-insect cell system, as a tool in cell and molecular biology, agriculture, and animal health, has been achieved. The versatility and recent applications of this popular expression system has been demonstrated by both academia and industry and it is clear that this cell-based system has been widely accepted for biotechnological applications. Numerous small to midsize startup biotechnology companies in North America and the Europe are currently using the baculovirus-insect cell technology to produce custom recombinant proteins for research and commercial applications. The recent breakthroughs using the baculovirus-insect cell-based system for the development of several commercial products that will impact animal and human health will further enhance interest in this technology by pharma. Clearly, future progress in novel cell and engineering advances will lead to fundamental scientific discoveries and serve to enhance the utility and applications of this baculovirus-insect cell system.

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

    Science.gov (United States)

    Wollin, Klaus-M; Gorlitz, Bernd-D

    2004-01-01

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

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

    Science.gov (United States)

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

    2015-11-17

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

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

    International Nuclear Information System (INIS)

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

  17. Cell Culture, Technology: Enhancing the Culture of Diagnosing Human Diseases.

    Science.gov (United States)

    Hudu, Shuaibu Abdullahi; Alshrari, Ahmed Subeh; Syahida, Ahmad; Sekawi, Zamberi

    2016-03-01

    Cell culture involves a complex of processes of cell isolation from their natural environment (in vivo) and subsequent growth in a controlled environmental artificial condition (in vitro). Cells from specific tissues or organs are cultured as short term or established cell lines which are widely used for research and diagnosis, most specially in the aspect of viral infection, because pathogenic viral isolation depends on the availability of permissible cell cultures. Cell culture provides the required setting for the detection and identification of numerous pathogens of humans, which is achieved via virus isolation in the cell culture as the "gold standard" for virus discovery. In this review, we summarized the views of researchers on the current role of cell culture technology in the diagnosis of human diseases. The technological advancement of recent years, starting with monoclonal antibody development to molecular techniques, provides an important approach for detecting presence of viral infection. They are also used as a baseline for establishing rapid tests for newly discovered pathogens. A combination of virus isolation in cell culture and molecular methods is still critical in identifying viruses that were previously unrecognized. Therefore, cell culture should be considered as a fundamental procedure in identifying suspected infectious viral agent.

  18. Techniques for mammalian cell tissue culture.

    Science.gov (United States)

    Phelan, Mary C

    2006-05-01

    This unit opens with detailed discussions on the latest principles of sterile technique and preparation of culture media. Step-by-step protocols describe trypsinizing and subculturing monolayer cultures, passaging suspension cultures, freezing and thawing cells, counting cells using a hemacytometer, and preparing cells for transport. PMID:18770828

  19. 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.

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

    Science.gov (United States)

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

    2015-01-01

    We have developed a new hybrid droplet-to-digital microfluidic platform (D2D) that integrates droplet-in-channel microfluidics with digital microfluidics (DMF) for performing multi-step assays. This D2D platform combines the strengths of the two formats-droplets-in-channel for facile generation of droplets containing single cells, and DMF for on-demand manipulation of droplets including control of different droplet volumes (pL-μL), creation of a dilution series of ionic liquid (IL), and parallel single cell culturing and analysis for IL toxicity screening. This D2D device also allows for automated analysis that includes a feedback-controlled system for merging and splitting of droplets to add reagents, an integrated Peltier element for parallel cell culture at optimum temperature, and an impedance sensing mechanism to control the flow rate for droplet generation and preventing droplet evaporation. Droplet-in-channel is well-suited for encapsulation of single cells as it allows the careful manipulation of flow rates of aqueous phase containing cells and oil to optimize encapsulation. Once single cell containing droplets are generated, they are transferred to a DMF chip via a capillary where they are merged with droplets containing IL and cultured at 30 °C. The DMF chip, in addition to permitting cell culture and reagent (ionic liquid/salt) addition, also allows recovery of individual droplets for off-chip analysis such as further culturing and measurement of ethanol production. The D2D chip was used to evaluate the effect of IL/salt type (four types: NaOAc, NaCl, [C2mim] [OAc], [C2mim] [Cl]) and concentration (four concentrations: 0, 37.5, 75, 150 mM) on the growth kinetics and ethanol production of yeast and as expected, increasing IL concentration led to lower biomass and ethanol production. Specifically, [C2mim] [OAc] had inhibitory effects on yeast growth at concentrations 75 and 150 mM and significantly reduced their ethanol production compared to cells grown

  1. Mammosphere culture of cancer stem cells in a microfluidic device

    Science.gov (United States)

    Saadin, Katayoon; White, Ian M.

    2012-03-01

    It is known that tumor-initiating cells with stem-like properties will form spherical colonies - termed mammospheres - when cultured in serum-free media on low-attachment substrates. Currently this assay is performed in commercially available 96-well trays with low-attachment surfaces. Here we report a novel microsystem that features on-chip mammosphere culture on low attachment surfaces. We have cultured mammospheres in this microsystem from well-studied human breast cancer cell lines. To enable the long-term culture of these unattached cells, we have integrated diffusion-based delivery columns that provide zero-convection delivery of reagents, such as fresh media, staining agents, or drugs. The multi-layer system consists of parallel cell-culture chambers on top of a low-attachment surface, connected vertically with a microfluidic reagent delivery layer. This design incorporates a reagent reservoir, which is necessary to reduce evaporation from the cell culture micro-chambers. The development of this microsystem will lead to the integration of mammosphere culture with other microfluidic functions, including circulating tumor cell recovery and high throughput drug screening. This will enable the cancer research community to achieve a much greater understanding of these tumor initiating cancer stem cells.

  2. Radiometric macrophage culture assay for rapid evaluation of antileprosy activity of rifampin

    Energy Technology Data Exchange (ETDEWEB)

    Mittal, A.; Seshadri, P.S.; Prasad, H.K.; Sathish, M.; Nath, I.

    1983-10-01

    The antileprosy effect of rifampin was evaluated by a newly developed rapid in vitro assay wherein 31 human-derived strains and 1 armadillo-derived strain of Mycobacterium leprae were maintained for 2 and 3 weeks, respectively, in murine and human macrophages in the presence of (3H)thymidine. Of these strains, 27 showed significant incorporation of the radiolabel in cultures of live bacilli as compared with control cultures of heat-killed bacilli of the same strain. Consistent and significant inhibition of (3H)thymidine uptake was observed in M. leprae resident cultures with 3 to 200 ng of rifampin per ml as compared with similar cultures without the drug. In general, an increase in percent inhibition was seen from 3 to 20 ng/ml, with marginal increases at 40, 50, and 100 ng/ml. M. leprae strains appear to be remarkably susceptible to this drug in the in vitro assay.

  3. Dynamized Preparations in Cell Culture

    Directory of Open Access Journals (Sweden)

    Ellanzhiyil Surendran Sunila

    2009-01-01

    Full Text Available Although reports on the efficacy of homeopathic medicines in animal models are limited, there are even fewer reports on the in vitro action of these dynamized preparations. We have evaluated the cytotoxic activity of 30C and 200C potencies of ten dynamized medicines against Dalton's Lymphoma Ascites, Ehrlich's Ascites Carcinoma, lung fibroblast (L929 and Chinese Hamster Ovary (CHO cell lines and compared activity with their mother tinctures during short-term and long-term cell culture. The effect of dynamized medicines to induce apoptosis was also evaluated and we studied how dynamized medicines affected genes expressed during apoptosis. Mother tinctures as well as some dynamized medicines showed significant cytotoxicity to cells during short and long-term incubation. Potentiated alcohol control did not produce any cytotoxicity at concentrations studied. The dynamized medicines were found to inhibit CHO cell colony formation and thymidine uptake in L929 cells and those of Thuja, Hydrastis and Carcinosinum were found to induce apoptosis in DLA cells. Moreover, dynamized Carcinosinum was found to induce the expression of p53 while dynamized Thuja produced characteristic laddering pattern in agarose gel electrophoresis of DNA. These results indicate that dynamized medicines possess cytotoxic as well as apoptosis-inducing properties.

  4. Dynamized preparations in cell culture.

    Science.gov (United States)

    Sunila, Ellanzhiyil Surendran; Kuttan, Ramadasan; Preethi, Korengath Chandran; Kuttan, Girija

    2009-06-01

    Although reports on the efficacy of homeopathic medicines in animal models are limited, there are even fewer reports on the in vitro action of these dynamized preparations. We have evaluated the cytotoxic activity of 30C and 200C potencies of ten dynamized medicines against Dalton's Lymphoma Ascites, Ehrlich's Ascites Carcinoma, lung fibroblast (L929) and Chinese Hamster Ovary (CHO) cell lines and compared activity with their mother tinctures during short-term and long-term cell culture. The effect of dynamized medicines to induce apoptosis was also evaluated and we studied how dynamized medicines affected genes expressed during apoptosis. Mother tinctures as well as some dynamized medicines showed significant cytotoxicity to cells during short and long-term incubation. Potentiated alcohol control did not produce any cytotoxicity at concentrations studied. The dynamized medicines were found to inhibit CHO cell colony formation and thymidine uptake in L929 cells and those of Thuja, Hydrastis and Carcinosinum were found to induce apoptosis in DLA cells. Moreover, dynamized Carcinosinum was found to induce the expression of p53 while dynamized Thuja produced characteristic laddering pattern in agarose gel electrophoresis of DNA. These results indicate that dynamized medicines possess cytotoxic as well as apoptosis-inducing properties. PMID:18955237

  5. Comparison of culture and a novel 5' Taq nuclease assay for direct detection of Campylobacter fetus subsp. venerealis in clinical specimens from cattle.

    Science.gov (United States)

    McMillen, Lyle; Fordyce, Geoffry; Doogan, Vivienne J; Lew, Ala E

    2006-03-01

    A Campylobacter fetus subsp. venerealis-specific 5' Taq nuclease PCR assay using a 3' minor groove binder-DNA probe (TaqMan MGB) was developed based on a subspecies-specific fragment of unknown identity (S. Hum, K. Quinn, J. Brunner, and S. L. On, Aust. Vet. J. 75:827-831, 1997). The assay specifically detected four C. fetus subsp. venerealis strains with no observed cross-reaction with C. fetus subsp. fetus-related Campylobacter species or other bovine venereal microflora. The 5' Taq nuclease assay detected approximately one single cell compared to 100 and 10 cells in the conventional PCR assay and 2,500 and 25,000 cells from selective culture from inoculated smegma and mucus, respectively. The respective detection limits following the enrichments from smegma and mucus were 5,000 and 50 cells/inoculum for the conventional PCR compared to 500 and 50 cells/inoculum for the 5' Taq nuclease assay. Field sampling confirmed the sensitivity and the specificity of the 5' Taq nuclease assay by detecting an additional 40 bulls that were not detected by culture. Urine-inoculated samples demonstrated comparable detection of C. fetus subsp. venerealis by both culture and the 5' Taq nuclease assay; however, urine was found to be less effective than smegma for bull sampling. Three infected bulls were tested repetitively to compare sampling tools, and the bull rasper proved to be the most suitable, as evidenced by the improved ease of specimen collection and the consistent detection of higher levels of C. fetus subsp. venerealis. The 5' Taq nuclease assay demonstrates a statistically significant association with culture (chi2 = 29.8; P < 0.001) and significant improvements for the detection of C. fetus subsp. venerealis-infected animals from crude clinical extracts following prolonged transport. PMID:16517880

  6. Comparison of Culture and a Novel 5′ Taq Nuclease Assay for Direct Detection of Campylobacter fetus subsp. venerealis in Clinical Specimens from Cattle

    Science.gov (United States)

    McMillen, Lyle; Fordyce, Geoffry; Doogan, Vivienne J.; Lew, Ala E.

    2006-01-01

    A Campylobacter fetus subsp. venerealis-specific 5′ Taq nuclease PCR assay using a 3′ minor groove binder-DNA probe (TaqMan MGB) was developed based on a subspecies-specific fragment of unknown identity (S. Hum, K. Quinn, J. Brunner, and S. L. On, Aust. Vet. J. 75:827-831, 1997). The assay specifically detected four C. fetus subsp. venerealis strains with no observed cross-reaction with C. fetus subsp. fetus-related Campylobacter species or other bovine venereal microflora. The 5′ Taq nuclease assay detected approximately one single cell compared to 100 and 10 cells in the conventional PCR assay and 2,500 and 25,000 cells from selective culture from inoculated smegma and mucus, respectively. The respective detection limits following the enrichments from smegma and mucus were 5,000 and 50 cells/inoculum for the conventional PCR compared to 500 and 50 cells/inoculum for the 5′ Taq nuclease assay. Field sampling confirmed the sensitivity and the specificity of the 5′ Taq nuclease assay by detecting an additional 40 bulls that were not detected by culture. Urine-inoculated samples demonstrated comparable detection of C. fetus subsp. venerealis by both culture and the 5′ Taq nuclease assay; however, urine was found to be less effective than smegma for bull sampling. Three infected bulls were tested repetitively to compare sampling tools, and the bull rasper proved to be the most suitable, as evidenced by the improved ease of specimen collection and the consistent detection of higher levels of C. fetus subsp. venerealis. The 5′ Taq nuclease assay demonstrates a statistically significant association with culture (χ2 = 29.8; P < 0.001) and significant improvements for the detection of C. fetus subsp. venerealis-infected animals from crude clinical extracts following prolonged transport. PMID:16517880

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

    Science.gov (United States)

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

    2016-02-01

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

  8. Expanding intestinal stem cells in culture

    NARCIS (Netherlands)

    Heo, Inha; Clevers, Hans

    2015-01-01

    Culturing intestinal stem cells into 3D organoids results in heterogeneous cell populations, reflecting the in vivo cell type diversity. In a recent paper published in Nature, Wang et al. established a culture condition for a highly homogeneous population of intestinal stem cells.

  9. Multizone paper platform for 3D cell cultures.

    Directory of Open Access Journals (Sweden)

    Ratmir Derda

    Full Text Available In vitro 3D culture is an important model for tissues in vivo. Cells in different locations of 3D tissues are physiologically different, because they are exposed to different concentrations of oxygen, nutrients, and signaling molecules, and to other environmental factors (temperature, mechanical stress, etc. The majority of high-throughput assays based on 3D cultures, however, can only detect the average behavior of cells in the whole 3D construct. Isolation of cells from specific regions of 3D cultures is possible, but relies on low-throughput techniques such as tissue sectioning and micromanipulation. Based on a procedure reported previously ("cells-in-gels-in-paper" or CiGiP, this paper describes a simple method for culture of arrays of thin planar sections of tissues, either alone or stacked to create more complex 3D tissue structures. This procedure starts with sheets of paper patterned with hydrophobic regions that form 96 hydrophilic zones. Serial spotting of cells suspended in extracellular matrix (ECM gel onto the patterned paper creates an array of 200 micron-thick slabs of ECM gel (supported mechanically by cellulose fibers containing cells. Stacking the sheets with zones aligned on top of one another assembles 96 3D multilayer constructs. De-stacking the layers of the 3D culture, by peeling apart the sheets of paper, "sections" all 96 cultures at once. It is, thus, simple to isolate 200-micron-thick cell-containing slabs from each 3D culture in the 96-zone array. Because the 3D cultures are assembled from multiple layers, the number of cells plated initially in each layer determines the spatial distribution of cells in the stacked 3D cultures. This capability made it possible to compare the growth of 3D tumor models of different spatial composition, and to examine the migration of cells in these structures.

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

    Science.gov (United States)

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

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

    OpenAIRE

    Hulkower, Keren I.; Herber, Renee L.

    2011-01-01

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

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

    Science.gov (United States)

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

    2015-05-15

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

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

    Science.gov (United States)

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

    2016-07-15

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

  14. Genotoxicity determinations of coriander drop and extract of Coriander Sativum cultured fibroblast of rat embryo by comet assay

    International Nuclear Information System (INIS)

    The single cell gel electrophoresis (SCGE) or comet assay is a quick, simple and sensitive technique for measuring DNA damage in cell nucleus. It is well known that medicinal herbs play an important role in the life of human beings, thus it is essential to determine their safety as public health is concerned. In this study the genotoxicity of Coriander drop, herbal pharmaceutical product, and the extract of Coriander sativum were examined in cultured fibroblast of rat embryo using comet assay. The thirteen to fifteen days old rat embryos were lysed with tripsin and after certain steps it was centrifuged and then cultured. After three to five passages, different concentrations of each product were applied to the fibroblasts. Lysing, electrophoresis, neutralization and staining were carried out. Finally the slides were analyzed with fluorescence microscope. In the test groups the results indicated that coriander drop at different doses showed some fragmentation of DNA but this damage as a result was deemed to be not significant. However, in the case of Coriander sativum extract the results showed no mutagenic effects in comparison with the positive control group (p<0.05). In conclusion, these herbal products did not show any magnetic effect according to our test, but further genotoxicity assays are recommended. (author)

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

    Science.gov (United States)

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

    2009-01-01

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

  16. PDMS/glass microfluidic cell culture system for cytotoxicity tests and cells passage

    DEFF Research Database (Denmark)

    Ziolkowska, K.; Jedrych, E.; Kwapiszewski, R.;

    2010-01-01

    In this paper, hybrid (PDMS/glass) microfluidic cell culture system (MCCS) integrated with the concentration gradient generator (CGG) is presented. PDMS gas permeability enabled cells' respiration in the fabricated microdevices and excellent glass hydrophilicity allowed successful cells' seeding....... The human lung carcinoma cells (A549) were cultured in the microdevice for several days. The growth and proliferation of cells was monitored using an inverted fluorescence microscope. After the cells' confluence was achieved in the microchambers, the novel method of cells' passaging in the designed...... microdevice was developed and successfully tested. The MCCS microdevice is fully reusable, i.e. it can be used several times for various cell culture and cytotoxic experiments. The suitability of designed MCCS for cell-based cytotoxicity assay application was verified using 1,4-dioxane as a model toxic agent...

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

    OpenAIRE

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

    2012-01-01

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

  18. 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

  19. Miniature Bioreactor System for Long-Term Cell Culture

    Science.gov (United States)

    Gonda, Steve R.; Kleis, Stanley J.; Geffert, Sandara K.

    2010-01-01

    A prototype miniature bioreactor system is designed to serve as a laboratory benchtop cell-culturing system that minimizes the need for relatively expensive equipment and reagents and can be operated under computer control, thereby reducing the time and effort required of human investigators and reducing uncertainty in results. The system includes a bioreactor, a fluid-handling subsystem, a chamber wherein the bioreactor is maintained in a controlled atmosphere at a controlled temperature, and associated control subsystems. The system can be used to culture both anchorage-dependent and suspension cells, which can be either prokaryotic or eukaryotic. Cells can be cultured for extended periods of time in this system, and samples of cells can be extracted and analyzed at specified intervals. By integrating this system with one or more microanalytical instrument(s), one can construct a complete automated analytical system that can be tailored to perform one or more of a large variety of assays.

  20. Assessment of potential anti-cancer stem cell activity of marine algal compounds using an in vitro mammosphere assay

    OpenAIRE

    de la Mare, Jo-Anne; Sterrenberg, Jason N; Sukhthankar, Mugdha G; Chiwakata, Maynard T; Denzil R. Beukes; Blatch, Gregory L.; Edkins, Adrienne L.

    2013-01-01

    Background The cancer stem cell (CSC) theory proposes that tumours arise from and are sustained by a subpopulation of cells with both cancer and stem cell properties. One of the key hallmarks of CSCs is the ability to grow anchorage-independently under serum-free culture conditions resulting in the formation of tumourspheres. It has further been reported that these cells are resistant to traditional chemotherapeutic agents. Methods In this study, the tumoursphere assay was validated in MCF-7 ...

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

    Institute of Scientific and Technical Information of China (English)

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

    2012-01-01

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

  2. 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.

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

    Institute of Scientific and Technical Information of China (English)

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

    2006-01-01

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

  4. Scaling and automation of a high-throughput single-cell-derived tumor sphere assay chip.

    Science.gov (United States)

    Cheng, Yu-Heng; Chen, Yu-Chih; Brien, Riley; Yoon, Euisik

    2016-10-01

    Recent research suggests that cancer stem-like cells (CSCs) are the key subpopulation for tumor relapse and metastasis. Due to cancer plasticity in surface antigen and enzymatic activity markers, functional tumorsphere assays are promising alternatives for CSC identification. To reliably quantify rare CSCs (1-5%), thousands of single-cell suspension cultures are required. While microfluidics is a powerful tool in handling single cells, previous works provide limited throughput and lack automatic data analysis capability required for high-throughput studies. In this study, we present the scaling and automation of high-throughput single-cell-derived tumor sphere assay chips, facilitating the tracking of up to ∼10 000 cells on a chip with ∼76.5% capture rate. The presented cell capture scheme guarantees sampling a representative population from the bulk cells. To analyze thousands of single-cells with a variety of fluorescent intensities, a highly adaptable analysis program was developed for cell/sphere counting and size measurement. Using a Pluronic® F108 (poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol)) coating on polydimethylsiloxane (PDMS), a suspension culture environment was created to test a controversial hypothesis: whether larger or smaller cells are more stem-like defined by the capability to form single-cell-derived spheres. Different cell lines showed different correlations between sphere formation rate and initial cell size, suggesting heterogeneity in pathway regulation among breast cancer cell lines. More interestingly, by monitoring hundreds of spheres, we identified heterogeneity in sphere growth dynamics, indicating the cellular heterogeneity even within CSCs. These preliminary results highlight the power of unprecedented high-throughput and automation in CSC studies.

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

    Directory of Open Access Journals (Sweden)

    Yomo Tetsuya

    2006-06-01

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

  6. Cell Culture as an Alternative in Education.

    Science.gov (United States)

    Nardone, Roland M.

    1990-01-01

    Programs that are intended to inform and provide "hands-on" experience for students and to facilitate the introduction of cell culture-based laboratory exercises into the high school and college laboratory are examined. The components of the CellServ Program and the Cell Culture Toxicology Training Programs are described. (KR)

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

    Directory of Open Access Journals (Sweden)

    Haifeng Geng

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

  8. In vitro culture of human thyroid cells

    International Nuclear Information System (INIS)

    Procedures for establishing primary cultures of human thyroid tissue are described. Tissues removed surgically from patients with papillary carcinoma (PC), follicular adenoma (FA), or hyperthyroidism were grown in culture. In addition, normal cells were separated from the margins of excised tumors and were also cultured. For each gram of thyroid tissue cultured, more than 1 x 105 cells attached to culture dishes. A mixture of 2.5 % fetal bovine serum supplemented with insulin, hydrocortisone, transferrin, glycl-1-histidyl-L-lysine acetate, somatostatin and epidermal growth factor was added to nutrient media containing equal parts of Ham's F-12 and minimum essential medium (αMEM). Complete medium selectively supported epithelial cell growth while restricting fibroblast cell growth, especially during the first two weeks of the primary culture. Cells were stimulated with thyroid stimulating hormone (TSH) and produced raised levels of cAMP and thyroid hormone (T3). Culture conditions that affected the response of cells to X-rays were identified. During the culture period, first and second passage cells were compared for differences in their radiosensitivities. In all cases, cells showed differences in their responses to radiation depending on the cell passage number. However, results of replicate experiments of first passage cells that were exposed to X-rays showed good agreement between experiments. This technique makes it possible to quantitate the effects of chemical and physical cytotoxic agents on proliferating human thyroid epithelial cells. (author)

  9. A microwell cell culture platform for the aggregation of pancreatic β-cells.

    Science.gov (United States)

    Bernard, Abigail B; Lin, Chien-Chi; Anseth, Kristi S

    2012-08-01

    Cell-cell contact between pancreatic β-cells is important for maintaining survival and normal insulin secretion. Various techniques have been developed to promote cell-cell contact between β-cells, but a simple yet robust method that affords precise control over three-dimensional (3D) β-cell cluster size has not been demonstrated. To address this need, we developed a poly(ethylene glycol) (PEG) hydrogel microwell platform using photolithography. This microwell cell-culture platform promotes the formation of 3D β-cell aggregates of defined sizes from 25 to 210 μm in diameter. Using this platform, mouse insulinoma 6 (MIN6) β-cells formed aggregates with cell-cell adherin junctions. These naturally formed cell aggregates with controllable sizes can be removed from the microwells for macroencapsulation, implantation, or other biological assays. When removed and subsequently encapsulated in PEG hydrogels, the aggregated cell clusters demonstrated improved cellular viability (>90%) over 7 days in culture, while the β-cells encapsulated as single cells maintained only 20% viability. Aggregated MIN6 cells also exhibited more than fourfold higher insulin secretion in response to a glucose challenge compared with encapsulated single β-cells. Further, the cell aggregates stained positively for E-cadherin, indicative of the formation of cell junctions. Using this hydrogel microwell cell-culture method, viable and functional β-cell aggregates of specific sizes were created, providing a platform from which other biologically relevant questions may be answered. PMID:22320435

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

    International Nuclear Information System (INIS)

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

  11. DNA MUTAGENESIS IN PANAX GINSENG CELL CULTURES

    Directory of Open Access Journals (Sweden)

    Kiselev K.V.

    2012-08-01

    Full Text Available At the present time, it is well documented that plant tissue culture induces a number of mutations and chromosome rearrangements termed “somaclonal variations”. However, little is known about the nature and the molecular mechanisms of the tissue culture-induced mutagenesis and the effects of long-term subculturing on the rate and specific features of the mutagenesis. The aim of the present study was to investigate and compare DNA mutagenesis in different genes of Panax ginseng callus cultures of different age. It has previously been shown that the nucleotide sequences of the Agrobacterium rhizogenes rolC locus and the selective marker nptII developed mutations during long-term cultivation of transgenic cell cultures of P. ginseng. In the present work, we analyzed nucleotide sequences of selected plant gene families in a 2-year-old and 20-year-old P. ginseng 1c cell culture and in leaves of cultivated P. ginseng plants. We analysed sequence variability between the Actin genes, which are a family of house-keeping genes; the phenylalanine ammonia-lyase (PAL and dammarenediol synthase (DDS genes, which actively participate in the biosynthesis of ginsenosides; and the somatic embryogenesis receptor kinase (SERK genes, which control plant development. The frequency of point mutations in the Actin, PAL, DDS, and SERK genes in the 2-year-old callus culture was markedly higher than that in cultivated plants but lower than that in the 20-year-old callus culture of P. ginseng. Most of the mutations in the 2- and 20-year-old P. ginseng calli were A↔G and T↔C transitions. The number of nonsynonymous mutations was higher in the 2- and 20-year-old callus cultures than the number of nonsynonymous mutations in the cultivated plants of P. ginseng. Interestingly, the total number of N→G or N→C substitutions in the analyzed genes was 1.6 times higher than the total number of N→A or N→T substitutions. Using methylation-sensitive DNA fragmentation

  12. Automated low dose assay system for survival measurements of mammalian cells in vitro

    International Nuclear Information System (INIS)

    It has been suggested that at low doses of radiation both surviving cells (S) and inactivated cells (K) should be identified to obtain accurate data. One way to achieve this is to microscopically examine individual cells attached to a culture vessel, record their positions and observe and classify subsequent cell growth. In this way most systematic errors (counting, pipetting, diluting, etc.) are eliminated and since both S and K cells are scored, statistical accuracy (binomial) is also improved. For this purpose the authors have developed a semi automatic low dose assay system (ALDAS) whereby a microscope state was modified and equipped with two stepping motors under computer control. The computer automatically scans tissue culture flasks in which cells were plated after irradiation. When a cell is observed, the operator assumes command of the stage monitor, centres the cell in the field of view using a ''joystick'' control and signals the computer to record the cell's X-Y coordinates. After one week of incubation each cell location is revisited automatically and the operator scores the cells as S or K

  13. A duplex PCR-based assay for measuring the amount of bacterial contamination in a nucleic acid extract from a culture of free-living protists.

    Directory of Open Access Journals (Sweden)

    Alan O Marron

    Full Text Available BACKGROUND: Cultures of heterotrophic protists often require co-culturing with bacteria to act as a source of nutrition. Such cultures will contain varying levels of intrinsic bacterial contamination that can interfere with molecular research and cause problems with the collection of sufficient material for sequencing. Measuring the levels of bacterial contamination for the purposes of molecular biology research is non-trivial, and can be complicated by the presence of a diverse bacterial flora, or by differences in the relative nucleic acid yield per bacterial or eukaryotic cell. PRINCIPAL FINDINGS: Here we describe a duplex PCR-based assay that can be used to measure the levels of contamination from marine bacteria in a culture of loricate choanoflagellates. By comparison to a standard culture of known target sequence content, the assay can be used to quantify the relative proportions of bacterial and choanoflagellate material in DNA or RNA samples extracted from a culture. We apply the assay to compare methods of purifying choanoflagellate cultures prior to DNA extraction, to determine their effectiveness in reducing bacterial contamination. Together with measurements of the total nucleic acid concentration, the assay can then be used as the basis for determining the absolute amounts of choanoflagellate DNA or RNA present in a sample. CONCLUSIONS: The assay protocol we describe here is a simple and relatively inexpensive method of measuring contamination levels in nucleic acid samples. This provides a new way to establish quantification and purification protocols for molecular biology and genomics in novel heterotrophic protist species. Guidelines are provided to develop a similar protocol for use with any protistan culture. This assay method is recommended where qPCR equipment is unavailable, where qPCR is not viable because of the nature of the bacterial contamination or starting material, or where prior sequence information is insufficient

  14. Assay for inorganic pyrophosphate in chondrocyte culture using anion-exchange high-performance liquid chromatography and radioactive orthophosphate labeling

    International Nuclear Information System (INIS)

    A method is described for determination of inorganic pyrophosphate (PPi) in cell culture medium and in rabbit articular chondrocytes grown in the presence of radioactive orthophosphate (32Pi). Intra- and extracellular 32PPi formed was measured using high-performance liquid chromatographic (HPLC) separation of the PPi from orthophosphate (Pi) and other phosphate-containing compounds. The chromatographic separation on a weak anion-exchange column is based on the extent to which various phosphate compounds form complexes with Mg2+ at low pH and the rate at which such formation occurs. These complexes are eluted more readily than the uncomplexed compounds. Best results were obtained using a simultaneous gradient of Mg2+ ions and ionic strength. In this case separation of small amounts of PPi from a large excess of Pi was possible without prior removal of Pi or extraction of the PPi fraction. The assay is also useful for measurement of inorganic pyrophosphatase activity. The sensitivity of the assay depends on the specific activity of the added 32Pi and on the culture conditions, but is comparable with the most sensitive of the enzymatic assays. Sample preparation, particularly deproteinization, proved to be of importance. The losses of PPi which occur during procedures of this sort due to hydrolysis and coprecipitation were quantitated

  15. Culture of Cells from Amphibian Embryos.

    Science.gov (United States)

    Stanisstreet, Martin

    1983-01-01

    Describes a method for in vitro culturing of cells from amphibian early embryos. Such cells can be used to demonstrate such properties of eukaryote cells as cell motility, adhesion, differentiation, and cell sorting into tissues. The technique may be extended to investigate other factors. (Author/JN)

  16. Assay and purification of Fv fragments in fermenter cultures: design and evaluation of generic binding reagents.

    Science.gov (United States)

    Berry, M J; Wattam, T A; Willets, J; Lindner, N; de Graaf, T; Hunt, T; Gani, M; Davis, P J; Porter, P

    1994-01-01

    Fv fragments whose genes have been cloned using common PCR primers carry identical peptide motifs at their termini. We have raised antibodies against the C-terminal motif of the VH chain GQGTTVTVSS and evaluated their utility as reagents for the assay and purification of Fvs in the fermenter culture. Three different Fvs were included in the investigation. We found that the motif was exposed and available for capture when Fv fragments were blotted onto nitrocellulose paper or adsorbed directly onto microtiter plates. In contrast, the motif was either partially or totally obscured when the Fv was complexed with immobilised antigen or when free in solution. This reactivity profile enabled us to develop a general-purpose assay for Fv protein, but not a general-purpose assay for monitoring active Fv. The apparent inaccessibility of the C-terminus of VH conflicts with currently held views on the three-dimensional structure of these molecules. PMID:7508476

  17. Assay and purification of Fv fragments in fermenter cultures: design and evaluation of generic binding reagents.

    Science.gov (United States)

    Berry, M J; Wattam, T A; Willets, J; Lindner, N; de Graaf, T; Hunt, T; Gani, M; Davis, P J; Porter, P

    1994-01-01

    Fv fragments whose genes have been cloned using common PCR primers carry identical peptide motifs at their termini. We have raised antibodies against the C-terminal motif of the VH chain GQGTTVTVSS and evaluated their utility as reagents for the assay and purification of Fvs in the fermenter culture. Three different Fvs were included in the investigation. We found that the motif was exposed and available for capture when Fv fragments were blotted onto nitrocellulose paper or adsorbed directly onto microtiter plates. In contrast, the motif was either partially or totally obscured when the Fv was complexed with immobilised antigen or when free in solution. This reactivity profile enabled us to develop a general-purpose assay for Fv protein, but not a general-purpose assay for monitoring active Fv. The apparent inaccessibility of the C-terminus of VH conflicts with currently held views on the three-dimensional structure of these molecules.

  18. In vitro BALB/3T3 cell transformation assay of nonoxynol-9 and 1,4-dioxane

    Energy Technology Data Exchange (ETDEWEB)

    Sheu, C.W.; Moreland, F.M.; Lee, J.K.; Dunkel, V.C.

    1988-01-01

    The spermicidal surfactant nonoxynol-9 (Igepal CO-630, GAF Corp.) and a potential impurity, 1,4-dioxane, were tested in the in vitro cell transformation assay using BALB/3T3 cells. Two treatment periods, 48 hr and 13 days, were used. Nonoxynol-9, tested at levels up to 10 /sup +/g/ml, did not induce transformation, whereas dioxane was very active in the induction type II foci in the cultured BALB/3T3 cells.

  19. 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.

  20. Development of a shaker culture of Buffalo green monkey kidney cells: potential use for detection of enteroviruses.

    OpenAIRE

    Goldstein, G.; Guskey, L E

    1982-01-01

    Buffalo green monkey kidney cells were adapted to grow as shaker cultures. Replication of environmental and clinical isolates of poliovirus, coxsackievirus, and echovirus in these cultures was analyzed by plaque assay and compared with replication in Buffalo green monkey kidney cell monolayers and HEp-2 cell shaker cultures. Dose-response tests with various concentrations of Mahoney type 1 poliovirus indicated that Buffalo green monkey kidney cell shaker cultures could detect as little as 1 P...

  1. Cell Suspension Culture of Neem Tree

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    The establishment of suspension culture system for neem (Azadirachta indica A. Juss) cells and the suspension culture condition was studied. It shows that the neem cell suspension culture system was best in B5 liquid medium, 2.0~4.0mg/L NAA with direct spill method. Based on the integrated analysis of cell biomass, Azadirachtin content and productivity, the optimum culture conditions were B5 liquid medium, 2.0-4.0 mg/L NAA, 3% sucrose at 25 ℃. The optimum rotating speed of the shaker and broth content d...

  2. Cell-free assay measuring repair DNA synthesis in human fibroblasts

    International Nuclear Information System (INIS)

    Osmotic disruption of confluent cultured human fibroblasts that have been irradiated or exposed to chemical carcinogens allows the specific measurement of repair DNA synthesis using dTTP as a precursor. Fibroblasts similarly prepared from various xeroderma pigmentosum cell lines show the deficiencies of uv-induced DNA synthesis predicted from in vivo studies, while giving normal responses to methylmethanesulfonate. A pyrimidine-dimer-specific enzyme, T4 endonuclease V, stimulated the rate of uv-induced repair synthesis with normal and xeroderma pigmentosum cell lines. This system should prove useful for identifying agents that induce DNA repair, and cells that respond abnormally to such induction. It should also be applicable to an in vitro complementation assay with repair-defective cells and proteins obtained from repair-proficient cells. Finally, by using actively growing fibroblasts and thymidine in the system, DNA replication can be measured and studied in vitro

  3. Callus and cell suspension cultures of carnation

    DEFF Research Database (Denmark)

    Engvild, Kjeld Christensen

    1972-01-01

    . Cell suspension cultures worked best in media containing 2,4-D in which they had a doubling time of about 2 days. Filtered suspensions were successfully plated on agar in petri dishes, but division was never observed in single cells. The cultures initiated roots at higher concentrations of IAA or NAA...

  4. [Effects of beryllium chloride on cultured cells].

    Science.gov (United States)

    Sakaguchi, T; Sakaguchi, S; Nakamura, I; Kagami, M

    1984-05-01

    The effects of beryllium on cultured cells were investigated. Three cell-lines (HeLa-S3, Vero, HEL-R66) were used in these experiments and they were cultured in Eagle's MEM plus 5 or 10% FBS (Fetal Bovine Serum) containing beryllium in various concentrations. HeLa cells or Vero cells were able to grow in the medium with 10 micrograms Be/ml (1.1 mM). On the other hand, the growth of HEL cells were strongly inhibited, even when cultured in the medium with 1 microgram Be/ml (1.1 X 10(-1) mM) and the number of living cells showed markedly low level as compared to that of the control samples cultured in the medium without beryllium. The cytotoxic effects of beryllium on these cells, which were cultured for three days in the medium with beryllium, were observed. None of cytotoxic effects were found on HeLa cells cultured with 0.5 micrograms/ml (5.5 X 10(-2) mM) and on Vero cells cultured with 0.05 micrograms Be/ml (5.5 X 10(-3) mM), while HEL cells received cytotoxic effects even when cultured in the medium containing 0.05 micrograms Be/ml (5.5 X 10(-3) mM), and these effects on the cells appeared strong when cultured in the medium without FBS. It was revealed from these experiments that HEL cells are very sensitive in terms of toxic effects of beryllium. Therefore, there cells can be used for the toxicological study on low level concentrations of the metal.

  5. A spheroid-based 3-D culture model for pancreatic cancer drug testing, using the acid phosphatase assay

    International Nuclear Information System (INIS)

    Current therapy for pancreatic cancer is multimodal, involving surgery and chemotherapy. However, development of pancreatic cancer therapies requires a thorough evaluation of drug efficacy in vitro before animal testing and subsequent clinical trials. Compared to two-dimensional culture of cell monolayer, three-dimensional (3-D) models more closely mimic native tissues, since the tumor microenvironment established in 3-D models often plays a significant role in cancer progression and cellular responses to the drugs. Accumulating evidence has highlighted the benefits of 3-D in vitro models of various cancers. In the present study, we have developed a spheroid-based, 3-D culture of pancreatic cancer cell lines MIAPaCa-2 and PANC-1 for pancreatic drug testing, using the acid phosphatase assay. Drug efficacy testing showed that spheroids had much higher drug resistance than monolayers. This model, which is characteristically reproducible and easy and offers rapid handling, is the preferred choice for filling the gap between monolayer cell cultures and in vivo models in the process of drug development and testing for pancreatic cancer

  6. A spheroid-based 3-D culture model for pancreatic cancer drug testing, using the acid phosphatase assay

    Directory of Open Access Journals (Sweden)

    Z. Wen

    2013-08-01

    Full Text Available Current therapy for pancreatic cancer is multimodal, involving surgery and chemotherapy. However, development of pancreatic cancer therapies requires a thorough evaluation of drug efficacy in vitro before animal testing and subsequent clinical trials. Compared to two-dimensional culture of cell monolayer, three-dimensional (3-D models more closely mimic native tissues, since the tumor microenvironment established in 3-D models often plays a significant role in cancer progression and cellular responses to the drugs. Accumulating evidence has highlighted the benefits of 3-D in vitro models of various cancers. In the present study, we have developed a spheroid-based, 3-D culture of pancreatic cancer cell lines MIAPaCa-2 and PANC-1 for pancreatic drug testing, using the acid phosphatase assay. Drug efficacy testing showed that spheroids had much higher drug resistance than monolayers. This model, which is characteristically reproducible and easy and offers rapid handling, is the preferred choice for filling the gap between monolayer cell cultures and in vivo models in the process of drug development and testing for pancreatic cancer.

  7. A spheroid-based 3-D culture model for pancreatic cancer drug testing, using the acid phosphatase assay

    Energy Technology Data Exchange (ETDEWEB)

    Wen, Z.; Liao, Q.; Hu, Y.; You, L.; Zhou, L.; Zhao, Y. [Department of General Surgery, Peking Union Medical College Hospital, Chinese Academy of Medical Science and Peking Union Medical College, Tsinghua University, Beijing (China)

    2013-08-10

    Current therapy for pancreatic cancer is multimodal, involving surgery and chemotherapy. However, development of pancreatic cancer therapies requires a thorough evaluation of drug efficacy in vitro before animal testing and subsequent clinical trials. Compared to two-dimensional culture of cell monolayer, three-dimensional (3-D) models more closely mimic native tissues, since the tumor microenvironment established in 3-D models often plays a significant role in cancer progression and cellular responses to the drugs. Accumulating evidence has highlighted the benefits of 3-D in vitro models of various cancers. In the present study, we have developed a spheroid-based, 3-D culture of pancreatic cancer cell lines MIAPaCa-2 and PANC-1 for pancreatic drug testing, using the acid phosphatase assay. Drug efficacy testing showed that spheroids had much higher drug resistance than monolayers. This model, which is characteristically reproducible and easy and offers rapid handling, is the preferred choice for filling the gap between monolayer cell cultures and in vivo models in the process of drug development and testing for pancreatic cancer.

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

    Directory of Open Access Journals (Sweden)

    Uma Basavanna

    2013-01-01

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

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

    Science.gov (United States)

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

    2014-04-01

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

  10. Dynamic culture improves cell reprogramming efficiency.

    Science.gov (United States)

    Sia, Junren; Sun, Raymond; Chu, Julia; Li, Song

    2016-06-01

    Cell reprogramming to pluripotency is an inefficient process and various approaches have been devised to improve the yield of induced pluripotent stem cells. However, the effect of biophysical factors on cell reprogramming is not well understood. Here we showed that, for the first time, dynamic culture with orbital shaking significantly improved the reprogramming efficiency in adherent cells. Manipulating the viscosity of the culture medium suggested that the improved efficiency is mainly attributed to convective mixing rather than hydrodynamic shear stress. Temporal studies demonstrated that the enhancement of reprogramming efficiency required the dynamic culture in the middle but not early phase. In the early phase, fibroblasts had a high proliferation rate, but as the culture became over-confluent in the middle phase, expression of p57 was upregulated to inhibit cell proliferation and consequently, cell reprogramming. Subjecting the over confluent culture to orbital shaking prevented the upregulation of p57, thus improving reprogramming efficiency. Seeding cells at low densities to avoid over-confluency resulted in a lower efficiency, and optimal reprogramming efficiency was attained at a high seeding density with dynamic culture. Our findings provide insight into the underlying mechanisms of how dynamic culture condition regulate cell reprogramming, and will have broad impact on cell engineering for regenerative medicine and disease modeling.

  11. Measurement and analysis of calcium signaling in heterogeneous cell cultures.

    Science.gov (United States)

    Richards, Gillian R; Jack, Andrew D; Platts, Amy; Simpson, Peter B

    2006-01-01

    High-content imaging platforms capable of studying kinetic responses at a single-cell level have elevated kinetic recording techniques from labor-intensive low-throughput experiments to potential high-throughput screening assays. We have applied this technology to the investigation of heterogeneous cell cultures derived from primary neural tissue. The neuronal cultures mature into a coupled network and display spontaneous oscillations in intracellular calcium, which can be modified by the addition of pharmacological agents. We have developed algorithms to perform Fourier analysis and quantify both the degree of synchronization and the effects of modulators on the oscillations. Functional and phenotypic experiments can be combined using this approach. We have used post-hoc immunolabeling to identify subpopulations of cells in cocultures and to dissect the calcium responses of these cells from the population response. The combination of these techniques represents a powerful tool for drug discovery.

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

    International Nuclear Information System (INIS)

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-10-31

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

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

    Directory of Open Access Journals (Sweden)

    Pooja Pardhanani

    2011-10-01

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

  15. Mesenchymal stem cell-conditioned medium accelerates skin wound healing: An in vitro study of fibroblast and keratinocyte scratch assays

    International Nuclear Information System (INIS)

    We have used in vitro scratch assays to examine the relative contribution of dermal fibroblasts and keratinocytes in the wound repair process and to test the influence of mesenchymal stem cell (MSC) secreted factors on both skin cell types. Scratch assays were established using single cell and co-cultures of L929 fibroblasts and HaCaT keratinocytes, with wound closure monitored via time-lapse microscopy. Both in serum supplemented and serum free conditions, wound closure was faster in L929 fibroblast than HaCaT keratinocyte scratch assays, and in co-culture the L929 fibroblasts lead the way in closing the scratches. MSC-CM generated under serum free conditions significantly enhanced the wound closure rate of both skin cell types separately and in co-culture, whereas conditioned medium from L929 or HaCaT cultures had no significant effect. This enhancement of wound closure in the presence of MSC-CM was due to accelerated cell migration rather than increased cell proliferation. A number of wound healing mediators were identified in MSC-CM, including TGF-β1, the chemokines IL-6, IL-8, MCP-1 and RANTES, and collagen type I, fibronectin, SPARC and IGFBP-7. This study suggests that the trophic activity of MSC may play a role in skin wound closure by affecting both dermal fibroblast and keratinocyte migration, along with a contribution to the formation of extracellular matrix.

  16. Mesenchymal stem cell-conditioned medium accelerates skin wound healing: An in vitro study of fibroblast and keratinocyte scratch assays

    Energy Technology Data Exchange (ETDEWEB)

    Walter, M.N.M. [Institute for Science and Technology in Medicine, Keele University RJAH Orthopaedic Hospital, Oswestry, SY10 7AG (United Kingdom); School of Life and Health Science, Aston University, Aston Triangle, Birmingham, B4 7EJ (United Kingdom); Wright, K.T.; Fuller, H.R. [Institute for Science and Technology in Medicine, Keele University RJAH Orthopaedic Hospital, Oswestry, SY10 7AG (United Kingdom); MacNeil, S. [Kroto Research Institute and Centre for Nanoscience and Technology, Sheffield University, Sheffield, S1 2UE (United Kingdom); Johnson, W.E.B., E-mail: w.e.johnson@aston.ac.uk [School of Life and Health Science, Aston University, Aston Triangle, Birmingham, B4 7EJ (United Kingdom)

    2010-04-15

    We have used in vitro scratch assays to examine the relative contribution of dermal fibroblasts and keratinocytes in the wound repair process and to test the influence of mesenchymal stem cell (MSC) secreted factors on both skin cell types. Scratch assays were established using single cell and co-cultures of L929 fibroblasts and HaCaT keratinocytes, with wound closure monitored via time-lapse microscopy. Both in serum supplemented and serum free conditions, wound closure was faster in L929 fibroblast than HaCaT keratinocyte scratch assays, and in co-culture the L929 fibroblasts lead the way in closing the scratches. MSC-CM generated under serum free conditions significantly enhanced the wound closure rate of both skin cell types separately and in co-culture, whereas conditioned medium from L929 or HaCaT cultures had no significant effect. This enhancement of wound closure in the presence of MSC-CM was due to accelerated cell migration rather than increased cell proliferation. A number of wound healing mediators were identified in MSC-CM, including TGF-{beta}1, the chemokines IL-6, IL-8, MCP-1 and RANTES, and collagen type I, fibronectin, SPARC and IGFBP-7. This study suggests that the trophic activity of MSC may play a role in skin wound closure by affecting both dermal fibroblast and keratinocyte migration, along with a contribution to the formation of extracellular matrix.

  17. Autofluorescence of viable cultured mammalian cells.

    Science.gov (United States)

    Aubin, J E

    1979-01-01

    The autofluorescence other than intrinsic protein emission of viable cultured mammalian cells has been investigated. The fluorescence was found to originate in discrete cytoplasmic vesicle-like regions and to be absent from the nucleus. Excitation and emission spectra of viable cells revealed at least two distinct fluorescent species. Comparison of cell spectra with spectra of known cellular metabolites suggested that most, if not all, of the fluorescence arises from intracellular nicotinamide adenine dinucleotide (NADH) and riboflavin and flavin coenzymes. Various changes in culture conditions did not affect the observed autofluorescence intensity. A multiparameter flow system (MACCS) was used to compare the fluorescence intensities of numerous cultured mammalian cells.

  18. 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.

  19. Expression of CD44 in Cultured Human Trabecular Meshwork Cells

    Institute of Scientific and Technical Information of China (English)

    Zhongguo Li; Hong Zhang

    2004-01-01

    Purpose:To determine whether cultured human trabecular meshwork cells express CD44 and to discuss their possible relationship with primary open angle glaucoma.Methods:Human trabecular meshwork cells were cultured in DMEM/F12 media. Total RNAs from the cells were extracted with Trizol reagent. Messenger RNA expression of CD44 in human trabecular meshwork cells was examined by using reverse transcriptasepolymerase chain reaction ( RT-PCR ) analysis. Expression of CD44 was confirmed by Western-blotting and immunofiuorescent microscopy. Effect of CD44-specific antisense oligonucleotide on adhesion of trabecular meshwork cells to hyaluronate was determined by MTT assay.Results:A single RT-PCR product whose size was 471bp was obtained.A band about 80kD was stained by Western-blot. Immunofiuorescent examination of expression of CD44 on the cell surface was positive and reactions were mainly localized in cell membranes.Adhesion of trabecular meshwork cells to hyaluronate was inhibited by CD44-specific antisense oligonucleotide.Conclusions: Cultured human trabecular meshwork cells express CD44. CD44 may play a role in pathogenesis of primary open angle glaucoma. Eye Science 2004;20:52-56.

  20. Cell Culture for Production of Insecticidal Viruses.

    Science.gov (United States)

    Reid, Steven; Chan, Leslie C L; Matindoost, Leila; Pushparajan, Charlotte; Visnovsky, Gabriel

    2016-01-01

    While large-scale culture of insect cells will need to be conducted using bioreactors up to 10,000 l scale, many of the main challenges for cell culture-based production of insecticidal viruses can be studied using small-scale (20-500 ml) shaker/spinner flasks, either in free suspension or using microcarrier-based systems. These challenges still relate to the development of appropriate cell lines, stability of virus strains in culture, enhancing virus yields per cell, and the development of serum-free media and feeds for the desired production systems. Hence this chapter presents mainly the methods required to work with and analyze effectively insect cell systems using small-scale cultures. Outlined are procedures for quantifying cells and virus and for establishing frozen cells and virus stocks. The approach for maintaining cell cultures and the multiplicity of infection (MOI) and time of infection (TOI) parameters that should be considered for conducting infections are discussed.The methods described relate, in particular, to the suspension culture of Helicoverpa zea and Spodoptera frugiperda cell lines to produce the baculoviruses Helicoverpa armigera nucleopolyhedrovirus, HearNPV, and Anticarsia gemmatalis multicapsid nucleopolyhedrovirus, AgMNPV, respectively, and the production of the nonoccluded Oryctes nudivirus, OrNV, using an adherent coleopteran cell line. PMID:27565495

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

    Science.gov (United States)

    Bausinger, Julia; Speit, Günter

    2014-11-01

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

  2. A Comparative Study Of The Indirect Fluorescent Antibody Assay And Culture Method In Symptomatic Pulmonary Nocardiosis

    Directory of Open Access Journals (Sweden)

    Eshraghi S

    2004-09-01

    Full Text Available Background: Pulmonary Nocardiosis is an infrequent infection whose incidence seems to be on the rise due to a higher degree of clinical suspicion and to an increasing number of immunosuppressive factors. The present investigation was carried out to detect Nocardiosis in immunocompromised patients confined in the pulmonary ward of Tehran’s Shariati Training Hospital through the use of indirect immunofluorescence assay (IFA and bacterial culture methods. The comparison of the two methods and the correlation between the antibody titer and the statistical and epidemiological data were also investigated. Materials and Methods: 101 patients with advanced symptomatic pulmonary infection were studied in the course of a twenty-month period. Individual patients’ sputum, BAL (bronchoalveolar lavage and blood sera were tested. From each sample three thin smears were prepared for microscopic observations. The samples were cultured in Sabouraud’s dextrose, blood and paraffin agar. The detection of antibody against Nocardia asteroides was carried out in all study groups, using the IFA method. The medical history of patients was also obtained through questionnaires for further analysis. Results: Nocardia asteroides was isolated from only one patient suffering from Wagner vasculitis with an antibody titer of in serum. The 41 patients suspected for Nocardiosis with an antibody titer ranging from to , detected by IFA method, included 26 (63.4% men and 15 (14.8% women. The age of the patients varied from 7-80 years. Those with reasonable antibody titers included 15 (36.5% housewives and 9 (21.9% workers. Furthermore, in-vitro investigation for the differentiation of the isolates was performed and confirmed the notion that the organism which grew on the primary media was, indeed, the Nocardia asteroids complex. Conclusion: Our results revealed that the broncho-pulmonary infections, which occur in high-risk patients -T-cell deficiencies, long term

  3. A standardized and reproducible protocol for serum-free monolayer culturing of primary paediatric brain tumours to be utilized for therapeutic assays.

    Science.gov (United States)

    Sandén, Emma; Eberstål, Sofia; Visse, Edward; Siesjö, Peter; Darabi, Anna

    2015-01-01

    In vitro cultured brain tumour cells are indispensable tools for drug screening and therapeutic development. Serum-free culture conditions tentatively preserve the features of the original tumour, but commonly comprise neurosphere propagation, which is a technically challenging procedure. Here, we define a simple, non-expensive and reproducible serum-free cell culture protocol for establishment and propagation of primary paediatric brain tumour cultures as adherent monolayers. The success rates for establishment of primary cultures (including medulloblastomas, atypical rhabdoid tumour, ependymomas and astrocytomas) were 65% (11/17) and 78% (14/18) for sphere cultures and monolayers respectively. Monolayer culturing was particularly feasible for less aggressive tumour subsets, where neurosphere cultures could not be generated. We show by immunofluorescent labelling that monolayers display phenotypic similarities with corresponding sphere cultures and primary tumours, and secrete clinically relevant inflammatory factors, including PGE2, VEGF, IL-6, IL-8 and IL-15. Moreover, secretion of PGE2 was considerably reduced by treatment with the COX-2 inhibitor Valdecoxib, demonstrating the functional utility of our newly established monolayer for preclinical therapeutic assays. Our findings suggest that this culture method could increase the availability and comparability of clinically representative in vitro models of paediatric brain tumours, and encourages further molecular evaluation of serum-free monolayer cultures.

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

    Science.gov (United States)

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

    2010-06-01

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

  5. Impedimetric quantification of cells encapsulated in hydrogel cultured in a paper-based microchamber.

    Science.gov (United States)

    Lei, Kin Fong; Huang, Chia-Hao; Tsang, Ngan-Ming

    2016-01-15

    Recently, 3D cell culture technique was proposed to provide a more physiologically-meaningful environment for cell-based assays. With the development of microfluidics technology, cellular response can be quantified by impedance measurement technique in a real-time and non-invasive manner. However, handling of these microfluidic systems requires a trained engineering personnel and the operation is not compatible to traditional biological research laboratories. In this work, we incorporated the impedance measurement technique to paper-based 3D cell culture model and demonstrated non-invasive quantification of cells encapsulated in hydrogel during the culture course. A cellulose filter paper was patterned with an array of circular microchambers. Cells were encapsulated in hydrogel and loaded to the microchambers for culturing cells in 3D environment. At the preset schedule during the culture course, the paper was placed on a glass substrate with measurement electrodes for the impedance measurement. Cells in each microchamber was represented by impedance magnitude and cell proliferation could be studied over time. Also, conventional bio-assay was performed to further confirm the feasibility of the impedimetric quantification of cells encapsulated in hydrogel cultured in the paper-based microchamber. This technique provides a convenient, fast, and non-invasive approach to monitor cells cultured in 3D environment. It has potential to be developed for routine 3D cell culture protocol in biological research laboratories. PMID:26592655

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

    Directory of Open Access Journals (Sweden)

    Horváth Gábor V

    2010-01-01

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

  7. 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.

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

    Directory of Open Access Journals (Sweden)

    Novielli Nicole M

    2011-10-01

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

  9. Methods for Maintaining Insect Cell Cultures

    OpenAIRE

    Lynn, Dwight E.

    2002-01-01

    Insect cell cultures are now commonly used in insect physiology, developmental biology, pathology, and molecular biology. As the field has advanced from methods development to a standard procedure, so has the diversity of scientists using the technique. This paper describes methods that are effective for maintaining various insect cell lines. The procedures are differentiated between loosely or non-attached cell strains, attached cell strains, and strongly adherent cell strains.

  10. Spheroid-Formation (Colonosphere) Assay for in Vitro Assessment and Expansion of Stem Cells in Colon Cancer.

    Science.gov (United States)

    Shaheen, Sameerah; Ahmed, Mehreen; Lorenzi, Federica; Nateri, Abdolrahman S

    2016-08-01

    Colorectal cancers (CRCs) form a disorganized hierarchy of heterogeneous cell populations on which current chemotherapy regimens fail to exert their distinctive cytotoxicity. A small sub-population of poorly differentiated cancer stem-like cells (CSCs), also known as cancer initiating cells, may exhibit embryonic and/or adult stem-cell gene expression signatures. Self-renewal and survival signals are also dominant over differentiation in CSCs. However, inducers of differentiation exclusive to CSC may affect cellular pathways required for the formation and progression of a tumor, which are not utilized in normal adult stem-cells. Nevertheless, assays for targeting CSCs have been hindered by expanding and maintaining rare CSCs in vitro. However, CRC-CSCs are able to form floating spheroids (known as colonospheres) 3-dimentinionally (3D) in a serum-free defined medium. Therefore, great efforts have been paid to improve colonosphere forming assay as a preclinical model to study tumor biology and to conduct drug screening in cancer research. The 3D-colonosphere culture model may also represent in vivo conditions for the spontaneous aggregation of cancer cells in spheroids. This protocol describes the development of an enrichment/culture assay using CRC-CSCs to facilitate colorectal cancer research through immunofluorescence staining of colonospheres. We have developed colonospheres from HCT116 CRC cell line to compare and link CRC-CSC markers to the NANOG expression level using an immunofluorescence assay. Our data also show that the immunostaining assay of colonosphere is a useful method to explore the role and dynamics of CRC-CSCs division between self-renewal and cell lineage differentiation of cancer cells. In principle, this method is applicable to a variety of primary cells and cell lines of epithelial origin. Furthermore, this protocol may also allow screening of libraries of compounds to identify bona fide CRC-CSC differentiation inducers. PMID:27207017

  11. In vitro assays for determining the metastatic potential of melanoma cell lines with characterized in vivo invasiveness.

    Science.gov (United States)

    Chandrasekaran, Siddarth; Giang, Ut-Binh T; Xu, Lei; DeLouise, Lisa A

    2016-10-01

    The metastatic potential of cancer cells is an elusive property that is indicative of the later stages of cancer progression. The ability to distinguish between poorly and highly metastatic cells is invaluable for understanding the basic biology of cancer and to develop more treatments. In this paper, we exploit a A375 melanoma cell line series (A375P, A375MA1, A375MA2) that vary in metastatic potential, to demonstrate an in vitro screening assay using polydimethylsiloxane (PDMS) microbubble well arrays that can distinguish these cell lines by their growth characteristics in including morphology, migratory potential, and clonogenic potential. These cell lines cannot be distinguished by their growth characteristics when cultured on standard tissue culture plastic or planar PDMS. Results show that the more metastatic cell lines (A375MA1, A375MA2) have a higher proliferative potential and a distinctive radial spreading growth pattern out of the microbubble well. The A375MA2 cell line also has a higher tendency to form multicellular spheroids. The ability to successfully correlate the metastatic potential of cancer cells with their growth characteristics is essential first step toward developing a high-throughput screening assay to identify aggressive tumor cells in primary samples. The capability to culture and recover aggressive cells from microbubble wells will enable identification of candidate metastatic biomarkers which has immense clinical significance. PMID:27620628

  12. 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.

  13. Establishment, characterization, and toxicological application of loggerhead sea turtle (Caretta caretta) primary skin fibroblast cell cultures.

    Science.gov (United States)

    Webb, Sarah J; Zychowski, Gregory V; Bauman, Sandy W; Higgins, Benjamin M; Raudsepp, Terje; Gollahon, Lauren S; Wooten, Kimberly J; Cole, Jennifer M; Godard-Codding, Céline

    2014-12-16

    Pollution is a well-known threat to sea turtles but its impact is poorly understood. In vitro toxicity testing presents a promising avenue to assess and monitor the effects of environmental pollutants in these animals within the legal constraints of their endangered status. Reptilian cell cultures are rare and, in sea turtles, largely derived from animals affected by tumors. Here we describe the full characterization of primary skin fibroblast cell cultures derived from biopsies of multiple healthy loggerhead sea turtles (Caretta caretta), and the subsequent optimization of traditional in vitro toxicity assays to reptilian cells. Characterization included validating fibroblast cells by morphology and immunocytochemistry, and optimizing culture conditions by use of growth curve assays with a fractional factorial experimental design. Two cell viability assays, MTT and lactate dehydrogenase (LDH), and an assay measuring cytochrome P4501A (CYP1A) expression by quantitative PCR were optimized in the characterized cells. MTT and LDH assays confirmed cytotoxicity of perfluorooctanoic acid at 500 μM following 72 and 96 h exposures while CYP1A5 induction was detected after 72 h exposure to 0.1-10 μM benzo[a]pyrene. This research demonstrates the validity of in vitro toxicity testing in sea turtles and highlights the need to optimize mammalian assays to reptilian cells. PMID:25384208

  14. Culture and transfection of axolotl cells.

    Science.gov (United States)

    Denis, Jean-François; Sader, Fadi; Ferretti, Patrizia; Roy, Stéphane

    2015-01-01

    The use of cells grown in vitro has been instrumental for multiple aspects of biomedical research and especially molecular and cellular biology. The ability to grow cells from multicellular organisms like humans, squids, or salamanders is important to simplify the analyses and experimental designs to help understand the biology of these organisms. The advent of the first cell culture has allowed scientists to tease apart the cellular functions, and in many situations these experiments help understand what is happening in the whole organism. In this chapter, we describe techniques for the culture and genetic manipulation of an established cell line from axolotl, a species widely used for studying epimorphic regeneration.

  15. Responses of the L5178Y mouse lymphoma cell forward mutation assay. V: 27 coded chemicals

    Energy Technology Data Exchange (ETDEWEB)

    McGregor, D.B.; Brown, A.G.; Howgate, S.; McBride, D.; Riach, C. (Inveresk Research International Limited, Musselburgh (Scotland)); Caspary, W.J. (National Inst. of Health, Research Triangle Park, NC (United States))

    1991-01-01

    Twenty-seven chemicals were tested for their mutagenic potential in the L5178Y tk{sup +}/tk{sup {minus}} mouse lymphoma cell forward mutation assay. Cultures were exposed to the chemicals for 4 hr, then cultured for 2 days before plating in soft agar with or without trifluorothymidine (TFT), 3 {mu}g/ml. The chemicals were tested at least twice. Statistically significant responses were obtained with acid orange 10, aniline, benzaldehyde o-chloroaniline, chlorodibromomethane, cytembena, 1,2-dibromo-4-(1,2-dibromomethyl) cyclohexane, dieldrin, lithocholic acid, oxytetracycline, phenazopyridine HCl, 1phenyl-3-methyl-5-pyrazolone, sodium diethyldithiocarbamate, solvent yellow 14, tetraethylthiuram disulfide (disulfiram), 2,4-toluene diisocyanate, and 2,6-toluene diisocyanate. Apart from phenazopyridine HCl, acid orange 10, and solvent yellow 14, rat liver S9 mix was not a requirement for the mutagenic activity of these compounds.

  16. Long term maintenance of myeloid leukemic stem cells cultured with unrelated human mesenchymal stromal cells

    Directory of Open Access Journals (Sweden)

    Sawa Ito

    2015-01-01

    Full Text Available Mesenchymal stromal cells (MSCs support the growth and differentiation of normal hematopoietic stem cells (HSCs. Here we studied the ability of MSCs to support the growth and survival of leukemic stem cells (LSCs in vitro. Primary leukemic blasts isolated from the peripheral blood of 8 patients with acute myeloid leukemia (AML were co-cultured with equal numbers of irradiated MSCs derived from unrelated donor bone marrow, with or without cytokines for up to 6 weeks. Four samples showed CD34+CD38− predominance, and four were predominantly CD34+CD38+. CD34+ CD38− predominant leukemia cells maintained the CD34+ CD38− phenotype and were viable for 6 weeks when co-cultured with MSCs compared to co-cultures with cytokines or medium only, which showed rapid differentiation and loss of the LSC phenotype. In contrast, CD34+ CD38+ predominant leukemic cells maintained the CD34+CD38+ phenotype when co-cultured with MSCs alone, but no culture conditions supported survival beyond 4 weeks. Cell cycle analysis showed that MSCs maintained a higher proportion of CD34+ blasts in G0 than leukemic cells cultured with cytokines. AML blasts maintained in culture with MSCs for up to 6 weeks engrafted NSG mice with the same efficiency as their non-cultured counterparts, and the original karyotype persisted after co-culture. Chemosensitivity and transwell assays suggest that MSCs provide pro-survival benefits to leukemic blasts through cell–cell contact. We conclude that MSCs support long-term maintenance of LSCs in vitro. This simple and inexpensive approach will facilitate basic investigation of LSCs and enable screening of novel therapeutic agents targeting LSCs.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2008-06-23

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

  18. Biodegradable Mg corrosion and osteoblast cell culture studies

    International Nuclear Information System (INIS)

    Magnesium (Mg) is a biodegradable metal that has significant potential advantages as an implant material. In this paper, corrosion and cell culture experiments were performed to evaluate the biocompatibility of Mg. The corrosion current and potential of a Mg disk were measured in different physiological solutions including deionized (DI) water, phosphate-buffered saline (PBS), and McCoy's 5A culture medium. The corrosion currents in the PBS and in the McCoy's 5A-5% FBS media were found to be higher than in DI water, which is expected because corrosion of Mg occurs faster in a chloride solution. Weight loss, open-circuit potential, and electrochemical impedance spectroscopy measurements were also performed. The Mg specimens were also characterized using an environmental scanning electron microscope and energy-dispersive X-ray analysis (EDAX). The X-ray analysis showed that in the cell culture media a passive interfacial layer containing oxygen, chloride, phosphate, and potassium formed on the samples. U2OS cells were then co-cultured with a Mg specimen for up to one week. Cytotoxicity results of magnesium using MTT assay and visual observation through cell staining were not significantly altered by the presence of the corroding Mg sample. Further, bone tissue formation study using von Kossa and alkaline phosphatase staining indicates that Mg may be suitable as a biodegradable implant material.

  19. Inhibitory effect of mycoplasma-released arginase. Activity in mixed-lymphocyte and tumour cell cultures

    DEFF Research Database (Denmark)

    Claesson, M H; Tscherning, T; Nissen, Mogens Holst;

    1990-01-01

    Non-fermenting mycoplasma species deplete culture media for arginine through arginase activity linked to their arginine deiminase pathway, resulting in proliferation arrest and cell death in mycoplasma-contaminated cell cultures. The presence of only 2-3 Mycoplasma (M.) arginini-contaminated T...... assayed in MLC and cell proliferation culture. SDS-PAGE followed by western blotting and reaction with antisera raised against non-fermenting mycoplasma species demonstrated a band at 43 kDa common for these micro-organisms....... cells in a one-way allogeneic mixed-lymphocyte culture (MLC) significantly inhibits development of cytotoxic T-cell activity. Likewise, strong degrees of inhibition are observed after addition of nanogram doses of M. arginini extracts (MAE) to MLC or cell proliferation cultures. M. arginini-induced cell...

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

    International Nuclear Information System (INIS)

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

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

    Science.gov (United States)

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

    2010-02-01

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

  2. Sponge cell culture? A molecular identification method for sponge cells

    NARCIS (Netherlands)

    Sipkema, D.; Heilig, G.H.J.; Akkermans, A.D.L.; Osinga, R.; Tramper, J.; Wijffels, R.H.

    2003-01-01

    Dissociated sponge cells are easily confused with unicellular organisms. This has been an obstacle in the development of sponge-cell lines. We developed a molecular detection method to identify cells of the sponge Dysidea avara in dissociated cell cultures. The 18S ribosomal RNA gene from a Dysidea

  3. Cell culture models for study of differentiated adipose cells

    OpenAIRE

    Clynes, Martin

    2014-01-01

    Adipose cells are an important source of mesenchymal stem cells and are important for direct use in research on lipid metabolism and obesity. In addition to use of primary cultures, there is increasing interest in other sources of larger numbers of cells, using approaches including induced pluripotent stem cell differentiation and viral immortalisation.

  4. Inverse dose rate effect in tumour cells measured by the comet assay

    International Nuclear Information System (INIS)

    Reduction of the dose rate of low LET radiation from high (Gy/min) to low (Gy/h) usually leads to a reduced effect as measured by the survival methods. If the dose rate is reduced, cells are able to repair sublethal damage even during irradiation. During the last few years a comet assay has been widely used to measure DNA damage induction and repair in single cells. In our study we used the alkaline version of the comet assay for comparison of high (0.833 Gy/min) and low dose rate (0.0707 Gy/min) effects on DNA damage and repair in R1 rat rhabdomyosarcoma and Me45 human malignant melanoma cells. Cells gathered from exponential culture by trypsynization were suspended in a growth medium and irradiated at room temperature, with 5 Gy of photons X generated by linear accelerator at both dose rates. Comets were analysed automatically using self-made software for measurement of percentage DNA in the tail, and tail moment and inertia. Our results show that tail inertia is the best parameter expressing DNA damage and repair. Although the level of DNA damage induced by low dose rate was comparable with that induced by a high dose rate, the damage induced by the low dose rate are repair more slowly than after high dose rate irradiation. This inverse dose rate effect suggest that nature of damage can differ in both groups. (author)

  5. Digital microfluidics for automated hanging drop cell spheroid culture.

    Science.gov (United States)

    Aijian, Andrew P; Garrell, Robin L

    2015-06-01

    Cell spheroids are multicellular aggregates, grown in vitro, that mimic the three-dimensional morphology of physiological tissues. Although there are numerous benefits to using spheroids in cell-based assays, the adoption of spheroids in routine biomedical research has been limited, in part, by the tedious workflow associated with spheroid formation and analysis. Here we describe a digital microfluidic platform that has been developed to automate liquid-handling protocols for the formation, maintenance, and analysis of multicellular spheroids in hanging drop culture. We show that droplets of liquid can be added to and extracted from through-holes, or "wells," and fabricated in the bottom plate of a digital microfluidic device, enabling the formation and assaying of hanging drops. Using this digital microfluidic platform, spheroids of mouse mesenchymal stem cells were formed and maintained in situ for 72 h, exhibiting good viability (>90%) and size uniformity (% coefficient of variation digital microfluidic platform provides a viable tool for automating cell spheroid culture and analysis. PMID:25510471

  6. Evaluation of new transport medium for detection of herpes simplex virus by culture and direct enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Ogburn, J R; Hoffpauir, J T; Cole, E; Hood, K; Michael, D; Nguyen, T; Raden, S; Raju, B; Reisinger, V; Oefinger, P E

    1994-12-01

    The transport medium Multi-Microbe Media (M4) was evaluated prospectively by culture and direct enzyme-linked immunosorbent assay (ELISA) for detection of herpes simplex virus from 473 specimens. In addition, 377 specimens in Bartels Viral Transport Medium were evaluated. By using culture as a "gold standard," the ELISA sensitivity was approximately 85%, while the specificities exceeded 96% for both media.

  7. Age and sex correlation of Chlamydia trachomatis infections evaluated by the culture technique and by an enzyme immunosorbent assay, IDEIA

    DEFF Research Database (Denmark)

    Østergaard, Lars; Lundemose, AG; Birkelund, Svend;

    1990-01-01

    tested by the tissue-culture technique, and the results were confirmed by the IDEIA (Boots-Celltech) enzyme-linked immunosorbent assay kit (EIA) for detection of C. trachomatis. The original smear was used for both culture and EIA. The EIA test was evaluated to have a sensitivity of 90...

  8. Development of a shaker culture of Buffalo green monkey kidney cells: potential use for detection of enteroviruses.

    Science.gov (United States)

    Goldstein, G; Guskey, L E

    1982-08-01

    Buffalo green monkey kidney cells were adapted to grow as shaker cultures. Replication of environmental and clinical isolates of poliovirus, coxsackievirus, and echovirus in these cultures was analyzed by plaque assay and compared with replication in Buffalo green monkey kidney cell monolayers and HEp-2 cell shaker cultures. Dose-response tests with various concentrations of Mahoney type 1 poliovirus indicated that Buffalo green monkey kidney cell shaker cultures could detect as little as 1 PFU in an inoculum of 0.2 ml. These data suggest that Buffalo green monkey kidney cell shaker cultures can be effectively used for the detection of small quantities of enteroviruses from environmental sources. PMID:6289745

  9. Melphalan metabolism in cultured cells

    International Nuclear Information System (INIS)

    Procedures are presented for the adaptation of reversed-phase-HPLC methods to accomplish separation and isolation of the cancer therapeutic drug melphalan (L-phenylalanine mustard) and its metabolic products from whole cells. Five major degradation products of melphalan were observed following its hydrolysis in phosphate buffer in vitro. The two most polar of these products (or modifications of them) were also found in the cytosol of Chinese hamster CHO cells. The amounts of these two polar products (shown not to be mono- or dihydroxymelphalan) were significantly changed by the pretreatment of cells with ZnC12, one being increased in amount while the other was reduced to an insignificant level. In ZnC12-treated cells, there was also an increased binding of melphalan (or its derivatives) to one protein fraction resolved by gel filtration-HPLC. These observations suggest that changes in polar melphalan products, and perhaps their interaction with a protein, may by involved in the reduction of melphalan cytotoxicity observed in ZnC12-treated cells. While ZnC12 is also known to increase the level of glutathione in cells, no significant amounts of glutathione-melphalan derivatives of the type formed non-enzymatically in vitro could be detected in ZnC12-treated or untreated cells. Formation of derivatives of melphalan with glutathione catabolic products in ZnC12-treated cells has not yet been eliminated, however. 17 refs., 5 figs., 1 tab

  10. Endothelial cells stimulate growth of normal and cancerous breast epithelial cells in 3D culture

    Directory of Open Access Journals (Sweden)

    Magnusson Magnus K

    2010-07-01

    Full Text Available Abstract Background Epithelial-stromal interaction provides regulatory signals that maintain correct histoarchitecture and homeostasis in the normal breast and facilitates tumor progression in breast cancer. However, research on the regulatory role of the endothelial component in the normal and malignant breast gland has largely been neglected. The aim of the study was to investigate the effects of endothelial cells on growth and differentiation of human breast epithelial cells in a three-dimensional (3D co-culture assay. Methods Breast luminal and myoepithelial cells and endothelial cells were isolated from reduction mammoplasties. Primary cells and established normal and malignant breast cell lines were embedded in reconstituted basement membrane in direct co-culture with endothelial cells and by separation of Transwell filters. Morphogenic and phenotypic profiles of co-cultures was evaluated by phase contrast microscopy, immunostaining and confocal microscopy. Results In co-culture, endothelial cells stimulate proliferation of both luminal- and myoepithelial cells. Furthermore, endothelial cells induce a subpopulation of luminal epithelial cells to form large acini/ducts with a large and clear lumen. Endothelial cells also stimulate growth and cloning efficiency of normal and malignant breast epithelial cell lines. Transwell and gradient co-culture studies show that endothelial derived effects are mediated - at least partially - by soluble factors. Conclusion Breast endothelial cells - beside their role in transporting nutrients and oxygen to tissues - are vital component of the epithelial microenvironment in the breast and provide proliferative signals to the normal and malignant breast epithelium. These growth promoting effects of endothelial cells should be taken into consideration in breast cancer biology.

  11. Bench-top validation testing of selected immunological and molecular Renibacterium salmoninarum diagnostic assays by comparison with quantitative bacteriological culture

    Science.gov (United States)

    Elliott, D.G.; Applegate, L.J.; Murray, A.L.; Purcell, M.K.; McKibben, C.L.

    2013-01-01

    No gold standard assay exhibiting error-free classification of results has been identified for detection of Renibacterium salmoninarum, the causative agent of salmonid bacterial kidney disease. Validation of diagnostic assays for R. salmoninarum has been hindered by its unique characteristics and biology, and difficulties in locating suitable populations of reference test animals. Infection status of fish in test populations is often unknown, and it is commonly assumed that the assay yielding the most positive results has the highest diagnostic accuracy, without consideration of misclassification of results. In this research, quantification of R. salmoninarum in samples by bacteriological culture provided a standardized measure of viable bacteria to evaluate analytical performance characteristics (sensitivity, specificity and repeatability) of non-culture assays in three matrices (phosphate-buffered saline, ovarian fluid and kidney tissue). Non-culture assays included polyclonal enzyme-linked immunosorbent assay (ELISA), direct smear fluorescent antibody technique (FAT), membrane-filtration FAT, nested polymerase chain reaction (nested PCR) and three real-time quantitative PCR assays. Injection challenge of specific pathogen-free Chinook salmon, Oncorhynchus tshawytscha (Walbaum), with R. salmoninarum was used to estimate diagnostic sensitivity and specificity. Results did not identify a single assay demonstrating the highest analytical and diagnostic performance characteristics, but revealed strengths and weaknesses of each test.

  12. Bench-top validation testing of selected immunological and molecular Renibacterium salmoninarum diagnostic assays by comparison with quantitative bacteriological culture.

    Science.gov (United States)

    Elliott, D G; Applegate, L J; Murray, A L; Purcell, M K; McKibben, C L

    2013-09-01

    No gold standard assay exhibiting error-free classification of results has been identified for detection of Renibacterium salmoninarum, the causative agent of salmonid bacterial kidney disease. Validation of diagnostic assays for R. salmoninarum has been hindered by its unique characteristics and biology, and difficulties in locating suitable populations of reference test animals. Infection status of fish in test populations is often unknown, and it is commonly assumed that the assay yielding the most positive results has the highest diagnostic accuracy, without consideration of misclassification of results. In this research, quantification of R. salmoninarum in samples by bacteriological culture provided a standardized measure of viable bacteria to evaluate analytical performance characteristics (sensitivity, specificity and repeatability) of non-culture assays in three matrices (phosphate-buffered saline, ovarian fluid and kidney tissue). Non-culture assays included polyclonal enzyme-linked immunosorbent assay (ELISA), direct smear fluorescent antibody technique (FAT), membrane-filtration FAT, nested polymerase chain reaction (nested PCR) and three real-time quantitative PCR assays. Injection challenge of specific pathogen-free Chinook salmon, Oncorhynchus tshawytscha (Walbaum), with R. salmoninarum was used to estimate diagnostic sensitivity and specificity. Results did not identify a single assay demonstrating the highest analytical and diagnostic performance characteristics, but revealed strengths and weaknesses of each test.

  13. Flux analysis of mammalian cell culture

    NARCIS (Netherlands)

    Martens, D.E.; Tramper, J.

    2010-01-01

    Animal cells are used for the production of vaccines and pharmaceutical proteins. The increase in demand for these products requires an increase in volumetric productivity of animal cell culture processes, which can be attained through an increase in biomass concentration and/or specific productivit

  14. Lethal impacts of cigarette smoke in cultured tobacco cells

    Directory of Open Access Journals (Sweden)

    Kawano Tomonori

    2011-07-01

    Full Text Available Abstract Background In order to understand and generalize the toxic mechanism of cigarette smoke in living cells, comparison of the data between animal systems and other biological system such as microbial and plant systems is highly beneficial. Objective By employing the tobacco cells as model materials for cigarette smoke toxicity assay, the impacts of the combustion by-products such as nitrogen oxides could be highlighted as the toxic impacts of the plant-derived endogenous chemicals could be excluded in the plant cells. Methods Cigarette smoke-induced cell death was assessed in tobacco cell suspension cultures in the presence and absence of pharmacological inhibitors. Results Cigarette smoke was effective in induction of cell death. The smoke-induced cell death could be partially prevented by addition of nitric oxide (NO scavenger, suggesting the role for NO as the cell death mediator. Addition of NO donor to tobacco cells also resulted in development of partial cell death further confirming the role of NO as cell death mediator. Members of reactive oxygen species and calcium ion were shown to be protecting the cells from the toxic action of smoke-derived NO.

  15. [CO-CULTURE OF BOAR SPERMATOGONIAL CELLS WITH SERTOLI CELLS].

    Science.gov (United States)

    Savchenkova, I P; Vasil'eva, S A

    2016-01-01

    In the present study, we developed in vitro culture conditions using co-culture of boar spermatogonial cells with Sertoli cells. Testes from 60-day-old crossbred boar were used. A spermatogonia-enriched culture was achieved by enzymatic digestion method and purification by density gradient centrifugation using a discontinuous Percoll gradient and differentiated adherence technique. Lipid drops were detected in isolated Sertoli cells by Oil Red O staining. We have found that the cultivation of boar spermatogonia in the presence of Sertoli cells (up to 35 days) leads to their differentiation as well as in vivo in testis. Association of cells in groups, formation of chains and suspension clusters of the spermatogenic cells were observed on the 10th day. Spermatogonial cellular colonies were noted at the same time. These cellular colonies were analyzed for the expression of genes: Nanog and Plzf in RT PCR. The expression of the Nanog gene in the experimental cellular clones obtained by short-term culture of spermatogonial cells in the presence of Sertoli cells was 200 times higher than the expression of this gene in the freshly isolated spermatogonial cells expression was found in freshly isolated germ cells and in cellular clones derived in vitro. We have found that, in the case of longer cultivation of these cells on Sertoli cells, in vitro process of differentiation of germ cells and formation of single mobile boar spermatozoa occurs at 30-33 days. Cellular population is heterogeneous at this stage. Spermatogenic differentiation in vitro without Sertoli cells stays on the 7th day of cultivation. The results show that co-culture of boar spermatogonia-enriched cells with Sertoli cells can induce their differentiation into spermatozoa in vitro and facilitate obtaining of porcine germ cell culture. PMID:27228660

  16. Increasing cell culture population doublings for long-term growth of finite life span human cell cultures

    Energy Technology Data Exchange (ETDEWEB)

    Stampfer, Martha R.; Garbe, James C.

    2016-06-28

    Cell culture media formulations for culturing human epithelial cells are herein described. Also described are methods of increasing population doublings in a cell culture of finite life span human epithelial cells and prolonging the life span of human cell cultures. Using the cell culture media disclosed alone and in combination with addition to the cell culture of a compound associated with anti-stress activity achieves extended growth of pre-stasis cells and increased population doublings and life span in human epithelial cell cultures.

  17. Increasing cell culture population doublings for long-term growth of finite life span human cell cultures

    Energy Technology Data Exchange (ETDEWEB)

    Stampfer, Martha R; Garbe, James C

    2015-02-24

    Cell culture media formulations for culturing human epithelial cells are herein described. Also described are methods of increasing population doublings in a cell culture of finite life span human epithelial cells and prolonging the life span of human cell cultures. Using the cell culture media disclosed alone and in combination with addition to the cell culture of a compound associated with anti-stress activity achieves extended growth of pre-stasis cells and increased population doublings and life span in human epithelial cell cultures.

  18. In Situ SUMOylation and DeSUMOylation Assays: Fluorescent Methods to Visualize SUMOylation and DeSUMOylation in Permeabilized Cells.

    Science.gov (United States)

    Yuasa, Eri; Saitoh, Hisato

    2016-01-01

    This chapter deals with the fluorescence detection of SUMOylation and deSUMOylation in semi-intact cultured human cells, the so-called "in situ SUMOylation assay" and the "in situ deSUMOylation assay," respectively. In the in situ SUMOylation assay, the recombinant green-fluorescence protein fused to the SUMO1 (GFP-SUMO1) protein is used to visualize the nuclear rim, nucleolus, and nuclear bodies. These GFP signals represent cellular regions where SUMOylation efficiently takes place. If the recombinant SUMO-specific protease SENP1-catalytic domain is added after in situ SUMOylation, GFP signals can be erased. Therefore, the in situ SUMOylation assay can be used to assess deSUMOylation enzymatic activity. PMID:27631804

  19. Optimisation of the Factor VIII yield in mammalian cell cultures by reducing the membrane bound fraction

    DEFF Research Database (Denmark)

    Kolind, Mille Petersen; Nørby, Peder Lisby; Berchtold, Martin Werner;

    2011-01-01

    of active membrane bound rFVIII to the culture medium. Moreover, the attachment of rFVIII to cell membranes of un-transfected HEK293 cells was studied in the presence of compounds that competes for interactions between rFVIII and PS. Competitive assays between iodinated rFVIII (¹²5I-rFVIII) and annexin V...

  20. Phenotypic characterization of bovine memory cells responding to mycobacteria in IFNγ enzyme linked immunospot assays.

    Science.gov (United States)

    Blunt, Laura; Hogarth, Philip J; Kaveh, Daryan A; Webb, Paul; Villarreal-Ramos, Bernardo; Vordermeier, Hans Martin

    2015-12-16

    Bovine tuberculosis (bTB) remains a globally significant veterinary health problem. Defining correlates of protection can accelerate the development of novel vaccines against TB. As the cultured IFNγ ELISPOT (cELISPOT) assay has been shown to predict protection and duration of immunity in vaccinated cattle, we sought to characterize the phenotype of the responding T-cells. Using expression of CD45RO and CD62L we purified by cytometric cell sorting four distinct CD4(+) populations: CD45RO(+)CD62L(hi), CD45RO(+)CD62L(lo), CD45RO(-)CD62L(hi) and CD45RO(-)CD62L(lo) (although due to low and inconsistent cell recovery, this population was not considered further in this study), in BCG vaccinated and Mycobacterium bovis infected cattle. These populations were then tested in the cELISPOT assay. The main populations contributing to production of IFNγ in the cELISPOT were of the CD45RO(+)CD62L(hi) and CD45RO(+)CD62L(lo) phenotypes. These cell populations have been described in other species as central and effector memory cells, respectively. Following in vitro culture and flow cytometry we observed plasticity within the bovine CD4(+) T-cell phenotype. Populations switched phenotype, increasing or decreasing expression of CD45RO and CD62L within 24h of in vitro stimulation. After 14 days all IFNγ producing CD4(+) T cells expressed CD45RO regardless of the original phenotype of the sorted population. No differences were detected in behavior of cells derived from BCG-vaccinated animals compared to cells derived from naturally infected animals. In conclusion, although multiple populations of CD4(+) T memory cells from both BCG vaccinated and M. bovis infected animals contributed to cELISPOT responses, the dominant contributing population consists of central-memory-like T cells (CD45RO(+)CD62L(hi)).

  1. A 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

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

    Science.gov (United States)

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

    2014-12-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    1999-01-01

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

  4. Use of integrated cell culture-PCR to evaluate the effectiveness of poliovirus inactivation by chlorine.

    Science.gov (United States)

    Blackmer, F; Reynolds, K A; Gerba, C P; Pepper, I L

    2000-05-01

    Current standards, based on cell culture assay, indicate that poliovirus is inactivated by 0.5 mg of free chlorine per liter after 2 min; however, integrated cell culture-PCR detected viruses for up to 8 min of exposure to the same chlorine concentration, requiring 10 min for complete inactivation. Thus, the contact time for chlorine disinfection of poliovirus is up to five times greater than previously thought.

  5. 21 CFR 864.2280 - Cultured animal and human cells.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Cultured animal and human cells. 864.2280 Section... (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products § 864.2280 Cultured animal and human cells. (a) Identification. Cultured animal and human cells are in...

  6. Pinoresinol from Ipomoea cairica cell cultures.

    Science.gov (United States)

    Páska, Csilla; Innocenti, Gabbriella; Ferlin, Mariagrazia; Kunvári, Mónika; László, Miklós

    2002-10-01

    Ipomoea cairica cell cultures produced a tetrahydrofuran lignan, (+)-pinoresinol, identified by UV, IR, MS and NMR methods, not yet found in the intact plant, and new in the Convolvulaceae family. Pinoresinol was found to have antioxidant and Ca2+ antagonist properties. As it could be requested for its biological activity, we examined the possibility to raise the pinoresinol yield of I. cairica cultures, as well as we continued investigations on lignans' response to optimization.

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

    International Nuclear Information System (INIS)

    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 H2O2), 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)

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

    OpenAIRE

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

    2014-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-08-15

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

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

    International Nuclear Information System (INIS)

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

  11. Effects of different Helicobacter pylori culture filtrates on growth of gastric epithelial cells

    Institute of Scientific and Technical Information of China (English)

    Yan-Guo Yan; Gang Zhao; Jin-Ping Ma; Shi-Rong Cai; Wen-Hua Zhan

    2008-01-01

    AIM: To study the effects of different Helicobacter pylori (H py/orl) culture filtrates on growth of gastric epithelial cells.METHODS: Broth culture filtrates of H pylori were prepared. Gastric epithelial cells were treated with the filtrates, and cell growth was determined by growth curve and flow cytometry. DNA damage of gastric epithelial cells was measured by single-cell microgel electrophoresis.RESULTS: Gastric epithelial cells proliferated actively when treated by CagA-gene-positive broth culture filtrates, and colony formation reached 40%. The number of cells in S phase increased compared to controls. Comet assay showed 41.2% comet cells in GES-1 cells treated with CagA-positive filtrates (P<0.05).CONCLUSION: CagA-positive filtrates enhance the changes in morphology and growth characteristics of human gastric epithelial tumor cells. DNA damage maybe one of the mechanisms involved in the growth changes.

  12. Culture of human cell lines by a pathogen-inactivated human platelet lysate.

    Science.gov (United States)

    Fazzina, R; Iudicone, P; Mariotti, A; Fioravanti, D; Procoli, A; Cicchetti, E; Scambia, G; Bonanno, G; Pierelli, L

    2016-08-01

    Alternatives to the use of fetal bovine serum (FBS) have been investigated to ensure xeno-free growth condition. In this study we evaluated the efficacy of human platelet lysate (PL) as a substitute of FBS for the in vitro culture of some human cell lines. PL was obtained by pools of pathogen inactivated human donor platelet (PLT) concentrates. Human leukemia cell lines (KG-1, K562, JURKAT, HL-60) and epithelial tumor cell lines (HeLa and MCF-7) were cultured with either FBS or PL. Changes in cell proliferation, viability, morphology, surface markers and cell cycle were evaluated for each cell line. Functional characteristics were analysed by drug sensitivity test and cytotoxicity assay. Our results demonstrated that PL can support growth and expansion of all cell lines, although the cells cultured in presence of PL experienced a less massive proliferation compared to those grown with FBS. We found a comparable percentage of viable specific marker-expressing cells in both conditions, confirming lineage fidelity in all cultures. Functionality assays showed that cells in both FBS- and PL-supported cultures maintained their normal responsiveness to adriamycin and NK cell-mediated lysis. Our findings indicate that PL is a feasible serum substitute for supporting growth and propagation of haematopoietic and epithelial cell lines with many advantages from a perspective of process standardization, ethicality and product safety. PMID:25944665

  13. Dexamethasone Modulation on Cultured Human Retinal Pigment Epithelial Cell

    Institute of Scientific and Technical Information of China (English)

    Bing Liu; Yannian; Hui Yusheng Wang; Hong Wei

    2001-01-01

    Purpose: Dexamethasone(DEX) was tested for its ability to modulate human retinal pigment epithelium (hRPE) cell proliferation in cell culture. Methods: DEX in different concentrations was added to cultured hRPE cells. The effects were measured with MTT method, 3H-thymidine(3H-TdR) incorporation and flow cytometw. Results: DEX increased survival rate and DNA synthesis from 32 mg/L to 320 mg/L under hRPE culture conditions, but paradoxically reduced them at 1 000 mg/L and 3 200 mg/L in dose and time dependent fashion by both MTT assay and 3 H-TdR incorporation. The cell numbers in S phase and G2/M phase increased 28.32 % at DEX concentration 320 mg/L, in contrast, reduced 41.84 % at 1 000 mg/L. Conclusion: DEX increased proliferation from 32 mg/L to 320 mg/L, and inhibited proliferation at concentrations greater than 320 mg/L. The inhibiting effect of DEX may happen in s phase and G2/M phase. Eye Sciernce 2001; 17:27 ~ 30.

  14. Growth regulation of cultured human nevus cells.

    Science.gov (United States)

    Mancianti, M L; Györfi, T; Shih, I M; Valyi-Nagy, I; Levengood, G; Menssen, H D; Halpern, A C; Elder, D E; Herlyn, M

    1993-03-01

    Cells isolated from congenital melanocytic nevi and cultured in vitro have growth characteristics that resemble their premalignant stage in situ. A serum-free, chemically defined medium has been developed that allows continuous growth of established nevus cultures for up to several months. Like primary melanoma cells, nevus cells in high-calcium-containing W489 medium require insulin for growth. In contrast to melanoma cells, nevus cells in serum-free medium require the presence of alpha-melanocyte-stimulating hormone, which enhanced intracellular levels of cyclic adenosine monophosphate. In contrast to the requirements of normal human melanocytes from newborn foreskin, congenital nevus cells grow with less dependency on basic fibroblast growth factor (bFGF). Nevus cultures contain bFGF-like activity, and they express bFGF mRNA. Nevic cells of compound nevi also express bFGF mRNA in situ but only in the junctional areas. These results indicate that bFGF plays an important growth regulatory role for nevus cells in vitro and in vivo. PMID:8440904

  15. A direct radioreceptor assay for human growth hormone in serum using cultured human lymphocytes

    International Nuclear Information System (INIS)

    Lesniak and co-workers have developed a radioreceptor assay (RRA) for hGH by using cultured human lymphocytes containing binding sites highly specific for hGH only. Because of the relatively low affinity constant of the receptors for hGH, the experimental sensitivity of the RRA (3.5ng/ml) was distinctly better than that of the RIA (0.5ng/ml) used. This imposes limitations on the use of the assay when hGH is to be measured in serum from normal individuals - particularly in pediatrics. When, for reasons of sensitivity, relatively large amounts of serum (0.1ml=15.4%) are added to the incubate, ''unspecific displacement'' of labelled hGH occurs. This unspecific displacement could not be avoided completely by modifications of the assay conditions. Experimentally, we were able to show that sera taken at different time intervals from patients without measurable hGH (RIA) exhibit a constant unspecific displacement. Furthermore, standard curves run with different hGH-free (RIA) sera were parallel. Thus, if a series of samples from the same individual were available in which one contained hGH (RIA) below the sensitivity threshold of the RRA, the unspecific displacement could be estimated. Also, for each set of samples, an individual standard curve could be constructed whose slope would be given by the measured standard curve. With this approach, immunoassayable and receptorassayable hGH concentrations were compared in 154 samples from children. The overall RRA/RIA ratio (0.76) was below unity (p<0.05); after injection of hGH the ratio was indistinguishable from unity. (author)

  16. Real-time PCR assay is superior to other methods for the detection of mycoplasma contamination in the cell lines of the National Cell Bank of Iran.

    Science.gov (United States)

    Molla Kazemiha, Vahid; Bonakdar, Shahin; Amanzadeh, Amir; Azari, Shahram; Memarnejadian, Arash; Shahbazi, Shirin; Shokrgozar, Mohammad Ali; Mahdian, Reza

    2016-08-01

    Mycoplasmas are the most important contaminants of cell cultures throughout the world. They are considered as a major problem in biological studies and biopharmaceutical economic issues. In this study, our aim was to find the best standard technique as a rapid method with high sensitivity, specificity and accuracy for the detection of mycoplasma contamination in the cell lines of the National Cell Bank of Iran. Thirty cell lines suspected to mycoplasma contamination were evaluated by five different techniques including microbial culture, indirect DNA DAPI staining, enzymatic mycoalert(®) assay, conventional PCR and real-time PCR. Five mycoplasma-contaminated cell lines were assigned as positive controls and five mycoplasma-free cell lines as negative controls. The enzymatic method was performed using the mycoalert(®) mycoplasma detection kit. Real-time PCR technique was conducted by PromoKine diagnostic kits. In the conventional PCR method, mycoplasma genus-specific primers were designed to analyze the sequences based on a fixed and common region on 16S ribosomal RNA with PCR product size of 425 bp. Mycoplasma contamination was observed in 60, 56.66, 53.33, 46.66 and 33.33 % of 30 different cell cultures by real-time PCR, PCR, enzymatic mycoalert(®), indirect DNA DAPI staining and microbial culture methods, respectively. The analysis of the results of the different methods showed that the real-time PCR assay was superior the other methods with the sensitivity, specificity, accuracy, predictive value of positive and negative results of 100 %. These values were 94.44, 100, 96.77, 100 and 92.85 % for the conventional PCR method, respectively. Therefore, this study showed that real-time PCR and PCR assays based on the common sequences in the 16S ribosomal RNA are reliable methods with high sensitivity, specificity and accuracy for detection of mycoplasma contamination in cell cultures and other biological products.

  17. Real-time PCR assay is superior to other methods for the detection of mycoplasma contamination in the cell lines of the National Cell Bank of Iran.

    Science.gov (United States)

    Molla Kazemiha, Vahid; Bonakdar, Shahin; Amanzadeh, Amir; Azari, Shahram; Memarnejadian, Arash; Shahbazi, Shirin; Shokrgozar, Mohammad Ali; Mahdian, Reza

    2016-08-01

    Mycoplasmas are the most important contaminants of cell cultures throughout the world. They are considered as a major problem in biological studies and biopharmaceutical economic issues. In this study, our aim was to find the best standard technique as a rapid method with high sensitivity, specificity and accuracy for the detection of mycoplasma contamination in the cell lines of the National Cell Bank of Iran. Thirty cell lines suspected to mycoplasma contamination were evaluated by five different techniques including microbial culture, indirect DNA DAPI staining, enzymatic mycoalert(®) assay, conventional PCR and real-time PCR. Five mycoplasma-contaminated cell lines were assigned as positive controls and five mycoplasma-free cell lines as negative controls. The enzymatic method was performed using the mycoalert(®) mycoplasma detection kit. Real-time PCR technique was conducted by PromoKine diagnostic kits. In the conventional PCR method, mycoplasma genus-specific primers were designed to analyze the sequences based on a fixed and common region on 16S ribosomal RNA with PCR product size of 425 bp. Mycoplasma contamination was observed in 60, 56.66, 53.33, 46.66 and 33.33 % of 30 different cell cultures by real-time PCR, PCR, enzymatic mycoalert(®), indirect DNA DAPI staining and microbial culture methods, respectively. The analysis of the results of the different methods showed that the real-time PCR assay was superior the other methods with the sensitivity, specificity, accuracy, predictive value of positive and negative results of 100 %. These values were 94.44, 100, 96.77, 100 and 92.85 % for the conventional PCR method, respectively. Therefore, this study showed that real-time PCR and PCR assays based on the common sequences in the 16S ribosomal RNA are reliable methods with high sensitivity, specificity and accuracy for detection of mycoplasma contamination in cell cultures and other biological products. PMID:25742733

  18. Electrophoretic mobilities of cultured human embryonic kidney cells in various buffers

    Science.gov (United States)

    1985-01-01

    Data on the electrophoretic mobility distributions of cells in the new D-1 buffer and the interlaboratory standardization of urokinase assay methods are presented. A table of cell strains and recent data on cell dispersal methods are also included. It was decided that glycerol in A-1 electrophoretic mobility data on cultured human embryonic kidney cells subjected to electrophoresis in this buffer. The buffer composition is presented.

  19. [Development of three-dimensional breast cancer cell culture drug resistance model].

    Science.gov (United States)

    Xu, Hong; Liu, Wei; Zhang, Xiu-Zhen; Hou, Liang; Lu, Ying-Jin; Chen, Pei-Pei; Zhang, Can; Feng, Di; Kong, Li; Wang, Xiu-Li

    2016-04-25

    The aim of the present study was to develop three-dimensional (3D) culture model, a more pathologically relevant model, of human breast cancer for drug resistance study. MCF-7 cells were embedded within collagen gel to establish 3D culture model. Cellular morphology was observed using Carmine and HE staining. Cell proliferation was evaluated by CCK-8 assay, and cell activity was detected by Live/Dead staining kit. Drug sensitivities of the 3D culture to doxorubicin, carboplatin, 5-fluorouracil were assayed and compared with those of monolayer (2D) culture. In addition, the levels of drug resistance-related genes P-glycoprotein (P-gp), mrp2 mRNA expressions were detected by real time RT-PCR. Expression level of P-gp protein was detected by Western blot. The results showed that MCF-7 cells in 3D culture formed a number of cell aggregates, and most of them displayed good cell viability. The IC50 values of doxorubicin, carboplatin, 5-fluorouracil were all increased significantly in 3D culture compared with those in 2D culture. Moreover, compared with MCF-7 cells in 2D culture, the cells in 3D culture showed increased mRNA levels of P-gp and mrp2, as well as up-regulated protein expression of P-gp. These results suggest that in vitro collagen-embedded culture system of human breast cancer cells represents an improved pathologically relevant 3D microenvironment for breast cancer cells, providing a robust tool to explore the mechanism of drug resistance of cancer cells. PMID:27108905

  20. General overview of neuronal cell culture.

    Science.gov (United States)

    Gordon, Jennifer; Amini, Shohreh; White, Martyn K

    2013-01-01

    In this introductory chapter, we provide a general overview of neuronal cell culture. This is a rapidly evolving area of research and we provide an outline and contextual framework for the different chapters of this book. These chapters were all contributed by scientists actively working in the field who are currently using state-of-the-art techniques to advance our understanding of the molecular and cellular biology of the central nervous system. Each chapter provides detailed descriptions and experimental protocols for a variety of techniques ranging in scope from basic neuronal cell line culturing to advanced and specialized methods.

  1. Enhanced growth medium and method for culturing human mammary epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Stampfer, Martha R. (7290 Sayre Dr., Oakland, CA 94611); Smith, Helene S. (5693 Cabot Dr., Oakland, CA 94611); Hackett, Adeline J. (82 Evergreen Dr., Orinda, CA 94563)

    1983-01-01

    Methods are disclosed for isolating and culturing human mammary epithelial cells of both normal and malignant origin. Tissue samples are digested with a mixture including the enzymes collagenase and hyaluronidase to produce clumps of cells substantially free from stroma and other undesired cellular material. Growing the clumps of cells in mass culture in an enriched medium containing particular growth factors allows for active cell proliferation and subculture. Clonal culture having plating efficiencies of up to 40% or greater may be obtained using individual cells derived from the mass culture by plating the cells on appropriate substrates in the enriched media. The clonal growth of cells so obtained is suitable for a quantitative assessment of the cytotoxicity of particular treatment. An exemplary assay for assessing the cytotoxicity of the drug adriamycin is presented.

  2. Effect of radiofrequency radiation in cultured mammalian cells: A review.

    Science.gov (United States)

    Manna, Debashri; Ghosh, Rita

    2016-01-01

    The use of mobile phone related technologies will continue to increase in the foreseeable future worldwide. This has drawn attention to the probable interaction of radiofrequency electromagnetic radiation with different biological targets. Studies have been conducted on various organisms to evaluate the alleged ill-effect on health. We have therefore attempted to review those work limited to in vitro cultured cells where irradiation conditions were well controlled. Different investigators have studied varied endpoints like DNA damage, cell cycle arrest, reactive oxygen species (ROS) formation, cellular morphology and viability to weigh the genotoxic effect of such radiation by utilizing different frequencies and dose rates under various irradiation conditions that include continuous or pulsed exposures and also amplitude- or frequency-modulated waves. Cells adapt to change in their intra and extracellular environment from different chemical and physical stimuli through organized alterations in gene or protein expression that result in the induction of stress responses. Many studies have focused on such effects for risk estimations. Though the effects of microwave radiation on cells are often not pronounced, some investigators have therefore combined radiofrequency radiation with other physical or chemical agents to observe whether the effects of such agents were augmented or not. Such reports in cultured cellular systems have also included in this review. The findings from different workers have revealed that, effects were dependent on cell type and the endpoint selection. However, contradictory findings were also observed in same cell types with same assay, in such cases the specific absorption rate (SAR) values were significant.

  3. Genetic reprogramming of human amniotic cells with episomal vectors: neural rosettes as sentinels in candidate selection for validation assays

    Directory of Open Access Journals (Sweden)

    Patricia G. Wilson

    2014-11-01

    Full Text Available The promise of genetic reprogramming has prompted initiatives to develop banks of induced pluripotent stem cells (iPSCs from diverse sources. Sentinel assays for pluripotency could maximize available resources for generating iPSCs. Neural rosettes represent a primitive neural tissue that is unique to differentiating PSCs and commonly used to identify derivative neural/stem progenitors. Here, neural rosettes were used as a sentinel assay for pluripotency in selection of candidates to advance to validation assays. Candidate iPSCs were generated from independent populations of amniotic cells with episomal vectors. Phase imaging of living back up cultures showed neural rosettes in 2 of the 5 candidate populations. Rosettes were immunopositive for the Sox1, Sox2, Pax6 and Pax7 transcription factors that govern neural development in the earliest stage of development and for the Isl1/2 and Otx2 transcription factors that are expressed in the dorsal and ventral domains, respectively, of the neural tube in vivo. Dissociation of rosettes produced cultures of differentiation competent neural/stem progenitors that generated immature neurons that were immunopositive for βIII-tubulin and glia that were immunopositive for GFAP. Subsequent validation assays of selected candidates showed induced expression of endogenous pluripotency genes, epigenetic modification of chromatin and formation of teratomas in immunodeficient mice that contained derivatives of the 3 embryonic germ layers. Validated lines were vector-free and maintained a normal karyotype for more than 60 passages. The credibility of rosette assembly as a sentinel assay for PSCs is supported by coordinate loss of nuclear-localized pluripotency factors Oct4 and Nanog in neural rosettes that emerge spontaneously in cultures of self-renewing validated lines. Taken together, these findings demonstrate value in neural rosettes as sentinels for pluripotency and selection of promising candidates for advance

  4. In vitro cell culture lethal dose submitted to gamma radiation

    Energy Technology Data Exchange (ETDEWEB)

    Moreno, Carolina S.; Rogero, Sizue O.; Rogero, Jose Roberto [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil)], e-mail: carolina_sm@hotmail.com; Ikeda, Tamiko I.; Cruz, Aurea S. [Instituto Adolfo Lutz, Sao Paulo, SP (Brazil)

    2009-07-01

    The present study was designed to evaluate the in vitro effect of gamma radiation in cell culture of mouse connective tissue exposed to different doses of gamma radiation and under several conditions. The cell viability was analyzed by neutral red uptake methodology. This assay was developed for establish a methodology to be used in the future in the study of resveratrol radioprotection. Resveratrol (3,4',5- trihydroxystilbene), a phenolic phytoalexin that occurs naturally in some spermatophytes, such as grapevines, in response to injury as fungal infections and exposure to ultraviolet light. In the wines this compound is found at high levels and is considered one of the highest antioxidant constituents. The intense antioxidant potential of resveratrol provides many pharmacological activities including cardioprotection, chemoprevention and anti-tumor effects. Our results demonstrated that {sup 60}Co gamma radiation lethal dose (LD50) on NCTC clone 929 cells was about 340Gy. (author)

  5. In vitro cell culture lethal dose submitted to gamma radiation

    International Nuclear Information System (INIS)

    The present study was designed to evaluate the in vitro effect of gamma radiation in cell culture of mouse connective tissue exposed to different doses of gamma radiation and under several conditions. The cell viability was analyzed by neutral red uptake methodology. This assay was developed for establish a methodology to be used in the future in the study of resveratrol radioprotection. Resveratrol (3,4',5- trihydroxystilbene), a phenolic phytoalexin that occurs naturally in some spermatophytes, such as grapevines, in response to injury as fungal infections and exposure to ultraviolet light. In the wines this compound is found at high levels and is considered one of the highest antioxidant constituents. The intense antioxidant potential of resveratrol provides many pharmacological activities including cardioprotection, chemoprevention and anti-tumor effects. Our results demonstrated that 60Co gamma radiation lethal dose (LD50) on NCTC clone 929 cells was about 340Gy. (author)

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

    Science.gov (United States)

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

    2013-10-01

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

  7. An Approach for Assessing the Signature Quality of Various Chemical Assays when Predicting the Culture Media Used to Grow Microorganisms

    Energy Technology Data Exchange (ETDEWEB)

    Holmes, Aimee E.; Sego, Landon H.; Webb-Robertson, Bobbie-Jo M.; Kreuzer, Helen W.; Anderson, Richard M.; Unwin, Stephen D.; Weimar, Mark R.; Tardiff, Mark F.; Corley, Courtney D.

    2013-02-01

    We demonstrate an approach for assessing the quality of a signature system designed to predict the culture medium used to grow a microorganism. The system was comprised of four chemical assays designed to identify various ingredients that could be used to produce the culture medium. The analytical measurements resulting from any combination of these four assays can be used in a Bayesian network to predict the probabilities that the microorganism was grown using one of eleven culture media. We evaluated combinations of the signature system by removing one or more of the assays from the Bayes network. We measured and compared the quality of the various Bayes nets in terms of fidelity, cost, risk, and utility, a method we refer to as Signature Quality Metrics

  8. Malignant monoblasts can function as effector cells in natural killer cell and antibody-dependent cellular cytotoxicity assays

    DEFF Research Database (Denmark)

    Hokland, P; Hokland, M; Ellegaard, J

    1981-01-01

    This is the first report describing natural killer (NK) and antibody-dependent cellular cytotoxicity (ADCC) of malignant monoblasts. Pure acute monoblastic leukemia was diagnosed in bone marrow aspirations from two patients by use of conventional cytochemical methods as well as multiple immunolog...... no modulation was seen in ADCC. These findings are discussed in the light of our present knowledge of lymphoid NK cells. Udgivelsesdato: 1981-May...... techniques including detection of ALL antigens and terminal transferase. The malignant cells were subsequently found to be potent effectors in NK and ADCC assays. Addition of partially purified alpha-interferon to the in vitro cultures was found to have an enhancing effect on NK activity, whereas...

  9. Cell Cycle Progression of Human Cells Cultured in Rotating Bioreactor

    Science.gov (United States)

    Parks, Kelsey

    2009-01-01

    Space flight has been shown to alter the astronauts immune systems. Because immune performance is complex and reflects the influence of multiple organ systems within the host, scientists sought to understand the potential impact of microgravity alone on the cellular mechanisms critical to immunity. Lymphocytes and their differentiated immature form, lymphoblasts, play an important and integral role in the body's defense system. T cells, one of the three major types of lymphocytes, play a central role in cell-mediated immunity. They can be distinguished from other lymphocyte types, such as B cells and natural killer cells by the presence of a special receptor on their cell surface called T cell receptors. Reported studies have shown that spaceflight can affect the expression of cell surface markers. Cell surface markers play an important role in the ability of cells to interact and to pass signals between different cells of the same phenotype and cells of different phenotypes. Recent evidence suggests that cell-cycle regulators are essential for T-cell function. To trigger an effective immune response, lymphocytes must proliferate. The objective of this project is to investigate the changes in growth of human cells cultured in rotating bioreactors and to measure the growth rate and the cell cycle distribution for different human cell types. Human lymphocytes and lymphoblasts will be cultured in a bioreactor to simulate aspects of microgravity. The bioreactor is a cylindrical culture vessel that incorporates the aspects of clinostatic rotation of a solid fluid body around a horizontal axis at a constant speed, and compensates gravity by rotation and places cells within the fluid body into a sustained free-fall. Cell cycle progression and cell proliferation of the lymphocytes will be measured for a number of days. In addition, RNA from the cells will be isolated for expression of genes related in cell cycle regulations.

  10. Cell-surface glycoproteins of human sarcomas: differential expression in normal and malignant tissues and cultured cells

    Energy Technology Data Exchange (ETDEWEB)

    Rettig, W.F.; Garin-Chesa, P.; Beresford, H.R.; Oettgen, H.F.; Melamed, M.R.; Old, L.J.

    1988-05-01

    Normal differentiation and malignant transformation of human cells are characterized by specific changes in surface antigen phenotype. In the present study, the authors have defined six cell-surface antigens of human sarcomas and normal mesenchymal cells, by using mixed hemadsorption assays and immunochemical methods for the analysis of cultured cells and immunohistochemical staining for the analysis of normal tissues and > 200 tumor specimens. Differential patterns of F19, F24, G171, G253, S5, and Thy-1 antigen expression were found to characterize (i) subsets of cultured sarcoma cell lines, (ii) cultured fibroblasts derived from various organs, (iii) normal resting and activated mesenchymal tissues, and (iv) sarcoma and nonmesenchymal tumor tissues. These results provide a basic surface antigenic map for cultured mesenchymal cells and mesenchymal tissues and permit the classification of human sarcomas according to their antigenic phenotypes.

  11. Integrated biosensors for cell culture monitoring

    OpenAIRE

    De Micheli, Giovanni; Boero, Cristina; Olivo, Jacopo; Carrara, Sandro

    2014-01-01

    Biosensors for endogenous compounds, such as glucose and lactate, are applied to monitor cell cultures. Cells can be cultivated for several purposes, such as understanding and modeling some biological mechanisms, the development of new drugs and therapies, and in the field of regenerative medicine. We have realized a self-contained monitoring system with remote readout. Metabolite detection is based on oxidases immobilized onto carbon nanotubes. We calibrate the system for glucose and lactate...

  12. Irradiation Response of Adipose-derived Stem Cells under Three-dimensional Culture Condition

    Institute of Scientific and Technical Information of China (English)

    DU Ya Rong; PAN Dong; CHEN Ya Xiong; XUE Gang; REN Zhen Xin; LI Xiao Man; ZHANG Shi Chuan; HU Bu Rong

    2015-01-01

    Objective Adipose tissue distributes widely in human body. The irradiation response of the adipose cells in vivo remains to be investigated. In this study we investigated irradiation response of adipose-derived stem cells (ASCs) under three-dimensional culture condition. Methods ASCs were isolated and cultured in low attachment dishes to form three-dimensional (3D) spheres in vitro. The neuronal differentiation potential and stem-liked characteristics was monitored by using immunofluoresence staining and flow cytometry in monolayer and 3D culture. To investigate the irradiation sensitivity of 3D sphere culture, the fraction of colony survival and micronucleus were detected in monolayer and 3D culture. Soft agar assays were performed for measuring malignant transformation for the irradiated monolayer and 3D culture. Results The 3D cultured ASCs had higher differentiation potential and an higher stem-like cell percentage. The 3D cultures were more radioresistant after either high linear energy transfer (LET) carbon ion beam or low LET X-ray irradiation compared with the monolayer cell. The ASCs’ potential of cellular transformation was lower after irradiation by soft agar assay. Conclusion These findings suggest that adipose tissue cell are relatively genomic stable and resistant to genotoxic stress.

  13. Cell culture from sponges: pluripotency and immortality

    NARCIS (Netherlands)

    Caralt Bosch, de S.; Uriz, M.J.; Wijffels, R.H.

    2007-01-01

    Sponges are a source of compounds with potential pharmaceutical applications. In this article, methods of sponge cell culture for production of these bioactive compounds are reviewed, and new approaches for overcoming the problem of metabolite supply are examined. The use of embryos is proposed as a

  14. 3D culture for cardiac cells.

    Science.gov (United States)

    Zuppinger, Christian

    2016-07-01

    This review discusses historical milestones, recent developments and challenges in the area of 3D culture models with cardiovascular cell types. Expectations in this area have been raised in recent years, but more relevant in vitro research, more accurate drug testing results, reliable disease models and insights leading to bioartificial organs are expected from the transition to 3D cell culture. However, the construction of organ-like cardiac 3D models currently remains a difficult challenge. The heart consists of highly differentiated cells in an intricate arrangement.Furthermore, electrical “wiring”, a vascular system and multiple cell types act in concert to respond to the rapidly changing demands of the body. Although cardiovascular 3D culture models have been predominantly developed for regenerative medicine in the past, their use in drug screening and for disease models has become more popular recently. Many sophisticated 3D culture models are currently being developed in this dynamic area of life science. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel.

  15. Nanotechnology, Cell Culture and Tissue Engineering

    Directory of Open Access Journals (Sweden)

    Kazutoshi Haraguchi

    2011-01-01

    Full Text Available We have fabricated new types of polymer hydrogels and polymer nanocomposites, i.e., nanocomposite gels (NC gels and soft, polymer nanocomposites (M-NCs: solid, with novel organic/inorganic network structures. Both NC gels and M-NCs were synthesized by in-situ free-radical polymerization in the presence of exfoliated clay platelets in aqueous systems and were obtained in various forms such as film, sheet, tube, coating, etc. and sizes with a wide range of clay contents. Here, disk-like inorganic clay nanoparticles act as multi-functional crosslinkers to form new types of network systems. Both NC gels and M-NCs have extraordinary optical and mechanical properties including ultra-high reversible extensibility, as well as a number of new characteristics relating to optical anisotropy, polymer/clay morphology, biocompatibility, stimuli-sensitive surfaces, micro-patterning, etc. For examples, the biological testing of medical devices, comprised of a sensitization test, an irritation test, an intracutaneous test and an in vitro cytotoxicity test,was carried out for NC gels and M-NCs. The safety of NC gels and M-NCs was confirmed in all tests. Also, the interaction of living tissue with NC gel was investigated in vivo by implantation in live goats; neither inflammation nor concrescence occurred around the NC gels. Furthermore, it was found that both N-NC gels consisting of poly(N-isopropylacrylamide(PNIPA/clay network and M-NCs consisting of poly(2-methoxyethyacrylate(PMEA/clay network show characteristic cell culture and subsequent cell detachment on their surfaces, although it was almost impossible to culture cells on conventional, chemically-crosslinked PNIPA hydrogels and chemically crossslinked PMEA, regardless of their crosslinker concentration. Various kinds of cells, such ashumanhepatoma cells (HepG2, normal human dermal fibroblast (NHDF, and human umbilical vein endothelial cells (HUVEC, could be cultured to be confluent on the surfaces of N

  16. Cell culture models using rat primary alveolar type I cells.

    Science.gov (United States)

    Downs, Charles A; Montgomery, David W; Merkle, Carrie J

    2011-10-01

    There is a lack of cell culture models using primary alveolar type I (AT I) cells. The purpose of this study was to develop cell culture models using rat AT I cells and microvascular endothelial cells from the lung (MVECL). Two types of model systems were developed: single and co-culture systems; additionally a 3-dimensional model system was developed. Pure AT I cell (96.3 ± 2.7%) and MVECL (97.9 ± 1.1%) preparations were used. AT I cell morphology, mitochondrial number and distribution, actin filament arrangement and number of apoptotic cells at confluence, and telomere attrition were characterized. AT I cells maintained their morphometric characteristics through at least population doubling (PD) 35, while demonstrating telomere attrition through at least PD 100. Furthermore, AT I cells maintained the expression of their specific markers, T1α and AQ-5, through PD 42. For the co-cultures, AT I cells were grown on the top and MVECL were grown on the bottom of fibronectin-coated 24-well Transwell Fluroblok™ filter inserts. Neither cell type transmigrated the 1 μm pores. Additionally, AT I cells were grown in a thick layer of Matrigel(®) to create a 3-dimensional model in which primary AT I cells form ring-like structures that resemble an alveolus. The development of these model systems offers the opportunities to investigate AT I cells and their interactions with MVECL in response to pharmacological interventions and in the processes of disease, repair and regeneration. PMID:21624488

  17. A biocompatible micro cell culture chamber (microCCC) for the culturing and on-line monitoring of eukaryote cells

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Petronis, Sarunas; Jørgensen, A M;

    2006-01-01

    culture chip compared to cell culture flasks. The cell culture chip could without further modification support cell growth of two other cell lines. Light coming from the microscope lamp during optical recordings of the cells was the only external factor identified, that could have a negative effect...

  18. A Microfluidic Platform for High-throughput Single-cell Isolation and Culture.

    Science.gov (United States)

    Lin, Ching-Hui; Chang, Hao-Chen; Hsu, Chia-Hsien

    2016-01-01

    Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency. PMID:27341146

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

    Directory of Open Access Journals (Sweden)

    Valverde Mahara

    1999-01-01

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

  20. Cell Culture Microfluidic Biochips: Experimental Throughput Maximization

    DEFF Research Database (Denmark)

    Minhass, Wajid Hassan; Pop, Paul; Madsen, Jan;

    2011-01-01

    Microfluidic biochips offer a promising alternative to a conventional biochemical laboratory, integrating all necessary functionalities on-chip in order to perform biochemical applications. Researchers have started to propose computer-aided design tools for the synthesis of such biochips. Our foc...... metaheuristic for experimental design generation for the cell culture microfluidic biochips, and we have evaluated our approach using multiple experimental setups....... in this paper is on the optimization of how a biochemical application is performed on a biochip. In this paper, we consider cell culture biochips, where several cell colonies are exposed to soluble compounds and monitored in real-time to determine the right combination of factors that leads to the desired...

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

    Science.gov (United States)

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

    2003-09-01

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

  2. Detection of cell wall mannoprotein Mp1p in culture supernatants of Penicillium marneffei and in sera of penicilliosis patients

    OpenAIRE

    Cao, Liang; Chan, King-Man; Chen, Daliang; Vanittanakom, Nongnuch; Lee, Cindy; Chan, Che-Man; Sirisanthana, Thira; Tsang, Dominic N. C.; Yuen, Kwok-Yung

    1999-01-01

    Mannoproteins are important and abundant structural components of fungal cell walls. The MP1 gene encodes a cell wall mannoprotein of the pathogenic fungus Penicillium marneffei. In the present study, we show that Mp1p is secreted into the cell culture supernatant at a level that can be detected by Western blotting. A sensitive enzyme-linked immunosorbent assay (ELISA) developed with antibodies against Mp1p was capable of detecting this protein from the cell culture supernatant of P. marneffe...

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

    Science.gov (United States)

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

    2015-11-01

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

  4. 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.

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

    International Nuclear Information System (INIS)

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

  6. Evaluation of the cytotoxicity of geosmin and 2-methylisoborneol using cultured human, monkey, and dog cells.

    Science.gov (United States)

    Mochida, Kyo

    2009-03-01

    The cytotoxicity of musty odor-emitting substances, geosmin (GM) and 2-methylisoborneol, at a concentration of 10 ng/L - 300 mg/L was investigated using cultured mammalian cells. These two compounds exhibited no cytotoxicity in either the colony-formation of human KB cells or WST-1 assays of human-, monkey-, and dog-derived cells. These results suggest that the maximum concentration (700 ng/L) of GM found in the water of Lake Shinji is not toxic.

  7. The pesticide methoxychlor decreases myotube formation in cell culture by slowing myoblast proliferation.

    Science.gov (United States)

    Steffens, Bradley W; Batia, Lyn M; Baarson, Chad J; Choi, Chang-Kun Charles; Grow, Wade A

    2007-08-01

    We studied the effect of the estrogenic pesticide methoxychlor (MXC) on skeletal muscle development using C2C12 cell culture. Myoblast cultures were exposed to various concentrations of MXC at various times during the process of myoblast fusion into myotubes. We observed that MXC exposure decreased myotube formation. In addition, we observed myoblasts with cytoplasmic vacuoles in cultures exposed to MXC. Because cytoplasmic vacuoles can be characteristic of cell death, apoptosis assays and trypan blue exclusion assays were performed. We found no difference in the frequency of apoptosis or in the frequency of cell death for cultures exposed to MXC and untreated cultures. Collectively, these results indicate that MXC exposure decreases myotube formation without causing cell death. In contrast, when cell proliferation was assessed, untreated cultures had a myoblast proliferation rate 50% greater than cultures exposed to MXC. We conclude that MXC decreases myotube formation at least in part by slowing myoblast proliferation. Furthermore, we suggest that direct exposure to MXC could affect skeletal muscle development in animals or humans, in addition to the defects in reproductive development that have previously been reported.

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

    Science.gov (United States)

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

    2011-08-01

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

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

    Science.gov (United States)

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

    2011-08-01

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

  10. Prevention and Detection of Mycoplasma Contamination in Cell Culture

    Directory of Open Access Journals (Sweden)

    Parvaneh Farzaneh

    2012-01-01

    Full Text Available One of the main problems in cell culture is mycoplasma infection. It can extensively affectcell physiology and metabolism. As the applications of cell culture increase in research,industrial production and cell therapy, more concerns about mycoplasma contaminationand detection will arise. This review will provide valuable information about: 1. the waysin which cells are contaminated and the frequency and source of mycoplasma species incell culture; 2. the ways to prevent mycoplasma contamination in cell culture; 3. the importanceof mycoplasma tests in cell culture; 4. different methods to identify mycoplasmacontamination; 5. the consequences of mycoplasma contamination in cell culture and 6.available methods to eliminate mycoplasma contamination. Awareness about the sourcesof mycoplasma and pursuing aseptic techniques in cell culture along with reliable detectionmethods of mycoplasma contamination can provide an appropriate situation to preventmycoplasma contamination in cell culture.

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

    Directory of Open Access Journals (Sweden)

    Elena Serena

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

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

    Science.gov (United States)

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

    2006-01-01

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

  13. Sulforaphane induces DNA single strand breaks in cultured human cells

    Energy Technology Data Exchange (ETDEWEB)

    Sestili, Piero, E-mail: piero.sestili@uniurb.it [Dipartimento di Scienze Biomolecolari, Via Maggetti, 21, Universita degli Studi di Urbino ' Carlo Bo' , 61029 Urbino, PU (Italy); Paolillo, Marco [Dipartimento di Scienze Biomolecolari, Via Maggetti, 21, Universita degli Studi di Urbino ' Carlo Bo' , 61029 Urbino, PU (Italy); Lenzi, Monia [Dipartimento di Farmacologia, Universita degli Studi di Bologna, Via Irnerio 48, 40126 Bologna (Italy); Colombo, Evelin; Vallorani, Luciana; Casadei, Lucia; Martinelli, Chiara [Dipartimento di Scienze Biomolecolari, Via Maggetti, 21, Universita degli Studi di Urbino ' Carlo Bo' , 61029 Urbino, PU (Italy); Fimognari, Carmela [Dipartimento di Farmacologia, Universita degli Studi di Bologna, Via Irnerio 48, 40126 Bologna (Italy)

    2010-07-07

    Sulforaphane (SFR), an isothiocyanate from cruciferous vegetables, possesses growth-inhibiting and apoptosis-inducing activities in cancer cell lines. Recently, SFR has been shown to promote the mitochondrial formation of reactive oxygen species (ROS) in human cancer cell lines. The present study was undertaken to see whether SFR-derived ROS might cause DNA damage in cultured human cells, namely T limphoblastoid Jurkat and human umbilical vein endothelial cells (HUVEC). 1-3 h treatments with 10-30 {mu}M SFR elicited intracellular ROS formation (as assayed with dihydrorhodamine, DHR, oxidation) as well as DNA breakage (as assessed with fast halo assay, FHA). These effects lacked cell-type specificity, since could be observed in both Jurkat and HUVEC. Differential-pH FHA analysis of damaged DNA showed that SFR causes frank DNA single strand breaks (SSBs); no DNA double strand breaks (DSBs) were found within the considered treatment times (up to 3 h). SFR-derived ROS were formed at the mitochondrial respiratory chain (MRC) level: indeed rotenone or myxothiazol (MRC Complex I and III inhibitors, respectively) abrogated ROS formation. Furthermore ROS were not formed in Jurkat cells pharmacologically depleted of respiring mitochondria (MRC-/Jurkat). Formation of ROS was causally linked to the induction of SSBs: indeed all the experimental conditions capable of preventing ROS formation also prevented the damage of nuclear DNA from SFR-intoxicated cells. As to the toxicological relevance of SSBs, we found that their prevention slightly but significantly attenuated SFR cytotoxicity, suggesting that high-dose SFR toxicity is the result of a complex series of events among which GSH depletion seems to play a pivotal role. In conclusion, the present study identifies a novel mechanism contributing to SFR toxicity which - since DNA damage is a prominent mechanism underlying the cytotoxic activity of established antineoplastic agents - might help to exploit the therapeutic value

  14. Artifacts by marker enzyme adsorption on nanomaterials in cytotoxicity assays with tissue cultures

    Science.gov (United States)

    Wohlleben, Wendel; Kolle, Susanne N.; Hasenkamp, Laura-Carolin; Böser, Alexander; Vogel, Sandra; von Vacano, Bernhard; van Ravenzwaay, Ben; Landsiedel, Robert

    2011-07-01

    We used precision cut lung slices (PCLS) to study the cytotoxicity of cobalt ferrite nanomaterials with and without bovine serum albumin (BSA) stabilization. Using mitochondrial activity as an indicator of cytotoxicity (WST-1 assay) increasing concentrations of cobalt ferrite nanomaterial caused increasing levels of cytotoxicity in PCLS irrespective of BSA stabilization. However, there was no increase in released lactate dehydrogenase (LDH) levels caused by BSA stabilized nanomaterial indicating concentration depended cytotoxictiy. Moreover, non-stabilized nanomaterial caused a decrease of background LDH levels in the PCLS culture supernatant confirmed by complementary methods. Direct characterization of the protein corona of extracted nanomaterial shows that the LDH decrease is due to adsorption of LDH onto the surface of the non-stabilized nanomaterial, correlated with strong agglomeration. Preincubation with serum protein blocks the adsorption of LDH and stabilizes the nanomaterial at low agglomeration. We have thus demonstrated the cytotoxicity of nanomaterials in PCLS does not correlate with disrupted membrane integrity followed by LDH release. Furthermore, we found that intracellular enzymes such as the marker enzyme LDH are able to bind onto surfaces of nanomaterial and thereby adulterate the detection of toxic effects. A replacement of BSA by LDH or a secondary LDH-on-BSA-corona were not observed, confirming earlier indications that the protein corona exchange rate are slow or vanishing on inorganic nanomaterial. Thus, the method(s) to assess nanomaterial-mediated effects have to be carefully chosen based on the cellular effect and possible nano-specific artifacts.

  15. Differences in the primary culture, purification and biological characteristics between endothelial cells and smooth muscle cells from rat aorta

    Institute of Scientific and Technical Information of China (English)

    Shaobo Hu; Zifang Song; Qichang Zheng; Jun Nie

    2009-01-01

    Objective: To investigate the differences of primary culture, purification and biological characteristics between endothelial cells and smooth muscle cells from rat aorta. Methods: Endothelial cells were obtained using the vascular ring adherence, collagenase digestion method and an improved vascular ring adherence method, while smooth muscle cells were separated from tissue sections of rat aorta. Clones of endothelial cells were selected by limiting dilution assay. Both cell types were identified using specific cell immunofluorescent markers,and phase contrast microscopy was used to observe the morphological disparity between endothelial cells and smooth muscle cells at the single cell and colony level. Cell proliferation was determined by the cell counting kit-8. Differences between endothelial cells and smooth muscle cells were evaluated in trypsin digestion 6me, attachment time and recovery after cryopreservation. Results: Endothelial cells were obtained by all three methods. The improved vascular ring method provided the most reproducible results. Cells were in good condition, and of high purity. Smooth muscle cells were cultured successfully by the tissue fragment culture method. Clonal expansion of singleendothelial cells was attained. The two cell types expressed their respective specific markers, and the rate of proliferation of smooth muscle cells exceeded that of endothelial cells. Endothelial cells were more sensitive to trypsin digestion than smooth muscle cells. In addition, they had a shorter adherence time and better recovery following cryopreservation than smooth muscle cells. Conclusion: The improved vascular ring method was optimal for yielding endothelial cells. Limiting dilution is a novel and valid method for purifying primary endothelial cells from rat aorta. Primary rat endothelial cell and vascular smooth muscle cell cultures exhibited different morphological characteristics, proliferation rate, adherence time, susceptibility to trypsin

  16. Rhinacanthus nasutus protects cultured neuronal cells against hypoxia induced cell death.

    Science.gov (United States)

    Brimson, James M; Tencomnao, Tewin

    2011-07-26

    Rhinacanthus nasutus (L.) Kurz (Acanthaceae) is an herb native to Thailand and Southeast Asia, known for its antioxidant properties. Hypoxia leads to an increase in reactive oxygen species in cells and is a leading cause of neuronal damage. Cell death caused by hypoxia has been linked with a number of neurodegenerative diseases including some forms of dementia and stroke, as well as the build up of reactive oxygen species which can lead to diseases such as Huntington's disease, Parkinson's disease and Alzeheimer's disease. In this study we used an airtight culture container and the Mitsubishi Gas Company anaeropack along with the MTT assay, LDH assay and the trypan blue exlusion assay to show that 1 and 10 µg mL⁻¹ root extract of R. nasutus is able to significantly prevent the death of HT-22 cells subjected to hypoxic conditions, and 0.1 to 10 µg mL⁻¹ had no toxic effect on HT-22 under normal conditions, whereas 100 µg mL⁻¹ reduced HT-22 cell proliferation. We also used H₂DCFDA staining to show R. nasutus can reduce reactive oxygen species production in HT-22 cells.

  17. Rhinacanthus nasutus Protects Cultured Neuronal Cells against Hypoxia Induced Cell Death

    Directory of Open Access Journals (Sweden)

    James M. Brimson

    2011-07-01

    Full Text Available Rhinacanthus nasutus (L. Kurz (Acanthaceae is an herb native to Thailand and Southeast Asia, known for its antioxidant properties. Hypoxia leads to an increase in reactive oxygen species in cells and is a leading cause of neuronal damage. Cell death caused by hypoxia has been linked with a number of neurodegenerative diseases including some forms of dementia and stroke, as well as the build up of reactive oxygen species which can lead to diseases such as Huntington’s disease, Parkinson’s disease and Alzeheimer’s disease. In this study we used an airtight culture container and the Mitsubishi Gas Company anaeropack along with the MTT assay, LDH assay and the trypan blue exlusion assay to show that 1 and 10 µg mL−1 root extract of R. nasutus is able to significantly prevent the death of HT-22 cells subjected to hypoxic conditions, and 0.1 to 10 µg mL−1 had no toxic effect on HT-22 under normal conditions, whereas 100 µg mL−1 reduced HT-22 cell proliferation. We also used H2DCFDA staining to show R. nasutus can reduce reactive oxygen species production in HT-22 cells.

  18. 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...

  19. [In vitro evaluation of the chemosensitivity of malignant gastrointestinal tumors by stem cell assay].

    Science.gov (United States)

    Scheithauer, W; Temsch, E M; Schieder, H; Funovics, J; Schiessel, R; Grabner, G

    1984-01-01

    The Human Tumor Stem Cell Assay, originally described by Hamburger and Salmon, was shown to be a useful in-vitro technique for predicting response or lack of response in individual patients' tumors. In the present study 34 GI-tumors were assayed for evaluation of in-vitro growth characteristics and sensitivity-patterns to standard chemotherapeutic drugs as well as to recombinant interferon alpha-2(rIF). Sufficient growth for evaluation of anticancer drug activity (greater than 30 colonies/control plate) was obtained in 56% of specimens: 2/9 colorectal, 0/3 stomach, 0/3 pancreatic tumors and 1/4 hepatomas revealed a 50% (or more) decrease of TCFUs, that was considered the minimum for in-vitro efficacy. Our results suggest a very limited overall activity of rIF in gastrointestinal malignancies. Only 1 pancreatic cancer (of 18 evaluable specimens) showed a significant decrease of colony formation (70%), when 100 U of interferon/ml were added to the culture system.

  20. A rapid and sensitive method for measuring N-acetylglucosaminidase activity in cultured cells.

    Directory of Open Access Journals (Sweden)

    Victor Mauri

    Full Text Available A rapid and sensitive method to quantitatively assess N-acetylglucosaminidase (NAG activity in cultured cells is highly desirable for both basic research and clinical studies. NAG activity is deficient in cells from patients with Mucopolysaccharidosis type IIIB (MPS IIIB due to mutations in NAGLU, the gene that encodes NAG. Currently available techniques for measuring NAG activity in patient-derived cell lines include chromogenic and fluorogenic assays and provide a biochemical method for the diagnosis of MPS IIIB. However, standard protocols require large amounts of cells, cell disruption by sonication or freeze-thawing, and normalization to the cellular protein content, resulting in an error-prone procedure that is material- and time-consuming and that produces highly variable results. Here we report a new procedure for measuring NAG activity in cultured cells. This procedure is based on the use of the fluorogenic NAG substrate, 4-Methylumbelliferyl-2-acetamido-2-deoxy-alpha-D-glucopyranoside (MUG, in a one-step cell assay that does not require cell disruption or post-assay normalization and that employs a low number of cells in 96-well plate format. We show that the NAG one-step cell assay greatly discriminates between wild-type and MPS IIIB patient-derived fibroblasts, thus providing a rapid method for the detection of deficiencies in NAG activity. We also show that the assay is sensitive to changes in NAG activity due to increases in NAGLU expression achieved by either overexpressing the transcription factor EB (TFEB, a master regulator of lysosomal function, or by inducing TFEB activation chemically. Because of its small format, rapidity, sensitivity and reproducibility, the NAG one-step cell assay is suitable for multiple procedures, including the high-throughput screening of chemical libraries to identify modulators of NAG expression, folding and activity, and the investigation of candidate molecules and constructs for applications in

  1. Mouse cell culture - Methods and protocols

    Directory of Open Access Journals (Sweden)

    CarloAlberto Redi

    2010-12-01

    Full Text Available The mouse is, out of any doubt, the experimental animal par excellence for many many colleagues within the scientific community, notably for those working in mammalian biology (in a broad sense, from basic genetic to modeling human diseases, starting at least from 1664 Robert Hooke experiments on air’s propertyn. Not surprising then that mouse cell cultures is a well established field of research itself and that there are several handbooks devoted to this discipline. Here, Andrew Ward and David Tosh provide a necessary update of the protocols currently needed. In fact, nearly half of the book is devoted to stem cells culture protocols, mainly embryonic, from a list of several organs (kidney, lung, oesophagus and intestine, pancreas and liver to mention some........

  2. Darwinian Evolution of Prions in Cell Culture*

    OpenAIRE

    Li, Jiali; Browning, Shawn; Mahal, Sukhvir P.; Oelschlegel, Anja M.; Weissmann, Charles

    2009-01-01

    Prions are infectious proteins consisting mainly of PrPSc, a β sheet-rich conformer of the normal host protein PrPC, and occur in different strains. Strain identity is thought to be encoded by PrPSc conformation. We found that biologically cloned prion populations gradually became heterogeneous by accumulating “mutants”, and selective pressures resulted in the emergence of different mutants as major constituents of the evolving population. Thus, when transferred from brain to cultured cells, ...

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

    Science.gov (United States)

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

    1980-04-01

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

  4. Effect of adrenotensin on cell proliferation is mediated by angiotensin Ⅱ in cultured rat mesangial cells

    Institute of Scientific and Technical Information of China (English)

    Hong XUE; Ping YUAN; Li ZHOU; Tai YAO; Yu HUANG; Li-min LU

    2009-01-01

    Aim: Both adrenomedullin (ADM) and adrenotensin (ADT) are derived from the same propeptide precursor, and both act as circulat- ing hormones and local paracrine mediators with multiple biological activities. Compared with ADM, little is known about how ADT achieves its functions. In the present study, we investigated the effect of ADT on cell proliferation and transforming growth factor-β (TGF-β) secretion in cultured renal mesangial cells (MCs) and determined whether angiotensin Ⅱ (Ang Ⅱ) was involved in mediating this process.Methods: Cell proliferation was measured by bromodeoxyuridine (BrdU) incorporation assay, Ang Ⅱ levels were assayed using an enzyme immunoassay, and real time PCR was used to measure Ang Ⅱ type 1 (AT1) receptor, Ang Ⅱ type 2 (AT2) receptor, angiotensino-gen (AGT), renin, angiotensin converting enzyme (ACE) and TGF-β1 mRNA levels. TGF-β1 and collagen type IV protein levels in cellmedia were measured using enzyme-linked immunoassays. Results: ADT treatment induced cell proliferation in a concentration-dependent manner; it also increased the levels of TGF-β1 mRNA and protein as well as collagen type Ⅳ excretion by cultured MCs. ADT treatment increased renin and AGT mRNAs as well as Ang Ⅱ protein, but did not affect the ACE mRNA level. ADT up-regulated angiotensin AT1 receptor mRNA, but not that of the AT2 receptor. The angiotensin AT1 receptor antagonist Iosartan blocked the effects of ADT-induced cell proliferation, TGF-β1 and collagen type Ⅳ synthe-sis and secretion.Conclusion: ADT has a stimulating role in cell proliferation in cultured MCs. Increases in the levels of Ang II and the AT1 receptor after ADT treatment mediate the stimulating effects of ADT on cell proliferation and extracellular matrix synthesis and secretion.

  5. Cytopathogenicity of Naegleria for cultured neuroblastoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Fulford, D.E.

    1985-01-01

    The cytopathic activity of live Naegleria amoebae and cell-free lysates of Naegleria for B-103 rat neuroblastoma cells was investigated using a /sup 51/Cr release assay. Live amoebae and cell-free lysates of N. fowleri, N. australiensis, N. lovaniensis, and N. gruberi all induced sufficient damage to radiolabeled B-103 cells to cause a significant release of chromium. The cytotoxic activity present in the cell-free lysates of N. fowleri can be recovered in the supernatant fluid following centrifugation at 100,000xg and precipitation of the 100,000xg supernatant fluid with ammonium sulfate. Initial characterization of the cytotoxic factor indicates that it is a heat labile, pH sensitive, soluble protein. The cytotoxic activity is abolished by either extraction, unaffected by repeated freeze-thawing, and is not sensitive to inhibitors of proteolytic enzymes. Phospholipase A activity was detected in the cytotoxic ammonium sulfate precipitable material, suggesting that this enzyme activity may have a role in the cytotoxic activity of the cell-free lysates.

  6. Prevention and Detection of Mycoplasma Contamination in Cell Culture

    OpenAIRE

    Parvaneh Farzaneh; Laleh Nikfarjam

    2011-01-01

    One of the main problems in cell culture is mycoplasma infection. It can extensively affect cell physiology and metabolism. As the applications of cell culture increase in research, industrial production and cell therapy, more concerns about mycoplasma contamination and detection will arise. This review will provide valuable information about: 1. the ways in which cells are contaminated and the frequency and source of mycoplasma species in cell culture; 2. the ways to prevent mycoplasma conta...

  7. Primary hemocyte culture of Penaeus monodon as an in vitro model for white spot syndrome virus titration, viral and immune related gene expression and cytotoxicity assays.

    Science.gov (United States)

    Jose, Seena; Mohandas, A; Philip, Rosamma; Bright Singh, I S

    2010-11-01

    Immortal cell lines have not yet been reported from Penaeus monodon, which delimits the prospects of investigating the associated viral pathogens especially white spot syndrome virus (WSSV). In this context, a method of developing primary hemocyte culture from this crustacean has been standardized by employing modified double strength Leibovitz-15 (L-15) growth medium supplemented with 2% glucose, MEM vitamins (1×), tryptose phosphate broth (2.95 gl⁻¹), 20% FBS, N-phenylthiourea (0.2 mM), 0.06 μg ml⁻¹ chloramphenicol, 100 μg ml⁻¹ streptomycin and 100 IU ml⁻¹ penicillin and hemolymph drawn from shrimp grown under a bio-secured recirculating aquaculture system (RAS). In this medium the hemocytes remained viable up to 8 days. 5-Bromo-2'-deoxyuridine (BrdU) labeling assay revealed its incorporation in 22 ± 7% of cells at 24h. Susceptibility of the cells to WSSV was confirmed by immunofluorescence assay using a monoclonal antibody against 28 kDa envelope protein of WSSV. A convenient method for determining virus titer as MTT(50)/ml was standardized employing the primary hemocyte culture. Expression of viral genes and cellular immune genes were also investigated. The cell culture could be demonstrated for determining toxicity of a management chemical (benzalkonium chloride) by determining its IC(50). The primary hemocyte culture could serve as a model for WSSV titration and viral and cellular immune related gene expression and also for investigations on cytotoxicity of aquaculture drugs and chemicals. PMID:20807537

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-08-06

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

  9. Efficient flotation of yeast cells grown in batch culture.

    Science.gov (United States)

    Palmieri, M C; Greenhalf, W; Laluce, C

    1996-05-01

    A fast flotation assay was used to select new floating yeast strains. The flotation ability did not seem to be directly correlated to total extracellular protein concentration of the culture. However, the hydrophobicity of the cell was definitely correlated to the flotation capacity. The Saccharomyces strains (FLT strains) were highly hydrophobic and showed an excellent flotation performance in batch cultures without additives (flotation agents) and with no need for a special flotation chamber or flotation column. A stable and well-organized structure was evident in the dried foam as shown by scanning electron microscopy which revealed its unique structure showing mummified cells (dehydrated) attached to each other. The attachment among the cells and the high protein concentration of the foams indicated that proteins might be involved in the foam formation. The floating strains (strains FLT) which were not flocculent and showed no tendency to aggregate, were capable of growing and producing ethanol in a synthetic medium containing high glucose concentration as a carbon source. The phenomenon responsible for flotation seems to be quite different from the flocculation phenomenon. PMID:18626952

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

    Science.gov (United States)

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

  11. A Versatile Bioreactor for Dynamic Suspension Cell Culture. Application to the Culture of Cancer Cell Spheroids.

    Science.gov (United States)

    Massai, Diana; Isu, Giuseppe; Madeddu, Denise; Cerino, Giulia; Falco, Angela; Frati, Caterina; Gallo, Diego; Deriu, Marco A; Falvo D'Urso Labate, Giuseppe; Quaini, Federico; Audenino, Alberto; Morbiducci, Umberto

    2016-01-01

    A versatile bioreactor suitable for dynamic suspension cell culture under tunable shear stress conditions has been developed and preliminarily tested culturing cancer cell spheroids. By adopting simple technological solutions and avoiding rotating components, the bioreactor exploits the laminar hydrodynamics establishing within the culture chamber enabling dynamic cell suspension in an environment favourable to mass transport, under a wide range of tunable shear stress conditions. The design phase of the device has been supported by multiphysics modelling and has provided a comprehensive analysis of the operating principles of the bioreactor. Moreover, an explanatory example is herein presented with multiphysics simulations used to set the proper bioreactor operating conditions for preliminary in vitro biological tests on a human lung carcinoma cell line. The biological results demonstrate that the ultralow shear dynamic suspension provided by the device is beneficial for culturing cancer cell spheroids. In comparison to the static suspension control, dynamic cell suspension preserves morphological features, promotes intercellular connection, increases spheroid size (2.4-fold increase) and number of cycling cells (1.58-fold increase), and reduces double strand DNA damage (1.5-fold reduction). It is envisioned that the versatility of this bioreactor could allow investigation and expansion of different cell types in the future. PMID:27144306

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

    Science.gov (United States)

    Dunn, Glynis

    2016-01-01

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

  13. Suitability of human Tenon's fibroblasts as feeder cells for culturing human limbal epithelial stem cells.

    Science.gov (United States)

    Scafetta, Gaia; Tricoli, Eleonora; Siciliano, Camilla; Napoletano, Chiara; Puca, Rosa; Vingolo, Enzo Maria; Cavallaro, Giuseppe; Polistena, Andrea; Frati, Giacomo; De Falco, Elena

    2013-12-01

    Corneal epithelial regeneration through ex vivo expansion of limbal stem cells (LSCs) on 3T3-J2 fibroblasts has revealed some limitations mainly due to the corneal microenvironment not being properly replicated, thus affecting long term results. Insights into the feeder cells that are used to expand LSCs and the mechanisms underlying the effects of human feeder cells have yet to be fully elucidated. We recently developed a standardized methodology to expand human Tenon's fibroblasts (TFs). Here we aimed to investigate whether TFs can be employed as feeder cells for LSCs, characterizing the phenotype of the co-cultures and assessing what human soluble factors are secreted. The hypothesis that TFs could be employed as alternative human feeder layer has not been explored yet. LSCs were isolated from superior limbus biopsies, co-cultured on TFs, 3T3-J2 or dermal fibroblasts (DFs), then analyzed by immunofluorescence (p63α), colony-forming efficiency (CFE) assay and qPCR for a panel of putative stem cell and epithelial corneal differentiation markers (KRT3). Co-cultures supernatants were screened for a set of soluble factors. Results showed that the percentage of p63α(+)LSCs co-cultured onto TFs was significantly higher than those on DFs (p = 0.032) and 3T3-J2 (p = 0.047). Interestingly, LSCs co-cultures on TFs exhibited both significantly higher CFE and mRNA expression levels of ΔNp63α than on 3T3-J2 and DFs (p < 0.0001), showing also significantly greater levels of soluble factors (IL-6, HGF, b-FGF, G-CSF, TGF-β3) than LSCs on DFs. Therefore, TFs could represent an alternative feeder layer to both 3T3-J2 and DFs, potentially providing a suitable microenvironment for LSCs culture. PMID:23832306

  14. Responses of the L5178Y mouse Lymphoma cell forward mutation assay. V: 27 coded chemicals.

    Science.gov (United States)

    McGregor, D B; Brown, A G; Howgate, S; McBride, D; Riach, C; Caspary, W J

    1991-01-01

    Twenty-seven chemicals were tested for their mutagenic potential in the L5178Y tk+/tk- mouse lymphoma cell forward mutation assay using procedures based upon those described by McGregor et al. (McGregor DB, Martin R, Cattanach P, Edwards I, McBride D, Caspary WJ (1987): Environ Mol Mutagen 9:143-160). Cultures were exposed to the chemicals for 4 hr, then cultured for 2 days before plating in soft agar with or without trifluorothymidine (TFT), 3 micrograms/ml. The chemicals were tested at least twice. Statistically significant responses were obtained with acid orange 10, aniline, benzaldehyde, o-chloroaniline, chlorodibromomethane, cytembena, 1,2-dibromo-4-(1,2-dibromomethyl) cyclohexane, dieldrin, lithocholic acid, oxytetracycline, phenazopyridine HCl, 1-phenyl-3-methyl-5-pyrazolone, sodium diethyldithiocarbamate, solvent yellow 14, tetraethylthiuram disulfide (disulfiram), 2,4-toluene diisocyanate, and 2,6-toluene diisocyanate. Apart from phenazopyridine HCl, acid orange 10, and solvent yellow 14, rat liver S9 mix was not a requirement for the mutagenic activity of these compounds. Chemical not identified as mutagens were N-4-acetylaminofluorene, chlorpheniramine maleate, chloropropamide, 1,4-dioxane, endrin, ethylene glycol, iron dextran, methapyrilene, sodium(2-ethylhexyl)alcohol PMID:1902415

  15. Single Cell Adhesion Assay Using Computer Controlled Micropipette

    OpenAIRE

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

    2014-01-01

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

  16. Reversible gelling culture media for in-vitro cell culture in three-dimensional matrices

    Science.gov (United States)

    An, Yuehuei H.; Mironov, Vladimir A.; Gutowska, Anna

    2000-01-01

    A gelling cell culture medium useful for forming a three dimensional matrix for cell culture in vitro is prepared by copolymerizing an acrylamide derivative with a hydrophilic comonomer to form a reversible (preferably thermally reversible) gelling linear random copolymer in the form of a plurality of linear chains having a plurality of molecular weights greater than or equal to a minimum gelling molecular weight cutoff, mixing the copolymer with an aqueous solvent to form a reversible gelling solution and adding a cell culture medium to the gelling solution to form the gelling cell culture medium. Cells such as chondrocytes or hepatocytes are added to the culture medium to form a seeded culture medium, and temperature of the medium is raised to gel the seeded culture medium and form a three dimensional matrix containing the cells. After propagating the cells in the matrix, the cells may be recovered by lowering the temperature to dissolve the matrix and centrifuging.

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

    OpenAIRE

    Huysamen, Cristal

    2005-01-01

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

  18. A comprehensive characterization of cell cultures and xenografts derived from a human verrucous penile carcinoma

    DEFF Research Database (Denmark)

    Muñoz, Juan J; Drigo, Sandra A; Kuasne, Hellen;

    2016-01-01

    , and cultivated in KSFM/DF12 medium. Cell cultures were evaluated at passage 5 (P5) using migration and invasion assays and were serially propagated, in vivo, in BALB/c nude mice until passage 3 (X1-X3). Immunophenotypic characterization of cultures and xenografts was performed. Genomic (CytoScan HD, Affymetrix...... xenograft origin. Cell cultures and xenografts retained the genomic alterations present in the parental tumor. Compared to VSCC, differentially expressed transcripts detected in all experimental conditions were associated with cellular morphology, movement, and metabolism and organization pathways......This study aimed to establish and characterize primary cell cultures and xenografts derived from penile carcinoma (PeCa) in order to provide experimental models for cellular processes and efficacy of new treatments. A verrucous squamous cell carcinoma (VSCC) was macrodissected, dissociated...

  19. Effects of deprivation of background environmental radiation on cultured human cells

    International Nuclear Information System (INIS)

    In this paper we present results from an experiment aimed at investigating whether living cells are influenced by background ionizing radiation. Parallel human cell cultures were set-up in two separate laboratories and maintained for several months under identical conditions but for a 80 x different level of background ionizing radiation. Periodically, the cell cultures were monitored for the onset of divergences in biochemical behavior, using two distinct cellular biology assays, namely micronuclei induction and activity of enzymes implicated in the management of oxidative stress. To reveal any subtle modifications, responses were also amplified by subjecting cell cultures to acute stress induced by exposure to moderately high doses of ionizing radiation. Compared to reference radiation background conditions, cultures maintained in a reduced background radiation environment handled the consequences of acute stress with diminished efficacy.

  20. Cell response of Chlamydomonas actinochloris culture to repeated microwave irradiation

    Directory of Open Access Journals (Sweden)

    OLESIA O. GRYGORIEVA

    2015-05-01

    Full Text Available Abstract. Grygorieva OO, Berezovsjka MA, Dacenko OI. 2015. Cell response of Chlamydomonas actinochloris culture to repeated microwave irradiation. Nusantara Bioscience 7: 38-42. Two cultures of Chlamydomonas actinochloris Deason et Bold in the lag-phase were exposed to the microwave irradiation. One of them (culture 1 was not treated beforehand, whereas the other (culture 2 was irradiated by microwaves 2 years earlier. The measurement of cell quantity as well as measurement of change of intensities and spectra of cultures photoluminescence (PL in the range of chlorophyll a emission was regularly conducted during the cell cultures development. Cell concentration of culture 1 exposed to the microwave irradiation for the first time has quickly restored while cell concentration of culture 2 which was irradiated repeatedly has fallen significantly. The following increasing of cell concentration of culture 2 is negligible. Cell concentration reaches the steady-state level that is about a half of the cell concentration of control culture. Initially the PL efficiency of cells of both cultures decreases noticeable as a result of irradiation. Then there is the monotonic increase to the values which are significantly higher than the corresponding values in the control cultures. The ratio of the intensities at the maxima of the main emission bands of chlorophyll for control samples of both cultures remained approximately at the same level. At the same time effect of irradiation on the cell PL spectrum appears as a temporary reduction of this magnitude.

  1. A long-term flow cytometry assay to analyze the role of specific genes of Drosophila melanogaster S2 cells in surviving genotoxic stress

    NARCIS (Netherlands)

    Yi, Xia; Lemstra, Willy; Vos, Michel J.; Shang, Yongfeng; Kampinga, Harm H.; Su, Tin Tin; Sibon, Ody C. M.

    2008-01-01

    Drosophila S2 cells are easy to manipulate and culture and are a versatile model system for high-throughput screens such as genome-wide siRNA screens to find genes involved in stress or therapy resistance or for screening through large compound libraries to identify cytotoxins. Clonogenic assays are

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

    Science.gov (United States)

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

    2016-01-01

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

  3. Ghrelin regulates cell cycle-related gene expression in cultured hippocampal neural stem cells.

    Science.gov (United States)

    Chung, Hyunju; Park, Seungjoon

    2016-08-01

    We have previously demonstrated that ghrelin stimulates the cellular proliferation of cultured adult rat hippocampal neural stem cells (NSCs). However, little is known about the molecular mechanisms by which ghrelin regulates cell cycle progression. The purpose of this study was to investigate the potential effects of ghrelin on cell cycle regulatory molecules in cultured hippocampal NSCs. Ghrelin treatment increased proliferation assessed by CCK-8 proliferation assay. The expression levels of proliferating cell nuclear antigen and cell division control 2, well-known cell-proliferating markers, were also increased by ghrelin. Fluorescence-activated cell sorting analysis revealed that ghrelin promoted progression of cell cycle from G0/G1 to S phase, whereas this progression was attenuated by the pretreatment with specific inhibitors of MEK/extracellular signal-regulated kinase 1/2, phosphoinositide 3-kinase/Akt, mammalian target of rapamycin, and janus kinase 2/signal transducer and activator of transcription 3. Ghrelin-induced proliferative effect was associated with increased expression of E2F1 transcription factor in the nucleus, as determined by Western blotting and immunofluorescence. We also found that ghrelin caused an increase in protein levels of positive regulators of cell cycle, such as cyclin A and cyclin-dependent kinase (CDK) 2. Moreover, p27(KIP1) and p57(KIP2) protein levels were reduced when cell were exposed to ghrelin, suggesting downregulation of CDK inhibitors may contribute to proliferative effect of ghrelin. Our data suggest that ghrelin targets both cell cycle positive and negative regulators to stimulate proliferation of cultured hippocampal NSCs. PMID:27325242

  4. Insect cell culture in research: Indian scenario.

    Science.gov (United States)

    Sudeep, A B; Mourya, D T; Mishra, A C

    2005-06-01

    Insect cell cultures are widely used in viral diagnosis and biotechnology, for the production of recombinant proteins, viral pesticides and vaccines as well as in basic research in genetics, molecular biology, biochemistry, endocrinology and virology. Following KRP Singh's pioneering research in 1967, a large number of cell lines from diptera, hemiptera, and lepidopteran insects were established and characterized in India. With the availability of the modern tools in molecular biology and the advancements made in biotechnology, the indigenous cell lines may prove useful in creating a future without biohazardous chemical pesticides as well as producing life saving pharmaceuticals and vaccines for many diseases. This review summarizes information gathered regarding the insect cell lines established so far in India. It also covers the familiarization of the well characterized continuous cell lines and their potential applications. Special attention is given to virus susceptibility of the cell lines, the yield of virus with a comparative analysis with other conventional systems. The potential applications of dipteran and lepidopteran cell lines in agriculture and biotechnology are also briefly discussed for prospective studies.

  5. Cytotoxicity effects of amiodarone on cultured cells.

    Science.gov (United States)

    Golli-Bennour, Emna El; Bouslimi, Amel; Zouaoui, Olfa; Nouira, Safa; Achour, Abdellatif; Bacha, Hassen

    2012-07-01

    Amiodarone is a potent anti-arrhythmic drug used for the treatment of cardiac arrhythmias. Although, the effects of amiodarone are well characterized on post-ischemic heart and cardiomyocytes, its toxicity on extra-cardiac tissues is still poorly understood. To this aim, we have monitored the cytotoxicity effects of this drug on three cultured cell lines including hepatocytes (HepG2), epithelial cells (EAhy 926) and renal cells (Vero). We have investigated the effects of amiodarone on (i) cell viabilities, (ii) heat shock protein expressions (Hsp 70) as a parameter of protective and adaptive response and (iii) oxidative damage.Our results clearly showed that amiodarone inhibits cell proliferation, induces an over-expression of Hsp 70 and generates significant amount of reactive oxygen species as measured by lipid peroxidation occurrence. However, toxicity of amiodarone was significantly higher in renal and epithelial cells than in hepatocytes. Vitamin E supplement restores the major part of cell mortalities induced by amiodarone showing that oxidative damage is the predominant toxic effect of the drug.Except its toxicity for the cardiac system, our findings demonstrated that amiodarone can target other tissues. Therefore, kidneys present a high sensibility to this drug which may limit its use with subjects suffering from renal disorders.

  6. Application of an integrated LC-UV-MS-NMR platform to the identification of secondary metabolites from cell cultures: benzophenanthridine alkaloids from elicited Eschscholzia californica (california poppy) cell cultures†

    OpenAIRE

    Gathungu, Rose M.; John T. Oldham; Bird, Susan S.; Lee-Parsons, Carolyn W. T.; Vouros, Paul; Kautz, Roger

    2012-01-01

    Plant cell and tissue cultures are a scalable and controllable alternative to whole plants for obtaining natural products of medical relevance. Cultures can be optimized for high yields of desired metabolites using rapid profiling assays such as HPLC. We describe an approach to establishing a rapid assay for profiling cell culture expression systems using a novel microscale LC-UV-MS-NMR platform, designed to acquire both MS and NMR each at their optimal sensitivity, by using nanosplitter MS f...

  7. Theoretical basis for reducing time-lines to the determination of positive Mycobacterium tuberculosis cultures using thymidylate kinase (TMK assays

    Directory of Open Access Journals (Sweden)

    Wayengera Misaki

    2009-03-01

    Full Text Available Abstract Background In vitro culture of pathogens on growth media forms a "pillar" for both infectious disease diagnosis and drug sensitivity profiling. Conventional cultures of Mycobacterium tuberculosis (M.tb on Lowenstein Jensen (LJ medium, however, take over two months to yield observable growth, thereby delaying diagnosis and appropriate intervention. Since DNA duplication during interphase precedes microbial division, "para-DNA synthesis assays" could be used to predict impending microbial growth. Mycobacterial thymidylate kinase (TMKmyc is a phosphotransferase critical for the synthesis of the thymidine triphosphate precursor necessary for M.tb DNA synthesis. Assays based on high-affinity detection of secretory TMKmyc levels in culture using specific antibodies are considered. The aim of this study was to define algorithms for predicting positive TB cultures using antibody-based assays of TMKmyc levels in vitro. Methods and results Systems and chemical biology were used to derive parallel correlation of "M.tb growth curves" with "TMKmyc curves" theoretically in four different scenarios, showing that changes in TMKmyc levels in culture would in each case be predictive of M.tb growth through a simple quadratic curvature, |tmk| = at2+ bt + c, consistent with the "S" pattern of microbial growth curves. Two drug resistance profiling scenarios are offered: isoniazid (INH resistance and sensitivity. In the INH resistance scenario, it is shown that despite the presence of optimal doses of INH in LJ to stop M.tb proliferation, bacilli grow and the resulting phenotypic growth changes in colonies/units are predictable through the TMKmyc assay. According to our current model, the areas under TMKmyc curves (AUC, calculated as the integral ∫(at2+ bt + cdt or ~1/3 at3+ 1/2 bt2+ct could directly reveal the extent of prevailing drug resistance and thereby aid decisions about the usefulness of a resisted drug in devising "salvage combinations" within

  8. Organotypic culture in three dimensions prevents radiation-induced transformation in human lung epithelial cells

    Science.gov (United States)

    El-Ashmawy, Mariam; Coquelin, Melissa; Luitel, Krishna; Batten, Kimberly; Shay, Jerry W.

    2016-08-01

    The effects of radiation in two-dimensional (2D) cell culture conditions may not recapitulate tissue responses as modeled in three-dimensional (3D) organotypic culture. In this study, we determined if the frequency of radiation-induced transformation and cancer progression differed in 3D compared to 2D culture. Telomerase immortalized human bronchial epithelial cells (HBECs) with shTP53 and mutant KRas expression were exposed to various types of radiation (gamma, +H, 56Fe) in either 2D or 3D culture. After irradiation, 3D structures were dissociated and passaged as a monolayer followed by measurement of transformation, cell growth and expression analysis. Cells irradiated in 3D produced significantly fewer and smaller colonies in soft agar than their 2D-irradiated counterparts (gamma P = 0.0004 +H P = 0.049 56Fe P culture conditions did not affect cell killing, the ability of cells to survive in a colony formation assay, and proliferation rates after radiation—implying there was no selection against cells in or dissociated from 3D conditions. However, DNA damage repair and apoptosis markers were increased in 2D cells compared to 3D cells after radiation. Ideally, expanding the utility of 3D culture will allow for a better understanding of the biological consequences of radiation exposure.

  9. Rapid method for culturing embryonic neuron-glial cell cocultures

    DEFF Research Database (Denmark)

    Svenningsen, Åsa Fex; Shan, Wei-Song; Colman, David R;

    2003-01-01

    A streamlined, simple technique for primary cell culture from E17 rat tissue is presented. In an attempt to standardize culturing methods for all neuronal cell types in the embryo, we evaluated a commercial medium without serum and used similar times for trypsinization and tested different surfaces...... for plating. In 1 day, using one method and a single medium, it is possible to produce robust E17 cultures of dorsal root ganglia (DRG), cerebellum, and enteric plexi. Allowing the endogenous glial cells to repopulate the cultures saves time compared with existing techniques, in which glial cells are added...... to cultures first treated with antimitotic agents. It also ensures that all the cells present in vivo will be present in the culture. Myelination commences after approximately 2 weeks in culture for dissociated DRG and 3-4 weeks in cerebellar cultures. In enteric cultures, glial wrapping of the enteric...

  10. Discarded human fetal tissue and cell cultures for transplantation research

    International Nuclear Information System (INIS)

    A feasibility study has been performed to explore the utility of various tissues from discarded human abortuses for transplantation and related research. Specifically, aborted fetuses plus parental blood samples and all relevant clinical data were obtained through a local hospital complex. Whenever possible, pancreas, skin and skeletal muscle, heart, liver, kidney, cartilage and lung tissues were removed, dissociated and subfractionated for cryopreservation, characterization and cultivation trials in vitro. Existing protocols for these manipulations were compared and improved upon as required. Clonal culture, cell aggregate maintenance techniques and use of feeder cell populations have been utilized where appropriate to develop quantitative comparative data. Histological and biochemical assays were applied both to evaluate separation/cultivation methods and to identify optimal culture conditions for maintaining functional cells. Immunochemical and molecular biological procedures were applied to study expression of Major Histocompatibility Vomplex (MHC) class 1 and 11 molecules on cell lines derived. Tissue and cell culture populations were examined for infections with bacteria, ftingi, mycoplasma, HIV, CMV, hepatitis B and other viruses. Only 1% of the abortuses tested were virally infected. Cytogenetic analyses confin-ned the normal diploid status in the vast majority (>98%) of lines tested. A total of over 250 abortuses have been obtained and processed. Only 25 were found to be contaminated with bacteria or fungi and unsuitable for further cultivation trials. A total of over 200 cell populations were isolated, characterized and cryopreserved for further study. Included were kidney, lung, liver and epidermal epithelia: cartilage-derived cells from the spine and epiphyses plus myogenic myoblasts. Selected lines have been immortalized using HPV I 6E6/E7 sequences. Epithelia from the liver and pancreas and cardiac myocytes were the most problematic in that initial

  11. Comparative study on the stem cell phenotypes of C6 cells under different culture conditions

    Institute of Scientific and Technical Information of China (English)

    ZHANG Suo-jun; YE Fei; XIE Rui-fan; HU Feng; WANG Bao-feng; WAN Feng; GUO Dong-sheng; LEI Ting

    2011-01-01

    Background Glioma stem cell (GSC) hypothesis posits that a subpopulation of cells within gliomas have true clonogenic and tumorigenic potential. Significantly, a more controversial correlate to GSC is that cells in different culture conditions might display distinct stem cell properties. Considering these possibilities, we applied an approach comparing stem cell characteristics of C6 glioma cells under different culture conditions.Methods C6 cells were cultured under three different growth conditions, i.e., adherent growth in conventional 10% serum medium, non-adherent spheres growth in serum-free medium, as well as adherent growth on laminin-coated flask in serum-free medium. Growth characteristics were detected contrastively through neurosphere formation assay and cell cycle analysis. Markers were determined by immunofluorescence, relative-quantitative reverse transcription (RT)-PCR,Western blotting and flow cytometry. Side population cells were analyzed via flow cytometry. Tumor models were detected by magnetic resonance imaging and hematoxylin & eosin staining. Data analyses were performed with SPSS software (17.0).Results C6 cells (C6-Adh, C6-SC-Sph and C6-SC-Adh) showed distinctive growth patterns and proliferation capacity.Compared to suspending C6-SC-Sph, adherent C6-Adh and C6-SC-Adh displayed higher growth ratio. C6-SC-Sph and C6-SC-Adh showed enhanced capability of neurosphere formation and self-renewal. High side population ratio was detected in C6-SC-Sph and C6-SC-Adh. CD133 was not detected in all three kinds of cells. Conversely, Nestin and β-Ⅲ-tubulin were demonstrated positive, nonetheless with no statistical significance (P >0.05). Interestingly, lower expression of glial fibrillary acidic protein was demonstrated in C6-SC-Sph and C6-SC-Adh. C6-Adh, C6-SC-Sph and C6-SC-Adh were all displayed in situ oncogenicity, while statistical difference of survival time was not confirmed.Conclusions C6 glioma cell line is endowed with some GSC

  12. Experimental Study of Rat Beta Islet Cells Cultured under Simulated Microgravity Conditions

    Institute of Scientific and Technical Information of China (English)

    ChunSONG; Xiu-QingDUAN; XiLI; Li-OuHAN; PingXU; Chun-FangSONG:; Lian-HongJIN

    2004-01-01

    To observe the effects of simulated microgravity on beta islet cell culture, we have compared the survival rates and the insulin levels of the isolated rat islet cells cultured at micro- and normal gravity conditions. The survival rates of the cells cultured were determined by acridine orange-propidium iodide double-staining on day 3,7 and 14. The morphology of the cells was observed by electron microscopy.Insulin levels were measured by radio immuno assays. Our results show that the cell number cultured underthe microgravity condition is significantly higher than that under the routine condition (P<0.01). Some tubular structure shown by transmission electron microscopy, possibly for the transport of nutrients, were formed intercellularly in the microgravity cultured group on day 7. There were also abundant secretion particles and mitochondria in the cytoplasm of the cells. Scanning electron microscopy showed that there were holes formed between each islet, possibly connecting with the nutrient transport tubules. The microgravity cultured group also has higher insulin levels in the media as compared with the control group (P<0.01). Our results indicate that microgravity cultivation of islet cells has advantages over the routine culture methods.

  13. Experimental Study of Rat Beta Islet Cells Cultured under Simulated Microgravity Conditions

    Institute of Scientific and Technical Information of China (English)

    Chun SONG; Xiu-Qing DUAN; Xi LI; Li-Ou HAN; Ping XU; Chun-Fang SONG; Lian-Hong JIN

    2004-01-01

    To observe the effects of simulated microgravity on beta islet cell culture, we have compared the survival rates and the insulin levels of the isolated rat islet cells cultured at micro- and normal gravity conditions. The survival rates of the cells cultured were determined by acridine orange-propidium iodide double-staining on day 3, 7 and 14. The morphology of the cells was observed by electron microscopy.Insulin levels were measured by radio immuno assays. Our results show that the cell number cultured under the microgravity condition is significantly higher than that under the routine condition (P<0.01). Some tubular structure shown by transmission electron microscopy, possibly for the transport of nutrients, were formed intercellularly in the microgravity cultured group on day 7. There were also abundant secretion particles and mitochondria in the cytoplasm of the cells. Scanning electron microscopy showed that there were holes formed between each islet, possibly connecting with the nutrient transport tubules. The microgravity cultured group also has higher insulin levels in the media as compared with the control group(P<0.01). Our results indicate that microgravity cultivation of islet cells has advantages over the routine culture methods.

  14. Development of Scalable Culture Systems for Human Embryonic Stem Cells

    OpenAIRE

    Azarin, Samira M.; Palecek, Sean P.

    2010-01-01

    The use of human pluripotent stem cells, including embryonic and induced pluripotent stem cells, in therapeutic applications will require the development of robust, scalable culture technologies for undifferentiated cells. Advances made in large-scale cultures of other mammalian cells will facilitate expansion of undifferentiated human embryonic stem cells (hESCs), but challenges specific to hESCs will also have to be addressed, including development of defined, humanized culture media and su...

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

    Science.gov (United States)

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

    2011-01-01

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

  16. Single cell adhesion assay using computer controlled micropipette.

    Directory of Open Access Journals (Sweden)

    Rita Salánki

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

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

    Science.gov (United States)

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

    2012-01-01

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

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

    International Nuclear Information System (INIS)

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

  19. PHYTOCHEMICAL STUDY OF CELL CULTURE JATROPHA CURCAS

    Directory of Open Access Journals (Sweden)

    KOMAR RUSLAN

    2011-01-01

    Full Text Available Jatropha curcas belongs to the Euphorbiaceae family which has potential economically. This plant has been reported to contain toxic compounds such as curcin and phorbol ester and its derivatives. These compounds may become a problem if J. curcas will be explored as a source of biofuel. In order to provide safety plants, the research on the study of phytochemical and initiation of cell and organ culture have been carried out. J curcas which has been collected from different regions in Indonesia showed to contain relatively the same profile of chemical contents. Dominant compounds that were detected by GCMS are hidrocarbon such as 2-heptenal, decadienal, hexsadecane, pentadecane, cyclooctane etc, fatty acid such as oktadecanoate acid, etthyl linoleate, ethyl stearate, heksadecanoate acid and steroid such as stigmasterol, fucosterol, sitosterol. No phorbol ester and its derivatives have been detected yet by the GCMS method. Callus and suspension cultures of J. curcas have been established to be used for further investigation.

  20. EFFECTS OF PDGF-BB ON INTRACELLULAR CALCIUM CONCENTRATION AND PROLIFERATION IN CULTURED GLOMERULAR MESANGIAL CELLS

    Institute of Scientific and Technical Information of China (English)

    WEN Li-ping; ZHANG Chong; BIAN Fan; ZOU Jun; JIANG Geng-ru; ZHU Han-wei

    2006-01-01

    Objective To investigate the relationship between the alteration of intracellular calcium concentration and proliferation in cultured glomerular mesangial cells. Methods Rat mesangial cells were cultured.Intracellular calcium concentrations were measured by confocal Laser Scanning Microscopy and Fura-3 fluorescence dyeing techniques. Cell growth was measured by MTT assay. Results PDGF-BB increased intracellular calcium concentrations in a dose-dependent manner, and at the same time promote the proliferation of mesangial cells. After preincubation with calcium channel blocker nifedipine or angiotensin converting enzyme inhibitor captopril, both the increase of intracellular calcium concentrations and cell proliferations induced by PDGF-BB were inhibited. Tripterigium Wilfordii Glycosides (TMG) significantly inhibited the mesangial cell proliferations, but it had no significant effect on intracellular calcium concentrations. Conclusion There was a positive relationship between the elevation of intracellular calcium concentration and cell proliferation in glomerular mesangial cells, but the increase of in- tracellular calcium concentrations wasn't the only way for proliferation.

  1. How do culture media influence in vitro perivascular cell behavior?

    Science.gov (United States)

    Huber, Birgit; Volz, Ann-Cathrin; Kluger, Petra Juliane

    2015-12-01

    Perivascular cells are multilineage cells located around the vessel wall and important for wall stabilization. In this study, we evaluated a stem cell media and a perivascular cell-specific media for the culture of primary perivascular cells regarding their cell morphology, doubling time, stem cell properties, and expression of cell type-specific markers. When the two cell culture media were compared to each other, perivascular cells cultured in the stem cell medium had a more elongated morphology and a faster doubling rate and cells cultured in the pericyte medium had a more typical morphology, with several filopodia, and a slower doubling rate. To evaluate stem cell properties, perivascular cells, CD146(-) cells, and mesenchymal stem cells (MSCs) were differentiated into the adipogenic, osteogenic, and chondrogenic lineages. It was seen that perivascular cells, as well as CD146(-) cells and MSCs, cultured in stem cell medium showed greater differentiation than cells cultured in pericyte-specific medium. The expression of pericyte-specific markers CD146, neural/glial antigen 2 (NG2), platelet-derived growth factor receptor-β (PDGFR-β), myosin, and α-smooth muscle actin (α-SMA) could be found in both pericyte cultures, as well as to varying amounts in CD146(-) cells, MSCs, and endothelial cells. The here presented work shows that perivascular cells can adapt to their in vitro environment and cell culture conditions influence cell functionality, such as doubling rate or differentiation behavior. Pericyte-specific markers were shown to be expressed also from cells other than perivascular cells. We can further conclude that CD146(+) perivascular cells are inhomogeneous cell population probably containing stem cell subpopulations, which are located perivascular around capillaries.

  2. Comparison of peptide nucleic acid fluorescence in situ hybridization assays with culture-based matrix-assisted laser desorption/ionization-time of flight mass spectrometry for the identification of bacteria and yeasts from blood cultures and cerebrospinal fluid cultures.

    Science.gov (United States)

    Calderaro, A; Martinelli, M; Motta, F; Larini, S; Arcangeletti, M C; Medici, M C; Chezzi, C; De Conto, F

    2014-08-01

    Peptide nucleic acid fluorescence in situ hybridization (PNA FISH) is a molecular diagnostic tool for the rapid detection of pathogens directly from liquid media. The aim of this study was to prospectively evaluate PNA FISH assays in comparison with culture-based matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) identification, as a reference method, for both blood and cerebrospinal fluid (CSF) cultures, during a 1-year investigation. On the basis of the Gram stain microscopy results, four different PNA FISH commercially available assays were used ('Staphylococcus aureus/CNS', 'Enterococcus faecalis/OE', 'GNR Traffic Light' and 'Yeasts Traffic Light' PNA FISH assays, AdvanDx). The four PNA FISH assays were applied to 956 positive blood cultures (921 for bacteria and 35 for yeasts) and 11 CSF cultures. Among the 921 blood samples positive for bacteria, PNA FISH gave concordant results with MALDI-TOF MS in 908/921 (98.64%) samples, showing an agreement of 99.4% in the case of monomicrobial infections. As regards yeasts, the PNA FISH assay showed a 100% agreement with the result obtained by MALDI-TOF MS. When PNA FISH assays were tested on the 11 CSF cultures, the results agreed with the reference method in all cases (100%). PNA FISH assays provided species identification at least one work-day before the MALDI-TOF MS culture-based identification. PNA FISH assays showed an excellent efficacy in the prompt identification of main pathogens, yielding a significant reduction in reporting time and leading to more appropriate patient management and therapy in cases of sepsis and severe infections.

  3. 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.

  4. Universal lab-on-a-chip platform for complex, perfused 3D cell cultures

    Science.gov (United States)

    Sonntag, F.; Schmieder, F.; Ströbel, J.; Grünzner, S.; Busek, M.; Günther, K.; Steege, T.; Polk, C.; Klotzbach, U.

    2016-03-01

    The miniaturization, rapid prototyping and automation of lab-on-a-chip technology play nowadays a very important role. Lab-on-a-chip technology is successfully implemented not only for environmental analysis and medical diagnostics, but also as replacement of animals used for the testing of substances in the pharmaceutical and cosmetics industries. For that purpose the Fraunhofer IWS and partners developed a lab-on-a-chip platform for perfused cell-based assays in the last years, which includes different micropumps, valves, channels, reservoirs and customized cell culture modules. This technology is already implemented for the characterization of different human cell cultures and organoids, like skin, liver, endothelium, hair follicle and nephron. The advanced universal lab-on-a-chip platform for complex, perfused 3D cell cultures is divided into a multilayer basic chip with integrated micropump and application-specific 3D printed cell culture modules. Moreover a technology for surface modification of the printed cell culture modules by laser micro structuring and a complex and flexibly programmable controlling device based on an embedded Linux system was developed. A universal lab-on-a-chip platform with an optional oxygenator and a cell culture module for cubic scaffolds as well as first cell culture experiments within the cell culture device will be presented. The module is designed for direct interaction with robotic dispenser systems. This offers the opportunity to combine direct organ printing of cells and scaffolds with the microfluidic cell culture module. The characterization of the developed system was done by means of Micro-Particle Image Velocimetry (μPIV) and an optical oxygen measuring system.

  5. Engineering and exploitation of a fluorescent HIV-1 gp120 for live cell CD4 binding assays

    International Nuclear Information System (INIS)

    The HIV-1 envelope glycoprotein, gp120, binds the host cell receptor, CD4, in the initial step of HIV viral entry and infection. This process is an appealing target for the development of inhibitory drugs and neutralizing antibodies. To study gp120 binding and intracellular trafficking, we engineered a fluorescent fusion of the humanized gp120 JRFL HIV-1 variant and GFP. Gp120-sfGFP is glycosylated with human sugars, robustly expressed, and secreted from cultured human cells. Protein dynamics, quality control, and trafficking can be visualized in live cells. The fusion protein can be readily modified with different gp120 variants or fluorescent proteins. Finally, secreted gp120-sfGFP enables a sensitive and easy binding assay that can quantitatively screen potential inhibitors of gp120-CD4 binding on live cells via fluorescence imaging or laser scanning cytometry. This adaptable research tool should aid in studies of gp120 cell biology and the development of novel anti-HIV drugs. - Highlights: • Development of fluorescent protein labeled HIV-1 envelope gp120. • Imaging of gp120 dynamics and trafficking in live cells. • Quantitative visual assay of antibody-mediated inhibition of gp120 binding to CD4 on live cells

  6. Engineering and exploitation of a fluorescent HIV-1 gp120 for live cell CD4 binding assays

    Energy Technology Data Exchange (ETDEWEB)

    Costantini, Lindsey M. [Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 (United States); Irvin, Susan C. [Department of Pediatrics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 (United States); Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 (United States); Kennedy, Steven C. [Department of Pediatrics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 (United States); Guo, Feng [Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 (United States); Goldstein, Harris; Herold, Betsy C. [Department of Pediatrics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 (United States); Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 (United States); Snapp, Erik L., E-mail: erik-lee.snapp@einstein.yu.edu [Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 (United States)

    2015-02-15

    The HIV-1 envelope glycoprotein, gp120, binds the host cell receptor, CD4, in the initial step of HIV viral entry and infection. This process is an appealing target for the development of inhibitory drugs and neutralizing antibodies. To study gp120 binding and intracellular trafficking, we engineered a fluorescent fusion of the humanized gp120 JRFL HIV-1 variant and GFP. Gp120-sfGFP is glycosylated with human sugars, robustly expressed, and secreted from cultured human cells. Protein dynamics, quality control, and trafficking can be visualized in live cells. The fusion protein can be readily modified with different gp120 variants or fluorescent proteins. Finally, secreted gp120-sfGFP enables a sensitive and easy binding assay that can quantitatively screen potential inhibitors of gp120-CD4 binding on live cells via fluorescence imaging or laser scanning cytometry. This adaptable research tool should aid in studies of gp120 cell biology and the development of novel anti-HIV drugs. - Highlights: • Development of fluorescent protein labeled HIV-1 envelope gp120. • Imaging of gp120 dynamics and trafficking in live cells. • Quantitative visual assay of antibody-mediated inhibition of gp120 binding to CD4 on live cells.

  7. Electrochemical sensors, MTT and immunofluorescence assays for monitoring the proliferation effects of cissus populnea extracts on Sertoli cells

    Directory of Open Access Journals (Sweden)

    McGee Dennis

    2011-05-01

    Full Text Available Abstract Background We describe the development of an electrochemical sensor array for monitoring the proliferation effects of cissus populnea plant extracts on TM4 Sertoli cells. Methods The proliferation activities of the extracts on Sertoli cells were studied using a high-throughput electrochemical sensor array (DOX-96 and the analytical sensor characteristics were compared with conventional colorimetric MTT (3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide assay and fluorescence spectroscopy. Results This work shows that there is a definite positive trend in the proliferation effect of the extract of Cissus populnea on the TM4 Sertoli cells. All of the three techniques confirmed that the most effective concentration for the proliferation is 10 ppm. At this concentration, the proliferation effect was established around 120% for both DOX-96 and MTT techniques, whereas fluorescence assays showed a higher level (120-150%. DOX-96 showed a lower limit of detection (1.25 × 10(4 cells/ml; whereas the LOD recorded for both MTT and fluorescence techniques was 2.5 × 10(4 cells/ml. Visual examination of the cells by means of confocal fluorescence microscopy confirmed the proliferation of Sertoli cells as was determined using the MTT assay. This investigation provides a confident interpretation of the results and proved that the most effective concentration for the proliferation using Cissus populnea plant extract is 10 ppm. Conclusions Overall, the DOX results compared well with the conventional methods of checking proliferation of cells. The fascinating feature of the sensor array is the ability to provide continuous proliferation experiments with no additional reagents including 96 simultaneous electrochemical experiments. The use of the DOX-96 could reduce a typical bioassay time by 20-fold. Thus the DOX-96 can be used as both a research tool and for practical cell culture monitoring.

  8. Sensitivity of immunofluorescence with monoclonal antibodies for detection of Chlamydia trachomatis inclusions in cell culture.

    OpenAIRE

    Stephens, R S; Kuo, C C; Tam, M R

    1982-01-01

    Monoclonal antibodies which recognize the species-specific major outer membrane protein antigen of Chlamydia trachomatis were used for immunofluorescence staining of chlamydial inclusions in cell culture. A total of 115 clinical specimens were inoculated onto replicate HeLa 229 cell monolayers and assayed for chlamydial inclusions by immunofluorescence staining and Giemsa staining. Of the isolates, 38 were detected by immunofluorescence staining on passage 1 and 1 was detected on passage 2; 2...

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

    DEFF Research Database (Denmark)

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

    2005-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1988-12-01

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

  11. Polyamines and the Cell Cycle of Catharanthus roseus Cells in Culture 1

    Science.gov (United States)

    Maki, Hisae; Ando, Satoshi; Kodama, Hiroaki; Komamine, Atsushi

    1991-01-01

    Investigation was made on the effect of partial depletion of polyamines (PAs), induced by treatment with inhibitors of the biosynthesis of PAs, on the distribution of cells at each phase of the cell cycle in Catharanthus roseus (L.) G. Don. cells in suspension cultures, using flow cytometry. More cells treated with inhibitors of arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) were accumulated in the G1 phase than those in the control, while the treatment with an inhibitor of spermidine (SPD) synthase showed no effect on the distribution of cells. The endogenous levels of the PAs, putrescine (PUT), SPD, and spermine (SPM), were determined during the cell cycle in synchronous cultures of C. roseus. Two peaks of endogenous level of PAs, in particular, of PUT and SPD, were observed during the cell cycle. Levels of PAs increased markedly prior to synthesis of DNA in the S phase and prior to cytokinesis. Activities of ADC and ODC were also assayed during the cell cycle. Activities of ADC was much higher than that of ODC throughout the cell cycle, but both activities of ODC and ADC changed in concert with changes in levels of PAs. Therefore, it is suggested that these enzymes may regulate PA levels during the cell cycle. These results indicate that inhibitors of PUT biosynthesis caused the suppression of cell proliferation by prevention of the progression of the cell cycle, probably from the G1 to the S phase, and PUT may play more important roles in the progression of the cell cycle than other PAs. PMID:16668290

  12. Feasibility of Primary Tumor Culture Models and Preclinical Prediction Assays for Head and Neck Cancer: A Narrative Review

    International Nuclear Information System (INIS)

    Primary human tumor culture models allow for individualized drug sensitivity testing and are therefore a promising technique to achieve personalized treatment for cancer patients. This would especially be of interest for patients with advanced stage head and neck cancer. They are extensively treated with surgery, usually in combination with high-dose cisplatin chemoradiation. However, adding cisplatin to radiotherapy is associated with an increase in severe acute toxicity, while conferring only a minor overall survival benefit. Hence, there is a strong need for a preclinical model to identify patients that will respond to the intended treatment regimen and to test novel drugs. One of such models is the technique of culturing primary human tumor tissue. This review discusses the feasibility and success rate of existing primary head and neck tumor culturing techniques and their corresponding chemo- and radiosensitivity assays. A comprehensive literature search was performed and success factors for culturing in vitro are debated, together with the actual value of these models as preclinical prediction assay for individual patients. With this review, we aim to fill a gap in the understanding of primary culture models from head and neck tumors, with potential importance for other tumor types as well

  13. Feasibility of Primary Tumor Culture Models and Preclinical Prediction Assays for Head and Neck Cancer: A Narrative Review

    Energy Technology Data Exchange (ETDEWEB)

    Dohmen, Amy J. C., E-mail: a.dohmen@nki.nl [Department of Head and Neck Surgery and Oncology, The Netherlands Cancer Institute-Antoni van Leeuwenhoek, Plesmanlaan 121, Amsterdam 1066 CX (Netherlands); Department of Cell Biology, the Netherlands Cancer Institute-Antoni van Leeuwenhoek, Plesmanlaan 121, Amsterdam 1066 CX (Netherlands); Swartz, Justin E. [Department of Otorhinolaryngology-Head and Neck Surgery, University Medical Center Utrecht, Heidelberglaan 100, Utrecht 3508 GA (Netherlands); Van Den Brekel, Michiel W. M. [Department of Head and Neck Surgery and Oncology, The Netherlands Cancer Institute-Antoni van Leeuwenhoek, Plesmanlaan 121, Amsterdam 1066 CX (Netherlands); Willems, Stefan M. [Department of Pathology, University Medical Center Utrecht, Heidelberglaan 100, Utrecht 3508 GA (Netherlands); Spijker, René [Medical library, Academic Medical Center, Amsterdam 1100 DE (Netherlands); Dutch Cochrane Centre, Julius Center for Health Sciences and Primary Care, University Medical Center Utrecht, Heidelberglaan 100, Utrecht 3508 GA (Netherlands); Neefjes, Jacques [Department of Cell Biology, the Netherlands Cancer Institute-Antoni van Leeuwenhoek, Plesmanlaan 121, Amsterdam 1066 CX (Netherlands); Zuur, Charlotte L. [Department of Head and Neck Surgery and Oncology, The Netherlands Cancer Institute-Antoni van Leeuwenhoek, Plesmanlaan 121, Amsterdam 1066 CX (Netherlands)

    2015-08-28

    Primary human tumor culture models allow for individualized drug sensitivity testing and are therefore a promising technique to achieve personalized treatment for cancer patients. This would especially be of interest for patients with advanced stage head and neck cancer. They are extensively treated with surgery, usually in combination with high-dose cisplatin chemoradiation. However, adding cisplatin to radiotherapy is associated with an increase in severe acute toxicity, while conferring only a minor overall survival benefit. Hence, there is a strong need for a preclinical model to identify patients that will respond to the intended treatment regimen and to test novel drugs. One of such models is the technique of culturing primary human tumor tissue. This review discusses the feasibility and success rate of existing primary head and neck tumor culturing techniques and their corresponding chemo- and radiosensitivity assays. A comprehensive literature search was performed and success factors for culturing in vitro are debated, together with the actual value of these models as preclinical prediction assay for individual patients. With this review, we aim to fill a gap in the understanding of primary culture models from head and neck tumors, with potential importance for other tumor types as well.

  14. Cocultivation of umbilical cord blood CD34+ cells with retro-transduced hMSCs leads to effective amplification of long-term culture-initiating cells

    Institute of Scientific and Technical Information of China (English)

    Chun-Gang Xie; Jin-Fu Wang; Ying Xiang; Li-Yan Qiu; Bing-Bing Jia; Li-Juan Wang; Guo-Zhong Wang; Guo-Ping Huang

    2006-01-01

    AIM: To establish a novel coculture system for ex vivo expansion of umbilical cord blood(UCB) hematopoietic progenitors using thrombopoietin (TPO)/Flt-3 ligand(FL)-transduced human marrow-derived mesenchymal stem cells (tfhMSCs) as feeder.METHODS: UCB CD34+ cells were isolated and cultured using four culture systems in serum-containing or serumfree medium. Suitable aliquots of cultured cells were used to monitor cell production, clonogenic activity,and long-term culture-initiating culture (LTC-IC) output.Finally, the severe-combined immunodeficient (SCID)mouse-repopulating cell (SRC) assay was performed to confirm ability of the cultured cells to reconstitute longterm hematopoiesis.RESULTS: There were no significant differences in the number of total nucleated cells among different culture systems in serum-containing medium during 21-d culture. However, on d 14, the outputs of CD34+ cells,CFU-C and CFU-GEMM in tfhMSCs coculture system were significantly enhanced. LTC-IC assay demonstrated that the tfhMSCs coculture system had the most powerful activity. The severe-combined immunodeficient (SCID)mouse repopulating cell (SRC) assay confirmed extensive ability of the expanded cells to reconstitute long-term hematopoiesis. Furthermore, PCR analysis demonstrated the presence of human hematopoietic cells in the bone marrow and peripheral blood cells of NOD/SCID mice.CONCLUSION: The TPO/FL-transduced hMSCs, in combination with additive cytokines, can effectively expand hematopoietic progenitors from UCB in vitro and the tfhMSCs coculture system may be a suitable system for ex vivo manipulation of primitive progenitor cells under contact culture conditions.

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

    Science.gov (United States)

    Jiang, Shibo; Radigan, Lin; Zhang, Li

    2000-03-01

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

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

    NARCIS (Netherlands)

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

    2012-01-01

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

  17. Evaluation of diffusion in gel entrapment cell culture within hollow fibers

    Institute of Scientific and Technical Information of China (English)

    Dan-Qing Wu; Guo-Liang Zhang; Chong Shen; Qian Zhao; Hui Li; Qin Meng

    2005-01-01

    AIM: To investigate diffusion in mammalian cell culture by gel entrapment within hollow fibers.METHODS: Freshly isolated rat hepatocytes or human oral epidermoid carcinoma (KB) cells were entrapped in type Ⅰ collagen solutions and statically cultured inside microporous and ultrafiltration hollow fibers. During the culture time collagen gel contraction, cell viability and specific function were assessed. Effective diffusion coefficients of glucose in cell-matrix gels were determined by lag time analysis in a diffusion cell.R ESULTS: Significant gel contractions occurred in the collagen gels by entrapment of either viable hepatocytes or KB cells. And the gel contraction caused a significant reduction on effective diffusion coefficient of glucose. The cell viability assay of both hepatocytes and KB cells statically cultured in hollow fibers by collagen entrapment further confirmed the existence of the inhibited mass transfer by diffusion. Urea was secreted about 50% more by hepatocytes entrapped in hollow fibers with pore size of 0.1 pm than that in hollow fibers with MWCO of 100 ku.CONCLUSION: Cell-matrix gel and membrane pore size are the two factors relevant to the limited mass transfer by diffusion in such gel entrapment of mammalian cell culture.

  18. Cardiac Cells Beating in Culture: A Laboratory Exercise

    Science.gov (United States)

    Weaver, Debora

    2007-01-01

    This article describes how to establish a primary tissue culture, where cells are taken directly from an organ of a living animal. Cardiac cells are taken from chick embryos and transferred to culture dishes. These cells are not transformed and therefore have a limited life span. However, the unique characteristics of cardiac cells are maintained…

  19. Growth of cultured porcine retinal pigment epithelial cells

    DEFF Research Database (Denmark)

    Wiencke, A.K.; Kiilgaard, Jens Folke; Nicolini, Jair;

    2003-01-01

    To establish and characterize cultures of porcine retinal pigment epithelial (pRPE) cells in order to produce confluent monolayers of cells for transplantation.......To establish and characterize cultures of porcine retinal pigment epithelial (pRPE) cells in order to produce confluent monolayers of cells for transplantation....

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

    Science.gov (United States)

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

    2016-06-01

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

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

    DEFF Research Database (Denmark)

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

    2012-01-01

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

  2. Comparative Analysis of Cultural Isolation and Pcr Based Assay for Detection of Campylobacter Jejuni In Food and Faecal Samples

    OpenAIRE

    Singh, Harkanwaldeep; Rathore, R. S.; Singh, Satparkash; Cheema, Pawanjit Singh

    2011-01-01

    In the present study, the efficacy of polymerase chain reaction (PCR) based on mapA gene of C. jejuni was tested for detection of Campylobacter jejuni in naturally infected as well as spiked faecal and food samples of human and animal origin. Simultaneously, all the samples were subjected to the cultural isolation of organism and biochemical characterization. The positive samples resulted in the amplification of a DNA fragment of size ~589 bp in PCR assay whereas the absence of such amplicon ...

  3. Comparative analysis of cultural isolation and PCR based assay for detection of Campylobacter jejuni in food and faecal samples

    OpenAIRE

    Harkanwaldeep Singh; Rathore, R. S.; Satparkash Singh; Pawanjit Singh Cheema

    2011-01-01

    In the present study, the efficacy of polymerase chain reaction (PCR) based on mapA gene of C. jejuni was tested for detection of Campylobacter jejuni in naturally infected as well as spiked faecal and food samples of human and animal origin. Simultaneously, all the samples were subjected to the cultural isolation of organism and biochemical characterization. The positive samples resulted in the amplification of a DNA fragment of size ~589 bp in PCR assay whereas the absence of such amplicon ...

  4. Control of MRSA infection and colonisation in an intensive care unit by GeneOhm MRSA assay and culture methods

    OpenAIRE

    Valle Claudia; Pasca Maria; De Vitis Debora; Marzani Federico; Emmi Vincenzo; Marone Piero

    2009-01-01

    Abstract Background Methicillin-resistant Staphylococcus aureus (MRSA) is one of the major nosocomial pathogens. Due to the diffusion of MRSA strains in both hospital and community settings, prevention and control strategies are receiving increased attention. Approximately 25% to 30% of the population is colonised with S. aureus and 0.2% to 7% with MRSA. The BD GeneOhm MRSA real-time PCR assay offers quicker identification of MRSA-colonised patients than do culture methods. Methods Ninety-fiv...

  5. A novel mast cell co-culture microfluidic chip for the electrochemical evaluation of food allergen.

    Science.gov (United States)

    Jiang, Hui; Jiang, Donglei; Zhu, Pei; Pi, Fuwei; Ji, Jian; Sun, Chao; Sun, Jiadi; Sun, Xiulan

    2016-09-15

    In this study a novel cell-to-cell electrochemical microfluidic chip was developed for qualitative and quantitative analysis of food allergen. Microfluidic cell culture, food allergen-induced cell morphological changes, and cell metabolism measurements were performed simultaneously using the aforementioned device. RBL-2H3 mast cells and ANA-1 macrophages have been used within a cell co-culture model to observe their allergic response when they are introduced to the antigen stimulus. Two cell cultivation microfluidic channels are located in the microfluidic chip, which is fabricated with four groups of gold electrodes, with an additional "capillary". In order to detect the allergic response, the cells were stimulated with dinitrophenylated bovine serum albumin (DNP-BSA) without anti-DNP IgE incubation. When exocytosis occurs, the cell-secreted inflammatory cytokines were measured by enzyme-linked immuno sorbent assay (ELISA) and cell impedance changes were detected using cell-based electrochemical assay. Results indicate that the real-time cell allergic response are accurately monitored by this electrochemical microfluidic chip, which provides a general example of rapidly prototyped low-cost biosensor technology for applications in both food allergen detection and investigation. PMID:27108255

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

    Science.gov (United States)

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

    2001-01-01

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

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

    Science.gov (United States)

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

    1999-06-01

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

  8. Development of primary cell culture from Scylla serrata: Primary cell cultures from Scylla serrata

    OpenAIRE

    Sashikumar, Anu; Desai, P. V.

    2008-01-01

    This paper reports for the first time, the Primary cell culture of hepatopancreas from edible crab Scylla serrata using crab saline, L-15 (Leibovitz), 1 × L-15 + crab saline, 2 × L-15 + crab saline, 3 × L-15 and citrate buffer without any serum. We could isolate and maintain E (Embryonalzellen), F (Fibrenzellen), B (Blasenzellen), R (Restzellen) and G (Granular cells). Upon seeding the hepatopancreatic E, F, B, and R cells showed different survival pattern over time than granular cells. A mod...

  9. Metabolic flux rewiring in mammalian cell cultures.

    Science.gov (United States)

    Young, Jamey D

    2013-12-01

    Continuous cell lines (CCLs) engage in 'wasteful' glucose and glutamine metabolism that leads to accumulation of inhibitory byproducts, primarily lactate and ammonium. Advances in techniques for mapping intracellular carbon fluxes and profiling global changes in enzyme expression have led to a deeper understanding of the molecular drivers underlying these metabolic alterations. However, recent studies have revealed that CCLs are not necessarily entrenched in a glycolytic or glutaminolytic phenotype, but instead can shift their metabolism toward increased oxidative metabolism as nutrients become depleted and/or growth rate slows. Progress to understand dynamic flux regulation in CCLs has enabled the development of novel strategies to force cultures into desirable metabolic phenotypes, by combining fed-batch feeding strategies with direct metabolic engineering of host cells. PMID:23726154

  10. Phosphatidylinositol species of suspension cultured plant cells

    Energy Technology Data Exchange (ETDEWEB)

    Heim, S.; Wagner, K.G.

    Suspension cultured Nicotiana tabacum and Catharanthus roseus cells were labeled with (/sup 3/H)inositol, the phospholipid fraction extracted and separated by thin layer chromatography. Three different solvent systems and reference compounds were used to assign the different /sup 3/H-labeled species by autoradiography. The ratio of (/sup 3/H)inositol incorporation into PI, PIP and PIP/sub 2/ was found to be 95:4:1; with some preparations a lyso-PI band was obtained which incorporated about a tenth of the label of the PIP band. With Catharanthus roseus cells a very faint band between PI and lyso-PI was detected which could not be assigned to a reference compound.

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

    International Nuclear Information System (INIS)

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

  12. Lack of FasL expression in cultured human retinal pigment epithelial cells

    DEFF Research Database (Denmark)

    Kaestel, C G; Madsen, H O; Prause, J U;

    2001-01-01

    Retinal pigment epithelial (RPE) cells have been proposed to play a part in maintaining the eye as an immune privileged organ. However, our knowledge of the implicated mechanism is still sparse. Fas ligand (FasL) expression of RPE cells is generally recognized to be essential for the immune...... privilege of the eye, but due to contradictory published results, it is unclear whether RPE cells express this molecule. The purpose of this study was to investigate the expression of FasL in RPE cells in vitro and in vivo. Cultured human fetal and adult RPE cells were examined by flow cytometry, Western...... blotting, RT-PCR and RNase Protection assay for FasL expression. Additionally, sections of ocular tissue were stained for FasL by immunohistochemistry. None of the used methods indicated FasL expression in cultured fetal or adult RPE cells of various passages. However, RPE cells in vivo, as judged from...

  13. Development of a pneumatically driven active cover lid for multi-well microplates for use in perfusion three-dimensional cell culture

    Science.gov (United States)

    Huang, Song-Bin; Chou, Dean; Chang, Yu-Han; Li, Ke-Cing; Chiu, Tzu-Keng; Ventikos, Yiannis; Wu, Min-Hsien

    2015-12-01

    Before microfluidic-based cell culture models can be practically utilized for bioassays, there is a need for a transitional cell culture technique that can improve conventional cell culture models. To address this, a hybrid cell culture system integrating an active cover lid and a multi-well microplate was proposed to achieve perfusion 3-D cell culture. In this system, a microfluidic-based pneumatically-driven liquid transport mechanism was integrated into the active cover lid to realize 6-unit culture medium perfusion. Experimental results revealed that the flow of culture medium could be pneumatically driven in a flow-rate uniform manner. We used the system to successfully perform a perfusion 3-D cell culture of mesenchymal stem cells (MSCs) for up to 16 days. Moreover, we investigated the effects of various cell culture models on the physiology of MSCs. The physiological nature of MSCs can vary with respect to the cell culture model used. Using the perfusion 3-D cell culture format might affect the proliferation and osteogenic differentiation of MSCs. Overall, we have developed a cell culture system that can achieve multi-well microplate-based perfusion 3-D cell culture in an efficient, cost-effective, and user-friendly manner. These features could facilitate the widespread application of perfusion cell culture models for cell-based assays.

  14. Effects of Ginsenoside Rb1 on proliferation of Schwann cells in culture

    Institute of Scientific and Technical Information of China (English)

    胡晞棠; 陈晓翔; 熊良俭

    2002-01-01

    Objective: To investigate the effects of Ginsenoside Rb1 on the proliferation of Schwann cells in culture.Methods: Applying MTT assay and Thymidine incorporation assay, the effects of Ginsenoside Rb1 on the proliferation of Schwann cells isolated from the sciatic nerve of adult rat were studied.Results: Ginsenoside Rb1 (10 μg/ml) significantly induced Schwann cell proliferation, the effect was similar to NGF (50 μg/ml). At high concentrations of Ginsenoside Rb1 (1 mg/ml), the proliferation of Schwann cells was significantly inhibited. Conclusions: Ginsenoside Rb1 at the optimal concentrations is found to be effective in inducing the proliferation of Schwann cells, but at higher concentrations the drug is cytotoxic for Schwann cells.

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

    International Nuclear Information System (INIS)

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2003-01-01

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

  17. Recombinant Protein Production and Insect Cell Culture and Process

    Science.gov (United States)

    Spaulding, Glenn F. (Inventor); Goodwin, Thomas J. (Inventor); OConnor, Kim C. (Inventor); Francis, Karen M. (Inventor); Andrews, Angela D. (Inventor); Prewett, Tracey L. (Inventor)

    1997-01-01

    A process has been developed for recombinant production of selected polypeptides using transformed insect cells cultured in a horizontally rotating culture vessel modulated to create low shear conditions. A metabolically transformed insect cell line is produced using the culture procedure regardless of genetic transformation. The recombinant polypeptide can be produced by an alternative process using virtually infected or stably transformed insect cells containing a gene encoding the described polypeptide. The insect cells can also be a host for viral production.

  18. Assessment of cell death studies by monitoring hydrogen peroxide in cell culture.

    Science.gov (United States)

    Hirsch, Irina; Prell, Erik; Weiwad, Matthias

    2014-07-01

    Hydrogen peroxide (H2O2) has been widely used to study the oxidative stress response. However, H2O2 is unstable and easily decomposes into H2O and O2. Consequently, a wide range of exposure times and treatment concentrations has been described in the literature. In the present study, we established a ferrous oxidation-xylenol orange (FOX) assay, which was originally described for food and body liquids, as a method for the precise quantification of H2O2 concentrations in cell culture media. We observed that the presence of FCS and high cell densities significantly accelerate the decomposition of H2O2, therefore acting as a protection against cell death by accidental necrosis. PMID:24747006

  19. Label-free imaging to study phenotypic behavioural traits of cells in complex co-cultures

    Science.gov (United States)

    Suman, Rakesh; Smith, Gabrielle; Hazel, Kathryn E. A.; Kasprowicz, Richard; Coles, Mark; O'Toole, Peter; Chawla, Sangeeta

    2016-02-01

    Time-lapse imaging is a fundamental tool for studying cellular behaviours, however studies of primary cells in complex co-culture environments often requires fluorescent labelling and significant light exposure that can perturb their natural function over time. Here, we describe ptychographic phase imaging that permits prolonged label-free time-lapse imaging of microglia in the presence of neurons and astrocytes, which better resembles in vivo microenvironments. We demonstrate the use of ptychography as an assay to study the phenotypic behaviour of microglial cells in primary neuronal co-cultures through the addition of cyclosporine A, a potent immune-modulator.

  20. Axitinib affects cell viability and migration of a primary foetal lung adenocarcinoma culture.

    Science.gov (United States)

    Menna, Cecilia; De Falco, Elena; Pacini, Luca; Scafetta, Gaia; Ruggieri, Paola; Puca, Rosa; Petrozza, Vincenzo; Ciccone, Anna Maria; Rendina, Erino Angelo; Calogero, Antonella; Ibrahim, Mohsen

    2014-01-01

    Fetal lung adenocarcinoma (FLAC) is a rare variant of lung adenocarcinoma. Studies regarding FLAC have been based only on histopathological observations, thus representative in vitro models of FLAC cultures are unavailable. We have established and characterized a human primary FLAC cell culture, exploring its biology, chemosensitivity, and migration. FLAC cells and specimen showed significant upregulation of VEGF165 and HIF-1α mRNA levels. This observation was confirmed by in vitro chemosensitivity and migration assay, showing that only Axitinib was comparable to Cisplatin treatment. We provide a suitable in vitro model to further investigate the nature of this rare type of cancer. PMID:24380379

  1. Noncytotoxic T cell clones obtained from a human mixed leukocyte culture.

    Science.gov (United States)

    Chu, M H; Wee, S L; Bach, F H

    1990-02-01

    Peripheral blood mononuclear cells from a DQW-1 homozygous individual were cocultured with irradiated lymphoblastoid cell line from a DQW-1 homozygous unrelated donor bearing BW35-DW1 haplotype. From T cell cloning of primary and twice-stimulated mixed leukocyte cultures (MLC), 7 and 11 T cell clones were obtained respectively. None of the 18 clones showed specific cytotoxic activity against the alloantigen of the stimulator cell as well as natural killer (NK)-like activity against K562 cells. However, most T cell clones from both primary and re-stimulated MLC demonstrated moderate cytotoxic activity in lectin-dependent cell-mediated cytolysis (LDCC) assay. Screening assay for cell-mediated lympholysis (CML) performed on growing microcultures obtained from restimulated MLC cloning confirmed the non-cytotoxic status of these T cell clones by showing that 41 out of 44 growing microcultures were not cytotoxic against the stimulator cell; the other 3 clones lyzed the target cell mildly. The cells from all 5 T cell clones detected for indirect fluorescence expressed CD3 and CD4 surface markers. Taken together, the results suggested that proliferation-regulating T cell subsets or factor(s) may be generated during the course of MLCs under the present responder-stimulator combination, and may suppress the development of alloreactive cytotoxic T cells and NK-like cells. PMID:2144231

  2. ENRICHMENT AND CHARACTERIZATION OF THYMUS-REPOPULATING CELLS IN STROMA-DEPENDENT CULTURES OF RAT BONE-MARROW

    NARCIS (Netherlands)

    PRAKAPAS, Z; DENOYELLE, M; DARGEMONT, C; KROESE, FGM; THIERY, JP; DEUGNIER, MA

    1993-01-01

    The bone marrow precursor cells seeding the thymus have been difficult to investigate using fresh bone marrow and in vivo thymus reconstitution assays. We have therefore designed a short-term bone marrow culture system allowing the study of thymus-repopulating cells in the marrow microenvironment. L

  3. Protection of cultured mammalian cells by rebamipide

    Energy Technology Data Exchange (ETDEWEB)

    Antoku, Shigetoshi; Aramaki, Ryoji [Kyushu Univ., Fukuoka (Japan). Faculty of Medicine; Tanaka, Hisashi; Kusumoto, Naotoshi

    1997-06-01

    Rebamipide which is used as a drug for gastritis and stomach ulcer has large capability for OH radical scavenging. It is expected that rebamipide has protective effect against ionizing radiations. The present paper deals with protective effect of rebamipide for cultured mammalian cells exposed to ionizing radiations. As rebamipide is insoluble in water, three solvents were used to dissolve. Rebamipide dissolved in dimethyl sulfoxide (DMSO), dimethyl formamide (DMFA) and 0.02 N NaOH was added to the cells in Eagle`s minimum essential medium (MEM) supplemented with 10% fetal calf serum and the cells were irradiated with X-rays. After irradiation, the cells were trypsinized, plated in MEM with 10% fetal calf serum and incubated for 7 days in a CO{sub 2} incubator to form colonies. Rebamipide dissolved in 0.02 N NaOH exhibited the protective effect expected its OH radical scavenging capability. However, the protective effect of rebamipide dissolved in DMSO was about half of that expected by its radical scavenging capability and that of rebamipide dissolved in DMFA was not observed. Uptake of rebamipide labeled with {sup 14}C increased with increasing contact time with rebamipide. These rebamipide mainly distributed in nucleus rather than cytoplasm. (author)

  4. Effect of primarily cultured human lung cancer-associated fibroblasts on radiosensitivity of lung cancer cells

    International Nuclear Information System (INIS)

    Objective: To investigate the effect of human lung cancer-associated fibroblasts (CAF) on the radiosensitivity of lung cancer cells when CAF is placed in direct contact co-culture with lung cancer cells. Methods: Human lung CAF was obtained from fresh human lung adenocarcinoma tissue specimens by primary culture and subculture and was then identified by immunofluorescence staining. The CAF was placed in direct contact co-culture with lung cancer A549 and H1299 cells, and the effects of CAF on the radiosensitivity of A549 and H1299 cells were evaluated by colony-forming assay. Results: The human lung CAF obtained by adherent culture could stably grow and proliferate, and it had specific expression of α-smooth muscle actin, vimentin, and fibroblast activation protein,but without expression of cytokeratin-18. The plating efficiency (PE, %) of A549 cells at 0 Gy irradiation was (20.0 ± 3.9)% when cultured alone versus (32.3 ± 5.5)% when co-cultured with CAF (t=3.16, P<0.05), and the PE of H1299 cells at 0 Gy irradiation was (20.6 ± 3.1)% when cultured alone versus (35.2 ± 2.3)% when co-cultured with CAF (t=6.55, P<0.05). The cell survival rate at 2 Gy irradiation (SF2) of A549 cells was 0.727 ±0.061 when cultured alone versus 0.782 ± 0.089 when co-cultured with CAF (t=0.88, P>0.05), and the SF2 of H1299 cells was 0.692 ±0.065 when cultured alone versus 0.782 ± 0.037 when co-cultured with CAF (t=2.08, P>0.05). The protection enhancement ratios of human lung CAF for A549 cells and H1299 cells were 1.29 and 1.25, respectively. Conclusions: Human lung CAF reduces the radiosensitivity of lung cancer cells when placed in direct contact co-culture with them, and the radioprotective effect may be attributed to CAF promoting the proliferation of lung cancer cells. (authors)

  5. Nerve Growth Factor Modulate Proliferation of Cultured Rabbit Corneal Endothelial Cells and Epithelial Cells

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    In order to investigate the effect of nerve growth factor (NGF) on the proliferation of rabbit corneal endothelial cells and epithelial cells, the in vitro cultured rabbit corneal endothelial cells and epithelial cells were treated with different concentrations of NGF.MTT assay was used to examine the clonal growth and proliferation of the cells by determining the absorbency values at 570nm. The results showed that NGF with three concentrations ranging from 5 U/mL to 500 U/mL enhanced the proliferation of rabbit corneal endothelial cells in a concentration-dependent manner.50 U/mL and 500 U/mL NGF got more increase of proliferation than that of 5 U/mL NGF did.Meanwhile, 50 U/mL and 500 U/mL NGF could promote the proliferation of the rabbit corneal epithelial cells significantly in a concentration-dependent manner. However, 5 U/mL NGF did not enhance the proliferation of epithelial cells. It was suggested that exogenous NGF can stimulate the proliferation of both rabbit corneal endothelial and epithelial cells, but the extent of modulation is different.

  6. 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.

  7. RBC invasion and invasion-inhibition assays using free merozoites isolated after cold treatment of Babesia bovis in vitro culture.

    Science.gov (United States)

    Ishizaki, Takahiro; Sivakumar, Thillaiampalam; Hayashida, Kyoko; Tuvshintulga, Bumduuren; Igarashi, Ikuo; Yokoyama, Naoaki

    2016-07-01

    Babesia bovis is an apicomplexan hemoprotozoan that can invade bovine red blood cells (RBCs), where it multiplies asexually. RBC invasion assays using free viable merozoites are now routinely used to understand the invasion mechanism of B. bovis, and to evaluate the efficacy of chemicals and antibodies that potentially inhibit RBC invasion by the parasite. The application of high-voltage pulses (high-voltage electroporation), a commonly used method to isolate free merozoites from infected RBCs, reduces the viability of the merozoites. Recently, a cold treatment of B. bovis in vitro culture was found to induce an effective release of merozoites from the infected RBCs. In the present study, we incubated in vitro cultures of B. bovis in an ice bath to liberate merozoites from infected RBCs and then evaluated the isolated merozoites in RBC invasion and invasion-inhibitions assays. The viability of the purified merozoites (72.4%) was significantly higher than that of merozoites isolated with high-voltage electroporation (48.5%). The viable merozoites prepared with the cold treatment also invaded uninfected bovine RBCs at a higher rate (0.572%) than did merozoites prepared with high-voltage electroporation (0.251%). The invasion-blocking capacities of heparin, a polyclonal rabbit antibody directed against recombinant B. bovis rhoptry associated protein 1, and B. bovis-infected bovine serum were successfully demonstrated in an RBC invasion assay with the live merozoites prepared with the cold treatment, suggesting that the targets of these inhibitors were intact in the merozoites. These findings indicate that the cold treatment technique is a useful tool for the isolation of free, viable, invasion-competent B. bovis merozoites, which can be effectively used for RBC invasion and invasion-inhibition assays in Babesia research. PMID:26965399

  8. In vitro androgenetic cultures of Hyoscyamus niger L., H. albus L. and alkaloid content assay

    Directory of Open Access Journals (Sweden)

    Maria Wesołwska

    2014-02-01

    Full Text Available In vitro cultures of Hyoscyamus niger L. and H. albus L. anthers were initiated which resulted in obtaining androgenectic plants and callus cultures. The leaves of these pants and the callus cultures were subjected to analysis (TLC, GC for the presence of alkaloids, derivatives of tropane. In the studied material, alkaloids of different qualitative and quantitative composition from that of ground-grown plants were found.

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

    Institute of Scientific and Technical Information of China (English)

    李建中; 章竹君; 金伯泉; 田方

    1996-01-01

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

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

    International Nuclear Information System (INIS)

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

  11. Induction of vascular endothelial phenotype and cellular proliferation from human cord blood stem cells cultured in simulated microgravity

    Science.gov (United States)

    Chiu, Brian; Z-M Wan, Jim; Abley, Doris; Akabutu, John

    2005-05-01

    Recent studies have demonstrated that stem cells derived from adult hematopoietic tissues are capable of trans-differentiation into non-hematopoietic cells, and that the culture in microgravity ( μg) may modulate the proliferation and differentiation. We investigated the application of μg to human umbilical cord blood stem cells (CBSC) in the induction of vascular endothelial phenotype expression and cellular proliferation. CD34+ mononuclear cells were isolated from waste human umbilical cord blood samples and cultured in simulated μg for 14 days. The cells were seeded in rotary wall vessels (RWV) with or without microcarrier beads (MCB) and vascular endothelial growth factor was added during culture. Controls consisted of culture in 1 G. The cell cultures in RWV were examined by inverted microscopy. Cell counts, endothelial cell and leukocyte markers performed by flow-cytometry and FACS scan were assayed at days 1, 4, 7 and at the termination of the experiments. Culture in RWV revealed significantly increased cellular proliferation with three-dimensional (3D) tissue-like aggregates. At day 4, CD34+ cells cultured in RWV bioreactor without MCB developed vascular tubular assemblies and exhibited endothelial phenotypic markers. These data suggest that CD34+ human umbilical cord blood progenitors are capable of trans-differentiation into vascular endothelial cell phenotype and assemble into 3D tissue structures. Culture of CBSC in simulated μg may be potentially beneficial in the fields of stem cell biology and somatic cell therapy.

  12. 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.

  13. Neural stem cell isolation and culture from C57BL/6 mice

    Directory of Open Access Journals (Sweden)

    S Koirala

    2015-07-01

    Full Text Available INTRODUCTION A widely used in vitro culture, the neurosphere assay (NSA has provided a means to retrospectively identify neural progenitor cells as well as to determine both their selfrenewal capacity. Objective of study was to isolate and compare growth of the embryonic neuronal stem cell and adult neuronal stem cells in presence of Epidermal Growth Factor (EGF and Fibroblastic Growth Factor (FGF2. MATERIALS AND METHODS Embryonic neuronal stem cell were collected from cortical plate of dorsal telencephalon of fifteen C57BL/6 transgenic mice using stereoscopic microscope on 11th gestational day (GD. Adult mammalian neuronal stem cells taken from subventricular zone (SVZ of the lateral ventricles and subgranular layer of the dentate gyrus of the hippocampus were cultured. The growth for the neurosphere was then observed in interval of 24 and 72 hours. RESULT The adult stem cell culture showed few intact cells with high amount of debris and 9% heterogeneous sphere after 24 hours while only 20 % was observed at the end of 72 hours. Higher proliferation rate was observed in embryonic neurospheres than the adult stem cell culture. CONCLUSION Presence of EGF and basic FGF2 is essential for culture of neurospheres.DOI: http://dx.doi.org/10.3126/jcmsn.v10i2.12946 Journal of College of Medical Sciences-Nepal, 2014, Vol.10(2; 1-3

  14. Multiplex profiling of cellular invasion in 3D cell culture models.

    Directory of Open Access Journals (Sweden)

    Gerald Burgstaller

    Full Text Available To-date, most invasion or migration assays use a modified Boyden chamber-like design to assess migration as single-cell or scratch assays on coated or uncoated planar plastic surfaces. Here, we describe a 96-well microplate-based, high-content, three-dimensional cell culture assay capable of assessing invasion dynamics and molecular signatures thereof. On applying our invasion assay, we were able to demonstrate significant effects on the invasion capacity of fibroblast cell lines, as well as primary lung fibroblasts. Administration of epidermal growth factor resulted in a substantial increase of cellular invasion, thus making this technique suitable for high-throughput pharmacological screening of novel compounds regulating invasive and migratory pathways of primary cells. Our assay also correlates cellular invasiveness to molecular events. Thus, we argue of having developed a powerful and versatile toolbox for an extensive profiling of invasive cells in a 96-well format. This will have a major impact on research in disease areas like fibrosis, metastatic cancers, or chronic inflammatory states.

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

    Institute of Scientific and Technical Information of China (English)

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

    2005-01-01

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

  16. A new microperfusion system for the cultivation of tumor-cells invitro - approach to integrate pharmacokinetic parameters in screening assays for cytostatic drugs.

    Science.gov (United States)

    Gimmel, S; Kinawi, A; Maurer, H

    1993-01-01

    By a newly introduced microperfusion system absorption and elimination rates can be simulated in vitro. This article describes the optimization of culture conditions (medium composition, membrane filters, pumping rates, and stirring speeds) of tumor cell lines (L1210, KB) maintained in suspension in an ultrafiltration-flat chamber. Viability and colony-forming ability are measured. Our results indicate that tumor cells can be cultured under serum-free conditions over a five hour incubation period with only minimal decrease in colony-forming ability. Survival of cells is independent from the pumping rate in the tested range, but is dependent of the stirring speed. Each cell line requires its own stirring speed. Ultrafiltration membranes with minimal nonspecific adsorption properties proved to be the best in terms of cell adsorption and toxicity to retain cells in the chamber. This system might improve the tumor cell colony assay for cytostatic drug screening. PMID:21573513

  17. The PCC assay can be used to predict radiosensitivity in biopsy cultures irradiated with different types of radiation.

    Science.gov (United States)

    Suzuki, Masao; Tsuruoka, Chizuru; Nakano, Takashi; Ohno, Tatsuya; Furusawa, Yoshiya; Okayasu, Ryuichi

    2006-12-01

    The aim of this study was to identify potential biomarkers for radiosensitivity using the relationship between cell killing and the yield of excess chromatin fragments detected with the premature chromosome condensation (PCC) technique. This method was applied to primary cultured cells obtained from biopsies from patients. Six primary culture biopsies were obtained from 6 patients with carcinoma of the cervix before starting radiotherapy. The cultures were irradiated with two different LET carbon-ion beams (LET = 13 keV/microm, 77.1+/-2.8 keV/microm) and 200 kV X-rays. The carbon-ion beams were produced by Heavy Ion Medical Accelerator in Chiba (HIMAC). PCC was performed using the polyethylene glycol-mediated cell fusion technique. The yield of excess chromatin fragments were measured by counting the number of unrejoined chromatin fragments detected with the PCC technique after a 24-h post-irradiation incubation period. Obtained results indicated that cultures which were more sensitive to killing were also more susceptible to the induction of excess chromatin fragments. Furthermore there was a good correlation between cell killing and excess chromatin fragments among the 6 cell cultures examined. There is also evidence that the induction of excess chromatin fragments increased with increasing LET as well as cell-killing effect in the same cell culture. The data reported here support the idea that the yield of excess chromatin fragments detected with the PCC technique might be useful for predicting the radiosensitivity of cells contained in tumor tissue, and to predict responses to different radiation types. PMID:17089052

  18. Arginine and glutamine supplementation to culture media improves the performance of various channel catfish immune cells.

    Science.gov (United States)

    Pohlenz, Camilo; Buentello, Alejandro; Mwangi, Waithaka; Gatlin, Delbert M

    2012-05-01

    Specific components of both the innate and adaptive immune systems of channel catfish were evaluated after supplementation of culture media with arginine (ARG) and/or glutamine (GLN). Primary cell cultures of head-kidney macrophages (MØ) were used for phagocytic and bactericidal assays against Edwardsiella ictaluri. Additionally, proliferation assays were conducted with naïve peripheral blood lymphocytes (PBL) exposed to non-specific mitogens. To indirectly assess amino acid utilization of both MØ and PBL, amino acid levels, with emphasis on ARG and GLN, were evaluated in the basal medium before and after activation or mitogenic exposure. After bactericidal and proliferation assays, the sum of the media free amino acid pool significantly (P Glutamine levels in medium decreased by 38% and ARG by 18% during the bactericidal assay. Also, decreases of 52 and 46% from initial values were found after the proliferation assay for GLN and ARG, respectively. Macrophage phagocytosis and killing ability was significantly (P < 0.05) enhanced by ARG supplementation to culture media regardless of GLN supplementation. Proliferation of naïve T- and B-lymphocytes upon mitogenic exposure was significantly (P < 0.05) enhanced by supplementing ARG and GLN to the media, but limited synergistic effects were observed. These results suggest that in vitro, ARG and GLN are important substrates and immunomodulators of both innate and adaptive responses in fish leukocytes, and further highlights the potential use of ARG and GLN as immunonutrients in aquafeeds.

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

    OpenAIRE

    Hazen, K C; LeMelle, W G

    1990-01-01

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

  20. Human placenta-derived mesenchymal progenitor cells support culture expansion of long-term culture-initiating cells from cord blood CD34+ cells

    Institute of Scientific and Technical Information of China (English)

    YiZhanga; ChangdongLi; XiaoxiaJiang; ShuangxiZhang; YingWu; BingLiu; PeihsienTang; NingMao

    2005-01-01

    Objective. Allogeneic transplantation with umbilical cord blood (UCB) in adult recipients is limited mainly by a low CD34+ cell dose. To overcome this shortcoming, human placenta as a novel source of human mesenchymal progenitor cell (MPC) was incorporated in an attempt to expand CD34+ ceils from UCB in vitro.Materials and Methods. Human placenta MPC was isolated and characterized by morphologic,immunophenotypical, and functional analysis. UCB CD34+ cells were expanded by coculturewith placeutal MPC. Suitable aliquots of cells were used to monitor cell production, elonogenie activity, and tong-term culture-initiating culture (LTC-IC) output. Finally, the immunoregulatory effect of placental MPC was evaluated by T-cell proliferation assay.Results. In its undifferentiated state, placental MPC displayed fibroblastoid morphology; was CD73, CD105, CD29, CD44, HLA-ABC, and CD166 positive; produced fibronectin, laminin,and vimentin; but was negative for CD14, CD31, CD34, CD45, HLA-DR, and α-smooth muscle actin. Functionally, it could be induced into adipocytes, osteocytes, and chondrocytes.In vitro expansion of UCB hematopoietic cells, when cocultured with placental MPC in the presence of eytokines, was significantly enhanced: CD34+ cells by 14.89±2.32 fold; colonyforming cell (CFC) by 36.73±5.79 told; and LTC-IC by 7.43±2.66 fold. Moreover, placental MPC could suppress T-cell proliferation induced by cellular stimuli.Conclusion. These results strongly suggest that human placental MPC may be a suitable feeder layer for expansion of hematopoietic progenitors from UCB in vitro.

  1. Three-Dimensional Cell Culture: A Breakthrough in Vivo

    Directory of Open Access Journals (Sweden)

    Delphine Antoni

    2015-03-01

    Full Text Available Cell culture is an important tool for biological research. Two-dimensional cell culture has been used for some time now, but growing cells in flat layers on plastic surfaces does not accurately model the in vivo state. As compared to the two-dimensional case, the three-dimensional (3D cell culture allows biological cells to grow or interact with their surroundings in all three dimensions thanks to an artificial environment. Cells grown in a 3D model have proven to be more physiologically relevant and showed improvements in several studies of biological mechanisms like: cell number monitoring, viability, morphology, proliferation, differentiation, response to stimuli, migration and invasion of tumor cells into surrounding tissues, angiogenesis stimulation and immune system evasion, drug metabolism, gene expression and protein synthesis, general cell function and in vivo relevance. 3D culture models succeed thanks to technological advances, including materials science, cell biology and bioreactor design.

  2. Control of MRSA infection and colonisation in an intensive care unit by GeneOhm MRSA assay and culture methods

    Science.gov (United States)

    2009-01-01

    Background Methicillin-resistant Staphylococcus aureus (MRSA) is one of the major nosocomial pathogens. Due to the diffusion of MRSA strains in both hospital and community settings, prevention and control strategies are receiving increased attention. Approximately 25% to 30% of the population is colonised with S. aureus and 0.2% to 7% with MRSA. The BD GeneOhm MRSA real-time PCR assay offers quicker identification of MRSA-colonised patients than do culture methods. Methods Ninety-five patients admitted to the Intensive Care Unit of IRCCS Policlinico San Matteo of Pavia (Italy) for a period > 24 h were screened for MRSA colonisation with both the culture method and the GeneOhm assay. Results Of the 246 nasal swabs collected from 95 patients, 36 samples were found to be positive by both methods (true-positive). 30% of colonised patients had developed the MRSA infection. Conclusion Our results show that the GeneOhm MRSA assay is a valuable diagnostic tool for detecting MRSA quickly in nasal swabs. This study confirms that colonisation represents a high risk factor for MRSA infection, and that good MRSA surveillance in an Intensive Care Unit is therefore an excellent way to prevent MRSA infection. PMID:19703294

  3. Control of MRSA infection and colonisation in an intensive care unit by GeneOhm MRSA assay and culture methods

    Directory of Open Access Journals (Sweden)

    Valle Claudia

    2009-08-01

    Full Text Available Abstract Background Methicillin-resistant Staphylococcus aureus (MRSA is one of the major nosocomial pathogens. Due to the diffusion of MRSA strains in both hospital and community settings, prevention and control strategies are receiving increased attention. Approximately 25% to 30% of the population is colonised with S. aureus and 0.2% to 7% with MRSA. The BD GeneOhm MRSA real-time PCR assay offers quicker identification of MRSA-colonised patients than do culture methods. Methods Ninety-five patients admitted to the Intensive Care Unit of IRCCS Policlinico San Matteo of Pavia (Italy for a period > 24 h were screened for MRSA colonisation with both the culture method and the GeneOhm assay. Results Of the 246 nasal swabs collected from 95 patients, 36 samples were found to be positive by both methods (true-positive. 30% of colonised patients had developed the MRSA infection. Conclusion Our results show that the GeneOhm MRSA assay is a valuable diagnostic tool for detecting MRSA quickly in nasal swabs. This study confirms that colonisation represents a high risk factor for MRSA infection, and that good MRSA surveillance in an Intensive Care Unit is therefore an excellent way to prevent MRSA infection.

  4. 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

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

    Science.gov (United States)

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

    2014-01-01

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

  6. Electrospinning of microbial polyester for cell culture

    Energy Technology Data Exchange (ETDEWEB)

    Kwon, Oh Hyeong [Department of Polymer Science and Engineering, Kumoh National Institute of Technology, 1 Yangho-dong, Gumi, Gyeongbuk 730-701 (Korea, Republic of); Lee, Ik Sang [Department of Polymer Science and Engineering, Kumoh National Institute of Technology, 1 Yangho-dong, Gumi, Gyeongbuk 730-701 (Korea, Republic of); Ko, Young-Gwang [Department of Polymer Science and Engineering, Kumoh National Institute of Technology, 1 Yangho-dong, Gumi, Gyeongbuk 730-701 (Korea, Republic of); Meng, Wan [Department of Polymer Science, Kyungpook National University, 1370 Sankyuk-dong, Buk-gu, Daegu 702-701 (Korea, Republic of); Jung, Kyung-Hye [Department of Polymer Science, Kyungpook National University, 1370 Sankyuk-dong, Buk-gu, Daegu 702-701 (Korea, Republic of); Kang, Inn-Kyu [Department of Polymer Science, Kyungpook National University, 1370 Sankyuk-dong, Buk-gu, Daegu 702-701 (Korea, Republic of); Ito, Yoshihiro [Kanagawa Academy of Science and Technology, KSP East 309, Sakado 3-2-1, Takatsu-ku, Kawasaki 213-0012 (Japan)

    2007-03-01

    Biodegradable and biocompatible poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a copolymer of microbial polyester, was fabricated as a nanofibrous mat by electrospinning. The specific surface area and the porosity of electrospun PHBV nanofibrous mat were determined. When the mechanical properties of flat film and electrospun PHBV nanofibrous mats were investigated, both the tensile modulus and strength of electrospun PHBV were less than those of cast PHBV film. However, the elongation ratio of nanofiber mat was higher than that of the cast film. The structure of electrospun nanofibers using PHBV-trifluoroethanol solutions depended on the solution concentrations. When x-ray diffraction patterns of bulk PHBV before and after electrospinning were compared, the crystallinity of PHBV was not significantly affected by the electrospinning process. Chondrocytes adhered and grew on the electrospun PHBV nanofibrous mat better than on the cast PHBV film. Therefore, the electrospun PHBV was considered to be suitable for cell culture.

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

    Science.gov (United States)

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

    2015-11-01

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

  8. Characterisation of the membrane transport of pilocarpine in cell suspension cultures of Pilocarpus microphyllus.

    Science.gov (United States)

    Andreazza, Nathalia Luiza; Abreu, Ilka Nacif; Sawaya, Alexandra Christine Helena Frankland; Mazzafera, Paulo

    2015-03-01

    Pilocarpine is an alkaloid obtained from the leaves of Pilocarpus genus, with important pharmaceutical applications. Previous reports have investigated the production of pilocarpine by Pilocarpus microphyllus cell cultures and tried to establish the alkaloid biosynthetic route. However, the site of pilocarpine accumulation inside of the cell and its exchange to the medium culture is still unknown. Therefore, the aim of this study was to determine the intracellular accumulation of pilocarpine and characterise its transport across membranes in cell suspension cultures of P. microphyllus. Histochemical analysis and toxicity assays indicated that pilocarpine is most likely stored in the vacuoles probably to avoid cell toxicity. Assays with exogenous pilocarpine supplementation to the culture medium showed that the alkaloid is promptly uptaken but it is rapidly metabolised. Treatment with specific ABC protein transporter inhibitors and substances that disturb the activity of secondary active transporters suppressed pilocarpine uptake and release suggesting that both proteins may participate in the traffic of pilocarpine to inside and outside of the cells. As bafilomicin A1, a specific V-type ATPase inhibitor, had little effect and NH4Cl (induces membrane proton gradient dissipation) had moderate effect, while cyclosporin A and nifedipine (ABC proteins inhibitors) strongly inhibited the transport of pilocarpine, it is believed that ABC proteins play a major role in the alkaloid transport across membranes but it is not the exclusive one. Kinetic studies supported these results. PMID:25474486

  9. Organ culture-cell culture system for studying multistage carcinogenesis in respiratory epithelium. [Mice

    Energy Technology Data Exchange (ETDEWEB)

    Steele, Vernon E.; Marchok, Ann C.; Nettesheim, Paul

    1977-01-01

    An organ culture-cell culture system was used to demonstrate carcinogen dose-dependent transformation of tracheal epithelial cells in vitro. Tracheal explants were exposed to MNNG (N-methyl-N/sup 1/-nitro-N-nitrosoguanidine) in organ culture. Outgrowths from these explants provided epithelial cell cultures. The numbers of long term epithelial cell cultures and cell lines that were established per explant increased as MNNG exposure concentration increased. At the present time, more cell lines derived from explants exposed to the highest MNNG concentration have produced palpable tumors than cell lines derived from explants exposed to lower MNNG concentrations. No cell lines were established from primaries derived from control explants. TPA (12-0-tetradecanoyl-phorbol-13-acetate), stimulates DNA synthesis in tracheal epithelium in organ culture in a manner simular to that described for mouse skin. Short exposures to TPA not only stimulated DNA synthesis earlier, but the stimulation was greater than that obtained with continuous exposure. At the present time, exposure of tracheal organ cultures to MNNG followed by TPA has resulted in an enhanced production of morphologically altered cells in primary epithelial cell cultures, than exposure to either agent alone.

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

    OpenAIRE

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

    2012-01-01

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

  11. 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.

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

    Science.gov (United States)

    Hunter, S A; Cochran, J R

    2016-01-01

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

  13. A Functional Assay for Putative Mouse and Human Definitive Endoderm using Chick Whole-Embryo Cultures

    DEFF Research Database (Denmark)

    Johannesson, Martina; Semb, Tor Henrik; Serup, Palle;

    2012-01-01

    Introduction: Embryonic stem cells (ESCs) represent a prospective cell source for treating degenerative diseases such as diabetes. Several studies have addressed the generation of definitive endoderm (DE) from this cell source by attempting to recapitulate the signaling events occurring during...

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

    Science.gov (United States)

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

    2016-01-01

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

  15. HAIR CELL-LIKE CELL GENERATION INDUCED BY NATURE CULTURE OF ADULT RAT AUDITORY EPITHELIUM

    Institute of Scientific and Technical Information of China (English)

    Liu Hui; Zhu Hongliang; Li Shengli; Yao Xiaobao; Wang Xiaoxia

    2006-01-01

    Objective To establish adult rat auditory epithelial cell culture and try to find precursor cells of auditory hair cells in vitro. Methods With refinement of culture media and techniques, cochlear sensory epithelial cells of adult rat were cultured. Immunocytochemistry and Bromodeoxyuridine (BrdU)labeling were used to detect properties and mitotic status of cultured cells. Results The cultured auditory epithelial cells showed a large, flat epithelial morphotype and expressed F-actin and cytokeratin, a subset of cells generated from auditory epithelium were labeled by calretinin, a specific marker of early hair cell. Conclusion Adult rat auditory epithelium can be induced to generate hair cell-like cells by nature culture, this phenomenon suggests that progenitor cells may exist in rat cochlea and they may give birth to new hair cells. Whether these progenitor cells are tissue specific stem cells is still need more study.

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

    OpenAIRE

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

    2003-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Pitipreya Suauam

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

  18. Particle Trajectories in Rotating Wall Cell Culture Devices

    Science.gov (United States)

    Ramachandran N.; Downey, J. P.

    1999-01-01

    Cell cultures are extremely important to the medical community since such cultures provide an opportunity to perform research on human tissue without the concerns inherent in experiments on individual humans. Development of cells in cultures has been found to be greatly influenced by the conditions of the culture. Much work has focused on the effect of the motions of cells in the culture relative to the solution. Recently rotating wall vessels have been used with success in achieving improved cellular cultures. Speculation and limited research have focused on the low shear environment and the ability of rotating vessels to keep cells suspended in solution rather than floating or sedimenting as the primary reasons for the improved cellular cultures using these devices. It is widely believed that the cultures obtained using a rotating wall vessel simulates to some degree the effect of microgravity on cultures. It has also been speculated that the microgravity environment may provide the ideal acceleration environment for culturing of cellular tissues due to the nearly negligible levels of sedimentation and shear possible. This work predicts particle trajectories of cells in rotating wall vessels of cylindrical and annular design consistent with the estimated properties of typical cellular cultures. Estimates of the shear encountered by cells in solution and the interactions with walls are studied. Comparisons of potential experiments in ground and microgravity environments are performed.

  19. Novel migrating mouse neural crest cell assay system utilizing P0-Cre/EGFP fluorescent time-lapse imaging

    Directory of Open Access Journals (Sweden)

    Kawakami Minoru

    2011-11-01

    Full Text Available Abstract Background Neural crest cells (NCCs are embryonic, multipotent stem cells. Their long-range and precision-guided migration is one of their most striking characteristics. We previously reported that P0-Cre/CAG-CAT-lacZ double-transgenic mice showed significant lacZ expression in tissues derived from NCCs. Results In this study, by embedding a P0-Cre/CAG-CAT-EGFP embryo at E9.5 in collagen gel inside a culture glass slide, we were able to keep the embryo developing ex vivo for more than 24 hours; this development was with enough NCC fluorescent signal intensity to enable single-cell resolution analysis, with the accompanying NCC migration potential intact and with the appropriate NCC response to the extracellular signal maintained. By implantation of beads with absorbed platelet-derived growth factor-AA (PDGF-AA, we demonstrated that PDGF-AA acts as an NCC-attractant in embryos. We also performed assays with NCCs isolated from P0-Cre/CAG-CAT-EGFP embryos on culture plates. The neuromediator 5-hydroxytryptamine (5-HT has been known to regulate NCC migration. We newly demonstrated that dopamine, in addition to 5-HT, stimulated NCC migration in vitro. Two NCC populations, with different axial levels of origins, showed unique distribution patterns regarding migration velocity and different dose-response patterns to both 5-HT and dopamine. Conclusions Although avian species predominated over the other species in the NCC study, our novel system should enable us to use mice to assay many different aspects of NCCs in embryos or on culture plates, such as migration, division, differentiation, and apoptosis.

  20. Nanocrystalline diamond: In vitro biocompatibility assessment by MG63 and human bone marrow cells cultures.

    Science.gov (United States)

    Amaral, M; Dias, A G; Gomes, P S; Lopes, M A; Silva, R F; Santos, J D; Fernandes, M H

    2008-10-01

    Nanocrystalline diamond (NCD) has a great potential for prosthetic implants coating. Nevertheless, its biocompatibility still has to be better understood. To do so, we employed several materials characterization techniques (SEM, AFM, micro-Raman spectroscopy) and cell culture assays using MG63 osteoblast-like and human bone marrow cells. Biochemical routines (MTT assays, Lowry's method, ALP activity) supported by SEM and confocal microscopy characterization were carried out. We used silicon nitride (Si3N4) substrates for NCD coatings based on a previous demonstration of the superior adhesion and tribological performance of these NCD coated ceramics. Results demonstrate an improved human osteoblast proliferation and the stimulation of differentiated markers, like ALP activity and matrix mineralization, compared with standard polystyrene tissue culture plates. The nanometric featuring of NCD, associated to its chemical affinity are key points for bone regeneration purposes. PMID:18085649

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

    Science.gov (United States)

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

    2016-01-01

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

  2. Usability and Applicability of Microfluidic Cell Culture Systems

    DEFF Research Database (Denmark)

    Hemmingsen, Mette

    , these devices still lack general implementation into biological research laboratories. In this project, the usability and applicability of microfluidic cell culture systems have been investigated. The tested systems display good properties regarding optics and compatibility with standard laboratory equipment......Microfluidic cell culture has been a research area with great attention the last decade due to its potential to mimic the in vivo cellular environment more closely compared to what is possible by conventional cell culture methods. Many exciting and complex devices have been presented providing...... possibilities for, for example, precise control of the chemical environment, 3D cultures, controlled co-culture of different cell types or automated, individual control of up to 96 cell culture chambers in one integrated system. Despite the great new opportunities to perform novel experimental designs...

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

    Directory of Open Access Journals (Sweden)

    Maria Charzyńska

    2014-02-01

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

  4. The IRIDICA BAC BSI Assay: Rapid, Sensitive and Culture-Independent Identification of Bacteria and Candida in Blood.

    Directory of Open Access Journals (Sweden)

    David Metzgar

    Full Text Available Bloodstream infection (BSI and sepsis are rising in incidence throughout the developed world. The spread of multi-drug resistant organisms presents increasing challenges to treatment. Surviving BSI is dependent on rapid and accurate identification of causal organisms, and timely application of appropriate antibiotics. Current culture-based methods used to detect and identify agents of BSI are often too slow to impact early therapy and may fail to detect relevant organisms in many positive cases. Existing methods for direct molecular detection of microbial DNA in blood are limited in either sensitivity (likely the result of small sample volumes or in breadth of coverage, often because the PCR primers and probes used target only a few specific pathogens. There is a clear unmet need for a sensitive molecular assay capable of identifying the diverse bacteria and yeast associated with BSI directly from uncultured whole blood samples. We have developed a method of extracting DNA from larger volumes of whole blood (5 ml per sample, amplifying multiple widely conserved bacterial and fungal genes using a mismatch- and background-tolerant PCR chemistry, and identifying hundreds of diverse organisms from the amplified fragments on the basis of species-specific genetic signatures using electrospray ionization mass spectrometry (PCR/ESI-MS. We describe the analytical characteristics of the IRIDICA BAC BSI Assay and compare its pre-clinical performance to current standard-of-care methods in a collection of prospectively collected blood specimens from patients with symptoms of sepsis. The assay generated matching results in 80% of culture-positive cases (86% when common contaminants were excluded from the analysis, and twice the total number of positive detections. The described method is capable of providing organism identifications directly from uncultured blood in less than 8 hours.The IRIDICA BAC BSI Assay is not available in the United States.

  5. The IRIDICA BAC BSI Assay: Rapid, Sensitive and Culture-Independent Identification of Bacteria and Candida in Blood

    Science.gov (United States)

    Rothman, Richard E.; Peterson, Stephen; Carroll, Karen C.; Zhang, Sean X.; Avornu, Gideon D.; Rounds, Megan A.; Carolan, Heather E.; Toleno, Donna M.; Moore, David; Hall, Thomas A.; Massire, Christian; Richmond, Gregory S.; Gutierrez, Jose R.; Sampath, Rangarajan; Ecker, David J.; Blyn, Lawrence B.

    2016-01-01

    Bloodstream infection (BSI) and sepsis are rising in incidence throughout the developed world. The spread of multi-drug resistant organisms presents increasing challenges to treatment. Surviving BSI is dependent on rapid and accurate identification of causal organisms, and timely application of appropriate antibiotics. Current culture-based methods used to detect and identify agents of BSI are often too slow to impact early therapy and may fail to detect relevant organisms in many positive cases. Existing methods for direct molecular detection of microbial DNA in blood are limited in either sensitivity (likely the result of small sample volumes) or in breadth of coverage, often because the PCR primers and probes used target only a few specific pathogens. There is a clear unmet need for a sensitive molecular assay capable of identifying the diverse bacteria and yeast associated with BSI directly from uncultured whole blood samples. We have developed a method of extracting DNA from larger volumes of whole blood (5 ml per sample), amplifying multiple widely conserved bacterial and fungal genes using a mismatch- and background-tolerant PCR chemistry, and identifying hundreds of diverse organisms from the amplified fragments on the basis of species-specific genetic signatures using electrospray ionization mass spectrometry (PCR/ESI-MS). We describe the analytical characteristics of the IRIDICA BAC BSI Assay and compare its pre-clinical performance to current standard-of-care methods in a collection of prospectively collected blood specimens from patients with symptoms of sepsis. The assay generated matching results in 80% of culture-positive cases (86% when common contaminants were excluded from the analysis), and twice the total number of positive detections. The described method is capable of providing organism identifications directly from uncultured blood in less than 8 hours. Disclaimer: The IRIDICA BAC BSI Assay is not available in the United States. PMID:27384540

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

    Institute of Scientific and Technical Information of China (English)

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

    2003-01-01

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

  7. A method for isolating and culturing placental cells from failed early equine pregnancies.

    Science.gov (United States)

    Rose, B V; Cabrera-Sharp, V; Firth, M J; Barrelet, F E; Bate, S; Cameron, I J; Crabtree, J R; Crowhurst, J; McGladdery, A J; Neal, H; Pynn, J; Pynn, O D; Smith, C; Wise, Z; Verheyen, K L P; Wathes, D C; de Mestre, A M

    2016-02-01

    Early pregnancy loss occurs in 6-10% of equine pregnancies making it the main cause of reproductive wastage. Despite this, reasons for the losses are known in only 16% of cases. Lack of viable conceptus material has inhibited investigations of many potential genetic and pathological causes. We present a method for isolating and culturing placental cells from failed early equine pregnancies. Trophoblast cells from 18/30 (60%) failed equine pregnancies of gestational ages 14-65 days were successfully cultured in three different media, with the greatest growth achieved for cells cultured in AmnioChrome™ Plus. Genomic DNA of a suitable quality for molecular assays was also isolated from 29/30 of these cases. This method will enable future investigations determining pathologies causing EPL. PMID:26907389

  8. Triple co-culture cell model as an in vitro model for oral particulate vaccine systems

    DEFF Research Database (Denmark)

    Nielsen, Line Hagner; De Rossi, C.; Lehr, C-M.;

    the immunostimulatory ability of particulate vaccine formulations designed for oral delivery. Levels of cytokine production in response to vaccine administration were measured following particulate vaccine administration, as an indication of dendritic cell and macrophage activation. Precursors of cubosomes containing...... with particle formulations. This was not the case when incubating with ovalbumin solution or blank. The ELISA screening assay showed production of a wide range of cytokines following culture incubation with cubosomes (with and without ovalbumin) and LPS solutions, indicative of a stimulatory effect......; this was not observed with ovalbumin and blank solution. An example of the results is shown in Figure 2 for IL-17A. An established co-culture of Caco-2, THP-1 and MUTZ-3 cells showed promise as an in vitro model for testing of oral vaccine formulations. Mobility of co-culture immune cells as well as cytokine production...

  9. Culturing and applications of rotating wall vessel bioreactor derived 3D epithelial cell models.

    Science.gov (United States)

    Radtke, Andrea L; Herbst-Kralovetz, Melissa M

    2012-04-03

    . The progression from a monolayer of epithelial cells to a fully differentiated 3-D aggregate varies based on cell type(1, 7-13). Periodic sampling from the bioreactor allows for monitoring of epithelial aggregate formation, cellular differentiation markers and viability (Figure 1D). Once cellular differentiation and aggregate formation is established, the cells are harvested from the bioreactor, and similar assays performed on 2-D cells can be applied to the 3-D aggregates with a few considerations (Figure 1E-G). In this work, we describe detailed steps of how to culture 3-D epithelial cell aggregates in the RWV bioreactor system and a variety of potential assays and analyses that can be executed with the 3-D aggregates. These analyses include, but are not limited to, structural/morphological analysis (confocal, scanning and transmission electron microscopy), cytokine/chemokine secretion and cell signaling (cytometric bead array and Western blot analysis), gene expression analysis (real-time PCR), toxicological/drug analysis and host-pathogen interactions. The utilization of these assays set the foundation for more in-depth and expansive studies such as metabolomics, transcriptomics, proteomics and other array-based applications. Our goal is to present a non-conventional means of culturing human epithelial cells to produce organotypic 3-D models that recapitulate the human in vivo tissue, in a facile and robust system to be used by researchers with diverse scientific interests.

  10. Bead array direct rRNA capture assay (rCapA for amplification free speciation of Mycobacterium cultures.

    Directory of Open Access Journals (Sweden)

    Hans de Ronde

    Full Text Available Mycobacterium cultures, from patients suspected of tuberculosis or nontuberculous mycobacteria (NTM infection, need to be identified. It is most critical to identify cultures belonging to the Mycobacterium tuberculosis complex, but also important to recognize clinically irrelevant or important NTM to allow appropriate patient management. Identification of M. tuberculosis can be achieved by a simple and cheap lateral flow assay, but identification of other Mycobacterium spp. generally requires more complex molecular methods. Here we demonstrate that a paramagnetic liquid bead array method can be used to capture mycobacterial rRNA in crude lysates of positive cultures and use a robust reader to identify the species in a direct and sensitive manner. We developed an array composed of paramagnetic beads coupled to oligonucleotides to capture 16 rRNA from eight specific Mycobacterium species and a single secondary biotinilated reporter probe to allow the captured rRNA to be detected. A ninth less specific bead and its associated reporter probe, designed to capture 23S rRNA from mycobacteria and related genera, is included as an internal control to confirm the presence of bacterial rRNA from a GC rich Gram variable genera. Using this rRNA capture assay (rCapA with the array developed we were already able to confirm the presence of members of the M. tuberculosis complex and to discriminate a range of NTM species. This approach is not based on DNA amplification and therefore does not require precautions to avoid amplicon contamination. Moreover, the new generation of stable and cost effective liquid bead readers provides the necessary multiplexing potential to develop a robust and highly discriminatory assay.

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

    Directory of Open Access Journals (Sweden)

    Lauren Forbes

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

  12. Ultrastructural study of long-term canine distemper virus infection in tissue culture cells.

    OpenAIRE

    Narang, H K

    1982-01-01

    The morphogenesis of canine distemper virus was studied in Vero cell cultures for 43 days post-inoculation. Active replication of the virus was observed by electron microscopy and assay from 12 h after inoculation on, and peak production was observed on days 5, 14, and 22. From day 28 on, constant but smaller amounts of infectious virus were detected. Two ultrastructural types of intracytoplasmic nucleoprotein filaments were observed; although they first appeared at different times, their sub...

  13. Evaluation of peste des petits ruminants cell culture vaccine in sheep and goats in Pakistan

    Directory of Open Access Journals (Sweden)

    Muhammad Asim

    2010-09-01

    Full Text Available The authors study the antibody response of a locally prepared live-attenuated peste des petits ruminants (PPR cell culture vaccine in sheep and goats. Antibodies were measured using the competitive enzyme-linked immuno-sorbent assay. The vaccine was found to be safe and produced high serological titres within 21 days post vaccination. The serological titres remained high for one year post vaccination.

  14. THE ALKALOID CYTISINE IN THE CELL CULTURE

    Directory of Open Access Journals (Sweden)

    Gazaliev A.M.

    2012-08-01

    Full Text Available Alkaloids are vegetative establishments of complex and original structure with nitrous heterocycles in the basis. For a long time they drew researchers’ attention because of their unique and specific physiological effect on alive organisms. Not all the representatives of the globe’s flora contain these unique substances. Alkaloid cytisine is to be found mainly in the plants of the fabaceous family - Fabaceae. For the cytisine production the seeds of Thermopsis lanceolata R.Br (T. lanceolata R.Br and Cytisus laburnum (C. laburnum are used as a raw material. The object of the research is T. lanceolata cell culture. Sterile sprouts are used at the first stage of the experiment. Callus genesis is accompanied with dedifferentiation. It leads to the cellular organization simplification. Based on an important property of a plant cell, such as totipotency, there appears the formation of the “de novo” biosynthetic device. The cultivation algorithm consists of two basic stages: (i the cultivation conditions optimization of callus with a high level of the primary metabolites biosynthesis (Aspartat – lysine; (ii the research of cultivation chemical and physical factors influence on the secondary metabolite (cytisine biosynthesis and accumulation. During the cultivation the Murashige and Skoog classical recipe of nutrient medium will be used. Optimization of the cultivation conditions will concern the phytohormones, macro- and micronutrients content, as the purpose of optimization is the production of the determined high-level competence embriogenical callus. The main problem is genetic heterogeneity of a cellular population and instability of morpho-physiological processes. The correct management of higher plants cells population is possible at the synchronization of a cellular cycle phases. The references analysis has shown that it is almost impossible to synchronize cellular cycles in the culture of plant tissue. The application of chemical

  15. The potential value of the neutral comet assay and γH2AX foci assay in assessing the radiosensitivity of carbon beam in human tumor cell lines

    International Nuclear Information System (INIS)

    Carbon ions (12C6+) are high linear energy transfer (LET) radiation characterized by higher relative biological effectiveness than low LET radiation. The assessment of tumour radiosensitivity would be particularly useful in optimizing the radiation dose during radiotherapy. The aim of the current study was to evaluate the potential value of the neutral comet assay and γH2AX foci assay in assessing 12C6+ radiosensitivity of tumour cells. The doses of 12C6+ and X-rays used in the present study were 2 and 4 Gy. The survival fraction, DNA double-strand breaks (DSB) and repair kinetics of DSB were assayed with clonogenic survival, neutral comet assay and γH2AX foci assay in human cervical carcinoma HeLa cells, hepatoma HepG2 cells, and mucoepidermoid carcinoma MEC-1 cells at the time points of 0.5, 4, 16 and 24 h after 12C6+ and X-rays irradiation. The survival fraction for 12C6+ irradiation was much more inhibited than for X-rays (p < 0.05) in all three tumour cell lines tested. Substantial amounts of residual damage, assessed by the neutral comet assay, were present after irradiation (p < 0.05). The highest residual damage was observed at 0.5 or 4 h, both for 12C6+ and X-ray irradiation. However, the residual damage in HeLa and MEC-1 cells was higher for 12C6+ than X-rays (p < 0.05). The strongest induction of γH2AX foci was observed after 30 min, for all three tumour cell lines (p < 0.01). The franction of γH2AX foci persisted for at least 24 h after 12C6+ irradiation; in HeLa cells and MEC-1 was higher than after X-ray irradiation (p < 0.05). The correlation coefficients between the clonogenic survival, neutral comet assay and γH2AX foci assay were not statistically significant, except for some tumour cells at individual irradiation doses and types. Our study demonstrated that the neutral comet assay and γ-H2AX foci assay could be used to assess the radiosensitivity of 12C6+ in human tumour cells

  16. Isolation and culture of larval cells from C. elegans.

    Directory of Open Access Journals (Sweden)

    Sihui Zhang

    Full Text Available Cell culture is an essential tool to study cell function. In C. elegans the ability to isolate and culture cells has been limited to embryonically derived cells. However, cells or blastomeres isolated from mixed stage embryos terminally differentiate within 24 hours of culture, thus precluding post-embryonic stage cell culture. We have developed an efficient and technically simple method for large-scale isolation and primary culture of larval-stage cells. We have optimized the treatment to maximize cell number and minimize cell death for each of the four larval stages. We obtained up to 7.8×10(4 cells per microliter of packed larvae, and up to 97% of adherent cells isolated by this method were viable for at least 16 hours. Cultured larval cells showed stage-specific increases in both cell size and multinuclearity and expressed lineage- and cell type-specific reporters. The majority (81% of larval cells isolated by our method were muscle cells that exhibited stage-specific phenotypes. L1 muscle cells developed 1 to 2 wide cytoplasmic processes, while L4 muscle cells developed 4 to 14 processes of various thicknesses. L4 muscle cells developed bands of myosin heavy chain A thick filaments at the cell center and spontaneously contracted ex vivo. Neurons constituted less than 10% of the isolated cells and the majority of neurons developed one or more long, microtubule-rich protrusions that terminated in actin-rich growth cones. In addition to cells such as muscle and neuron that are high abundance in vivo, we were also able to isolate M-lineage cells that constitute less than 0.2% of cells in vivo. Our novel method of cell isolation extends C. elegans cell culture to larval developmental stages, and allows use of the wealth of cell culture tools, such as cell sorting, electrophysiology, co-culture, and high-resolution imaging of subcellular dynamics, in investigation of post-embryonic development and physiology.

  17. Hemolymph analysis and evaluation of newly formulated media for culture of shrimp cells (Penaeus stylirostris).

    Science.gov (United States)

    Shimizu, C; Shike, H; Klimpel, K R; Burns, J C

    2001-06-01

    Creation of a shrimp cell line has been an elusive goal. This failure may be due to the composition of the cell culture medium, which may be inadequate to support primary cultured cells. Shrimp hemolymph should contain the nutritional components needed to support cell growth and division. We report here the comprehensive biochemical analysis of hemolymph from the blue shrimp, Penaeus stylirostris (Litopenaeus stylirostris) (see Holthuis, L. B. Shrimps and prawns of the world, in: FAO species catalog. Vol. 1. Rome: Food and Agriculture Organization of the United Nations; 1980), for free amino acids (FAAs), carbohydrates, electrolytes, metals, pH, and osmolality. Levels of hemolymph components were compared to 2xL-15 with 20% fetal bovine serum, a commonly used culture medium for crustacean cells. The FAAs, taurine and proline, and the metals, strontium and zinc, were significantly higher in hemolymph than in the 2 x L-15 medium. In contrast, other FAAs were up to 50 times higher in the 2 x L-15 medium than in the hemolymph. To mimic more closely the hemolymph composition, we created two new media based on either the 0.2 x L-15 or the M199 medium. We compared the microscopic appearance of cells cultured in these media and evaluated deoxyribonucleic acid (DNA) and protein synthesis by 3H-thymidine uptake and 35S-methionine uptake assays. The ovary cells of P. stylirostris cultured in either of the new media formed monolayers, while the cells cultured in 2 x L-15 medium did not. Despite these differences, there was no evidence of sustained DNA or protein synthesis with any of the media. Future studies to establish a shrimp cell line should focus on analysis of the cell cycle and on overcoming the molecular blocks to cell division. PMID:11515962

  18. Single cell dual adherent-suspension co-culture micro-environment for studying tumor-stromal interactions with functionally selected cancer stem-like cells.

    Science.gov (United States)

    Chen, Yu-Chih; Zhang, Zhixiong; Fouladdel, Shamileh; Deol, Yadwinder; Ingram, Patrick N; McDermott, Sean P; Azizi, Ebrahim; Wicha, Max S; Yoon, Euisik

    2016-08-01

    Considerable evidence suggests that cancer stem-like cells (CSCs) are critical in tumor pathogenesis, but their rarity and transience has led to much controversy about their exact nature. Although CSCs can be functionally identified using dish-based tumorsphere assays, it is difficult to handle and monitor single cells in dish-based approaches; single cell-based microfluidic approaches offer better control and reliable single cell derived sphere formation. However, like normal stem cells, CSCs are heavily regulated by their microenvironment, requiring tumor-stromal interactions for tumorigenic and proliferative behaviors. To enable single cell derived tumorsphere formation within a stromal microenvironment, we present a dual adherent/suspension co-culture device, which combines a suspension environment for single-cell tumorsphere assays and an adherent environment for co-culturing stromal cells in close proximity by selectively patterning polyHEMA in indented microwells. By minimizing dead volume and improving cell capture efficiency, the presented platform allows for the use of small numbers of cells (concept, we co-cultured single T47D (breast cancer) cells and primary cancer associated fibroblasts (CAF) on-chip for 14 days to monitor sphere formation and growth. Compared to mono-culture, co-cultured T47D have higher tumorigenic potential (sphere formation rate) and proliferation rates (larger sphere size). Furthermore, 96-multiplexed single-cell transcriptome analyses were performed to compare the gene expression of co-cultured and mono-cultured T47D cells. Phenotypic changes observed in co-culture correlated with expression changes in genes associated with proliferation, apoptotic suppression, tumorigenicity and even epithelial-to-mesechymal transition. Combining the presented platform with single cell transcriptome analysis, we successfully identified functional CSCs and investigated the phenotypic and transcriptome effects induced by tumor

  19. Comparison of the Binax NOW Flu A Enzyme Immunochromatographic Assay and R-Mix Shell Vial Culture for the 2003-2004 Influenza Season

    Science.gov (United States)

    Fader, Robert C.

    2005-01-01

    The Binax NOW Flu A enzyme immunochromatographic assay was compared to viral culture with R-Mix shell vials for 455 nasal-wash or nasal-aspirate specimens. The overall sensitivity, specificity, positive predictive value, and negative predictive value of the assay were 64.9%, 98.4%, 89.3%, and 93.2%, respectively. However, the assay sensitivity decreased significantly with increasing patient age. PMID:16333112

  20. Using Tissue Culture To Investigate Plant Cell Differentiation and Dedifferentiation.

    Science.gov (United States)

    Bozzone, Donna M.

    1997-01-01

    Describes an experimental project that uses plant tissue culture techniques to examine cell differentiation in the carrot. Allows students to gain experience in some important techniques and to explore fundamental questions about cell differentiation. (DDR)

  1. Systems Biology for Organotypic Cell Cultures

    Energy Technology Data Exchange (ETDEWEB)

    Grego, Sonia [RTI International, Research Triangle Park, NC (United States); Dougherty, Edward R. [Texas A & M Univ., College Station, TX (United States); Alexander, Francis J. [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Auerbach, Scott S. [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States); Berridge, Brian R. [GlaxoSmithKline, Research Triangle Park, NC (United States); Bittner, Michael L. [Translational Genomics Research Inst., Phoenix, AZ (United States); Casey, Warren [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States); Cooley, Philip C. [RTI International, Research Triangle Park, NC (United States); Dash, Ajit [HemoShear Therapeutics, Charlottesville, VA (United States); Ferguson, Stephen S. [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States); Fennell, Timothy R. [RTI International, Research Triangle Park, NC (United States); Hawkins, Brian T. [RTI International, Research Triangle Park, NC (United States); Hickey, Anthony J. [RTI International, Research Triangle Park, NC (United States); Kleensang, Andre [Johns Hopkins Univ., Baltimore, MD (United States). Center for Alternatives to Animal Testing; Liebman, Michael N. [IPQ Analytics, Kennett Square, PA (United States); Martin, Florian [Phillip Morris International, Neuchatel (Switzerland); Maull, Elizabeth A. [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States); Paragas, Jason [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Qiao, Guilin [Defense Threat Reduction Agency, Ft. Belvoir, VA (United States); Ramaiahgari, Sreenivasa [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States); Sumner, Susan J. [RTI International, Research Triangle Park, NC (United States); Yoon, Miyoung [The Hamner Inst. for Health Sciences, Research Triangle Park, NC (United States); ScitoVation, Research Triangle Park, NC (United States)

    2016-08-04

    Translating in vitro biological data into actionable information related to human health holds the potential to improve disease treatment and risk assessment of chemical exposures. While genomics has identified regulatory pathways at the cellular level, translation to the organism level requires a multiscale approach accounting for intra-cellular regulation, inter-cellular interaction, and tissue/organ-level effects. Tissue-level effects can now be probed in vitro thanks to recently developed systems of three-dimensional (3D), multicellular, “organotypic” cell cultures, which mimic functional responses of living tissue. However, there remains a knowledge gap regarding interactions across different biological scales, complicating accurate prediction of health outcomes from molecular/genomic data and tissue responses. Systems biology aims at mathematical modeling of complex, non-linear biological systems. We propose to apply a systems biology approach to achieve a computational representation of tissue-level physiological responses by integrating empirical data derived from organotypic culture systems with computational models of intracellular pathways to better predict human responses. Successful implementation of this integrated approach will provide a powerful tool for faster, more accurate and cost-effective screening of potential toxicants and therapeutics. On September 11, 2015, an interdisciplinary group of scientists, engineers, and clinicians gathered for a workshop in Research Triangle Park, North Carolina, to discuss this ambitious goal. Participants represented laboratory-based and computational modeling approaches to pharmacology and toxicology, as well as the pharmaceutical industry, government, non-profits, and academia. Discussions focused on identifying critical system perturbations to model, the computational tools required, and the experimental approaches best suited to generating key data. This consensus report summarizes the discussions held.

  2. Inhibition on Telomerase Activity and Cytotoxic Effects by Cisplatin in Cultured Human Choroidal Melanoma Cells

    Institute of Scientific and Technical Information of China (English)

    Hao Cheng; Zhongyao Wu; Jianliang Zheng; Guilan Lu; Jianhua Yan; Min Liu; Danping Huang; Jianxian Lin

    2003-01-01

    Purpose: To study the changes of telomerase activity and cytotoxic effects by Cisplatin;cis-dichlorodiamine platinum (CDDP) in cultured human choroidal melanoma.Material and Methods: The primary cultured human choroidal melanoma cells werecultured in the presence and absence of CDDP with different concentration and timerespectively. The toxic effects were evaluated by MTT and the level of telormarse wasdetected by PCR-ELISA assay. And the relationship between telomerase activity andcytotoxic effects were analyzed by a correlation analysis.Results: Following the increase of the concentration and the time of CDDP, graduallyrepressed telomerase activity was detected in cultured cells. Meanwhile, the restrain rateof the cells increased. The telomerase activity at 24h and 1μg/ml was repressedsignificantly compared with the control cells. However, the appearance of cell deathlagged behind the decreasing of telomerase.Conclusions: CDDP is an effective telomerase inhibitor in cultured choroidal melanomacells of human eyes, which presents concentration and time dependency and can causethe death of cultured cells.

  3. Mechanism of initial attachment of cells derived from human bone to commonly used prosthetic materials during cell culture.

    Science.gov (United States)

    Howlett, C R; Evans, M D; Walsh, W R; Johnson, G; Steele, J G

    1994-02-01

    The suitability of polymeric biomaterials as surfaces for the attachment and growth of cells has often been investigated in cell culture. In this study the contribution that serum fibronectin (Fn) or vitronectin (Vn) make to the attachment and spreading of cells cultured from explanted human bone (bone-derived cells) during the first 90 min of culture was determined for metallic and ceramic surfaces. The requirement for Fn or Vn for attachment and spreading of bone-derived cells onto stainless steel 316 (SS), titanium (Ti) and alumina (Al2O3) and to polyethyleneterephthalate (PET) was directly tested by selective removal of Fn or Vn from the serum prior to addition to the culture medium. Attachment and spreading of bone-derived cells onto SS, Ti and Al2O3 surfaces were reduced by 73-83% when the cells were seeded in medium containing serum from which the Vn had been removed. Cell attachment and spreading on these surfaces when seeded in medium containing Fn-depleted serum (which contained Vn) were not reduced to the same extent as in the medium containing Vn-depleted serum. The bone-derived cells failed to attach to the surfaces to the same extent when seeded in medium containing serum depleted of both Vn and Fn. Our results show that for human bone-derived cells, the attachment and spreading of cells onto SS, Ti and Al2O3 as well as PET during the first 90 min of a cell culture attachment assay are a function of adsorption of serum Vn onto the surface. PMID:7515290

  4. Microfluidically supported biochip design for culture of endothelial cell layers with improved perfusion conditions.

    Science.gov (United States)

    Raasch, Martin; Rennert, Knut; Jahn, Tobias; Peters, Sven; Henkel, Thomas; Huber, Otmar; Schulz, Ingo; Becker, Holger; Lorkowski, Stefan; Funke, Harald; Mosig, Alexander

    2015-01-01

    Hemodynamic forces generated by the blood flow are of central importance for the function of endothelial cells (ECs), which form a biologically active cellular monolayer in blood vessels and serve as a selective barrier for macromolecular permeability. Mechanical stimulation of the endothelial monolayer induces morphological remodeling in its cytoskeleton. For in vitro studies on EC biology culture devices are desirable that simulate conditions of flow in blood vessels and allow flow-based adhesion/permeability assays under optimal perfusion conditions. With this aim we designed a biochip comprising a perfusable membrane that serves as cell culture platform multi-organ-tissue-flow (MOTiF biochip). This biochip allows an effective supply with nutrition medium, discharge of catabolic cell metabolites and defined application of shear stress to ECs under laminar flow conditions. To characterize EC layers cultured in the MOTiF biochip we investigated cell viability, expression of EC marker proteins and cell adhesion molecules of ECs dynamically cultured under low and high shear stress, and compared them with an endothelial culture in established two-dimensionally perfused flow chambers and under static conditions. We show that ECs cultured in the MOTiF biochip form a tight EC monolayer with increased cellular density, enhanced cell layer thickness, presumably as the result of a rapid and effective adaption to shear stress by remodeling of the cytoskeleton. Moreover, endothelial layers in the MOTiF biochip express higher amounts of EC marker proteins von-Willebrand-factor and PECAM-1. EC layers were highly responsive to stimulation with TNFα as detected at the level of ICAM-1, VCAM-1 and E-selectin expression and modulation of endothelial permeability in response to TNFα/IFNγ treatment under flow conditions. Compared to static and two-dimensionally perfused cell culture condition we consider MOTiF biochips as a valuable tool for studying EC biology in vitro under

  5. Poly-L-lysine Prevents Senescence and Augments Growth in Culturing Mesenchymal Stem Cells Ex Vivo

    Directory of Open Access Journals (Sweden)

    June Seok Heo

    2016-01-01

    Full Text Available Mesenchymal stem cells (MSCs possess great therapeutic potential. Efficient in vitro expansion of MSCs is however necessary for their clinical application. The extracellular matrix (ECM provides structural and biochemical support to the surrounding cells, and it has been used as a coating substrate for cell culture. In this study, we have aimed to improve the functionality and stemness of MSCs during culture using poly-L-lysine (PLL. Functionality of MSCs was analysed by cell cycle analysis, differentiation assay, β-galactosidase staining, and RT-PCR. Furthermore, we assessed the global gene expression profile of MSCs on uncoated and PLL-coated plates. MSCs on PLL-coated plates exhibited a faster growth rate with increased S-phase and upregulated expression of the stemness markers. In addition, their osteogenic differentiation potential was increased, and genes involved in cell adhesion, FGF-2 signalling, cell cycle, stemness, cell differentiation, and cell proliferation were upregulated, compared to that of the MSCs cultured on uncoated plates. We also confirmed that MSCs on uncoated plates expressed higher β-galactosidase than the MSCs on PLL-coated plates. We demonstrate that PLL provides favourable microenvironment for MSC culture by reversing the replicative senescence. This method will significantly contribute to effective preparation of MSCs for cellular therapy.

  6. Establishment and characterization of a differentiated epithelial cell culture model derived from the porcine cervix uteri

    Directory of Open Access Journals (Sweden)

    Miessen Katrin

    2012-03-01

    Full Text Available Abstract Background Cervical uterine epithelial cells maintain a physiological and pathogen-free milieu in the female mammalian reproductive tract and are involved in sperm-epithelium interaction. Easily accessible, differentiated model systems of the cervical epithelium are not yet available to elucidate the underlying molecular mechanisms within these highly specialized cells. Therefore, the aim of the study was to establish a cell culture of the porcine cervical epithelium representing in vivo-like properties of the tissue. Results We tested different isolation methods and culture conditions and validated purity of the cultured cells by immunohistochemistry against keratins. We could reproducibly culture pure epithelial cells from cervical tissue explants. Based on a morphology score and the WST-1 Proliferation Assay, we optimized the growth medium composition. Primary porcine cervical cells performed best in conditioned Ham's F-12, containing 10% FCS, EGF and insulin. After cultivation in an air-liquid interface for three weeks, the cells showed a discontinuously multilayered phenotype. Finally, differentiation was validated via immunohistochemistry against beta catenin. Mucopolysaccharide production could be shown via alcian blue staining. Conclusions We provide the first suitable protocol to establish a differentiated porcine epithelial model of the cervix uteri, based on easily accessible cells using slaughterhouse material.

  7. Morphology, proliferation, and osteogenic differentiation of mesenchymal stem cells cultured on titanium, tantalum, and chromium surfaces

    DEFF Research Database (Denmark)

    Stiehler, Maik; Lind, M.; Mygind, Tina;

    2007-01-01

    interactions between human mesenchymal stem cells (MSCs) and smooth surfaces of titanium (Ti), tantalum (Ta), and chromium (Cr). Mean cellular area was quantified using fluorescence microscopy (4 h). Cellular proliferation was assessed by (3)H-thymidine incorporation and methylene blue cell counting assays (4...... other surfaces tested. Cells cultured on Cr demonstrated reduced spreading and proliferation. In conclusion, Ta metal, as an alternative for Ti, can be considered as a promising biocompatible material, whereas further studies are needed to fully understand the role of Cr and its alloys in bone implants...

  8. Uptake and Intracellular Activity of Moxifloxacin in Human Neutrophils and Tissue-Cultured Epithelial Cells

    OpenAIRE

    Pascual, Alvaro; García, Isabel; Ballesta, Sofía; Perea, Evelio J.

    1999-01-01

    The penetration by moxifloxacin of human neutrophils (polymorphonuclear leukocytes [PMN]) and tissue-cultured epithelial cells (McCoy cells) was evaluated by a fluorometric assay. At extracellular concentrations of 5 mg/liter, the cellular-to-extracellular concentration ratios (C/E) of moxifloxacin in PMN and McCoy cells were 10.9 ± 1.0 and 8.7 ± 1.0, respectively (20 min; 37°C). The uptake of moxifloxacin by PMN was rapid, reversible, nonsaturable (at extracellular concentrations ranging fro...

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

    International Nuclear Information System (INIS)

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

  10. Induced engulfment of Neisseria gonorrhoeae by tissue culture cells.

    OpenAIRE

    Richardson, W P; Sadoff, J C

    1988-01-01

    Engulfment of gonococci by mammalian tissue culture cells was examined as a model of the penetration of host cells in gonorrhea. Engulfment required viable organisms; killing the gonococci with heat or refrigeration abolished the process. Engulfment also required tissue culture cell microtubule- and microfilament-dependent movement; treating the cells with cytochalasin B (0.5 micrograms/ml) or demecolcine (Colcemid; Ciba-Geigy AG, Basel, Switzerland) (10 micrograms/ml) also prevented his proc...

  11. Acetylsalicylic acid induces programmed cell death in Arabidopsis cell cultures.

    Science.gov (United States)

    García-Heredia, José M; Hervás, Manuel; De la Rosa, Miguel A; Navarro, José A

    2008-06-01

    Acetylsalicylic acid (ASA), a derivative from the plant hormone salicylic acid (SA), is a commonly used drug that has a dual role in animal organisms as an anti-inflammatory and anticancer agent. It acts as an inhibitor of cyclooxygenases (COXs), which catalyze prostaglandins production. It is known that ASA serves as an apoptotic agent on cancer cells through the inhibition of the COX-2 enzyme. Here, we provide evidences that ASA also behaves as an agent inducing programmed cell death (PCD) in cell cultures of the model plant Arabidopsis thaliana, in a similar way than the well-established PCD-inducing agent H(2)O(2), although the induction of PCD by ASA requires much lower inducer concentrations. Moreover, ASA is herein shown to be a more efficient PCD-inducing agent than salicylic acid. ASA treatment of Arabidopsis cells induces typical PCD-linked morphological and biochemical changes, namely cell shrinkage, nuclear DNA degradation, loss of mitochondrial membrane potential, cytochrome c release from mitochondria and induction of caspase-like activity. However, the ASA effect can be partially reverted by jasmonic acid. Taking together, these results reveal the existence of common features in ASA-induced animal apoptosis and plant PCD, and also suggest that there are similarities between the pathways of synthesis and function of prostanoid-like lipid mediators in animal and plant organisms.

  12. Assessing Drug Efficacy in a Miniaturized Pancreatic Cancer In Vitro 3D Cell Culture Model.

    Science.gov (United States)

    Shelper, Todd B; Lovitt, Carrie J; Avery, Vicky M

    2016-09-01

    Pancreatic cancer continues to have one of the poorest prognoses among all cancers. The drug discovery efforts for this disease have largely failed, with no significant improvement in survival outcomes for advanced pancreatic cancer patients over the past 20 years. Traditional in vitro cell culture techniques have been used extensively in both basic and early drug discovery; however, these systems offer poor models to assess emerging therapeutics. More predictive cell-based models, which better capture the cellular heterogeneity and complexities of solid pancreatic tumors, are urgently needed not only to improve drug discovery success but also to provide insight into the tumor biology. Pancreatic tumors are characterized by a unique micro-environment that is surrounded by a dense stroma. A complex network of interactions between extracellular matrix (ECM) components and the effects of cell-to-cell contacts may enhance survival pathways within in vivo tumors. This biological and physical complexity is lost in traditional cell monolayer models. To explore the predictive potential of a more complex cellular system, a three-dimensional (3D) micro-tumor assay was evaluated. Efficacy of six current chemotherapeutics was determined against a panel of primary and metastatic pancreatic tumor cell lines in a miniaturized ECM-based 3D cell culture system. Suitability for potential use in high-throughput screening applications was assessed, including ascertaining the effects that miniaturization and automation had on assay robustness. Cellular health was determined by utilizing an indirect population-based metabolic activity assay and a direct imaging-based cell viability assay. PMID:27552143

  13. Establishment of a high content assay for the identification and characterisation of bioactivities in crude bacterial extracts that interfere with the eukaryotic cell cycle.

    Science.gov (United States)

    Jensen, Nickels A; Gerth, Klaus; Grotjohann, Tim; Kapp, Dieter; Keck, Matthias; Niehaus, Karsten

    2009-03-10

    High content microscopy as a screening tool to identify bioactive agents has provided researchers with the ability to characterise biological activities at the level of single cells. Here, we describe the development and the application of a high content screening assay for the identification and characterisation of cytostatic bioactivities from Myxobacteria extracts. In an automated microscopy assay Sf9 insect cells were visualised utilising the stains bisbenzimide Hoechst 33342, calcein AM, and propidium iodide. Imaging data were processed by the ScanR Analysis-software to determine the ploidy and vitality of each cell and to quantify cell populations. More than 98% of the Sf9 cells were viable and the culture consisted of diploid ( approximately 30%), tetraploid ( approximately 60%), polyploidic (colchicine, paclitaxel, and cytochalasin D induced changes in ploidy and vitality, which were characteristic for the respective bioactive substance. Furthermore, crude extracts from the chivosazole producing Myxobacterium Sorangium cellulosum So ce56 induced an increase of polyploid cells and a decrease in total cell count, while a mutant producing nearly no chivosazole triggered none of these effects. Purified chivosazole induced the same effects as the wild type extract. Similar effects have been observed for the reference compound cytochalasin D. On the basis of this assay, crude extracts of ten different Myxobacteria cultures were screened. Three extracts exhibited strong cytotoxic activities, further five extracts induced weak changes in the ploidy distribution, and two extracts showed no detectable effect within the assay. Therefore, this robust assay provides the ability to discover and characterise cytotoxic and cytostatic bioactivities in crude bacterial extracts. PMID:19111838

  14. Sensitivity to radiation of human normal, hyperthyroid, and neoplastic thyroid epithelial cells in primary culture

    International Nuclear Information System (INIS)

    Samples of thyroid tissue removed surgically from 63 patients were cultured in vitro and X-irradiated to investigate the radiosensitivities of various types of thyroid epithelial cells. A total of 76 samples were obtained, including neoplastic cells from patients with papillary carcinoma (PC) or follicular adenoma (FA), cells from hyperthyroidism (HY) patients, and normal cells from the surgical margins of PC and FA patients. Culturing of the cells was performed in a manner which has been shown to yield a predominance of epithelial cells. Results of colony formation assays indicated that cells from HY and FA patients were the least radiosensitive: when adjusted to the overall geometric mean plating efficiency of 5.5 %, the average mean lethal dose D0 was 97.6 cGy for HY cells, and 96.7 cGy and 94.3 cGy, respectively, for neoplastic and normal cells from FA patients. Cells from PC patients were more radiosensitive, normal cells having an adjusted average D0 of 85.0 cGy and PC cells a significantly (p = .001) lower average D0 of 74.4 cGy. After allowing for this variation by cell type, in vitro radiosensitivity was not significantly related to age at surgery (p = .82) or sex (p = .10). These results suggest that malignant thyroid cells may be especially radiosensitive. (author)

  15. Dissecting functions of the conserved oligomeric Golgi tethering complex using a cell-free assay.

    Science.gov (United States)

    Cottam, Nathanael P; Wilson, Katherine M; Ng, Bobby G; Körner, Christian; Freeze, Hudson H; Ungar, Daniel

    2014-01-01

    Vesicle transport sorts proteins between compartments and is thereby responsible for generating the non-uniform protein distribution along the eukaryotic secretory and endocytic pathways. The mechanistic details of specific vesicle targeting are not yet well characterized at the molecular level. We have developed a cell-free assay that reconstitutes vesicle targeting utilizing the recycling of resident enzymes within the Golgi apparatus. The assay has physiological properties, and could be used to show that the two lobes of the conserved oligomeric Golgi tethering complex play antagonistic roles in trans-Golgi vesicle targeting. Moreover, we can show that the assay is sensitive to several different congenital defects that disrupt Golgi function and therefore cause glycosylation disorders. Consequently, this assay will allow mechanistic insight into the targeting step of vesicle transport at the Golgi, and could also be useful for characterizing some novel cases of congenital glycosylation disorders.

  16. Cytotoxicity and genotoxicity assessment of Euphorbia hirta in MCF-7 cell line model using comet assay

    Institute of Scientific and Technical Information of China (English)

    Kwan Yuet Ping; Ibrahim Darah; Yeng Chen; Sreenivasan Sasidharan

    2013-01-01

    Objective:To evaluate the cytotoxicity and genotoxicity activity of Euphorbia hirta (E. hirta) in MCF-7 cell line model using comet assay. Methods: The cytotoxicity of E. hirta extract was investigated by employing brine shrimp lethality assay and the genotoxicity of E. hirta was assessed by using Comet assay. Results: Both toxicity tests exhibited significant toxicity result. In the comet assay, the E. hirta extract exhibited genotoxicity effects against MCF-7 DNA in a time-dependent manner by increasing mean percentage of DNA damage. The extract of E. hirta showed significant toxicity against brine shrimp with an LC50 value of 620.382 μg/mL (24 h). Comparison with positive control potassium dichromate signifies that cytotoxicity exhibited by the methanol extract might have moderate activity. Conclusion:The present work confirmed the cytotoxicity and genotoxicity of E. hirta. However, the observed toxicity of E. hirta extracts needs to be confirmed in additional studies.

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

    Science.gov (United States)

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

    2016-08-01

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

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

    Science.gov (United States)

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

    2016-08-01

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

  19. A microfluidic digital single-cell assay for the evaluation of anticancer drugs.

    Science.gov (United States)

    Wang, Yao; Tang, Xiaolong; Feng, Xiaojun; Liu, Chao; Chen, Peng; Chen, Dongjuan; Liu, Bi-Feng

    2015-02-01

    Digital single-cell assays hold high potentials for the analysis of cell apoptosis and the evaluation of chemotherapeutic reagents for cancer therapy. In this paper, a microfluidic hydrodynamic trapping system was developed for digital single-cell assays with the capability of monitoring cellular dynamics over time. The microfluidic chip was designed with arrays of bypass structures for trapping individual cells without the need for surface modification, external electric force, or robotic equipment. After optimization of the bypass structure by both numerical simulations and experiments, a single-cell trapping efficiency of ∼90 % was achieved. We demonstrated the method as a digital single-cell assay for the evaluation of five clinically established chemotherapeutic reagents. As a result, the half maximal inhibitory concentration (IC50) values of these compounds could be conveniently determined. We further modeled the gradual decrease of active drugs over time which was often observed in vivo after an injection to investigate cell apoptosis against chemotherapeutic reagents. The developed method provided a valuable means for cell apoptotic analysis and evaluation of anticancer drugs. PMID:25433683

  20. Comparison of defined culture systems for feeder cell free propagation of human embryonic stem cells

    OpenAIRE

    Akopian, Veronika; Andrews, Peter W.; Beil, Stephen; Benvenisty, Nissim; Brehm, Jennifer; Christie, Megan; Ford, Angela; Fox, Victoria; Gokhale, Paul J; Healy, Lyn; Holm, Frida; Hovatta, Outi; Knowles, Barbara B; Ludwig, Tenneille E; Ronald D G McKay

    2010-01-01

    There are many reports of defined culture systems for the propagation of human embryonic stem cells in the absence of feeder cell support, but no previous study has undertaken a multi-laboratory comparison of these diverse methodologies. In this study, five separate laboratories, each with experience in human embryonic stem cell culture, used a panel of ten embryonic stem cell lines (including WA09 as an index cell line common to all laboratories) to assess eight cell culture methods, with pr...

  1. Passage of bone-marrow-derived liver stem cells in a proliferating culture system

    Institute of Scientific and Technical Information of China (English)

    Yun-Feng Cai; Ji-Sheng Chen; Shu-Ying Su; Zuo-Jun Zhen; Huan-Wei Chen

    2009-01-01

    AIM: To explore the feasibility of passage of bonemarrow-derived liver stem cells (BDLSCs) in culture systems that contain cholestatic serum. METHODS: Whole bone marrow cells of rats were purified with conditioning selection media that contained 50 mL/L cholestatic serum. The selected BDLSCs were grown in a proliferating culture system and a differentiating culture system. The culture systems contained factors that stimulated the proliferation and differentiation of BDLSCs. Each passage of the proliferated stem cells was subjected to flow cytometry to detect stem cell markers. The morphology and phenotypic markers of BDLSCs were characterized using immunohistochemistry, reverse transcription polymerase chain reaction (RT-PCR) and electron microscopy. The metabolic functions of differentiated cells were also determined by glycogen staining and urea assay. RESULTS: The conditioning selection medium isolated BDLSCs directly from cultured bone marrow cells. The selected BDLSCs could be proliferated for six passages and maintained stable markers in our proliferating system. When the culture system was changed to a differentiating system, hepatocyte-like colony-forming units (H-CFUs) were formed. H-CFUs expressed markers of embryonic hepatocytes (alpha-fetoprotein, albumin and cytokeratin 8/18), biliary cells (cytokeratin 19), hepatocyte functional proteins (transthyretin and cytochrome P450-2b1), and hepatocyte nuclear factors 1α and -3β). They also had glycogen storage and urea synthesis functions, two of the critical features of hepatocytes. CONCLUSION: BDLSCs can be selected directly from bone marrow cells, and pure BDLSCs can be proliferated for six passages. The differentiated cells have hepatocyte-like phenotypes and functions. BDLSCs represent a new method to provide a readily available alternate source of cells for clinical hepatocyte therapy.

  2. Three-dimensional cell culturing by magnetic levitation for evaluating efficacy/toxicity of photodynamic therapy

    Science.gov (United States)

    Sabino, Luis G.; Menezes, Priscila F. C.; Bagnato, Vanderlei S.; Souza, Glauco; Killian, Thomas C.; Kurachi, Cristina

    2014-03-01

    We used three dimensional cell cultures (3D) based on the magnetic levitation method (MLM) to evaluate cytotoxicity of photodynamic therapy (PDT). First, we decorated Hep G2 and MDA-MB-321 cells with NanoShuttle by introducing it in the media and incubated overnight. Next day, we transferred the cells to a 6-well plate and placed a magnetic driver on the top of the plate to start levitation. We monitored the formation of the 3D cell culture by optical microscopy and after four days, we added the photosensitizer Photogem (PG) in the culture media in concentrations of 50, 25, 12.5, 6.25μg/ml. We incubated them for 24 hours, after that we washed the cultures with PBS and added fresh media. Samples were then illuminated for 600s using a 630nm LED-based device, generating light intensities of 30 mW/cm2 in a total light fluence of 18 J/cm2. Following the illumination, we added fresh media, and 30 hours later, the 3D structures were broken using a pipettor and the cells seeded in 96 well plates, 105 cells per well, with a magnetic drive placed on the bottom of the plate to create cell culture dots. After 24 hours, we used a MTT assay to evaluate PDT cytotoxicity. The PDT effect, evaluated by the half maximal effective concentration (EC50), in MDA-MB-231 cells (EC50 =3.14 μg/ml) is more aggressive compared to the effect of PDT in Hep G2 cells (EC50 = 7.48 μg/ml). It suggests that the cell culture structure and its interaction facilitated the PG uptake and consequently elevated the Photodynamic effect for MDA-MB-231.

  3. A simple assay for agonist-regulated Cl and K conductances in salt-secreting epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Venglarik, C.J.; Bridges, R.J.; Frizzell, R.A. (Univ. of Alabama, Birmingham (USA))

    1990-08-01

    We developed a convenient flux assay that permits simultaneous measurement of Cl and K conductance pathways in Cl-secreting epithelial cells. Monolayers of the colonic tumor cell line T84 were preloaded with 125I and 86Rb, and isotope effluxes were monitored by a sample-replace procedure. The adenosine 3',5'-cyclic monophosphate (cAMP)-mediated agonists forskolin and prostaglandin E2 increased I efflux with little effect on Rb efflux, whereas the Ca-mediated agonists ionomycin, A23187, and carbachol increased both I and Rb effluxes. Simultaneous determinations of I and Cl or Rb and K effluxes indicated that I and Rb provide good measures of the effluxes of Cl and K, respectively. Forskolin- and ionomycin-stimulated I effluxes were inhibited by the Cl-channel blockers diphenylamine-2-dicarboxylate (DPC), 5-nitro-2-(3-phenylpropyl-amino)benzoic acid (NPPB), and 2-(cyclopentyl-6,7-dichloro-2,3-dihydro-2-methyl-1-oxo-1H- inden-5-yl-oxy)acetic acid (IAA-94) and by high external K. The Rb efflux evoked by ionomycin was inhibited by the K-channel blockers Ba and charybdotoxin. These findings suggest that I and Rb effluxes provide qualitative estimates of agonist-stimulated Cl and K conductance pathways. Thus this method can provide a simple and relatively inexpensive screening assay for Cl and K conductances in cultured cells to assess the effects of agonist, blockers, or genetic manipulations.

  4. Characterization of Berberine on Human Cancer Cells in Culture

    OpenAIRE

    SZETO, Savio

    2002-01-01

    Berberine originates from a Chinese herbal medicine and possesses a wide variety of anti-cancer activities. In this study, the killing effect of berberine on nasopharyngeal carcinoma cells (NPC/HK1) was investigated. The trypan blue exclusion assay was used to assess the cytotoxic effect of berberine in this cell line. Berberine, at 5-200 µM, induced cell death in a dose-dependent manner. Treatment of cells with 200 µM berberine for 5 h yielded a lethal dose of 50% (LD50). The Comet Assay was...

  5. Optimization and standardization of the ''comet assay'' for analyzing the repair of DNA damage in cells

    International Nuclear Information System (INIS)

    Human tumor cells or isolated human peripheral blood lymphocytes were analyzed in the experiments. The amount of DNA damage and the effectiveness of DNA repair was measured after X-irradiation using the 'comet assay' technique. Results: In this presentation the influences of different methodological factors like agarose concentration, buffer pH, electrophoresis time, electric field strength on the applicability of the 'comet assay' are described in detail and optimum conditions for 'comet assay' experiments have been evaluated. Additionally the authors will show a comparison of different fluorescent DNA dyes pointing out their advantages or disadvantages for 'comet' analysis. The usefulness of this technique and its capabilities are exemplified by showing DNA repair kinetics of human lymphocytes of different healthy or radiosensitive donors after in-vitro irradiation with 2 Gy X-rays. Conclusions: This paper presents data on the optimization and standardization of the original 'comet assay' leading to an extremely fast and practicable protocol in the field of single cell gel electrophoresis. After irradiation with 0.1 Gy an increase in the amount of DNA damage can be measured with high statistical significance and the DNA repair capacity of individual cells after X-ray doses of 2 Gy can be analyzed with high reproducibility. The results comparing DNA repair capacities of different donors point out that the 'comet assay' may have the potential for the estimation of individual radiosensitivity. (orig./MG)

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

    Institute of Scientific and Technical Information of China (English)

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

    2011-01-01

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

  7. Development and evaluation of an ELIME assay to reveal the presence of Salmonella in irrigation water: Comparison with Real-Time PCR and the Standard Culture Method.

    Science.gov (United States)

    Volpe, G; Delibato, E; Fabiani, L; Pucci, E; Piermarini, S; D'Angelo, A; Capuano, F; De Medici, D; Palleschi, G

    2016-01-01

    A reliable, low-cost and easy-to-use ELIME (Enzyme-Linked-Immuno-Magnetic-Electrochemical) assay for detection of Salmonella enterica in irrigation water is presented. Magnetic beads (MBs), coupled to a strip of eight-magnetized screen-printed electrodes localized at the bottom of eight wells (8-well/SPE strip), effectively supported a sandwich immunological chain. Enzymatic by-product is quickly measured by chronoamperometry, using a portable instrument. With the goal of developing a method able to detect a wide range of Salmonella serotypes, including S. Napoli and S. Thompson strains responsible for various community alerts, different kinds of MBs, antibodies and blocking agents were tested. The final system employs MBs coated with a broad reactivity monoclonal antibody anti-salmonella and blocked with dry milk. For a simple and rapid assay these two steps were performed in a preliminary phase, while the two sequential incubations for the immuno-recognition events were merged in a single step of 1h. In parallel a Real-Time PCR (RTi-PCR) method, based on a specific locked nucleic acid (LNA) fluorescent probe and an internal amplification control (IAC), was carried out. The selectivity of the ELIME and RTi-PCR assays was proved by inclusivity and exclusivity tests performed analyzing different Salmonella serotypes and non-target microorganisms, most commonly isolated from environmental sources. Furthermore, both methods were applied to experimentally and not experimentally contaminated irrigation water samples. Results confirmed by the ISO culture method, demonstrated the effectiveness of ELIME and RTi-PCR assays to detect a low number of salmonella cells (1-10 CFU/L) reducing drastically the long analysis time usually required to reveal this pathogen.

  8. Analysis of micronuclei and microtubule arrangement to identify aneuploidy-inducing agents in cultured mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Degrassi, F.; Pisano, C. [Centre di Genetica Evoluzionistica, Rome (Italy); Tanzarella, C.; Antoccia, A.; Battistoni, A. [Universita La Sapienza, Rome (Italy)

    1993-12-31

    The development of in vitro test methods to detect environmental agents that might induce aneuploidy is of crucial importance in genotoxicity testing. Chromosome numerical changes may arise from damage to various cell structures and activities such as spindle components or kinetochore proteins as well as from damage to the chromosomes. Therefore, the development of effective assays to identify chromosome misdistribution in mammalian cell cultures requires the contribution of different research areas such as cytogenetics, molecular biology and cell biology. Recently, we have been working at the development of an in vitro test for aneuploidy-inducing agents combining the micronucleus assay with the immunofluorescent staining of kinetochores in micronuclei (MN). The assay has been standardized by analyzing the induction of MN containing kinetochores (CREST-positive MN) after a number of agents with different mechanism of action. Subsequently, the optimization of the assay has been carried out by introducing cytochalasin-B (cyt-B) in the test protocol in order to score MN in cells that have undergone one cell cycle. Finally, with the aim of providing an understanding of the mechanisms responsible for the production of CREST-positive MN we have analyzed the cellular structures involved in mitotic division by using specific antibodies in immunofluorescence studies.

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

    Institute of Scientific and Technical Information of China (English)

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

    2003-01-01

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

  10. Development of electrochemical reporter assay using HeLa cells transfected with vector plasmids encoding various responsive elements

    Energy Technology Data Exchange (ETDEWEB)

    Shiku, Hitoshi, E-mail: shiku@bioinfo.che.tohoku.ac.jp [Graduate School of Environmental Studies, Tohoku University, 6-6-11-604 Aramaki-Aoba, Sendai 980-8579 (Japan); Takeda, Michiaki; Murata, Tatsuya [Graduate School of Environmental Studies, Tohoku University, 6-6-11-604 Aramaki-Aoba, Sendai 980-8579 (Japan); Akiba, Uichi; Hamada, Fumio [Graduate School of Engineering and Resource Science, Akita University, 1-1 Tegata gakuen-machi, Akita 010-8502 (Japan); Matsue, Tomokazu, E-mail: matsue@bioinfo.che.tohoku.ac.jp [Graduate School of Environmental Studies, Tohoku University, 6-6-11-604 Aramaki-Aoba, Sendai 980-8579 (Japan)

    2009-04-27

    Electrochemical assay using HeLa cell lines transfected with various plasmid vectors encoding SEAP (secreted alkaline phosphatase) as the reporter has been performed by using SECM (scanning electrochemical microscopy). The plasmid vector contains different responsive elements that include GRE (glucocorticoid response elements), CRE (cAMP responsive elements), or {kappa}B (binding site for NF{kappa}B (nuclear factor kappa B)) upstream of the SEAP sequence. The transfected HeLa cells were patterned on a culture dish in a 4 x 4 array of circles of diameter 300 {mu}m by using the PDMS (poly(dimethylsiloxane)) stencil technique. The cellular array was first exposed to 100 ng mL{sup -1} dexamethasone, 10 ng mL{sup -1} forskolin, or 100 ng mL{sup -1} TNF-{alpha} (tumor necrosis factor {alpha}) after which it was further cultured in an RPMI culture medium for 6 h. After incubation, the cellular array was soaked in a measuring solution containing 4.7 mM PAPP (p-aminophenylphosphate) at pH 9.5, following which electrochemical measurements were performed immediately within 40 min. The SECM method allows parallel evaluation of different cell lines transfected with pGRE-SEAP, pCRE-SEAP, and pNF{kappa}B-SEAP patterned on the same solid support for detection of the oxidation current of PAP (p-aminophenol) flux produced from only 300 HeLa cells in each stencil pattern. The results of the SECM method were highly sensitive as compared to those obtained from the conventional CL (chemiluminescence) protocol with at least 5 x 10{sup 4} cells per well.

  11. Use of sensitive, broad-spectrum molecular assays and human airway epithelium cultures for detection of respiratory pathogens.

    Directory of Open Access Journals (Sweden)

    Krzysztof Pyrc

    Full Text Available Rapid and accurate detection and identification of viruses causing respiratory tract infections is important for patient care and disease control. Despite the fact that several assays are available, identification of an etiological agent is not possible in ~30% of patients suffering from respiratory tract diseases. Therefore, the aim of the current study was to develop a diagnostic set for the detection of respiratory viruses with sensitivity as low as 1-10 copies per reaction. Evaluation of the assay using a training clinical sample set showed that viral nucleic acids were identified in ~76% of cases. To improve assay performance and facilitate the identification of novel species or emerging strains, cultures of fully differentiated human airway epithelium were used to pre-amplify infectious viruses. This additional step resulted in the detection of pathogens in all samples tested. Based on these results it can be hypothesized that the lack of an etiological agent in some clinical samples, both reported previously and observed in the present study, may result not only from the presence of unknown viral species, but also from imperfections in the detection methods used.

  12. PECULIARITIES OF SECONDARY METABOLITES BIOSYNTHESIS IN PLANT CELL CULTURES

    Directory of Open Access Journals (Sweden)

    A.M. NOSOV

    2014-06-01

    Full Text Available metabolites formation in plant cell cultures of Panax spp., (ginsenosides; Dioscorea deltoidea (steroid glycosides; Ajuga reptans, Serratula coronata, Rhaponticum carthamoides (ecdisteroids; Polyscias spp., (triterpene glycosides, Taxus spp. (taxoids, Stevia rebaudiana (diterpene steviol-glycosides, Stephania glabra (alkaloids. They are some regular trends of secondary metabolites synthesis in the plant cell culture:It can be noted the stable synthesis of the compound promoting cell proliferation. Indeed, cell cultures of Dioscorea deltoidea were demonstrated to accumulate only furostanol glycosides, which promoted cell division. Furostanol glycoside content of Dioscorea strain DM-0.5 was up to 6 - 12% by dry biomass.Panax ginseng and P. japonicus plant cell cultures synthesize as minimum seven triterpene glycosides (ginsenosides, the productivity of these compounds was up to 6.0 - 8.0% on dry biomass.By contrast, the detectable synthesis of diterpene steviol-glycosides in cultivated cells of Stevia rebaudiana initiated in the mixotrophic cultures during chloroplast formation only.Despite these differences, or mainly due to them, plant cell cultures have become an attractive source of phytochemicals in alternative to collecting wild plants. It provides a guideline to bioreactor-based production of isoprenoids using undifferentiated plant cell cultures

  13. Follicle stimulating hormone increases spermatogonial stem cell colonization during in vitro co - culture

    Directory of Open Access Journals (Sweden)

    Reza Narenji Sani

    2013-03-01

    Full Text Available The complex process of spermatogenesis is regulated by various factors. Studies onspermatogonial stem cells(SCCshave provided very important tool to improve herd geneticand different field. 0.2 to 0.3 percent of total cells of seminiferous tubules is consist ofspermatogonial stem cells. To investigate and biomanipulation of these cells, proliferationand viability rate of cells should be increasedin vitro, at first. Follicle stimulating hormone(FSH has been suggested to play a determinant role in the survival of germ cells in additionto increasing spermatogonial proliferation. In this study, thein vitroeffects ofFSHonspermatogonial cell colony formation were investigated. Sertoli and spermatogonial cellswere isolated from 3-5 months old calves. The identity of theSertoli cells and spermatogonialstem cells were confirmed through immunocytochemistry and colony morphology,respectively. Co-cultured Sertoli and spermatogonial cells were treatedwithFSHin differentdose of10, 20 and 40 IU mL-1FSH, before colony assay.Results indicated that,FSHincreasedin vitrocolonization of spermatogonial cells in comparison with control group. In conclusion,usingFSHprovided proper bovine spermatogonial stem cell culture medium forin vitrostudy of these cells.

  14. Evaluating the role of low-speed centrifugation towards transfecting human peripheral blood mononuclear cell culture

    Directory of Open Access Journals (Sweden)

    M Majumdar

    2014-01-01

    Full Text Available The conventional method of transfection of suspension cells by chemical has proven to be very difficult. We present a new transfection protocol, wherein, low-speed centrifugation of cell culture plates immediately after adding the lipid: DNA complex significantly enhances the transfection efficiency. Peripheral blood mononuclear cells (PBMCs were transfected with BLOCK-iT™ Fluorescent Oligo (scrambled siRNA and lipofectamine complex using conventional and low-speed centrifugation modified transfection protocols. The efficiency of transfection was determined using flowcytometer and cell viability was checked using MTT assay. Incorporation of low-speed centrifugation significantly enhances the transfection efficiency of BLOCK-iT™ in the suspension culture of PBMCs as compared to conventional transfection method (99.8% vs 28.3%; P < 0.0001, even at a low concentration of 40 picomoles without affecting the cell viability. Centrifugation enhanced transfection (CET technique is simple, time-saving and novel application without compromising the cell viability in the context of recently popular RNA interference in suspension cultures of PBMCs. This undemanding modification might be applicable to a wide variety of cell lines and solve crucial problem of researchers working with RNA interference in suspension cultures.

  15. Cytotoxicity assessment of antibiofouling compounds and by-products in marine bivalve cell cultures.

    Science.gov (United States)

    Domart-Coulon, I; Auzoux-Bordenave, S; Doumenc, D; Khalanski, M

    2000-06-01

    Short-term primary cell cultures were derived from adult marine bivalve tissues: the heart of oyster Crassostrea gigas and the gill of clam Ruditapes decussatus. These cultures were used as experimental in vitro models to assess the acute cytotoxicity of an organic molluscicide, Mexel-432, used in antibiofouling treatments in industrial cooling water systems. A microplate cell viability assay, based on the enzymatic reduction of tetrazolium dye (MTT) in living bivalve cells, was adapted to test the cytotoxicity of this compound: in both in vitro models, toxicity thresholds of Mexel-432 were compared to those determined in vivo with classic acute toxicity tests. The clam gill cell model was also used to assess the cytotoxicity of by-products of chlorination, a major strategy of biofouling control in the marine environment. The applications and limits of these new in vitro models for monitoring aquatic pollutants were discussed, in reference with the standardized Microtox test. PMID:10806375

  16. A hybrid microfluidic-vacuum device for direct interfacing with conventional cell culture methods

    Directory of Open Access Journals (Sweden)

    Monuki Edwin S

    2007-09-01

    Full Text Available Abstract Background Microfluidics is an enabling technology with a number of advantages over traditional tissue culture methods when precise control of cellular microenvironment is required. However, there are a number of practical and technical limitations that impede wider implementation in routine biomedical research. Specialized equipment and protocols required for fabrication and setting up microfluidic experiments present hurdles for routine use by most biology laboratories. Results We have developed and validated a novel microfluidic device that can directly interface with conventional tissue culture methods to generate and maintain controlled soluble environments in a Petri dish. It incorporates separate sets of fluidic channels and vacuum networks on a single device that allows reversible application of microfluidic gradients onto wet cell culture surfaces. Stable, precise concentration gradients of soluble factors were generated using simple microfluidic channels that were attached to a perfusion system. We successfully demonstrated real-time optical live/dead cell imaging of neural stem cells exposed to a hydrogen peroxide gradient and chemotaxis of metastatic breast cancer cells in a growth factor gradient. Conclusion This paper describes the design and application of a versatile microfluidic device that can directly interface with conventional cell culture methods. This platform provides a simple yet versatile tool for incorporating the advantages of a microfluidic approach to biological assays without changing established tissue culture protocols.

  17. Biologic characteristics of fibroblast cells cultured from the knee ligaments

    Institute of Scientific and Technical Information of China (English)

    陈鸿辉; 唐毅; 李斯明; 沈雁; 刘向荣; 钟灿灿

    2002-01-01

    Objective: To culture fibroblast cells from the kneeligaments and to study the biological characteristics of thesecells.Methods: Cells of the anterior cruciate ligament(ACL) and the medial collateral ligament (MCL) fromNew Zealand white rabbit were cultured in vitro. Cellulargrowth and expression of the collagen were analyzed.Moreover, an in vitro wound closure model was establishedand the healing of the ACL and the MCL cells wascompared.Results: Maximal growth for all these cells wereobtained with Dulbecco's modified Eagle's mediumsupplemented with 10% fetal bovine serum, but RPMI 1640and Ham's F12 media were not suitable to maintain thesecells. Morphology of both ACL and MCL cells from NewZealand white rabbit was alike in vitro, but the MCL cellsgrew faster than the ACL cells. Both cell types producedsimilar amount of collagen in culture, but the ratio ofcollage type I to type III produced by ACL cells was higherthan that produced by MCL cells. Wound closure assayshowed that at 36 hours after injury, cell-free zones createdin the ACL cultures were occupied partially by the ACLcells; in contrast, the wounded zone in the MCL cultureswas almost completely covered by the cells.Conclusions: Although the ACL cells and the MCLcells from New Zealand white rabbit show similarappearance in morphology in culture, the cellular growthand the biochemical synthesis of collagen as well as thehealing in vitro were significantly different. Thesedifferences in intrinsic properties of the two types of cells invitro might contribute to the differential healing potentialsof these ligaments in vivo.

  18. LIF-free embryonic stem cell culture in simulated microgravity.

    Directory of Open Access Journals (Sweden)

    Yumi Kawahara

    Full Text Available BACKGROUND: Leukemia inhibitory factor (LIF is an indispensable factor for maintaining mouse embryonic stem (ES cell pluripotency. A feeder layer and serum are also needed to maintain an undifferentiated state, however, such animal derived materials need to be eliminated for clinical applications. Therefore, a more reliable ES cell culture technique is required. METHODOLOGY/PRINCIPAL FINDINGS: We cultured mouse ES cells in simulated microgravity using a 3D-clinostat. We used feeder-free and serum-free media without LIF. CONCLUSIONS/SIGNIFICANCE: Here we show that simulated microgravity allows novel LIF-free and animal derived material-free culture methods for mouse ES cells.

  19. 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

    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...

  20. Optimization of Betanodavirus culture and enumeration in striped snakehead fish cells.

    Science.gov (United States)

    Hick, Paul; Tweedie, Alison; Whittington, Richard J

    2011-05-01

    An optimized culture method for detection of infection of fish with the Red spotted grouper nervous necrosis virus (RGNNV) genotype of betanodavirus in striped snakehead (SSN-1, Channa striatus) cells is described. Inoculation of fish tissue homogenates at the same time or within 4 hr of seeding the SSN-1 cells was as sensitive as the method recommended by the World Organization for Animal Health, where homogenates were adsorbed onto an established cell monolayer. Such modification halved the time required and the costs of consumables, and reduced the potential for error when processing large numbers of samples. Positive culture results were obtained from 88.3% of 392 fish tissue homogenates in which RGNNV was detected using a quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay; 99.7% of 943 tissue homogenates, which were qRT-PCR negative, were cell culture negative. Cytopathic effect (CPE) was characterized by large intracytoplasmic vacuoles in 0.1-60% of cells. Detachment of affected cells from the culture surface resulting in progressive disruption of the monolayer occurred in 46.4% of primary cultures and 96.0% of subcultures of positive samples. Identification of CPE that did not disrupt the cell monolayer increased estimates of the 50% tissue culture infective dose (TCID(50)) by 1.07-2.79 logs (95% confidence interval). The predicted mean TCID(50)/ml was 3.3 logs higher when cells were inoculated less than 36 hr after subculture at less than 80% confluence compared to cells inoculated at greater than 80% confluence and more than 36 hr after subculture (P < 0.05).

  1. DNA Microarray Assay Helps to Identify Functional Genes Specific for Leukemia Stem Cells

    Directory of Open Access Journals (Sweden)

    Haojian Zhang

    2013-01-01

    Full Text Available Chronic myeloid leukemia (CML is a myeloproliferative disease derived from an abnormal hematopoietic stem cell (HSC and is consistently associated with the formation of Philadelphia (Ph chromosome. Tyrosine kinase inhibitors (TKIs are highly effective in treating chronic phase CML but do not eliminate leukemia stem cells (LSCs, which are believed to be related to disease relapse. Therefore, one major challenge in the current CML research is to understand the biology of LSCs and to identify the molecular difference between LSCs and its normal stem cell counterparts. Comparing the gene expression profiles between LSCs and normal HSCs by DNA microarray assay is a systematic and unbiased approach to address this issue. In this paper, we present a DNA microarray dataset for CML LSCs and normal HSCs to show that the microarray assay will benefit the current and future studies of the biology of CML stem cells.

  2. Bioactivities of Culture Supernatants from Retroviral Packaging Cells Carrying the Mouse Fas Ligand Gene

    Institute of Scientific and Technical Information of China (English)

    LIU Lingbo; ZOU Ping; GUO Rong; XIAO Juan; XU Zhiliang

    2001-01-01

    The bioactivities of culture supernatants from retroviral packaging cells carrying the mouse Fas ligand (mFasL) gene was investigated. FasLcDNA was cloned into PLXIN with an internal ribosome entry site to link two cistrons through gene recombination technology, PLXIN and the recombinant vector PLFIN were separately transfected into PA317 retrovirus packing cell line by lipofectamine 2000, and the resistant clones were selected with G418 selective medium. The integration of genome DNA was assayed by genomic DNA PCR. NIH3T3 cells were transduced by the culture supernatants from PA317 carrying the mFasLcDNA gene, and were selected with G418 selective medium, so as to select the PLFIN-PA317 clone capable of producing higher titer of supernatants. The levels of mFasL protein on NIH3T3 cells membrane were assayed by flow cytometry (FCM). The biological activity of mFasL on NIH3T3 cells membrane was investigated by the inducing apoptosis of Fas+ Yac-1 cells co-cultured with NIH3T3 cells expressing Fas ligand. To explore the direct mFasL cytotoxicity of culture supernatants from retroviral packaging cells carrying the mFasL gene, the culture supernatants from PLFIN-PA317 and PLXIN-PA317 were separately co-cultured with Yac-1cells in parallel. The recombinant PLFIN was successfully constructed. The highest titer of supernatants from twelve resistant clones was 8. 5 × 105 colony-forming-unit (CFU)/ml. The NIH3T3cells transfected by above supernatants had a higher level of mFasL (53.81±6.9 %), and significantly induced the apoptosis of Fas+ Yac-1 cells (56. 78±4.5 %), as both were cocultured for 5 h at1 : 1 ratio, whereas it is 7. 08±3.4 % in control group (P<0. 01). Supernatant from PLFINPA317 could also directly induce the apoptosis of Yac-1 within 5 h of incubation. Thus, the culture supernatants from PLFIN-PA317 possessed both infectivity and cytotoxicity of mFasL.

  3. Effects of external radiation in a co-culture model of endothelial cells and adipose-derived stem cells

    International Nuclear Information System (INIS)

    The inflammatory response clinically observed after radiation has been described to correlate with elevated expression of cytokines and adhesion molecules by endothelial cells. Therapeutic compensation for this microvascular compromise could be an important approach in the treatment of irradiated wounds. Clinical reports describe the potential of adipose-derived stem cells to enhance wound healing, but the underlying cellular mechanisms remain largely unclear. Human dermal microvascular endothelial cells (HDMEC) and human adipose-derived stem cells (ASC) were cultured in a co-culture setting and irradiated with sequential doses of 2 to 12 Gy. Cell count was determined 48 h after radiation using a semi-automated cell counting system. Levels of interleukin-6 (IL-6), basic fibroblast growth factor (FGF), intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were determined in the supernatants using enzyme-linked immunosorbent assay (ELISA). Irradiated HDMEC and ASC as well as non-irradiated co-cultures, HDMEC or ASC respectively were used as controls. Cell count was significantly reduced in irradiated co-cultures of HDMEC and ASC compared to non-irradiated controls. Levels of IL-6, FGF, ICAM-1 and VCAM-1 in the supernatants of the co-cultures were significantly less affected by external radiation in comparison to HDMEC. The increased expression of cytokines and adhesion molecules by HDMEC after external radiation is mitigated in the co-culture setting with ASC. These in vitro changes seem to support the clinical observation that ASC may have a stabilizing effect when injected into irradiated wounds

  4. Acute shear stress direction dictates adherent cell remodeling and verifies shear profile of spinning disk assays

    International Nuclear Information System (INIS)

    Several methods have been developed to quantify population level changes in cell attachment strength given its large heterogeneity. One such method is the rotating disk chamber or ‘spinning disk’ in which a range of shear forces are applied to attached cells to quantify detachment force, i.e. attachment strength, which can be heterogeneous within cell populations. However, computing the exact force vectors that act upon cells is complicated by complex flow fields and variable cell morphologies. Recent observations suggest that cells may remodel their morphology and align during acute shear exposure, but contrary to intuition, shear is not orthogonal to the radial direction. Here we theoretically derive the magnitude and direction of applied shear and demonstrate that cells, under certain physiological conditions, align in this direction within minutes. Shear force magnitude is also experimentally verified which validates that for spread cells shear forces and not torque or drag dominate in this assay, and demonstrates that the applied force per cell area is largely independent of initial morphology. These findings suggest that direct quantified comparison of the effects of shear on a wide array of cell types and conditions can be made with confidence using this assay without the need for computational or numerical modeling. (paper)

  5. Culture materials affect ex vivo expansion of hematopoietic progenitor cells.

    Science.gov (United States)

    LaIuppa, J A; McAdams, T A; Papoutsakis, E T; Miller, W M

    1997-09-01

    Ex vivo expansion of hematopoietic cells is important for applications such as cancer treatment, gene therapy, and transfusion medicine. While cell culture systems are widely used to evaluate the biocompatibility of materials for implantation, the ability of materials to support proliferation of primary human cells in cultures for reinfusion into patients has not been addressed. We screened a variety of commercially available polymer (15 types), metal (four types), and glass substrates for their ability to support expansion of hematopoietic cells when cultured under conditions that would be encountered in a clinical setting. Cultures of peripheral blood (PB) CD34+ cells and mononuclear cells (MNC) were evaluated for expansion of total cells and colony-forming unit-granulocyte monocyte (CFU-GM; progenitors committed to the granulocyte and/or monocyte lineage). Human hematopoietic cultures in serum-free medium were found to be extremely sensitive to the substrate material. The only materials tested that supported expansion at or near the levels of polystyrene were tissue culture polystyrene, Teflon perfluoroalkoxy, Teflon fluorinated ethylene propylene, cellulose acetate, titanium, new polycarbonate, and new polymethylpentene. MNC were less sensitive to the substrate materials than the primitive CD34+ progenitors, although similar trends were seen for expansion of the two cell populations on the substrates tested. CFU-GM expansion was more sensitive to substrate materials than was total cell expansion. The detrimental effects of a number of the materials on hematopoietic cultures appear to be caused by protein adsorption and/or leaching of toxins. Factors such as cleaning, sterilization, and reuse significantly affected the performance of some materials as culture substrates. We also used PB CD34+ cell cultures to examine the biocompatibility of gas-permeable cell culture and blood storage bags and several types of tubing commonly used with biomedical equipment

  6. Expression of vascular endothelial growth factor in cultured human dental follicle cells and its biological roles

    Institute of Scientific and Technical Information of China (English)

    Xue-peng CHEN; Hong QIAN; Jun-jie WU; Xian-wei MA; Ze-xu GU; Hai-yan SUN; Yin-zhong DUAN; Zuo-lin JIN

    2007-01-01

    Aim: To investigate the expression of vascular endothelial growth factor (VEGF) in cultured human dental follicle cells (HDFC), and to examine the roles of VEGF in the proliferation, differentiation, and apoptosis of HDFC in vitro. Methods: Immunocytochemistry, ELISA, and RT-PCR were used to detect the expression and transcription of VEGF in cultured HDFC. The dose-dependent and the time-course effect of VEGF on cell proliferation and alkaline phosphatase (ALP) activ-ity in cultured HDFC were determined by MTT assay and colorimetric ALP assay, respectively. The effect of specific mitogen-activated protein kinase (MAPK) inhibitors (PD98059 and U0126) on the VEGF-mediated HDFC proliferation was also determined by MTT assay. The effect of VEGF on HDFC apoptosis was measured by flow cytometry. Results: VEGF was transcribed and expressed in cultured HDFC. VEGF at 10-300 μg/L significantly increased HDFC proliferation and ALP activity compared to the control. Following 1, 3, 5, or 7 d of stimulation, VEGF induced a significant increase in HDFC proliferation compared with the corresponding control, while VEGF was effective at increasing ALP activity at the incubation time point of 3, 5, or 7 d. PD98059 and U0126 could attenuate the VEGF-mediated HDFC proliferation. Fewer apoptotic cells were observed in the VEGF-treated groups compared to the controls, although the difference was not statistically significant. Conclusion: VEGF is expressed in cultured HDFC, and at a proper concentration range can stimulate HDFC proliferation, induce HDFC to differentiate in a "cementoblast/osteoblast" pathway and protect HDFC from apoptosis. The MAPK signaling pathway might be involved in the VEGF-medi-ated HDFC proliferation.

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

    International Nuclear Information System (INIS)

    Application of the Single Cell Gel Electrophoresis (SCGE) Assay to Genotoxicity Evaluation in Plants and Animals. Recently, the importance of ionizing radiation and chemicals has been recognized since radio- and chemical therapy is directly related to the control of various diseases such as cancer. Radiation and the chemicals can cause biological damages while they have great applicability. It is of necessity to analyze rapidly, easily and accurately the biological effects, especially DNA damage due to those factors. Recently SCGE (single cell gel electrophoresis assay, alias comet assay) has been developed for the efficient evaluation of DNA damage. In this report, the comprehensive review will be given on the rationale, the technical applications and the advantages and shortcomings of SCGE assay. This method can be directly applied to study on toxicity, cancer, and aging in terms of the evaluation of DNA damages due to radiation and chemicals on human cellular level. It is also suggested that comet assay be used for testing genotoxicity of suspected substances, detecting irradiated foods, screening radioprotective candidates, and studying DNA repair process in various biological systems

  8. 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.

  9. Sensitive hepatocyte-mediated assay for the metabolism of nitrosamines to mutagens for mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Jones, C.A.; Huberman, E.

    1980-02-01

    A sensitive cell-mediated assay has been developed for testing mutagenesis in Chinese hamster V79 cells by carcinogenic nitrosamines. Mutations were characterized by resistance to ouabian and 6-thioguanine. Since V79 cells do not metabolize nitrosamines, mutagenesis in the V79 cells was tested in the presence of primary hepatocytes capable of metabolizing nitrosamines. The hepatocytes were isolated after collagenase and hyaluronidase digestion of liver slices. All seven liver carcinogens of the nine tested nitrosamines exhibited a mutagenic response in this cell-mediated assay. The potent liver carcinogens nitrosodimethylamine, nitrosodiethylamine, nitrosoethylmethylamine, and nitrosodipropylamine could be detected with doses as low as 1 ..mu..m. The noncarcinogenic nitrosodiphenylamine was not mutagenic. Nitrosomethoxymethylamine was the only nitrosamine that exhibited mutagenic activity in the absence of hepatocytes, and this activity was diminished in the presence of hepatocytes. It is suggested that the use of hepatocytes prepared by the slicing method for carcinogen metabolism and mutable V79 cells offers a highly sensitive assay for determining the mutagenic potential of carcinogenic nitrosamines and probably of other classes of hazardous chemicals occurring in the environment.

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

    Science.gov (United States)

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

    2009-01-01

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

  11. Culturing Schwann Cells from Neonatal Rats by Improved Enzyme Digestion Combined with Explants-culture Method.

    Science.gov (United States)

    Liu, Di; Liang, Xiao-Chun; Zhang, Hong

    2016-08-01

    Objective To develop an improved method for culturing Schwann cells(SCs) by using both enzyme digestion and explants-culture approaches and compared with traditional explants-culture method and general hemi-explants-culture method. Methods Bilaterally sciatic nerves and brachial plexus nerves were dissected from 3 to 5-day-old neonatal SD rats and explants-culture method,general hemi-explants-culture method,and improved enzyme digestion combined with explants-culture method were adopted to culture SCs,respectively. SCs were digested and passaged after 7 days in culture and counted under the microscope. The purity of SCs was identified by S-100 immunofluorescence staining. Results The SCs of improved method group grew fastest and the total number of cells obtained was(1.85±0.13)×10(6);the SCs of the hemi-explants-culture method group grew slower than the improved method group and the total number of cells obtained was (1.10±0.10)×10(6);the SCs of the explants-culture method group grew slowest and the total number of cells obtained was (0.77±0.03)×10(6).The total number of cells obtained showed significant difference among the three groups(Pculture method group,and (74.50±4.23)% in the explants-culture method group(Pculture method can obtain sufficient amount of high-purity SCs in a short time and thus may be applied in further research on peripheral nerve regeneration.

  12. Chemotherapy in heterogeneous cultures of cancer cells with interconversion

    International Nuclear Information System (INIS)

    Recently, the interconversion between differentiated and stem-like cancer cells has been observed. Here, we model the in vitro growth of heterogeneous cell cultures in the presence of interconversion from differentiated cancer cells to cancer stem cells (CSCs), showing that, by targeting only CSC with cytotoxic agents, it is not always possible to eradicate cancer. We have determined the kinetic conditions under which cytotoxic agents in in vitro heterogeneous cultures of cancer cells eradicate cancer. In particular, we have shown that the chemotherapeutic elimination of in vitro cultures of heterogeneous cancer cells is effective only if it targets all cancer cell types, and if the induced death rates for the different subpopulations of cancer cell types are large enough. The quantitative results of the model are compared and validated with experimental data. (paper)

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

    Science.gov (United States)

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

    2016-07-01

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

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

    Science.gov (United States)

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

    2016-01-01

    Mesenchymal stromal/stem cells (MSC) are promising candidates for the development of cell-based therapies for various diseases and are currently being evaluated in a number of clinical trials (Sharma et al., Transfusion 54:1418-1437, 2014; Ikebe and Suzuki, Biomed Res Int 2014:951512,